Fusion proteins comprising an interleukin-2 mutein and an antigen-binding fragment to serum albumin, and compositions and uses thereof

Abstract

A recombinant fusion protein, as well as compositions and uses thereof, is provided, which comprises an interleukin-2 mutein and an antigen-binding fragment for serum albumin. Due to its increased in vivo half-life, the recombinant fusion protein has the advantage of a relatively long administration cycle. Due to the selective activity of the recombinant fusion protein against regulatory T cells, the recombinant fusion protein can be effectively used for the treatment of various immune diseases and inflammatory neurological diseases, including inflammatory bowel disease and systemic lupus erythematosus. TIFF2025536327000083.tif71128

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to Korean Application No. 10-2022-0135135, filed October 19, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing This application contains a Sequence Listing, which has been submitted via EFS-Web in XML format and is incorporated herein by reference in its entirety. This XML copy, created on October 12, 2023, is named 2662-0008WO01_SEQL_ST26 and is 117,497 bytes in size.

[0003] 1. Field The present disclosure relates to fusion proteins comprising an interleukin-2 mutein and an antigen-binding fragment to serum albumin, and compositions and uses thereof. [Background technology]

[0004] 2. Description of Related Technology Autoimmune diseases are caused by abnormalities in the human immune system, resulting in an incorrect immune response to normal chemicals or certain cells. The human immune system has a powerful ability to recognize invading microorganisms and cancer cells as foreign antigens and attack and eliminate them, but it does not attack its own cells due to self-tolerance. However, when the immune system's self-tolerance is broken, autoreactive T cells that react to the body's own cells (or self-antigens) are activated and autoantibodies are produced, causing the body to constantly destroy its own cells and triggering inflammation and immune responses.

[0005] Although the causes of autoimmune diseases have not been clearly identified, the causes can be broadly divided into genetic, acquired, and environmental factors. Recent studies have shown that autoimmune diseases reduce the number of regulatory T cells (Treg cells), and therefore, increasing Treg cells is considered an important therapeutic strategy.

[0006] Treg cells are a class of CD4+CD25+ T cells that suppress the activity of several immune cells. Treg cells are important for maintaining immune system homeostasis, playing a major role in maintaining tolerance to self-antigens and regulating immune responses to foreign antigens. Defects in the number or function of Treg cells have been identified in many autoimmune and inflammatory diseases, including systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and graft-versus-host disease (GVHD). Therefore, the development of therapies that activate the number and function of Treg cells is of great interest.

[0007] Interleukin-2 (IL-2) is known to play an important role in the activation of Treg cells, and Treg cells express high and constant levels of IL-2Rαβγ, which is composed of the IL-2 receptor α subunit (IL-2RA, also known as CD25), the IL-2 receptor β subunit (IL-2RB, also known as CD122), and the IL-2 receptor γ subunit (IL-2RG, also known as CD132). The ILTOO Company is conducting clinical trials for autoimmune diseases such as type 1 diabetes and lupus by using low-dose IL-2 as a strategy to target only Treg cells. This is because Treg cells expressing IL-2Rαβγ respond to IL-2 at lower concentrations than many other immune cell types (Klatzmann D, 2015, Nat Rev Immunol. 15:283-94). However, there is a need to improve the safety, tolerability, and patient convenience of even these low-dose IL-2 administrations, thus necessitating the development of new mechanisms of non-toxic treatment.

[0008] IL-2 is a cytokine produced by CD4+ helper T cells that induces proliferation and activation of all T cells, including Treg cells and NK cells. Among immune cells, Treg cells express the IL-2 receptor (IL-2R) on their surface in the IL-2Rαβγ trimer, whereas CD4+ effector T cells, CD8+ effector T cells, and NK cells express the IL-2 receptor in the IL-2Rβγ form.

[0009] Therefore, if we develop an IL-2 mutein that selectively binds to the IL-2Rαβγ trimer rather than the IL-2Rβγ dimer, we can selectively activate Treg cells but not CD8+ T cells and NK cells, thereby stabilizing the immune system of patients with abnormally overactive autoimmune diseases.

[0010] There is a need to develop fusion proteins for improved IL-2m therapy and increased patient convenience.

[0011] Antigen-binding fragments that bind to serum albumin are provided in U.S. Patent Nos. 9,879,077 and 11,773,176, each of which is incorporated herein by reference in its entirety. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 9,879,077 [Patent Document 2] U.S. Patent No. 11,773,176 [Non-patent literature]

[0013] [Non-Patent Document 1] Klatzmann D, 2015, Nat Rev Immunol. 15:283-94 Summary of the Invention

[0014] overview Disclosed herein is a recombinant fusion protein comprising an interleukin-2 mutein (IL-2m) and an antigen-binding fragment (Fab) that binds to serum albumin. In some embodiments, IL-2m and the Fab against serum albumin are linked to each other by a linker. In some embodiments, the C-terminal region of the heavy chain of the Fab against serum albumin and IL-2m are linked to each other by a linker. In other embodiments, IL-2m, a linker, a heavy chain comprising the heavy chain region of the Fab against serum albumin, and a light chain comprising the light chain region of the Fab against serum albumin are linked by a non-covalent bond.

[0015] In some embodiments, the linker comprises 1 to 50 amino acids. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 16, 70-85, and 88. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 84.

[0016] The IL-2m, linker, and heavy chain may comprise the amino acid sequence of SEQ ID NO:3 or 4. The recombinant fusion protein may comprise the amino acid sequences of SEQ ID NO:3 or 4 and SEQ ID NO:13. The IL-2m may comprise an amino acid sequence having at least 90% identity to SEQ ID NO:1 or 2. The IL-2m may comprise the amino acid sequence of SEQ ID NO:1. The IL-2m may comprise the amino acid sequence of SEQ ID NO:2.

[0017] In some embodiments, the Fab comprises: (a) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025536327000002.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025536327000003.tif4128; (b) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000004.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000005.tif4128; (c) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO:26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000006.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000007.tif4128; (d) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000008.tif4128; and heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000009.tif4128; (e) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO:32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000010.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (f) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO:35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000011.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000012.tif4128 a heavy chain comprising a heavy chain variable domain comprising (g) light chain CDR1 containing the amino acid sequence of TIFF2025536327000013.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (h) light chain CDR1 containing the amino acid sequence of TIFF2025536327000014.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000015.tif4128; (i) light chain CDR1 containing the amino acid sequence of TIFF2025536327000016.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000017.tif4128; (j) light chain CDR1 containing the amino acid sequence of TIFF2025536327000018.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (k) light chain CDR1 containing the amino acid sequence of TIFF2025536327000019.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (l) light chain CDR1 containing the amino acid sequence of TIFF2025536327000020.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000021.tif4128 a light chain comprising a light chain variable domain comprising may include:

[0018] The Fab may comprise a heavy chain variable domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and a light chain variable domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0019] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. The heavy chain variable domain may comprise the amino acid sequence of SEQ ID NO:60. In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. The light chain variable domain may comprise the amino acid sequence of SEQ ID NO:67.

[0020] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:68. In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91. A Fab may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:91. A Fab may comprise a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO:68 linked to a heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:69 or 91 linked to a light chain variable domain.

[0021] Disclosed herein are nucleic acids encoding the recombinant fusion proteins disclosed herein.Further disclosed herein are expression vectors comprising the nucleic acids disclosed herein.Disclosed herein are cells transformed with the expression vectors disclosed herein.

[0022] Disclosed herein are compositions comprising the recombinant fusion proteins disclosed herein and a carrier. Further disclosed herein are pharmaceutical compositions comprising the recombinant fusion proteins disclosed herein and a pharmaceutically acceptable carrier. Disclosed herein are kits comprising the compositions or pharmaceutical compositions disclosed herein and a label containing instructions for use.

[0023] Disclosed herein is a method for preventing or treating an autoimmune disease or an inflammatory neurological disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition disclosed herein. Further disclosed herein is the use of a pharmaceutical composition for preventing or treating an autoimmune disease or an inflammatory neurological disease, the pharmaceutical composition comprising a recombinant fusion protein disclosed herein as an active ingredient. In some embodiments, the autoimmune disease is selected from the group consisting of Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematosus (SLE), asthma, edema, delayed-type allergy (type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemia-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy, and the inflammatory neurological disease is selected from the group consisting of amyotrophic lateral sclerosis and Parkinson's disease.

[0024] Additional aspects will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of presented aspects of the present disclosure.

[0025] According to some aspects of the present disclosure, a recombinant fusion protein comprises an interleukin-2 mutein and an antigen-binding fragment for serum albumin. In some embodiments, the recombinant fusion protein consists of a heavy chain containing 366 amino acids and a light chain having 215 amino acids. Furthermore, the recombinant fusion protein does not have glycosylation in the antigen-binding fragment for serum, but the IL-2 protein has one NO-glycosylation site. Thus, the recombinant fusion protein according to some aspects may contain glycosylation. [Brief explanation of the drawings]

[0026] These and other aspects, features, and advantages of particular embodiments of the present disclosure will become more apparent from the following description taken in conjunction with the accompanying drawings. [Figure 1A] For preparing recombinant fusion proteins, FIG. 1A shows an expression vector for the heavy chain, and FIG. 1B shows an expression vector for the light chain. [Figure 1B] See legend to Figure 1A. [Figure 2] The structure of SAFA-IL-2m (Fab-IL-2m) is shown. [Figure 3] Figures 3A and 3B show the size of certain aspects of SAFA-IL-2m and efavaleukin-α analog proteins at 1 μg / well under reducing (R), non-reducing with heat (NR(B)), and non-reducing without heat (NR(NB)) conditions by SDS-PAGE. [Figure 4A] 4A and 4B show the purity of SAFA-IL-2m2 and efavaleukin-α analog proteins by SE-HPLC. [Figure 4B] See legend to Figure 4A. [Figure 5A] 5A-5C show the binding ability of SAFA-IL-2m to human serum albumin, monkey serum albumin, and mouse serum albumin. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] 6A and 6B are graphs of the measurement of activity by SAFA-IL2m in the HEK-blue IL-2 reporter cell line. [Figure 7] 7A and 7B show the binding activity of SAFA-IL-2m to CD25 and CD122. [Figure 8] 1 shows a graph of SAFA-IL-2m dose response measurement of phospho-STAT5 signaling in regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells isolated from mouse spleens. [Figure 9] 1 shows a graph of SAFA-IL-2m dose response measurement of phospho-STAT5 signaling in regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells using human peripheral blood mononuclear cells. [Figure 10]1 shows a graph of Ki67 proliferation assay of SAFA-IL-2m dose response in regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells using human peripheral blood mononuclear cells. [Figure 11] Figure 1 shows graphs of the ratio of regulatory T cells to CD4+ Tconv cells, the ratio of regulatory T cells to CD8+ T cells, and the ratio of regulatory T cells to NK cells after a single intraperitoneal injection and isolation of cells from the spleen, inguinal lymph nodes, and mesenteric lymph nodes of SAFA-IL-2m-treated mice and the positive control group. [Figure 12] 1 is a graph of the immune response of SAFA-IL-2m in immunized marmosets. [Figure 13A] Figures 13A and 13B are graphs of Disease Activity Index (DAI) scores, changes in intestinal length, and cytokine levels in a mouse model with dextran sulfate sodium (DSS)-induced inflammatory bowel disease after administration of SAFA-IL-2m and a positive control to the mouse model. [Figure 13B] See legend to Figure 13A. [Figure 14] 14A and 14B show the time course of blood concentrations of SAFA-IL-2m after intravenous and subcutaneous administration of SAFA-IL-2m to monkeys. [Figure 15] 1 shows the time course of changes in blood concentrations of SAFA-IL-2m after intravenous administration of SAFA-IL-2m to mice. [Figure 16] 16A and 16B show the time course of regulatory T cell proliferation following subcutaneous administration of SAFA-IL-2m to monkeys. [Figure 17A] 17A and 17B show the biodistribution of SAFA-IL-2m after subcutaneous administration in a collagen-induced arthritis mouse model. [Figure 17B] See legend to Figure 17A. [Figure 18A]18A-18C show the levels of proteinuria score, lymphadenopathy score, and anti-dsDNA IgG levels in the blood after administration of SAFA-IL-2m to the MRL / lpr mouse model of systemic lupus erythematosus. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19A] Figures 19A and 19B show immunofluorescence and hematoxylin and eosin (H&E) staining for IgG targeting kidney tissue obtained from the MRL / lpr mouse model of systemic lupus erythematosus after administration of SAFA-IL-2m according to the present invention to the mouse model. [Figure 19B] See legend to Figure 19A. DETAILED DESCRIPTION OF THE INVENTION

[0027] Detailed Description The term "interleukin-2 mutein (IL-2m)" as used herein refers to a compound that inhibits CD8 + It refers to a mutant form of IL-2 that selectively binds to the IL-2 receptor trimer, i.e., IL-2Rαβγ, expressed on regulatory T cells rather than the IL receptor dimer, i.e., IL-2Rβγ, expressed on effector T cells or NK cells. In some embodiments, IL-2m can comprise the amino acid sequence of SEQ ID NO:2.

[0028] The IL-2m, linker, and heavy chain may comprise the amino acid sequence of SEQ ID NO:3 or 4. In some embodiments, the IL-2m, linker, and heavy chain may comprise the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:5. The recombinant fusion protein may comprise the amino acid sequences of SEQ ID NO:3 or 4 and SEQ ID NO:2. The IL-2m may comprise an amino acid sequence having at least 90% identity to SEQ ID NO:1 or 2. The IL-2m may comprise the amino acid sequence of SEQ ID NO:1. The IL-2m may comprise the amino acid sequence of SEQ ID NO:2.

[0029] The term "serum albumin," as used herein, refers to a protein that constitutes the basic substance of cells and plays an important role in maintaining osmotic pressure between blood vessels and tissues by retaining bodily fluids within blood vessels. Additionally, the term "antigen-binding fragment against serum albumin" refers to an anti-serum albumin antibody or antigen-binding fragment of an antibody molecule, each of which specifically binds to an epitope on serum albumin. Antigen-binding fragments of antibodies or antibody fragments refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, Fv, etc. Among antibody fragments, Fab has a structure that contains light and heavy chain variable regions, a light chain constant region, and the first heavy chain constant region (CH1), i.e., it has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 antibodies are generated by forming disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region. Recombinant techniques for producing Fv fragments are disclosed in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. Two-chain Fvs contain heavy-chain variable regions and light-chain variable regions that are non-covalently linked to each other, while single-chain Fvs (scFvs) generally contain heavy-chain variable regions and light-chain variable regions that are covalently linked via a peptide linker or directly linked at the C-terminus to form a dimer-like structure similar to two-chain Fvs. Such antibody fragments can be obtained using proteolytic enzymes (e.g., Fab fragments can be obtained by limited digestion of whole antibodies with papain, and F(ab') fragments can be obtained by pepsin cleavage) or can also be produced by recombinant genetic technology.

[0030] In some embodiments, the antigen-binding fragment against serum albumin may comprise a light chain region comprising the amino acid sequence of SEQ ID NO:13; and a heavy chain region comprising the amino acid sequence of SEQ ID NO:1.

[0031] In some embodiments, the Fab heavy chain may comprise the leader amino acid sequence of SEQ ID NO: 19, which is cleaved after production of the fusion protein, i.e., the final fusion protein does not contain SEQ ID NO: 19. The Fab heavy chain may comprise the leader amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:20.

[0032] In some embodiments, the Fab light chain may include the leader amino acid sequence of SEQ ID NO:21, which is cleaved after production of the fusion protein, ie, the final fusion protein does not contain SEQ ID NO:21.

[0033] The term "recombinant fusion protein," as used herein, refers to a protein in which two or more proteins are artificially linked. In some embodiments, this term refers to a protein in which an IL-2 protein is linked to an antigen-binding fragment of serum albumin, i.e., an anti-serum albumin Fab antibody fragment. Such a recombinant fusion protein can be obtained by chemical synthesis or by expression and purification using genetic recombination methods after determining each partner. In some embodiments, the recombinant fusion protein can be obtained by expressing a fusion gene (expression vector) in a cell expression system in which a gene sequence encoding an IL-2 protein is linked to a gene sequence encoding an anti-serum albumin Fab antibody fragment. In such a recombinant fusion protein, the IL-2 protein and the anti-serum albumin Fab antibody fragment can be linked to each other directly or via a connector such as a linker. In some embodiments, the recombinant fusion protein can be non-covalently linked to the IL-2 protein, a linker, a heavy chain comprising the heavy chain region of the antigen-binding fragment of serum albumin, and a light chain comprising the light chain region of the antigen-binding fragment of serum albumin. For example, the recombinant fusion protein can include a peptide having the amino acid sequence of SEQ ID NO:3 or 4 and a peptide having the amino acid sequence of SEQ ID NO:13.

[0034] The term "linker," as used herein, refers to a peptide inserted between proteins to enhance the activity of each protein. In this context, the proteins are linked by increasing their structural flexibility during the preparation of a recombinant fusion moiety by linking an IL-2 protein and an anti-serum albumin Fab antibody fragment. There are no limitations on the type or number of amino acids of the linker, as long as the linker can minimize immune reactions. For example, the linker can contain 1 to 20 amino acids, 1 to 15 amino acids, 1 to 10 amino acids, or 1 to 8 amino acids. In some embodiments, the linker can link the IL-2 protein to the C-terminus of the heavy chain region of the serum albumin antigen-binding fragment. For example, the linker can contain the amino acids of SEQ ID NO:84.

[0035] Disclosed herein is a recombinant fusion protein comprising an interleukin-2 mutein (IL-2m) and an antigen-binding fragment (Fab) that binds to serum albumin. In some embodiments, IL-2m and the Fab against serum albumin are linked to each other by a linker. In some embodiments, the C-terminal region of the heavy chain of the Fab against serum albumin and IL-2m are linked to each other by a linker. In other embodiments, IL-2m, a linker, a heavy chain comprising the heavy chain region of the Fab against serum albumin, and a light chain comprising the light chain region of the Fab against serum albumin are linked by a non-covalent bond.

[0036] In some embodiments, the linker comprises 1 to 50 amino acids. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 16, 70-85, and 88. In some embodiments, the linker has the amino acid sequence of SEQ ID NO: 84.

[0037] The IL-2m, linker, and heavy chain may comprise the amino acid sequence of SEQ ID NO:3 or 4. The recombinant fusion protein may comprise the amino acid sequence of SEQ ID NO:3 or 4 and SEQ ID NO:13. The IL-2m may comprise an amino acid sequence having at least 90% identity to SEQ ID NO:2. The IL-2m may comprise the amino acid sequence of SEQ ID NO:2. The IL-2m may comprise the amino acid sequence of SEQ ID NO:1.

[0038] In some embodiments, the Fab comprises:

[0001] A heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025536327000022.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025536327000023.tif4128;

[0002] A heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000024.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000025.tif4128;

[0003] A heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO:26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000026.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000027.tif4128;

[0004] A heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO:29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000028.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000029.tif4128;

[0005] A heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO:32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000030.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or

[0006] A heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO:35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000031.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000032.tif4128 a heavy chain comprising a heavy chain variable domain comprising

[0007] light chain CDR1 containing the amino acid sequence of TIFF2025536327000033.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40);

[0008] light chain CDR1 containing the amino acid sequence of TIFF2025536327000034.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000035.tif4128;

[0009] light chain CDR1 containing the amino acid sequence of TIFF2025536327000036.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000037.tif4128;

[0010] light chain CDR1 containing the amino acid sequence of TIFF2025536327000038.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49);

[0011] light chain CDR1 containing the amino acid sequence of TIFF2025536327000039.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or

[0012] light chain CDR1 containing the amino acid sequence of TIFF2025536327000040.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000041.tif4128 a light chain comprising a light chain variable domain comprising may include:

[0039] In some embodiments, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0040] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:68. In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0041] In some embodiments, the heavy chain of the Fab comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 6.

[0042] In some embodiments of the recombinant proteins disclosed herein, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0043] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60.

[0044] In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67.

[0045] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67.

[0046] In some embodiments, the Fab comprises a heavy chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60, and a light chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, or 66 or 67, respectively, or any combination of heavy chain and light chain variable domains disclosed herein. For example, a Fab may comprise a heavy chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:60, and a light chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:67.

[0047] In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:68.

[0048] In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:69 or 91.

[0049] In some embodiments, the recombinant fusion protein may comprise a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3 or 4. In some embodiments, the Fab comprises a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6 (V H -C H1 In some embodiments, the Fab comprises a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain). L -Cκ domain).

[0050] In some embodiments, the recombinant fusion protein may comprise a heavy chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 3 or 4; and a light chain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. The recombinant protein may have significantly improved pharmacokinetic properties while maintaining the inherent biological activity of IL-2m.

[0051] In some embodiments, the Fab is selected from the group consisting of SEQ ID NO:6 (V H -C H1 a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain),L -Cκ domain).

[0052] In some embodiments, the heavy chain variable domain (VH) may comprise a heavy chain complementarity determining region (HCDR) comprising the amino acid sequence of SEQ ID NO:35, 36, or 37. For example, the heavy chain variable domain (VH) may comprise the amino acid sequence of SEQ ID NO:60, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:60. The C-terminus of the heavy chain variable domain (VH) may be linked to the heavy chain constant 1 domain (CH1). Here, the heavy chain constant 1 domain (CH1) may comprise the amino acid sequence of SEQ ID NO:68, or a sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:68, although the embodiment is not particularly limited thereto.

[0053] In some embodiments, the light chain variable domain (VL) may comprise a light chain complementarity determining region (LCDR) comprising the amino acid sequence of SEQ ID NO:52, 53, and 54. For example, the light chain variable domain (VL) may comprise the amino acid sequence of SEQ ID NO:67, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or more sequence identity to the amino acid sequence of SEQ ID NO:67. The C-terminus of the light chain variable domain (VL) may be linked to a light chain constant domain (CL), wherein the light chain constant domain (CL) may comprise the amino acid sequence of SEQ ID NO:69 or 91, for example, a sequence having at least 80%, 85%, 90%, and 95% or more sequence identity to the amino acid sequence of SEQ ID NO:69 or 91, although the embodiment is not particularly limited thereto.

[0054] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, a polypeptide is considered "substantially identical" if its amino acid sequence has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity. The percent identity of two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison). For example, the length of the sequence aligned for comparison is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleic acid or polypeptide sequences at corresponding positions are then compared. The determination of percent identity between two sequences and the comparison of these sequences can be achieved using mathematical algorithms. As is well known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms available in commercial computer programs, including, in particular, BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAS for amino acid sequences.

[0055] In some embodiments, in the fusion proteins disclosed herein, the heavy and light chains of the Fab are non-covalently linked.

[0056] In some embodiments, the antigen-binding fragment that binds to serum albumin can be linked to IL-2m via a linker. For example, the linker can link IL-2m to any one region selected from the C-terminus of the constant 1 domain of the heavy chain (CH1), the N-terminus of the variable domain of the heavy chain (VH), the C-terminus of the constant domain of the light chain (CL), and the N-terminus of the variable domain of the light chain (VL). Furthermore, the linker can be modified for use as needed.

[0057] For example, the linker can be a polypeptide containing 5 to 400 amino acids, 5 to 200 amino acids, or 5 to 200 amino acids. Such peptide linkers can contain Gly, Asn, and Ser residues, and can also contain neutral amino acids such as Thr and Ala. Amino acid sequences suitable for peptide linkers are known in the art. Furthermore, the copy number "n" can be adjusted to optimize the linker to achieve appropriate separation between functional moieties or to maintain necessary interactions between moieties. Other linkers are known in the art, such as a GS linker, which not only adds polar amino acid residues to improve water solubility but also adds amino acid residues such as T and A to maintain flexibility.

[0058] Thus, the linker can be a flexible linker containing G, S, and / or T, A residues. The linker can be (GpSs) n or (SpGs) n where, independently, p is an integer from 1 to 10, s is 0 or an integer from 0 to 10, p + s is an integer less than or equal to 20, and n is an integer from 1 to 20. More specifically, examples of linkers include: TIFF2025536327000042.tif41159, where n can be an integer from 1 to 20, or from 1 to 10.

[0059] Furthermore, the linker may comprise the amino acid sequence of SEQ ID NO: 84, 85 or 88, although the embodiment is not particularly limited thereto.

[0060] In some embodiments, the linker comprises 1 to 50 amino acids. In some embodiments, the linker comprises the amino acid sequence of any one of SEQ ID NOs:16, 70-85, and 88. In some embodiments, the recombinant fusion protein may comprise (i) a heavy chain comprising the heavy chain fragment SL335 of SEQ ID NO:6, the linker of SEQ ID NO:84, and the IL-2m of SEQ ID NO:1; and a light chain comprising the light chain fragment SL335 of SEQ ID NO:69 or 91, or (ii) a heavy chain comprising the heavy chain fragment SL335 of SEQ ID NO:6, the linker of SEQ ID NO:84, and the IL-2m of SEQ ID NO:2; and a light chain comprising the light chain fragment SL335 of SEQ ID NO:69 or 91. The recombinant fusion protein may have significantly improved pharmacokinetic properties while maintaining the inherent biological activity of IL-2m.

[0061] In some embodiments, a recombinant fusion protein (SAFA-IL-2m) is prepared, comprising: an antigen-binding fragment that binds to serum albumin; and hIL-2m fused to the C-terminus of the constant 1 domain of the heavy chain (CH1) and the constant domain of the light chain (CL). The recombinant fusion protein retains the biological activity of each factor, specifically, the ability to bind to human serum albumin and the IL-2m receptor, respectively, thereby extending the in vivo half-life and improving therapeutic effects, such as promoting ovarian growth.

[0062] In some embodiments, the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0063] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67.

[0064] In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91.

[0065] In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, the Fab comprises a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO:68 linked to a heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:69 or 91 linked to a light chain variable domain.

[0066] Antigen-binding fragments of antibodies, or antibody fragments, refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, Fv, etc. The Fab antibody fragment has a structure containing light and heavy chain variable regions, a light chain constant region, and a heavy chain constant region (CH) that has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH domain. F(ab')2 antibodies are generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments, the minimum antibody fragment containing only the heavy and light chain variable regions, are described in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In two-chain Fvs, the heavy chain variable region and the light chain variable region are linked via a non-covalent bond. In single-chain Fvs (scFvs), the heavy chain variable region and the light chain variable region are generally linked at the C-terminus directly or via a covalent peptide linker. Therefore, single-chain Fvs (scFvs) can have a dimer-like structure, similar to two-chain Fvs. Such antibody fragments can be obtained using protease hydrolases (for example, Fab fragments can be obtained by cleaving whole antibodies with papain, and F(ab')2 fragments can be obtained by cleaving whole antibodies with pepsin), and they can also be produced by recombinant gene technology.

[0067] In some embodiments, an antigen-binding fragment against serum albumin may comprise a heavy chain region comprising the amino acid sequence of SEQ ID NO:6; and a light chain region comprising the amino acid sequence of SEQ ID NO:13. In some embodiments, a nucleic acid molecule encoding a heavy chain region comprising the amino acid sequence of SEQ ID NO:6 may have the nucleotide sequence of SEQ ID NO:7 or 8. In some embodiments, a nucleic acid molecule encoding a light chain region comprising the amino acid sequence of SEQ ID NO:13 may have the nucleotide sequence of SEQ ID NO:14 or 15.

[0068] The Fab may comprise a heavy chain variable domain comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and a light chain variable domain comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54.

[0069] In some embodiments, the heavy chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. The heavy chain variable domain may comprise the amino acid sequence of SEQ ID NO:60. In some embodiments, the light chain variable domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. The light chain variable domain may comprise the amino acid sequence of SEQ ID NO:67.

[0070] In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:55, 56, 57, 58, 59, or 60, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. In some embodiments, the heavy chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:68. In some embodiments, the light chain constant domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO:69 or 91. A Fab may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:6 and a light chain comprising the amino acid sequence of SEQ ID NO:91. A Fab may comprise a heavy chain constant 1 domain comprising the amino acid sequence of SEQ ID NO:68 linked to a heavy chain variable domain; and a light chain constant domain comprising the amino acid sequence of SEQ ID NO:69 or 91 linked to a light chain variable domain.

[0071] As used herein, the term "antibody" (singular or plural) is a term of the art and can be used interchangeably herein to refer to a molecule having an antigen-binding site that specifically binds to an antigen. Antibodies include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies.

[0072] An antibody can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ) can be.

[0073] As used herein, the terms "bioeffector moiety," "antigen-binding domain," "antigen-binding region," "antigen-binding site," and similar terms refer to the portion of a recombinant protein (e.g., the complementarity-determining region (CDR)) that contains the amino acid residues that confer the recombinant protein its specificity for an antigen. The antigen-binding region can be derived from any animal species, including cats, rodents (e.g., mice, rats, or hamsters), and humans.

[0074] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of either the light or heavy chain, typically the amino-terminal 110-120 amino acids in the mature heavy chain and approximately 90-115 amino acids in the mature light chain, which vary extensively in sequence among antibodies and are used to determine the binding and specificity of a particular antibody for its specific antigen. Sequence variability is concentrated in regions called complementarity-determining regions (CDRs), while more highly conserved regions within the variable domain are called framework regions (FRs). While not wishing to be bound by a particular mechanism or theory, the CDRs of the light and heavy chains are believed to be primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FR).

[0075] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody. The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0076] As used herein, the term "heavy chain (HC or CH)" refers to both a full-length heavy chain and a fragment thereof, the full-length heavy chain comprising a variable region domain VH containing an amino acid sequence with sufficient variable region (VR) sequence to confer specificity for an antigen, and three constant region domains CH1, CH2, and CH3. As used herein, the term "light chain (LC or CL)" refers to both a full-length light chain and a fragment thereof, the full-length light chain comprising a variable region domain VL containing an amino acid sequence with sufficient VR sequence to confer specificity for an antigen, and a constant region domain CL.

[0077] The heavy and light chain constant domains can be derived from IgG1 antibody constant domains, and in any one or more thereof, the cysteine, an amino acid used in the disulfide bond between the light and heavy chain domains, can be conserved, deleted, or substituted with an amino acid residue other than cysteine. For example, the heavy chain constant domain can comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:68, and the light chain constant domain can comprise an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:69 or 91. Deletion or substitution of cysteines within the domains can contribute to improved expression levels of recombinant proteins in transformed cells during the process of producing the above-mentioned recombinant proteins. In some embodiments, (i) one or more cysteines in the heavy chain constant domain and / or (ii) one or more cysteines in the light chain constant domain located at the interchain disulfide bond between the light chain and the heavy chain are conserved, deleted, and / or substituted with an amino acid residue other than cysteine.

[0078] As used herein, the terms "constant region" and "constant domain" are interchangeable and have the meanings generally used in the art. The constant region is an antibody portion, for example, the carboxyl-terminal portion of the light chain and / or heavy chain, which is not directly involved in binding the antibody to an antigen, but can exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence than the immunoglobulin variable domain.

[0079] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, and include subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0080] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, for example, kappa (Cκ) or lambda (Cλ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0081] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ), and the equilibrium association constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D is k off / k on is calculated from the quotient of A is k on / k off It is calculated from the quotient of k on refers to the association rate constant of, for example, an antibody to an antigen, and k off refers to, for example, the dissociation of an antibody against an antigen. on and k offcan be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0082] In some embodiments, the binding affinity of the recombinant fusion proteins disclosed herein is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold higher than that of human IL-2m, or any range therein, e.g., 2-fold to 10-fold higher.

[0083] As used herein, "epitope" is a term used in the art and refers to the localized region of an antigen that an antibody can specifically bind to.Epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or epitope can be, for example, a sequence of two or more non-contiguous regions of a polypeptide (single or multiple polypeptides) (stereoscopic, non-linear, discontinuous, or non-contiguous epitope).In some embodiments, the epitope that an antibody binds to can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange combined with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assay, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). In the case of X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson, A. (1976) J. Biol. Chem. 251:6300-6303).Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.,; US 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis techniques, see, for example, Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085. In some embodiments, the epitope of the antibody is determined using alanine scanning mutagenesis studies.

[0084] As used herein, the terms "immunospecifically bind," "immunospecifically recognize," "specifically bind," and "specifically recognize" are similar terms in the context of antibodies, and such binding refers to a molecule that binds to an antigen (e.g., an epitope, immune complex, or binding partner of an antigen-binding site) as understood by one of skill in the art. For example, a molecule that specifically binds to an antigen can generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, a molecule that immunospecifically binds to an antigen has a K A At least 2 logs, 2.5 logs, 3 logs, 4 logs or more greater than K A It binds to the antigen.

[0085] In some embodiments, molecules that immunospecifically bind to an antigen do not cross-react with other proteins under similar binding conditions. In some embodiments, molecules that immunospecifically bind to an antigen do not cross-react with other proteins. In some embodiments, provided herein are recombinant proteins that bind to a specified antigen with higher affinity than to another unrelated antigen. In certain embodiments, provided herein are recombinant proteins that bind to a specified antigen (e.g., human serum albumin) with 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more affinity than to another unrelated antigen, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion. In some embodiments, the degree of binding of a recombinant protein described herein to an unrelated protein is less than 10%, 15%, or 20% of the binding of an antibody to the specified antigen, as measured, for example, by radioimmunoassay.

[0086] In some embodiments, provided herein are recombinant proteins that bind to antigens of various species, such as cats, rodents (e.g., mice, rats, or hamsters), and humans. In some embodiments, provided herein are recombinant proteins that bind to human antigens with higher affinity than to antigens of other species. In certain embodiments, provided herein are recombinant proteins that bind to human antigens with 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more higher affinity than to other species, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion. In some embodiments, the recombinant proteins described herein that bind to human antigens will bind to antigen proteins of other species with less than 10%, 15%, or 20% of the binding of an antibody to human antigen proteins, as measured, for example, by radioimmunoassay, surface plasmon resonance, or equilibrium binding exclusion.

[0087] As used herein, the term "host cell" can be any type of cell, for example, a primary cell, a cell in culture, or a cell derived from a cell line. In some embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to, for example, possible mutations or environmental influences that may occur later, or the integration of the nucleic acid molecule into the host cell genome.

[0088] In some aspects, the recombinant proteins described herein can be described by their VL domain alone, their VH domain alone, their three VL CDRs alone, or their three VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910-8915, which is incorporated herein by reference in its entirety, describing the humanization of a murine anti-αvβ3 antibody by identifying complementary light or heavy chains from a human light or heavy chain library, respectively, to produce humanized antibody variants with similar or higher affinity than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a specific VL domain (or VH domain) to screen a library for complementary variable domains. The screen yielded 14 new partners for the specific VH domain and 13 new partners for the specific VL domain that were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a specific VH domain to screen a library (e.g., a human VL library) for complementary VL domains; the selected VL domains could then be used to guide the selection of additional complementary (e.g., human) VH domains.

[0089] In one aspect, provided herein is a recombinant protein that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VL CDR in the VL. In one aspect, provided herein is an antibody that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VH CDR in the VH. In one aspect, provided herein is an antibody that specifically binds to serum albumin (e.g., human serum albumin) and comprises a Chothia VL CDR in the VL and a Chothia VH CDR in the VH. In one embodiment, an antibody that specifically binds to serum albumin (e.g., human serum albumin) comprises one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In one embodiment, provided herein is an antibody that specifically binds to serum albumin and comprises a combination of Kabat and Chothia CDRs.

[0090] In certain aspects, the CDRs of an antibody can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.).

[0091] In some embodiments, the position of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied by 1, 2, 3, 4, 5, or 6 amino acid positions, so long as immunospecific binding to an antigen is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). For example, the positions defining the CDRs of an antibody described herein may be varied by shifting the N-terminal and / or C-terminal boundaries of the CDRs by 1, 2, 3, 4, 5, or 6 amino acids relative to the CDR positions of an antibody described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the length of one or more CDRs along the VH (e.g., CDR1, CDR2, or CDR3) and / or VL (e.g., CDR1, CDR2, or CDR3) regions of an antibody described herein may be varied (e.g., made shorter or longer) by 1, 2, 3, 4, 5, or more amino acids, so long as immunospecific binding to the antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).

[0092] In some embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be 1, 2, 3, 4, 5 or more amino acids shorter than one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be 1, 2, 3, 4, 5 or more amino acids longer than one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In other embodiments, the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be extended by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%).In other embodiments, the amino terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein may be shortened by 1, 2, 3, 4, 5 or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). In some embodiments, the carboxy terminus of a VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and / or VH CDR3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein, so long as immunospecific binding to an antigen(s) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%). Any method known in the art, for example, the binding assays and conditions described in the "Examples" section herein, can be used to determine whether immunospecific binding to an antigen(s) is maintained.

[0093] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0094] The recombinant proteins disclosed herein can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as glucose oxidase; iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In), and technetium (99 These include radioisotopes such as Tc; luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labeled antibodies can be used to detect antigen proteins.

[0095] Polynucleotides, vectors, and cells According to some aspects of the present disclosure, there are provided nucleic acids encoding the recombinant fusion proteins, expression vectors comprising the nucleic acids, and cells transformed with the expression vectors.

[0096] The nucleic acids, expression vectors, and transformed cells either directly contain the recombinant fusion proteins or nucleic acids encoding the recombinant fusion proteins, or use these, and therefore a description of the common features among them will be omitted.

[0097] For example, recombinant fusion proteins can be produced by isolating nucleic acids encoding the recombinant fusion proteins. After isolating the nucleic acids, the resulting nucleic acids can be inserted into replicable vectors for further cloning (i.e., DNA amplification) or further expression. In this regard, some aspects provide vectors containing nucleic acids.

[0098] As used herein, the term "nucleic acid" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the term "nucleotide," which is the basic structural unit in nucleic acids, includes not only natural nucleotides but also analogs in which the sugar or base moiety is modified.

[0099] Nucleic acid is also understood to include a nucleotide sequence that shows substantial identity to the nucleotide sequence. Substantial identity refers to a nucleotide sequence that shows at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology when the nucleotide sequence of the present disclosure and another sequence are aligned to correspond as closely as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0100] DNA encoding a recombinant fusion protein can be readily isolated or synthesized using conventional procedures (e.g., by using an oligonucleotide probe capable of specifically binding to DNA encoding the recombinant fusion protein). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0101] As used herein, the term "vector" refers to a plasmid vector as a means for expressing a target gene in a host cell, examples of which include plasmid vectors; cosmid vectors; viral vectors, such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, the nucleic acid encoding the recombinant fusion protein can be operably linked to a promoter.

[0102] As used herein, the phrase "operably linked" refers to a functional linkage between a control sequence for nucleic acid expression (e.g., a promoter, a signal sequence, or an array of binding sites for transcriptional regulators) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0103] In some embodiments, when prokaryotic cells are used as hosts, a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence are generally included. In one or more embodiments, for example, when eukaryotic cells are used as hosts, promoters derived from the genomes of mammalian cells (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and these promoters generally have polyadenylation sequences as transcription termination sequences. In one or more embodiments, the vector can be fused to other sequences to facilitate purification of recombinant fusion proteins expressed from the vector. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6xHis (hexahistidine; Qiagen, USA), etc. The vector may contain, as a selectable marker, an antibiotic resistance gene commonly used in the art, such as genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0104] Some aspects of the present disclosure provide cells transformed with the aforementioned vectors. Cells used to produce the recombinant fusion proteins of the present disclosure can be prokaryotic cells, yeast cells, or higher eukaryotic cells, but the embodiments are not particularly limited thereto. Prokaryotic host cells include Escherichia coli, Bacillus strains such as B. subtilis and B. thuringiensis, Streptomyces, Pseudomonas (e.g., P. putida), Proteus mirabilis, and Staphylococcus (e.g., S. carnosus). However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO (GS null CHO-K1), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, WI-38, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0105] As used herein, the term "transformation" refers to a molecular biological technique in which a DNA fragment or a plasmid carrying a foreign gene of a different type from that of the original cell enters a cell and combines with the DNA present in the original cell, thereby changing the genetic traits of the original cell. Transformation can also refer to the insertion of an expression vector containing a recombinant fusion protein gene into a host cell.

[0106] Disclosed herein are nucleic acid molecules that encode the recombinant proteins disclosed herein.

[0107] Disclosed herein are expression vectors that include the nucleic acid molecules disclosed herein.

[0108] Disclosed herein are cells transformed with the expression vectors disclosed herein.

[0109] The nucleic acids, expression vectors, and transformed cells either directly contain or use the above-mentioned recombinant proteins or nucleic acids encoding the recombinant proteins, and therefore a description common to them will be omitted.

[0110] For example, in some aspects, recombinant proteins can be produced by isolating nucleic acids encoding the recombinant proteins. The nucleic acids are isolated and inserted into replicable vectors for further cloning (DNA amplification) or further expression. Based on this, other aspects relate to vectors containing the nucleic acids.

[0111] As used herein, the term "nucleic acid" or "nucleic acid molecule" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides as the basic units of nucleic acids include not only naturally occurring nucleotides but also analogs having modified sugar or base moieties.

[0112] Nucleic acid is understood to include a nucleotide sequence that shows substantial identity to the nucleotide sequence. Substantial identity means a nucleotide sequence that shows at least 80% homology, more specifically at least 90% homology, and most specifically at least 95% homology when the nucleotide sequence of the present disclosure and another optional sequence are aligned to correspond to each other as closely as possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0113] DNA encoding a recombinant protein is readily isolated or synthesized using conventional processes (e.g., by using an oligonucleotide probe capable of specifically binding to DNA encoding the recombinant protein). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0114] As used herein, the term "vector" includes, as a means for expressing a target gene in a host cell, a plasmid vector; a cosmid vector; a viral vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. In a vector, a nucleic acid encoding a recombinant protein is operably linked to a promoter.

[0115] "Operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., promoter, signal sequence, array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence directs the transcription and / or translation of the other nucleic acid sequence.

[0116] When prokaryotic cells are used as hosts, a strong promoter capable of directing transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and transcription / translation termination sequences are generally included. For example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionein promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or mammalian virus promoters (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and polyadenylation sequences are generally used as transcription termination sequences. In some cases, vectors can be fused with additional sequences to facilitate purification of recombinant proteins expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6X His (hexahistidine; Quiagen, USA), etc. The vector contains, as a selectable marker, an antibiotic resistance gene commonly used in the art, such as genes resistant to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0117] In yet another aspect, the present disclosure provides a cell transformed with the above-described vector. The cells used to produce the recombinant proteins of the present disclosure can be, but are not limited to, prokaryotic cells, yeast cells, or higher eukaryotic cells. Prokaryotic host cells can be used, for example, Escherichia coli, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus). However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO (GS null CHO-K1), CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0118] As used herein, the term "transformation" refers to a molecular biological technique in which a DNA fragment or plasmid carrying a foreign gene of a different type from that of the original cell changes the genetic make-up of a cell by invading the cell and combining with the DNA in the original cell. Transformation refers to the insertion of an expression vector containing a gene for a recombinant protein into a host cell.

[0119] Provided herein are nucleic acid molecules comprising nucleotide sequences encoding the recombinant proteins described herein (e.g., variable light chain regions and / or variable heavy chain regions) that immunospecifically bind to an antigen, as well as vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Provided herein are polynucleotides comprising nucleotide sequences encoding any of the antibodies provided herein, as well as vectors comprising such polynucleotide sequences, e.g., expression vectors for their efficient expression in host cells, e.g., mammalian cells.

[0120] As used herein, an "isolated" polynucleotide or nucleic acid molecule is one that is separated from other nucleic acid molecules present in the nucleic acid molecule's natural source (e.g., in a mouse or human). Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium if produced by recombinant technology, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the term "substantially free" includes preparations of polynucleotides or nucleic acid molecules that have less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In some embodiments, the nucleic acid molecule(s) encoding the antibodies described herein are isolated or purified.

[0121] Provided herein are polynucleotides comprising nucleotide sequences encoding antibodies that immunospecifically bind to an antigenic polypeptide (e.g., human serum albumin) and that comprise an amino acid sequence described herein, as well as antibodies that compete with such antibodies (e.g., in a dose-dependent manner) for binding to the antigenic polypeptide or that bind to the same epitope as such antibodies.

[0122] Provided herein is a polynucleotide comprising a nucleotide sequence encoding the light chain or heavy chain of the antibody described herein.The polynucleotide can comprise a nucleotide sequence encoding the light chain comprising the VL FR and CDR of the antibody described herein.The polynucleotide can comprise a nucleotide sequence encoding the heavy chain comprising the VH FR and CDR of the antibody described herein.

[0123] Provided herein are polynucleotides comprising nucleotide sequences encoding recombinant proteins comprising three VH chain CDRs (e.g., comprising VL CDR1, VL CDR2, and VL CDR3 of an antibody against human serum albumin described herein) and a Fab comprising three VH chain CDRs (e.g., comprising VH CDR1, VH CDR2, and VH CDR3 of an antibody against human serum albumin described herein).

[0124] Provided herein is a polynucleotide comprising a nucleotide sequence encoding a recombinant protein comprising a VL domain.

[0125] In certain embodiments, the polynucleotide described herein comprises a nucleotide sequence encoding a recombinant protein provided herein comprising a light chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:61, 62, 63, 64, 65, 66, or 67), wherein the antibody immunospecifically binds to serum albumin.

[0126] In certain embodiments, the polynucleotides described herein comprise a nucleotide sequence encoding an antibody provided herein comprising a heavy chain variable region comprising an amino acid sequence described herein (e.g., SEQ ID NO:55, 56, 57, 58, 59, or 60), wherein the antibody immunospecifically binds to serum albumin.

[0127] In specific aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies comprising a light chain and a heavy chain (e.g., separate light and heavy chains). With respect to the light chain, in some embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding a κ light chain. In other embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding a λ light chain. In still other embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody described herein comprising a human κ light chain or a human λ light chain. In some embodiments, the polynucleotides provided herein comprise a nucleotide sequence encoding an antibody that immunospecifically binds to serum albumin, wherein the antibody comprises a light chain, and wherein the amino acid sequence of the VL domain can comprise the amino acid sequence set forth in SEQ ID NO:61, 62, 63, 64, 65, 66, or 67, and wherein the constant region of the light chain comprises the amino acid sequence of a κ light chain constant region.

[0128] Also provided herein are polynucleotides encoding antibodies or fragments thereof that are optimized, for example, by codon / RNA optimization, substitution with heterologous signal sequences, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding antibodies or fragments thereof (e.g., light chains, heavy chains, VH domains, or VL domains) for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA can be performed accordingly by adapting the optimization methods described in, for example, U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498. For example, potential splice sites and instability elements (e.g., A / T or A / U-rich elements) within RNA can be mutated without changing the amino acids encoded by the nucleic acid sequence to increase the stability of RNA for recombinant expression. This modification can take advantage of the degeneracy of the genetic code, for example, by using alternative codons for the same amino acid. In some embodiments, conservative variations, e.g., altering one or more codons to encode a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid, may be desirable.

[0129] In certain embodiments, optimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL or VH domains) described herein can hybridize to antisense (e.g., complementary) polynucleotides of non-optimized polynucleotide sequences encoding the antibodies or fragments thereof (e.g., VL or VH domains) described herein. In specific embodiments, optimized nucleotide sequences encoding the antibodies or fragments described herein hybridize to antisense polynucleotides of non-optimized polynucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency conditions. In some embodiments, optimized nucleotide sequences encoding the antibodies or fragments thereof described herein hybridize to antisense polynucleotides of non-optimized nucleotide sequences encoding the antibodies or fragments thereof described herein under high stringency, intermediate, or low stringency hybridization conditions. Information regarding hybridization conditions has been described, see, e.g., US 2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference.

[0130] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of polynucleotides can be determined.The nucleotide sequences encoding the antibodies and modified versions of these antibodies described herein can be determined using methods well known in the art, that is, nucleotide codons known to encode specific amino acids are assembled in such a way as to generate nucleic acids encoding antibodies.Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier G et al., (1994), BioTechniques 17: 242-246), which simply involves synthesizing overlapping oligonucleotides containing portions of the sequence encoding antibodies, annealing and ligating these oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0131] Alternatively, polynucleotides encoding the antibodies or fragments thereof described herein can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acid containing sequences encoding the light and / or heavy chains of the antibody. Such PCR amplification methods can be used to obtain nucleic acid containing sequences encoding the variable light and / or variable heavy chain regions of the antibody. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning, e.g., to generate chimeric and humanized antibodies.

[0132] If a clone containing a nucleic acid encoding a particular antibody or fragment thereof is not available but the sequence of the antibody molecule or fragment thereof is known, nucleic acid encoding the immunoglobulin or fragment can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell that expresses the antibody, such as hybridoma cells selected to express an antibody described herein, or nucleic acid isolated therefrom, e.g., polyA+ RNA) by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for the particular gene sequence to identify cDNA clones from the cDNA library that encode the antibody, for example. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using any method well known in the art.

[0133] DNA encoding the recombinant proteins described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the recombinant protein). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain synthesis of the recombinant protein in the recombinant host cells.

[0134] To generate antibodies, PCR primers containing the VH or VL nucleotide sequence, restriction sites, and flanking sequences for protecting the restriction sites can be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. In some embodiments, the vector for expressing the VH or VL domain contains an EF-1α promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. Then, using techniques known to those skilled in the art, the heavy chain conversion vector and the light chain conversion vector are co-transfected into a cell line to generate a stable or transient cell line expressing a full-length antibody, such as an IgG.

[0135] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the murine sequences, or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0136] Also provided are polynucleotides that hybridize to the polynucleotides encoding the antibodies described herein under high, intermediate, or low stringency hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize to the polynucleotides encoding the VH domains and / or VL domains provided herein under high, intermediate, or low stringency hybridization conditions.

[0137] Hybridization conditions are described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65°C; hybridization under highly stringent conditions may involve hybridization to filter-bound nucleic acid in 6xSSC at about 45°C, followed by one or more washes in 0.1xSSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, for example, see Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3.

[0138] Another aspect provides a recombinant vector comprising a gene encoding IL-2m and a nucleic acid encoding an antigen-binding fragment of serum albumin. Yet another aspect provides a cell transformed with the vector.

[0139] Disclosed herein are nucleic acid molecules encoding a heavy chain region comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 6. Disclosed herein are nucleic acid molecules encoding a heavy chain region comprising a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 7 or 8.

[0140] Disclosed herein are nucleic acid molecules encoding a light chain region comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Disclosed herein are nucleic acid molecules encoding a light chain region comprising a nucleotide sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14 or 15.

[0141] In some embodiments, disclosed herein are nucleic acids that each encode the heavy chain region of SEQ ID NO:6 and the light chain region of SEQ ID NO:13. In some embodiments, the nucleic acid encoding the heavy chain region of SEQ ID NO:6 may be represented by SEQ ID NO:7 or SEQ ID NO:8, and the nucleic acid encoding the light chain region of SEQ ID NO:13 may be represented by SEQ ID NO:14 or SEQ ID NO:15.

[0142] (a) the promoter; (b) a first nucleic acid molecule encoding a light chain that binds to serum albumin, and (c) a second nucleic acid molecule encoding the heavy chain and a biologically active effector moiety (e.g., IL-2m) and a linker. An expression vector comprising: Further disclosed herein is an expression vector wherein the promoter, first nucleic acid sequence, and second nucleic acid molecule are operably linked, and the second nucleic acid molecule can encode one, two, three, four, five, six, or more biologically active effector moieties and a linker.

[0143] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding a heavy chain variable domain as disclosed herein and a heavy chain constant domain as disclosed herein.

[0144] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding an IL18BP as disclosed herein, a heavy chain variable domain as disclosed herein, and a heavy chain constant domain as disclosed herein.

[0145] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding a light chain variable domain as disclosed herein and a light chain constant domain as disclosed herein.

[0146] Also disclosed herein is an expression vector comprising: (a) the promoter; and (b) a nucleic acid molecule encoding IL-2m as disclosed herein, a light chain variable domain as disclosed herein, and a light chain constant domain as disclosed herein. One, two, three, or more expression vectors or nucleic acid molecules can be expressed to produce a desired recombinant protein.

[0147] In some embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a heavy chain, wherein the heavy chain comprises a heavy chain variable domain and a heavy chain constant domain, wherein the heavy chain variable domain comprises: (1) a heavy chain complementarity-determining domain 1 (CDR1) comprising the amino acid sequence of SYGIS (SEQ ID NO: 22); a heavy chain complementarity-determining domain 2 (CDR2) comprising the amino acid sequence of TIFF2025536327000043.tif4128; and heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence of TIFF2025536327000044.tif4128; (2) a heavy chain CDR1 comprising the amino acid sequence of SYGIS (SEQ ID NO:22); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000045.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000046.tif4128; (3) a heavy chain CDR1 comprising the amino acid sequence of NYGIH (SEQ ID NO: 26); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000047.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000048.tif4128; (4) a heavy chain CDR1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 29); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000049.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000050.tif4128; (5) a heavy chain CDR1 comprising the amino acid sequence of AYWIA (SEQ ID NO: 32); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000051.tif4128; and a heavy chain CDR3 comprising the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (6) a heavy chain CDR1 comprising the amino acid sequence of AYSMN (SEQ ID NO: 35); a heavy chain CDR2 comprising the amino acid sequence of TIFF2025536327000052.tif4128; and Heavy chain CDR3 containing the amino acid sequence of TIFF2025536327000053.tif4128.

[0148] A second nucleic acid molecule or vector is disclosed herein, which comprises a nucleic acid sequence encoding a recombinant protein comprising an antigen-binding fragment comprising a light chain, wherein the light chain comprises a light chain variable domain and a light chain constant domain, wherein the light chain variable domain comprises: (7) light chain CDR1 containing the amino acid sequence of TIFF2025536327000054.tif4128; a light chain CDR2 comprising the amino acid sequence of GASRLES (SEQ ID NO:39); and a light chain CDR3 comprising the amino acid sequence of QQSDSVPVT (SEQ ID NO:40); (8) light chain CDR1 containing the amino acid sequence of TIFF2025536327000055.tif4128; a light chain CDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000056.tif4128; (9) light chain CDR1 containing the amino acid sequence of TIFF2025536327000057.tif4128; a light chain CDR2 comprising the amino acid sequence of DASNRAT (SEQ ID NO:45); and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000058.tif4128; (10) light chain CDR1 containing the amino acid sequence of TIFF2025536327000059.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (11) light chain CDR1 containing the amino acid sequence of TIFF2025536327000060.tif4128; a light chain CDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO:48); and a light chain CDR3 comprising the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (12) light chain CDR1 containing the amino acid sequence of TIFF2025536327000061.tif4128; a light chain CDR2 comprising the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence of TIFF2025536327000062.tif4128.

[0149] For example, a nucleic acid molecule encoding IL-2m can be ligated to the first or second nucleic acid molecule or vector described above.

[0150] In other embodiments, the first nucleic acid molecule can comprise a nucleic acid sequence encoding a Fab comprising: a heavy chain variable domain comprising (1) above and a light chain variable domain comprising (7) above; a heavy chain variable domain comprising (2) above and a light chain variable domain comprising (8) above; a heavy chain variable domain comprising (3) above and a light chain variable domain comprising (9) above; a heavy chain variable domain comprising (4) above and a light chain variable domain comprising (10) above; a heavy chain variable domain comprising (5) above and a light chain variable domain comprising (11) above; a heavy chain variable domain comprising (6) above and a light chain variable domain comprising (12) above; or any and all combinations of the heavy chain variable domains and light chain variable domains described above. In some embodiments, the first nucleic acid molecule comprises a nucleic acid sequence encoding a Fab(SL335) comprising: the heavy chain variable domain comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:35, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:37, and the light chain variable domain comprises a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:52, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:53, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:54. The first or second nucleic acid molecule can encode IL-2m.

[0151] In other embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:55, 56, 57, 58, 59, or 60. In some embodiments, the second nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a light chain variable domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:61, 62, 63, 64, 65, 66, or 67. A nucleic acid molecule encoding IL-2m can be ligated to the first or second nucleic acid molecule or vector.

[0152] In some embodiments, the first nucleic acid molecule or vector comprises a nucleic acid sequence encoding a Fab comprising a heavy chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:55, 56, 57, 58, 59, or 60, and a light chain variable domain comprising an amino acid sequence at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:61, 62, 63, 64, 65, or 66 or 67.

[0153] In some embodiments, the first nucleic acid molecule is SEQ ID NO:6 (V H -C H1 a heavy chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:13 (V domain); L-Cκ domain) and a light chain domain comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity thereto.

[0154] In some embodiments, the bioactive effector moiety is IL-2m. In some embodiments, the nucleic acid molecule encodes an IL-2m protein comprising an amino acid sequence having at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1 or 2.

[0155] Recombinant expression of an antibody or fragment thereof described herein that specifically binds (e.g., the heavy or light chain of an antibody described herein) involves construction of an expression vector containing a polynucleotide encoding the antibody or fragment. Once a polynucleotide encoding an antibody or fragment thereof described herein (e.g., a heavy or light chain variable domain) is obtained, vectors for the production of the antibody molecule can be generated by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or antibody fragment (e.g., a light or heavy chain) are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody or antibody fragment (e.g., a light or heavy chain) coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors containing a nucleotide sequence encoding an antibody molecule described herein, an antibody heavy or light chain, a heavy or light chain variable domain of an antibody or fragment thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors can contain, for example, nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of an antibody can be cloned into such a vector for expression of an entire heavy chain, an entire light chain, or both the entire heavy and light chains.

[0156] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques and the resulting cells can then be cultured by conventional techniques to produce an antibody described herein.

[0157] A variety of host-expression vector systems can be utilized to express the described antibody molecules. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also represent cells which, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the antibody molecules described herein in situ. These include microorganisms such as bacteria (e.g., Escherichia coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences; or mammalian cell systems (e.g., COS typhimurium) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells. In some embodiments, cells for expressing an antibody described herein (e.g., an antibody comprising any one of the CDRs of antibody pab1949 or pab2044) are CHO cells, e.g., CHO cells from CHO GS System™ (Lonza).In some embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines. In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells), particularly for expression of whole recombinant antibody molecules, are used for expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in combination with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In certain embodiments, the antibodies described herein are produced by CHO cells or NS0 cells. In some embodiments, expression of the nucleotide sequence encoding the antibodies described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0158] In bacterial systems, numerous expression vectors can be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when large quantities of such antibodies are produced for the preparation of pharmaceutical compositions of antibody molecules, a vector directing the expression of high-level fusion protein products that are easily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), in which the antibody coding sequence can be individually ligated into the vector in frame with the lac Z coding region to produce a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509). For example, pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to contain thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0159] In mammalian host cells, a number of viral-based expression systems can be utilized. When adenovirus is used as an expression vector, the antibody coding sequence of interest can be ligated into an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81: 3655-3659). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bitter G et al., (1987) Methods Enzymol 153:516-544).

[0160] In addition, a host cell strain can be selected that modulates the expression of the inserted sequence or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed foreign protein. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some embodiments, the recombinant proteins (e.g., antibodies comprising CDRs) described herein are produced in mammalian cells, such as CHO cells.

[0161] In some aspects, rather than using expression vectors containing viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker. After introduction of the foreign DNA / polynucleotide, engineered cells can be grown in an enriched medium for 1-2 days and then switched to a selective medium. The selectable marker on the recombinant plasmid confers resistance to the selection, allowing cells to stably integrate the plasmid into their chromosomes and grow to form foci that can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the antibodies or fragments thereof described herein. Such engineered cell lines can be particularly useful in screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0162] A number of selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-232), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-823) genes, which can be used in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-3570; O'Hare K et al., (1981) PNAS 78: 1527-1531); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-2076); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; and Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-215; and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-156).

[0163] Once an antibody molecule described herein is produced by recombinant expression, it can be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, chromatography (e.g., ion exchange, affinity, particularly by affinity for a specific antigen following Protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. Additionally, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0164] In specific embodiments, the antibodies described herein are isolated or purified. Generally, an isolated antibody is substantially free of other antibodies having antigen specificities different from the isolated antibody. For example, in some embodiments, preparations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of antibodies in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, antibodies that are substantially free of cellular material include preparations of antibodies that have less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, e.g., antibody preparations with different post-translationally modified forms of the antibody. When an antibody or fragment is recombinantly produced, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation.When an antibody or fragment is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the protein.Thus, such preparations of antibodies or fragments have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the antibody or fragment of interest.In some embodiments, the antibodies described herein are isolated or purified.

[0165] antibody production Yet another aspect provides a method for preparing a recombinant protein, comprising: (a) culturing cells; and (b) recovering the recombinant protein from the cultured cells. The cells can be cultured in a variety of media. Commercially available culture media can be used without limitation. Any other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature and pH, will be those previously used with the host cells selected for expression and will be apparent to those skilled in the art. The recombinant protein can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and purifying the resulting product, for example, by affinity chromatography. Other additional purification techniques, such as anion exchange or cation exchange chromatography, hydrophobic interaction chromatography, or hydroxylapatite chromatography, can also be used.

[0166] The recombinant proteins disclosed herein can be produced by any method known in the art for the synthesis of antibodies, for example, by chemical synthesis or recombinant expression technology. The methods described herein, unless otherwise indicated, use conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields, within the skill of the art. These techniques are described, for example, in the references cited herein and are fully explained in the literature. For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. ,See Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually updated); Current Protocols in Immunology, John Wiley & Sons (1987 and annually updated); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0167] In some embodiments, a recombinant protein described herein is an antibody (e.g., a recombinant antibody) that is prepared, expressed, produced, or isolated by any means involving, for example, synthesis of a DNA sequence or production via genetic engineering. In certain embodiments, such an antibody comprises a sequence (e.g., a DNA sequence or an amino acid sequence) that does not naturally occur within the antibody germline repertoire of an animal or mammal (e.g., a human) in vivo.

[0168] In some aspects, provided herein are methods for producing a recombinant protein disclosed herein, comprising culturing a cell or host cell as described herein. In some aspects, provided herein are methods for producing a recombinant protein, comprising expressing (e.g., recombinantly expressing) an antibody using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody described herein). In some embodiments, the cell is an isolated cell. In some embodiments, an exogenous polynucleotide has been introduced into the cell. In some embodiments, the method further comprises purifying the antibody obtained from the cell or host cell.

[0169] Antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including those known in the art and taught, for example, in Harlow E & Lane D, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al.,: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. For example, monoclonal antibodies can be recombinantly produced from host cells exogenously expressing the antibodies described herein.

[0170] A "monoclonal antibody," as used herein, is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody immunospecifically binds to an antigen (e.g., human serum albumin), e.g., as determined by ELISA or other antigen-binding or competitive binding assays known in the art or in the Examples provided herein. In certain embodiments, a monoclonal antibody may be a chimeric or humanized antibody. In certain embodiments, a monoclonal antibody is a monovalent or polyvalent (e.g., bivalent) antibody. In certain embodiments, a monoclonal antibody may be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies described herein can be made by hybridoma methods, e.g., as described in Kohler G & Milstein C (1975) Nature 256: 495, or can be isolated, e.g., from phage libraries, using techniques such as those described herein. Other methods for the preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, e.g., Chapter 11 in Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0171] Methods for producing and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters, or macaques, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the antigen used for immunization (e.g., human serum albumin). Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Some aspects of the present disclosure provide methods for preparing recombinant fusion proteins, including (a) culturing cells; and (b) recovering the recombinant fusion protein from the cultured cells. Cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other necessary supplements known to those skilled in the art can also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., will be readily applied to the host cells selected for expression and will be apparent to those skilled in the art. The recombinant fusion protein can be purified, for example, by centrifugation or ultrafiltration to remove impurities, and the resulting product can be purified, for example, using affinity chromatography, etc. Other purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc.

[0172] Compositions and Uses Thereof Disclosed herein is a composition comprising the recombinant fusion protein disclosed herein and a carrier. Further disclosed herein is a pharmaceutical composition comprising the recombinant fusion protein disclosed herein and a pharmaceutically acceptable carrier.

[0173] Disclosed herein is a method for preventing or treating an autoimmune disease or an inflammatory neurological disease in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition disclosed herein. Further disclosed herein is the use of a pharmaceutical composition for preventing or treating an autoimmune disease or an inflammatory neurological disease, the pharmaceutical composition comprising a recombinant fusion protein disclosed herein as an active ingredient. In some embodiments, the autoimmune disease is selected from the group consisting of Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematosus (SLE), asthma, edema, delayed-type allergy (type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemia-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy, and the inflammatory neurological disease is selected from the group consisting of amyotrophic lateral sclerosis and Parkinson's disease.

[0174] The present invention provides a pharmaceutical composition for preventing or treating an immune disease, which comprises a recombinant fusion protein as an active ingredient. The present invention also provides a method for preventing or treating an immune disease, which comprises administering the recombinant fusion protein to a subject. Details of the recombinant fusion protein are as described above.

[0175] The immune disease can be inflammatory bowel disease, systemic lupus erythematosus, amyotrophic lateral sclerosis, or Parkinson's disease.

[0176] In some embodiments, pharmaceutical compositions may contain recombinant fusion proteins for preventing or treating inflammatory bowel disease. Inflammatory bowel disease is a typical autoimmune disease that causes abdominal pain, diarrhea, and bleeding due to a severe chronic inflammatory response, and is classified as Crohn's disease and ulcerative colitis. Crohn's disease causes random inflammation throughout the digestive tract, while ulcerative colitis causes inflammation limited to the large intestine. The exact etiology is still unknown, and the disease is known to repeatedly worsen and improve without a clear treatment. Increasing the number of regulatory T cells has been shown to have disease-suppressing effects in animal models of various autoimmune diseases, but there are currently no approved therapeutic agents that increase the number of regulatory T cells in vivo. Therefore, recombinant fusion proteins according to some aspects can be effectively used to treat inflammatory bowel disease.

[0177] A pharmaceutical composition for preventing or treating systemic lupus, amyotrophic lateral sclerosis, or Parkinson's disease is provided, which comprises a recombinant fusion protein as an active ingredient.

[0178] In some embodiments, the pharmaceutical composition may comprise: (a) a pharmaceutically effective amount of the recombinant protein; and (b) a pharmaceutically acceptable carrier.

[0179] In some embodiments, the in vivo half-life of the pharmaceutical composition may exhibit a 2- to 20-fold increase when compared to that of human IL-2m. The in vivo half-life, when compared to that of human IL-2m, can be, for example, about a 2.5-fold to about 3.5-fold increase, about a 3.5-fold to about 6-fold increase, about a 4-fold to about 6-fold increase, about a 4.5-fold to about 6-fold increase, about a 5-fold to about 6-fold increase, about a 5.5-fold to about 6-fold increase, about a 3-fold to about 5.5-fold increase, about a 3.5-fold to about 5.5-fold increase, about a 4-fold to about 5.5-fold increase, about a 4.5-fold to about 5.5-fold increase, about a 5-fold to about 5.5-fold increase, about a 3-fold to about 5-fold increase, about a 3.5-fold to about 5-fold increase, about a 4-fold to about 5-fold increase, about a 4.5-fold to about 5-fold increase, about a 3-fold to about 4.5-fold increase, about a 3.5-fold to about 4.5-fold increase, about a 4-fold to about 4.5-fold increase, or any factor or factor range derived therefrom. In some embodiments, the in vivo half-life of human IL-2m can be assessed following subcutaneous injection of human IL-2m.

[0180] In some embodiments, pharmaceutical compositions can reduce the white blood cell level in blood.White blood cells can be, for example, neutrophils, monocytes, basophils, or a combination thereof.In some embodiments, the reduced white blood cell level can be sustained and maintained for up to 20 days after administration, up to 15 days after administration, up to 12 days after administration, up to 10 days after administration, up to 8 days after administration, up to 7 days after administration, or any range derived therefrom.

[0181] Pharmaceutical compositions for preventing or treating immune diseases according to some aspects can be administered via various routes to mammals, e.g., rats, mice, livestock, humans, etc. All administration methods can be performed, for example, orally or rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection.

[0182] Route of administration and dosage The pharmaceutical compositions of the present disclosure can be administered to a subject through a variety of routes of administration, including oral, transdermal, subcutaneous, intravenous, and intramuscular routes of administration.

[0183] The amount of the recombinant protein or composition disclosed herein that will be effective in the treatment and / or prevention of a condition will depend on the nature of the disease, and can be determined by standard clinical techniques.

[0184] In the present disclosure, the amount of the recombinant protein disclosed herein to be actually administered is determined in light of various relevant factors, including the disease to be treated, the selected administration route, the age, sex, and weight of the patient, and the severity of the disease, as well as the type of biologically active polypeptide as the active ingredient. Because the recombinant protein of the present disclosure has excellent sustainability in the blood, the number and frequency of administration of peptide preparations containing the recombinant protein of the present disclosure can be significantly reduced.

[0185] The pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, "pharmacologically effective amount" or "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that achieves the desired preventive or therapeutic effect. The effective dose level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, and co-administered drugs, as well as other factors well known in the medical field. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered simultaneously with existing therapeutic agents, separately, or sequentially, in one dose or in a few divided doses. Taking all factors into consideration, it is important to administer the composition in the minimum amount sufficient to achieve maximum effect without any side effects, and this amount can be easily determined by those skilled in the art. As used herein, the term "pharmacologically effective amount" refers to an amount sufficient to treat or induce the disease or condition described herein, for example, to induce superovulation or treat anovulation, hypogonadism, or polycystic ovary syndrome.

[0186] The appropriate dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, disease severity, drug formulation, administration route and duration, but can be appropriately selected by those skilled in the art. However, to achieve the desired effect, the pharmaceutical composition can be administered at a daily dose of 0.0001 mg / kg to 2,000 mg / kg, specifically 0.001 mg / kg to 2,000 mg / kg. Administration can be performed once a day or in several divided doses.

[0187] The exact dosage employed in the composition also depends on the route of administration and the severity of the disease, and should be determined according to the judgment of the doctor and the individual patient's circumstances.For example, the effective dosage can also vary depending on the administration means, target site, patient's physiological condition (including age, weight and health condition), other drugs administered, or whether treatment is preventive or therapeutic.Usually, the patient is human, but can also be non-human, such as pets, for example, dogs and cats.The treatment dosage is optimally titrated to optimize safety and effectiveness.

[0188] In certain embodiments, in vitro assays are employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0189] In some embodiments, the recombinant fusion protein may be administered at a dose of 0.001 mg / kg to 2,000 mg / kg, for example, 0.001 mg / kg to 0.01 mg / kg, 0.1 mg / kg to 1 mg / kg, 1.5 mg / kg to 2 mg / kg, 4 mg / kg to 10 mg / kg, 15 mg / kg to 20 mg / kg, 30 mg / kg to 40 mg / kg, 60 mg / kg to 80 mg / kg, 100 mg / kg to 200 mg / kg, or any dose or dose range derived therefrom.

[0190] Pharmaceutical compositions can be prepared in unit dosage form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients according to methods readily practiced by those skilled in the art to which this disclosure pertains, and in this case, the formulations can be in the form of solutions, suspensions, or emulsions in oily or aqueous vehicles, or in the form of extracts, suppositories, powders, granules, tablets, or capsules, and the formulations can further include dispersing agents or stabilizers.

[0191] Provided herein are compositions comprising the recombinant proteins described herein having a desired purity in physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Also disclosed herein are pharmaceutical compositions comprising the recombinant proteins described herein and pharmaceutically acceptable excipients. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used. Pharmaceutical compositions for preventing or treating immune diseases according to some aspects can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injectable solutions, each prepared according to methods in the art. For formulation, suitable carriers, excipients, or diluents commonly used in the preparation of pharmaceutical compositions can be included.

[0192] For use as carriers, excipients, and diluents, various compounds or mixtures can be used, including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0193] When forming the preparation, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. may be used for preparation.

[0194] Solid formulations for oral administration can be prepared by mixing the recombinant fusion protein with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0195] For use as a liquid preparation for oral administration, suspensions, oral liquids, emulsions, syrups, etc. can be used. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. can be included.

[0196] Preparations for parenteral administration may include sterile solutions, non-aqueous agents, suspensions, emulsions, lyophilized agents, and suppositories. Examples of non-aqueous agents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, glycerol, gelatin, and the like.

[0197] The dosage of the pharmaceutical composition for preventing or treating immune diseases according to some embodiments may vary depending on the patient's condition, patient weight, disease severity, drug form, administration route, and administration period, but can be appropriately selected by those skilled in the art. However, to achieve the desired effect, the dosage may be in the range of about 0.0001 mg / kg to about 2,000 mg / kg per day, or about 0.001 mg / kg to about 2,000 mg / kg per day. Administration may be once a day or several times a day.

[0198] Pharmaceutical compositions for treating diseases or inducing conditions can be administered via various routes to mammals, e.g., rats, mice, livestock, humans, etc. All modes of administration are contemplated, for example, oral, rectal or intravenous, intramuscular, subcutaneous, or intrathecal administration, or by intracerebroventricular injection.

[0199] Pharmaceutical compositions can be administered orally or parenterally.Specifically, pharmaceutical compositions can be administered parenterally, in which case it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.In some embodiments, it can be administered in the form of subcutaneous injection.When administered orally, proteins or peptides are digested, and therefore it is necessary to formulate oral compositions by coating the active ingredient or protecting it from degradation in the stomach.In addition, pharmaceutical compositions can be administered by any device that can deliver active substances to target cells.

[0200] The present invention provides a functional health food composition for preventing or treating immune diseases, the composition comprising a recombinant fusion protein as an active ingredient. The present invention also provides a functional health food composition for preventing or treating cancer, the composition comprising a recombinant fusion protein as an active ingredient. The details of the recombinant fusion protein, immune diseases, and cancer are as described above.

[0201] In some aspects, when a recombinant fusion protein is used as an additive in a health functional food for preventing or ameliorating immune diseases or cancer, the recombinant fusion protein may be added as is or may be used together with other foods or food ingredients, according to methods known in the art. The amount of the active ingredient to be mixed can be appropriately determined depending on the intended use, such as prevention, health, or treatment.

[0202] The health functional food may be in any form such as powder, granules, pills, tablets, or capsules, as well as in the form of a general food or drink.

[0203] The types of common foods are not particularly limited, and examples of common foods to which the substance can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, multivitamin supplements, etc., and may include all foods within common sense.

[0204] Generally, when preparing a food or beverage, the recombinant fusion protein can be added in an amount of 15 parts by weight or less, or 10 parts by weight or less, based on 100 parts by weight of the raw material. However, for long-term intake for health and hygiene purposes or health management purposes, the amount can be less than the above range. Furthermore, since the present disclosure uses fractions derived from natural products, there are no safety issues and amounts greater than the above range can be used.

[0205] In some aspects of the health functional food, the beverage can contain various flavorings or natural carbohydrates as additional ingredients, as in common beverages. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used as sweeteners. The proportion of natural carbohydrates in the beverage can range from about 0.01 g to 0.04 g, or about 0.02 g to 0.03 g per 100 mL of the beverage according to the present disclosure.

[0206] Furthermore, health functional food compositions for preventing or ameliorating immune diseases or cancer according to some aspects can contain various nutrients, vitamins, electrolytes, flavorings, colorants, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated beverages. Furthermore, the sleep-improving compositions of the present disclosure can contain natural fruit juices, fruit juice drinks, and fruit pulp for preparing vegetable drinks. Such ingredients can be used alone or in combination. The proportions of these additives are not limited, but are generally selected from the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition.

[0207] As described above, the recombinant fusion protein can exhibit an approximately 3.5-fold increased half-life in rats, for example, when compared to human recombinant IL-2m, and exhibits biological activity at a level similar to that of IL-2m not fused to SL335. Thus, the recombinant fusion protein can exhibit similar efficacy with less frequent administration, allowing patients to administer the drug at more convenient intervals.

[0208] kit Disclosed herein are kits comprising a composition or pharmaceutical composition disclosed herein and a label containing instructions for use.

[0209] Provided herein are kits containing one or more recombinant proteins or conjugates thereof described herein. Provided herein are kits containing a composition disclosed herein and a label containing instructions for its use. Provided herein are pharmaceutical packs or kits containing one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more recombinant proteins provided herein. In some embodiments, the kits contain the pharmaceutical compositions described herein and any prophylactic or therapeutic agents, such as those described herein. Optionally associated with such container(s) may be a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, reflecting agency approval of the manufacture, use, or sale for human administration. Also provided herein are kits that can be used in the above-described methods. In some embodiments, the kits contain a recombinant protein described herein, e.g., a purified recombinant protein, in one or more containers. In some embodiments, the kits described herein contain a substantially isolated antigen(s) (e.g., human serum albumin) that can be used as a control. In other embodiments, the kits described herein further include a control antibody that does not react with the serum albumin antigen. In other embodiments, the kits described herein include one or more elements for detecting binding of the recombinant protein to the serum albumin antigen (e.g., the recombinant protein can be conjugated to a detectable substrate such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be conjugated to a detectable substrate). In specific embodiments, the kits provided herein can include a recombinantly produced or chemically synthesized serum albumin antigen. The serum albumin antigen provided in the kit can also be bound to a solid support. In some embodiments, the detection means of the above-mentioned kits includes a solid support to which the serum albumin antigen is bound.Such kits can also include an unconjugated reporter-labeled anti-human antibody or anti-mouse / rat antibody. Upon binding of the antibody to serum albumin, the antigen can be detected by binding of the reporter-labeled antibody.

[0210] As noted above, recombinant fusion proteins according to some aspects, when fused to SL335 (SAFA-IL-2m), are expected to have an extended half-life compared to recombinant human IL-2m, and therefore may be similarly effective with fewer doses, thereby providing more convenient drug administration intervals for patients.

[0211] Reference will now be made in detail to the embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the embodiments may have different forms and should not be construed as limited to the description set forth herein. Accordingly, the embodiments will be described below with reference to the figures only to illustrate aspects of the present specification. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. A phrase such as "at least one," when preceding a list of elements, modifies the entire list of elements and not individual elements of the list.

[0212] Examples are presented below to aid in understanding the present disclosure. However, the following examples are presented merely to facilitate understanding of the present disclosure, and the contents of the present disclosure are not limited by the following examples. [Example]

[0213] Example 1. Preparation of a recombinant fusion protein containing an interleukin-2 mutein and an antigen-binding fragment to serum albumin 1-1. Preparation of expression vectors for interleukin-2 muteins and antigen-binding fragments against serum albumin An expression vector for a recombinant fusion protein containing an interleukin-2 mutein and an antibody fragment that binds to serum albumin was prepared. The SL335H-linker-IL-2m1 gene (SEQ ID NO:3) [in which the interleukin-2 mutein 1 gene (C125A) (IL-2m1, SEQ ID NO:1) and the heavy chain gene of the Fab antibody fragment (SL335) that binds to serum albumin (SEQ ID NO:6) are linked by a peptide linker (SEQ ID NO:84)] and the SL335L-linker-IL-2m2 gene [in which the interleukin-2 mutein 2 gene (N88R, V91D, M104S, C125A) (IL-2m2, SEQ ID NO:2) and the heavy chain gene of the Fab antibody fragment (SL335) that binds to serum albumin (SEQ ID NO:6) are linked by a peptide linker (SEQ ID NO:84)] were kindly provided by Cosmogenetech Co., Ltd. (South It was synthesized by the Korea Co., Ltd. after gene codon optimization (SEQ ID NO: 4).

[0214] To express the SL335H-linker-IL-2m1 gene and the SL335L-linker-IL-2m2 gene prepared by gene synthesis, the pD2535NT (ATUM, USA) expression vector, the SL335H-linker-IL-2m1 gene, and the SL335L-linker-IL-2m2 gene were each treated with Bbs I restriction enzyme (Takara, Japan) to cleave the Bbs I site, and then treated with T4 DNA ligase (Takara, Japan) and inserted into the expression vector.

[0215] The light chain gene of a serum albumin-binding Fab antibody fragment (SL335) was synthesized by ATUM Company (USA) after gene codon optimization and then inserted into the pD2359 expression vector (ATUM, USA).

[0216] FIG. 1A shows a heavy chain expression vector and FIG. 1B shows a light chain expression vector for preparing a recombinant fusion protein according to some aspects.

[0217] FIG. 2 shows the structure of the APB-R4 (SAFA-IL-2m2) protein prepared according to aspects.

[0218] 1-2. Preparation of transient expression cells ExpiCHO-S™ cells (Thermo Fisher Scientific) were added to a 125 ml culture flask containing expression medium (ExpiCHO Expression Medium, Thermo Fisher Scientific), and then cultured at 37°C, 140 rpm, 5% CO2, and 80% humidity. Then, for the preparation of transiently expressing cells, the cultured cells were diluted to 6.0 × 10 6 The cells were seeded at a concentration of 6a per ml and then transfected with the plasmid vectors (pD2535NT and pD2539) containing the genes prepared in Example 1-1. The transfected cells were then cultured in a shaking incubator at 37°C, 140 rpm, 5% CO2, and 80% humidity for 16 hours, after which they were treated with ExpiCHO feed and enhancer and then cultured under the same conditions as above. On day 6, D-glucose was added, and the resulting cells were cultured for 8 days at 37°C, 140 rpm, 5% CO2, and 80% humidity. After the culture was completed, the resulting culture medium was centrifuged at 4,000 rpm for 15 minutes at 4°C to separate the cells from the culture medium. The separated culture medium was then passed through a 0.2 μm filter paper to remove impurities.

[0219] 1-3. Preparation of stable cell lines Stable cell lines were prepared using HD-BIOP3 GS null CHO-K1 cells (Horizon Discovery) at 3.0 × 10 cells / mL in CD FortiCHO (Thermo Fisher Scientific) medium supplemented with 4 mM L-glutamine. 5The cells were seeded at a concentration of 1.0 × 10 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and humidity of 80% or higher. For transfection, the cultured cells were cultured at a concentration of 1.0 × 10 cells / ml. 6 The cells were seeded at a concentration of 1000 cells / ml and then transfected with the SAFA-IL-2m1 and SAFA-IL-2m2 plasmid vectors (pD2535NT and pD2539) prepared in Example 1-1 using OptiPRO SFM medium and Freestyle Max reagent (Invitrogen, Carlsbad, California). The transfected cells were then cultured for 2 days at 37°C, 5% CO2, and 80% or higher humidity. To select stable pools, the medium was replaced with L-glutamine-free CD FortiCHO medium by centrifugation, and the cells were treated with 50 μM methionine sulfoximine (MSX) (Sigma-Aldrich, St. St. Louis, Missouri) and 10 μg / ml puromycin (Thermo Fisher Scientific) every 2 days to remove cells not injected with the vector. Thereafter, the medium was replaced with CD FortiCHO medium containing both MSX and puromycin at intervals of 7 to 10 days by centrifugation, and the cell count was increased to 5.0 × 10 5 The cells were cultured for 21 days at a constant concentration of 1.0 × 10 cells / ml. When the viability recovered to 90% or more, the cells were cultured at 1.0 × 10 cells / ml. 7 A stock of cells / ml was prepared.

[0220] 1-4. Isolation and purification of SAFA-IL-2 muteins Protein samples present in the SAFA-IL-2m1 and SAFA-IL-2m2 stable cell lines of Examples 1-3 were purified by sequentially performing affinity chromatography (AC), multimodal anion exchange chromatography (MAEX), and cation exchange chromatography (CEX).

[0221] Specifically, to purify the SAFA-IL-2m1 and SAFA-IL-2m2 (APB-R4) proteins in Examples 1-3 by AC, the culture medium was equilibrated with 1x PBS (pH 7.4) using CaptureSelect CH1-XL resin (Thermo Fisher Scientific, USA) and then combined at a fluid velocity of 136 cm / hr, thereby eluting the protein with 25 mM sodium citrate, pH 5.0 buffer. The eluted protein solution was subjected to MAEX using Capto adhere ImpRes resin (Cytiva, USA) to remove impurities in FT mode at pH 6.0. The separated protein solution was finally combined with resin equilibrated with 20 mM L-histidine buffer, pH 7.0, by CEX using Capto SP ImpRes resin (Cytiva, USA), and the final protein was separated and purified using 20 mM L-histidine buffer, pH 7.0, 1 M NaCl. The proteins purified in this manner were designated SAFA-IL-2m1 and SAFA-IL-2m2 (APB-R4).

[0222] Example 2. Preparation of recombinant human IL-2 muteins 2-1. Preparation of expression vectors for IL-2 muteins and efavaleukin-α analogs An expression vector was prepared to express the efavaleukin-α (AMGEN, AMG-592) analog protein, a comparative substance for IL-2 muteins. The efavaleukin-α analog was synthesized by Cosmogenetech Co., Ltd. (South Korea) after gene codon optimization using public sequence information from a drug information site (KEGG, Kyoto Encyclopedia of Genes and Genomes, D11612) (SEQ ID NO: 9).

[0223] To express the synthesized efavaleukin-α analog gene, the pD2535NT expression vector (ATUM, USA) and the efavaleukin-α analog gene were treated with BbsI restriction enzyme (Takara, Japan) to cleave the BbsI site, and then treated with T4 DNA ligase (Takara, Japan) and inserted into the expression vector.

[0224] 2-2. Preparation of transient expression cells ExpiCHO-S™ cells (Thermo Fisher Scientific) were added to a 125 ml culture flask containing expression medium (ExpiCHO Expression Medium, Thermo Fisher Scientific), and then cultured at 37°C, 140 rpm, 5% CO2, and 80% humidity. Then, for the preparation of transiently expressing cells, the cultured cells were diluted to 6.0 × 10 6 The cells were seeded at a concentration of 6a per ml and then transfected with the gene-inserted plasmid vector (pD2535NT) prepared in Example 2-1. The transfected cells were then cultured in a shaking incubator at 37°C, 140 rpm, 5% CO2, and 80% humidity for 16 hours, after which they were treated with ExpiCHO feed and enhancer and then cultured under the same conditions as above. On day 6, D-glucose was added, and the resulting cells were cultured for 8 days at 37°C, 140 rpm, 5% CO2, and 80% humidity. After the culture was completed, the resulting culture medium was centrifuged at 4,000 rpm for 15 minutes at 4°C to separate the cells from the culture medium. The separated culture medium was then passed through a 0.2 μm filter paper to remove impurities.

[0225] 2-3. Preparation of stable cell lines Stable cell lines were prepared using HD-BIOP3 GS null CHO-K1 cells (Horizon Discovery) at 3.0 × 10 cells / mL in CD FortiCHO (Thermo Fisher Scientific) medium supplemented with 4 mM L-glutamine. 5The cells were seeded at a concentration of 1.0 × 10 cells / ml and cultured in a shaking incubator at 37°C, 5% CO2, and humidity of 80% or higher. For transfection, the cultured cells were cultured at a concentration of 1.0 × 10 cells / ml. 6 Cells were seeded at a concentration of 1000 cells / ml and then transfected with the efavaleukin-α analog plasmid vector (pD2535NT) prepared in Example 2-1 using OptiPRO SFM medium and Freestyle Max Reagent (Invitrogen, Carlsbad, California). The transfected cells were then cultured for 2 days at 37°C, 5% CO2, and 80% or higher humidity. To select stable pools, the medium was replaced with L-glutamine-free CD FortiCHO medium by centrifugation. The cells were treated with 50 μM methionine sulfoximine (MSX) (Sigma-Aldrich, St. St. Louis, Missouri) and 10 μg / ml puromycin (Thermo Fisher Scientific) every 2 days to remove cells not injected with the vector. The medium was then replaced with CD FortiCHO medium containing both MSX and puromycin by centrifugation every 7–10 days, and the cell number was increased to 5.0 × 10 each time. 5 The cells were cultured for 21 days at a constant concentration of 1.0 × 10 cells / ml. When the viability recovered to 90% or more, the cells were cultured at 1.0 × 10 cells / ml. 7 A stock of cells / ml was prepared.

[0226] 2-4. Isolation and purification of efavaleukin-α analogue protein To purify the efavaleukin-α analog protein of Examples 2-3 by AC, the culture medium was equilibrated with 1x PBS (pH 7.4) using CaptureSelect CH1-XL resin (Thermo Fisher Scientific, USA) and then combined at a fluid velocity of 240 cm / hr, thereby eluting the protein with 50 mM sodium citrate pH 3.0 buffer. The eluted protein solution was subjected to AEX using POROS™ XQ resin (Thermo Fisher Scientific, USA) to remove impurities in FT mode at pH 5.5. The separated protein solution was finally combined with the resin equilibrated with 25 mM sodium citrate pH 5.0 by CEX using CM Sepharose FF resin (Cytiva, USA), and the final protein was separated and purified using 5 mM sodium citrate pH 5.0, 1 M NaCl.

[0227] Example 3. Molecular characterization of SAFA-IL-2m 3-1. Check the size To confirm the size of the recombinant proteins in Examples 1 and 2, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on them. Specifically, protein samples were prepared under reducing and non-reducing conditions using non-reducing 4x SDS sample buffer (Thermo Fisher Scientific) and 2-mercaptoethanol. For non-reducing conditions, samples heated at 100°C for 5 minutes and unheated samples were prepared together to compare the heat-dependent protein shape and size. Protein size markers (SMOBio, Taiwan) were prepared to compare the protein size under each condition. The prepared protein samples were loaded onto a 4-15% Mini-protein TGX precast gel (15-well, Bio-Rad) at 1 μg or 2 μg per well, respectively, and then subjected to electrophoresis in tris-glycine SDS running buffer at 150 V for 1 hour. After completion of electrophoresis, the SDS-PAGE gel was stained with EZ-gel staining solution (DoGenBio, South Korea) for 1 h, followed by destaining with distilled water for 1 day.

[0228] FIG. 3A shows the results of size analysis of SAFA-IL-2m1 and SAFA-IL-2m2 proteins according to certain aspects at 1 μg per well by SDS-PAGE under reducing (R), non-reducing with heat (NR(B)), and non-reducing without heat (NR(NB)) conditions.

[0229] FIG. 3B shows the results of size analysis of SAFA-IL-2m2 and efavaleukin-α analog proteins according to certain aspects at 1 μg per well by SDS-PAGE under reducing (R), non-reducing with heat (NR(B)), and non-reducing without heat (NR(NB)) conditions.

[0230] As shown in Figure 3A, after 5 minutes of heating under non-reducing and reducing conditions for SAFA-IL-2m1 and SAFA-IL-2m2 proteins, a protein band was observed at 40 kDa for the heavy chain of SL335H-IL-2m1 and SL335H-IL-2m2, which is similar to the theoretical size of 39.6 kDa. A protein band was also observed at 26.3 kDa for the light chain of SL335L, which is similar to the theoretical size of 23.3 kDa. Under non-reducing conditions, no change in protein size due to the O-linked glycans present in the IL-2m protein was observed. On the other hand, under non-reducing conditions without heating, a highly purified protein band corresponding to the intact form of APB-R4, consisting of the heavy and light chains, was observed at 56.7 kDa, smaller than the theoretical size of 62.975 kDa, whereas the protein bands corresponding to the heavy and light chains were very weak.

[0231] For efavaleukin-α analogs, as shown in Figure 3B, under non-reducing and reducing conditions with heat application for 5 minutes, a protein band was observed at 43.3 kDa, which is similar to the theoretical size of 41.1 kDa. On the other hand, under non-reducing conditions without heat application, a highly purified protein band corresponding to the intact form of the efavaleukin-α analog with its heavy and light chains bound was observed at 69.8 kDa, which is lower than the theoretical size of 82.196 kDa. Furthermore, a protein band corresponding to the efavaleukin-α analog monomer without its heavy and light chains bound was very weakly detected.

[0232] 3-2. Purity check To determine the purity of the SAFA-IL-2m2 (APB-R4) and efavaleukin-α analog proteins purified in Examples 1-4 and 2-4, SE-HPLC was performed. First, an HPLC system equipped with a TSKgel G3000SWXL 7.8 × 300 mm (Tosoh Bioscience, Japan) column and an Alliance HPLC system (Waters, Milford, MA) was equilibrated with 300 mM sodium phosphate pH 7.0 buffer containing 100 mM KCl. The sample to be analyzed was prepared by dilution with 300 mM potassium phosphate pH 7.0, 100 mM KCl, and pH 7.0 buffer, and 50 μg of the prepared sample was loaded onto the column. SE-HPLC analysis was performed for 40 minutes at a flow rate of 0.5 ml / min and a maximum pressure of 1,000 psi, and the purity was measured at a wavelength of A280 nm.

[0233] 4A and 4B show the results of SE-HPLC analysis of the purity of SAFA-IL-2m2 and efavaleukin-α analog proteins according to certain aspects. All SAFA-IL-2m2 and efavaleukin-α analog proteins were confirmed to have a purity of 99% or higher.

[0234] Example 4. Confirmation of the biological properties of SAFA-IL-2m 4-1. Analysis of the binding ability of SAFA-IL-2m to human serum albumin, monkey serum albumin, and mouse serum albumin To evaluate the biological function of the SAFA-IL-2m2 protein purified in Examples 1-4, the binding ability of the SAFA-IL-2m2 protein to human serum albumin, monkey serum albumin, and mouse serum albumin was confirmed by ELISA. As shown in Figures 5A to 5C, it was confirmed that SAFA-IL-2m2 does not interfere with the cross-reactivity of SAFA with serum albumin.

[0235] 4-2. Confirmation of IL-2 receptor binding at the cellular level To evaluate the biological function of the SAFA-IL-2m2 protein purified in Examples 1-4, the IL-2 receptor-dependent activity of the SAFA-IL-2m2 protein was confirmed using the HEK-blue IL-2 reporter cell line expressing all of the IL-2 receptors α, β, and γ, as well as the HEK-blue IL-2 reporter cell line expressing both the IL-2 receptors β and γ. First, the protein sample from Example 1 was prepared by diluting it in PBS buffer mixed with 0.2% bovine serum albumin (BSA). Then, 2.777 × 10 cells were cultured. 5 The diluted protein sample was seeded onto the plate at a concentration of 1000 cells / ml. The mixed cells and protein were incubated in an incubator at 37°C and 5% CO2 for 21 hours. After incubation, the cell supernatant mixed with the protein was mixed with QUANTI-blue solution, and the resulting mixture was incubated in an incubator at 37°C and 5% CO2 for 1 hour. The absorbance was then measured at a wavelength of 655 nm.

[0236] As a result, the activity of SAFA-IL-2m2 was lower than that of the efavaleukin-α analog in cell lines expressing all of the IL-2 receptors α, β, and γ, as shown in Figure 6A, and the IL-2 mutant was demonstrated based on the result that SAFA-IL-2m2 showed no activity in cell lines expressing IL-2 receptors β and γ, as shown in Figure 6B.

[0237] 4-3. Confirmation of the binding degree of SAFA-IL-2m2 to the IL-2 receptor In Figures 7A and 7B, the binding ability of SAFA-IL-2m2 protein to IL-2 receptors α and β was confirmed by ELISA analysis.

[0238] As a result, it was confirmed that SAFA-IL-2m2, which has a small mutation in IL-2 receptor α, has lower binding ability compared to IL-2 wild-type and efavaleukin-α analogs, and that SAFA-IL-2m2, which has a large mutation in IL-2 receptor β, has no binding compared to IL-2 wild-type and efavaleukin-α analogs.

[0239] 4-4. Verification of the specific activity of regulatory T cells The SAFA-IL-2m2 protein purified in Examples 1-4 was intended to confer specificity, completely attenuating the ability of IL-2 to signal through the intermediate-affinity β and γ IL-2 receptor complexes, and attenuating the ability of IL-2 to signal through the high-affinity α, β, and γ IL-2 receptor complexes. In this regard, the ability of the SAFA-IL-2m2 protein to differentially induce signal transduction and activator of transcription 5 (STAT5) phosphorylation in regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells was examined. STAT5 is known to be involved in the downstream signaling cascade upon IL-2 binding to the IL-2 receptor.

[0240] Briefly, cells isolated from the spleens of C57BL / 6 mice (available from Orient Bio Co., Ltd.) were treated with SAFA-IL-2m2 at different concentrations for 15 minutes, fixed and permeabilized, washed, and then stained with a mixture of anti-CD45-BV510, anti-CD3-BV605, anti-CD4-PerCP / cy5.5, anti-CD8-BV786, anti-CD25-PE, anti-FOXP3-AF700, anti-NK1.1-AF488, and anti-PSTAT5-AF647 antibodies. Cells were then gated by flow cytometry into CD4+CD25+Foxp3+, CDF4+CD25+Foxp3-, CD3+CD8+, and CD4-CD8- groups for regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells, respectively. The data are expressed as the percentage of pSTAT5-positive cells in the gated population and are shown in Figure 8. It was confirmed that STAT5 phosphorylation was specifically increased by the concentration of SAFA-IL-2m2 protein only in regulatory T cells.

[0241] As shown in FIG. 9, phosphorylation of STAT5 using human peripheral blood mononuclear cells (PBMCs) was confirmed by flow cytometry in the same T cell populations as shown in FIG.

[0242] To put it simply, 3 x 10 6 Human PBMCs were incubated in an incubator at 37°C and 5% CO for 2 hours, treated with 100 ng / mL of anti-CD3 antibody, and then incubated in an incubator at 37°C and 5% CO for 2 days. Next, 2 x 10 human PBMCs were added to the 100 ng / mL anti-CD3 antibody solution. 6 The mixture was incubated in an incubator at 37°C and 5% CO for 5 days. 6Human PBMCs were treated with serial dilutions of test compounds for 15 minutes in an incubator at 37°C and 5% CO2. Cells were fixed, permeabilized, washed, and stained with a mixture of anti-CD45-PE / Cy7, anti-CD3-BV605, anti-CD4-FITC, anti-CD8-PE, anti-CD25-BV421, anti-FOXP3-AF700, anti-CD49b-CD510, and anti-pSTAT5-AF547 antibodies. Cells were then gated by flow cytometry into CD4+CD25+Foxp3+, CDF4+CD25+Foxp3-, CD3+CD8+, and CD4-CD8- groups for regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells, respectively. Data are presented as the percentage of pSTAT5-positive cells in the gated population and are shown in Figure 9. The control substance, efavaleukin-α analog, induced STAT5 phosphorylation not only in regulatory T cells but also in CD4+ Tconv cells, CD8+ T cells, and NK cells, whereas SAFA-IL-2m2 protein induced STAT5 phosphorylation only in regulatory T cells.

[0243] As shown in FIG. 10, changes in the Ki67 proliferation marker using human PBMCs were confirmed by flow cytometry in the same T cell populations as shown in FIG.

[0244] To put it simply, 3 x 10 6 Human PBMCs were incubated in an incubator at 37°C and 5% CO for 2 hours, treated with 100 ng / mL of anti-CD3 antibody, and then incubated in an incubator at 37°C and 5% CO for 2 days. Next, 2 x 10 human PBMCs were added to the 100 ng / mL anti-CD3 antibody solution. 6 The mixture was incubated in an incubator at 37°C and 5% CO for 5 days. 6Human PBMCs were treated with serial dilutions of test compounds for 2 days in an incubator at 37°C and 5% CO2. Cells were fixed, permeabilized, washed, and stained with a mixture of anti-CD45-PE / Cy7, anti-CD3-BV510, anti-CD4-FITC, anti-CD8-PerCP / Cy5.5, anti-CD25-APC, anti-FOXP3-eFluor450, anti-CD49b-BV510, and anti-ki67-PE antibodies. Cells were then gated by flow cytometry into CD4+CD25+Foxp3+, CDF4+CD25+Foxp3-, CD3+CD8+, and CD4-CD8- populations for regulatory T cells, CD4+ Tconv cells, CD8+ T cells, and NK cells, respectively. Data are presented as the percentage of Ki67-positive cells in the gated population and are shown in Figure 10. The control substance, efavaleukin-α analog, stimulated proliferation and proliferation not only in regulatory T cells but also in CD4+ Tconv cells, CD8+ T cells, and NK cells, whereas SAFA-IL-2m2 protein was observed to strongly induce proliferation and proliferation only in regulatory T cells.

[0245] As shown in Figure 11, changes in regulatory T cell proliferation were confirmed by flow cytometry after isolating cells from the spleen, inguinal lymph nodes, and mesenteric lymph nodes of C57BL / 6 mice upon a single subcutaneous injection.

[0246] As a result, as shown in Figure 11, it was confirmed that the ratios of regulatory T cells to CD4+ Tconv cells, regulatory T cells to CD8+ T cells, and regulatory T cells to NK cells increased in a SAFA-IL-2m2 concentration-dependent manner. Furthermore, it was confirmed that the formation of regulatory T cells was superior to that of the efavaleukin-α analog.

[0247] 4-5.Verification of the efficacy of SAFA-IL-2m2 Figure 12 shows the results of examining the immune response induced by administration of SAFA-IL-2m2 to marmosets (a type of primate) after immunization with keyhole limpet hemocyanin (KLH). The immune response was confirmed by ELISA for anti-KLH IgG and anti-KLH-IgM.

[0248] As a result, as shown in Figure 12, it was confirmed that in the group administered with KLH alone, the levels of anti-KLH-IgG and anti-KLH-IgM increased due to the KLH immune response, while no changes were observed in the group administered with SAFA-IL-2m2.

[0249] In Figures 13A and 13B, the efficacy of SAFA-IL-2m2 in a mouse model of DSS-induced inflammatory bowel disease was confirmed.

[0250] Briefly, as shown in Figure 13A, the disease activity index (DAI) was quantitatively evaluated based on the occurrence of intestinal symptoms and body weight changes after the start of administration of control substances (Humira, efavaleukin-α analogs) and SAFA-IL-2m2. The results showed that administration of SAFA-IL-2m2 at different concentrations tended to result in a statistically significant decrease in the DAI compared to the negative control group (vehicle), and also tended to result in a lower DIA compared to the positive control group (Humira, efavaleukin-α analogs).

[0251] As shown in Figure 13B, the results obtained by measuring the length of the mouse colon tissues excised at the end of the experiment showed that the negative control group showed a decrease in colon length compared to the normal group, while the SAFA-IL-2m2-treated groups maintained their colon length at each concentration. In this respect, this indicates that the treatment did not significantly reduce the total length of the colon.

[0252] Example 5. Pharmacokinetic evaluation of SAFA-IL-2m2 protein To measure the in vivo half-life of SAFA-IL-2m2, pharmacokinetic studies were performed in cynomolgus monkey and mouse models (C57BL / 6). Specifically, 12 healthy male monkeys (cynomolgus monkeys) were divided into four groups of three. SAFA-IL-2m2 was administered at a single intravenous dose of 0.1 mg / kg (Group 1), a single intravenous dose of 0.3 mg / kg (Group 2), a single subcutaneous dose of 0.1 mg / kg (Group 3), or a single subcutaneous dose of 0.3 mg / kg (Group 4). Subsequently, 1 mL of whole blood was collected according to a set blood collection schedule (e.g., single intravenous administration at 0, 0.25, 0.5, 1, 8, 24, 48, 96, 168, 240, 360, 528, 696, and 864 hours (14 time points in total); and single subcutaneous administration at 0, 1, 8, 12, 24, 48, 72, 96, 120, 168, 240, 360, 528, 696, and 864 hours (15 time points in total)). Serum was separated by centrifugation and stored in a cryogenic freezer (-70°C). ELISA was then performed to measure the concentration of SAFA-IL-2m2 in the serum. In a pharmacokinetic study using a mouse model, 30 female C57BL / 6 mice were intravenously injected with a single dose of 0.4 mg / kg SAFA-IL-2m2. After injection, blood samples were collected at 10 time points, including one time point before administration and nine time points at 0.25, 0.5, 2.5, 5, and 10 hours, and 1, 3, 5, and 7 days after administration. The concentration of SAFA-IL-2m2 present in mouse serum was measured by ELISA.

[0253] Next, after intravenous and subcutaneous administration of SAFA-IL-2m2 to monkeys, the pharmacokinetic parameters were determined using Phoenix WinNonlin software (ver. 1.4; Certara LP, Princeton, NJ, USA) based on the measurement of blood protein concentrations. As shown in Figures 14A and 14B, in the group administered intravenously at a dose of 0.1 mg / kg, the AUC last is 88,000 hr*ng / mL, and C max is 1,670 ng / mL, and t 1 / 2In the group administered intravenously at a dose of 0.3 mg / kg, AUC last is 397,000 hr*ng / mL, and C max is 6,730 ng / mL, and t 1 / 2 In the group administered subcutaneously at a dose of 0.1 mg / kg, AUC last is 113,000 hr*ng / mL, and C max is 772 ng / mL, and t 1 / 2 The AUC was 71.5 hr, and in the group administered subcutaneously at a dose of 0.3 mg / kg, last is 321,000 hr*ng / mL, and C max was 2,370 ng / mL, and t 1 / 2 was 114 hours.

[0254] Furthermore, after a single intravenous administration of SAFA-IL-2m2 to mice, the pharmacokinetic parameters were determined based on the blood protein concentration using Phoenix WinNonlin software (ver 8.3; Certara LP, Princeton, NJ, USA). As a result, as shown in Figure 15, SAFA-IL-2m2 exhibited an AUC of 54,098 hr*μg / mL. last , C0 of 5,827 μg / mL, and t of 8.3 hr 1 / 2 had.

[0255] Example 6. Verification of specific activity on regulatory T cells Similar to the specific activity of SAFA-IL-2m2 protein on regulatory T cells in the C57BL / 6 mouse model, we conducted experiments using three healthy male cynomolgus monkeys to verify similar effects. Specifically, SAFA-IL-2m2 was administered subcutaneously once at a dose of 0.3 mg / kg. Next, 2 mL of whole blood was collected according to a predetermined blood collection schedule (0, 48, 96, 168, and 360 hours), and changes in regulatory T cell proliferation were confirmed using a flow cytometer. By flow cytometry analysis, cells were gated into CD45+CD3+, CD45+CD3+CD4+CD25+FoxP3+, CD45+CD3+CD4+CD25-FoxP3-, CD45+CD3+CD4-CD8+, and CD45+CD3-CD16+ groups for total T cells, regulatory T cells, helper T cells, killer T cells, and natural killer T cells, respectively.

[0256] As shown in Figures 16A and 16B, it was observed that SAFA-IL-2m2 administration strongly increased proliferation only in regulatory T cells, but not in other T cells.

[0257] Example 7. Analysis of biodistribution of SAFA-IL-2m2 protein To evaluate the biodistribution of SAFA-IL-2m2 in inflammatory arthritis in a DBA / 1J mouse model with collagen-induced arthritis, SAFA-IL-2m2 was administered subcutaneously once to the mouse model. The time course of SAFA-IL-2m2 biodistribution was then examined by image analysis. Male DBA / 1J mice (8-9 weeks old) received a second immunization 21 days after the first immunization and were assigned to experimental groups of six mice. Clinical assessment of the arthritis activity index (CAI) was performed periodically from the day of group separation until the end of the experiment, with the development of inflammation monitored. At the time of the highest CAI, mice were subcutaneously injected with 3 mg / kg vehicle-CF750 (group 1), 6.5 mg / kg efavaleukin-α-CF750 (group 2), or 5 mg / kg SAFA-IL-2m2 (group 3). After anesthesia with an inhalation anesthetic, optical images were taken using an IVIS Lumina (PerkinElmer) at time points of 0, 1, 4, 6, 24, 48, and 72 hours. 72 hours after administration of the test substance, the liver, lungs, spleen, and kidneys were removed from the mice and analyzed for biodistribution.

[0258] As shown in Figures 17A and 17B, the total radiative efficiency of the SAFA-IL-2m2-treated group was higher than that of the positive control (efavaleukin-α-CD750) group at the arthritic paw site, confirming that SAFA-IL-2m2 was distributed by targeting the inflammation-induced arthritis site. Furthermore, when the total radiative efficiency of the excised tissues of the SAFA-IL-2m2-CF750-treated group was compared with that of the positive control (efavaleukin-α-CD750), the values ​​in the liver, lung, and spleen were relatively low, whereas the value in the kidney was high. This suggests that SAFA-IL-2m2-CF750 accumulated less in tissues other than the inflamed target site than the positive control (efavaleukin-α-CF750).

[0259] Example 8. Verification of the efficacy of SAFA-IL-2m2 using an MRL lupus mouse model We investigated the efficacy of SAFA-IL-2m2 in the MRL / lpr mouse model, a widely used model for evaluating the therapeutic effects of systemic lupus erythematosus (SLE). Specifically, normal mice (C57BL / 6) and MRL / lpr mice were administered mycophenolate mofetil (60 mg / kg) as a positive control and SAFA-IL-2m2 (400 μg / kg) as a negative control, along with vehicle, from 9 weeks of age. After the start of drug administration, proteinuria and lymphadenopathy scores were measured. Furthermore, to confirm DAI by quantitative analysis of autoantibodies in the mouse blood, anti-dsDNA IgG was measured in blood collected by cardiac blood collection after the experiment.

[0260] As shown in Figures 18A-18C, the positive control (mycophenolic acid) and SAFA-IL-2m2-treated groups demonstrated statistically significant reductions in proteinuria and lymphadenopathy scores compared with the negative control (vehicle, MRL / lpr). This confirms that the therapeutic effect on lupus is equivalent to or superior to that of mycophenolate mofetil, the standard treatment in clinical practice. Furthermore, the anti-dsDNA IgG concentration measurements also showed a trend toward a decrease in the positive control (mycophenolic acid) and SAFA-IL-2m2-treated groups compared with the negative control (vehicle, MRL / lpr), with a statistically significant decrease observed in the SAFA-IL-2m2-treated group. These results demonstrate that SAFA-IL-2m2 effectively suppresses autoantibody production and exhibits superior therapeutic efficacy compared with mycophenolate mofetil, the standard treatment used in clinical practice.

[0261] Furthermore, after the experiment, left kidney tissue was removed and subjected to hematoxylin-eosin (H&E) staining, and right kidney tissue was removed and subjected to immunofluorescence staining for IgG. As shown in Figures 19A and 19B, H&E staining confirmed that SAFA-IL-2m2 administration not only inhibited mesangial proliferation but also significantly reduced inflammatory cell infiltration in the glomeruli. Immunofluorescence staining for IgG confirmed that SAFA-IL-2m2 exerted a therapeutic effect on glomerulonephritis by reducing immune complex infiltration into the kidney. Based on the above results, the therapeutic effect of SAFA-IL-2m2 on lupus, as well as its efficacy in reducing the frequency of moderate to severe proteinuria and improving glomerulonephritis, were confirmed.

[0262] According to one or more embodiments, the recombinant fusion protein is a fusion of an IL-2 mutein and an anti-serum albumin antibody, and has the advantage of a relatively long administration cycle due to its increased in vivo half-life. Furthermore, due to its low immunogenicity, it does not cause side effects in the body, so the recombinant fusion protein can be effectively used to treat various immune diseases, including inflammatory bowel disease or systemic lupus erythematosus.

[0263] It should be understood that the embodiments described herein should be considered for illustrative purposes only, and not for purposes of limitation. The description of a feature or aspect within each embodiment should typically be considered applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the drawings, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure, as defined by the following claims.

[0264] All of the various aspects, embodiments, and options described herein can be combined in any and all variations. All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A recombinant fusion protein containing interleukin-2 mutein (IL-2m) and an antigen-binding fragment (Fab) that binds to serum albumin.

2. The recombinant fusion protein according to claim 1, wherein IL-2m and Fab for serum albumin are linked to each other by a linker, or the C-terminal region of the heavy chain of Fab for serum albumin and IL-2m are linked to each other by a linker.

3. The recombinant fusion protein according to claim 1, wherein IL-2m, a linker, a heavy chain containing the heavy chain region of Fab for serum albumin, and a light chain containing the light chain region of Fab for serum albumin are linked by a non-covalent bond.

4. The recombinant fusion protein according to claim 2, wherein the linker contains 1 to 50 amino acids.

5. The recombinant fusion protein according to claim 4, wherein the linker comprises one amino acid sequence from SEQ ID NO: 16, 70-85, and 88, or the amino acid sequence of SEQ ID NO:

84.

6. The recombinant fusion protein according to claim 5, wherein IL-2m, the linker, and the heavy chain comprise the amino acid sequence of SEQ ID NO:

4.

7. The recombinant fusion protein according to claim 1, comprising amino acid sequences of SEQ ID NO: 6 and 2.

8. The recombinant fusion protein according to claim 1, wherein IL-2m comprises an amino acid sequence having at least 90% identity with SEQ ID NO:

2.

9. The recombinant fusion protein according to claim 1, wherein IL-2m comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:

1.

10. Fab, (a) Heavy chain complementarity determination domain 1 (CDR1) containing the amino acid sequence of SYGIS (SEQ ID NO:22), Heavy chain complementarity determination domain 2 (CDR2), which includes the amino acid sequence of, and Heavy chain complementarity-determining domain 3 (CDR3) containing the amino acid sequence; (b) Heavy chain CDR1 containing the amino acid sequence of SYGIS (SEQ ID NO:22), Heavy chain CDR2 containing the amino acid sequence, and Heavy chain CDR3 containing the amino acid sequence; (c) Heavy chain CDR1 containing the amino acid sequence of NYGIH (SEQ ID NO:26), Heavy chain CDR2 containing the amino acid sequence, and Heavy chain CDR3 containing the amino acid sequence; (d) Heavy chain CDR1 containing the amino acid sequence of SYAMS (SEQ ID NO:29) Heavy chain CDR2 containing the amino acid sequence, and Heavy chain CDR3 containing the amino acid sequence; (e) Heavy chain CDR1 containing the amino acid sequence of AYWIA (SEQ ID NO:32), Heavy chain CDR2 containing the amino acid sequence, and Heavy chain CDR3 containing the amino acid sequence of LYSGSYSP (SEQ ID NO:34); or (f) Heavy chain CDR1 containing the amino acid sequence of AYSMN (SEQ ID NO:35), Heavy chain CDR2 containing the amino acid sequence, and Heavy chain CDR3 containing the amino acid sequence A heavy chain containing a heavy chain variable domain, (g) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of GASRLES (SEQ ID NO:39), and Light chain CDR3 containing the amino acid sequence QQSDSVPVT (SEQ ID NO:40); (h) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of AASSLQS (SEQ ID NO:42), and Light chain CDR3 containing the amino acid sequence; (i) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of DASNRAT (SEQ ID NO:45), and Light chain CDR3 containing the amino acid sequence; (j) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of GASSRAT (SEQ ID NO:48), and Light chain CDR3 containing the amino acid sequence of QQYGSSPRT (SEQ ID NO:49); (k) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of GASSRAT (SEQ ID NO:48), and Light chain CDR3 containing the amino acid sequence of QKYSSYPLT (SEQ ID NO:51); or (l) Light chain CDR1 containing the amino acid sequence, Light chain CDR2 containing the amino acid sequence of GASTGAT (SEQ ID NO:53), and Light chain CDR3 containing the amino acid sequence Light chain containing variable domains and including and / or The heavy chain variable domain comprises heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:35, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:36, and heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:37, and The light chain variable domain includes light chain CDR1 containing the amino acid sequence of SEQ ID NO:52, light chain CDR2 containing the amino acid sequence of SEQ ID NO:53, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:

54. The recombinant fusion protein according to claim 1.

11. The recombinant fusion protein according to claim 1, wherein the Fab comprises a heavy chain variable domain having an amino acid sequence having at least 90% identity with SEQ ID NO: 55, 56, 57, 58, 59, or 60, and / or an amino acid sequence of SEQ ID NO:

60.

12. The recombinant fusion protein according to claim 1, wherein the Fab comprises a light chain variable domain having an amino acid sequence having at least 90% identity with SEQ ID NO: 61, 62, 63, 64, 65, 66, or 67, and / or an amino acid sequence of SEQ ID NO:

67.

13. The recombinant fusion protein according to claim 1, wherein the Fab comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, 59, or 60, and the light chain variable domain comprising the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, or 67.

14. The recombinant fusion protein according to claim 1, wherein the Fab comprises a heavy chain constant domain having an amino acid sequence having at least 90% identity with SEQ ID NO:

68.

15. The recombinant fusion protein according to claim 1, wherein the Fab comprises a light chain constant domain having an amino acid sequence having at least 90% identity with SEQ ID NO: 69 or 91.

16. The recombinant fusion protein according to claim 1, wherein the Fab comprises a heavy chain containing the amino acid sequence of SEQ ID NO:6 and a light chain containing the amino acid sequence of SEQ ID NO:69 or 91.

17. The recombinant fusion protein according to claim 1, wherein the Fab comprises a heavy chain constant 1 domain containing an amino acid sequence of SEQ ID NO: 68 linked to the heavy chain variable domain; and a light chain constant domain containing an amino acid sequence of SEQ ID NO: 69 or 91 linked to the light chain variable domain.

18. A nucleic acid encoding a recombinant fusion protein according to any one of claims 1 to 17.

19. An expression vector comprising the nucleic acid described in claim 18.

20. Cells transformed with the expression vector according to claim 19.

21. A composition comprising a recombinant fusion protein according to any one of claims 1 to 17 and a carrier.

22. A pharmaceutical composition comprising a recombinant fusion protein according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

23. (I) A composition comprising (i) a recombinant fusion protein according to any one of claims 1 to 17 and a carrier, or (ii) a pharmaceutical composition comprising a recombinant fusion protein according to any one of claims 1 to 17 and a pharmaceutically acceptable carrier, and (II) a label comprising instructions for use.

24. A pharmaceutical composition for preventing or treating an autoimmune disease or inflammatory neurological disease, comprising a recombinant fusion protein according to any one of claims 1 to 17 as an active ingredient.

25. The pharmaceutical composition according to claim 24, wherein the autoimmune disease is inflammatory bowel disease.

26. The pharmaceutical composition according to claim 24, wherein the autoimmune disease is selected from the group consisting of Crohn's disease, ulcerative colitis, ankylosing spondylitis, systemic lupus erythematosus (SLE), asthma, edema, delayed-type allergy (type IV allergy), transplant rejection, graft-versus-host disease, autoimmune encephalomyelitis, multiple sclerosis, inflammatory bowel disease, cystic fibrosis, diabetic retinopathy, ischemic-reperfusion injury, vascular restenosis, glomerulonephritis, and gastrointestinal allergy, and the inflammatory neurological disease is selected from the group consisting of amyotrophic lateral sclerosis and Parkinson's disease.

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