Patent Application
20230174617
The present invention relates to a novel modified immunoglobulin Fc fusion protein and use thereof, and more specifically, relates to a novel modified immunoglobulin Fc fusion protein that has an increased half-life and is capable of minimizing side effects, such as ADCC and CDC, which may be caused by antibody Fc fragment; and use thereof.
An immunoglobulin (antibody) Fc domain is widely used as a carrier protein for various therapeutic and diagnostic proteins. An antibody includes two functionally independent moieties, that is, a variable domain that binds to an antigen and is known as “Fab”, and a constant domain that is associated with effector functions such as complement activation and attack by a phagocyte and is known as “Fc”. Fc has a long serum half-life, and Fab has a short life span (Capon et al., Nature 337: 525-531, 1989). When combined with a therapeutic protein or a peptide, the Fc domain can provide a longer half-life, or integrate functions such as the binding of an Fc receptor, the binding of protein A, the fixation of a complement, and the transfer of a placenta. In particular, the longer half-life of such an Fc domain is due to neonatal Fc receptor (hereinafter, referred to as “FcRn”) binding affinity.
Many techniques for fusing an Fc domain and a biologically active protein have been developed so far to increase the half-life of the biologically active protein. As a representative example thereof, etanercept (trade name: Enbrel®) that is a fusion protein in which TNFα receptor 2 is linked to the Fc domain of an IgG1 antibody is mentioned (U.S. Pat. No. 5,447,851). In addition to the example, there are also some cytokines and growth hormones fused to the Fc domain of an IgG antibody.
However, unlike the fusion of a cell surface receptor to an extracellular domain, the fusion of a water-soluble protein to IgG causes the reduction in a biological activity compared to a non-fusion cytokine or growth factor. A chimeric protein exists as a dimer, and causes steric hindrance in binding to a target molecule such as a receptor due to the presence of two active proteins close to each other. Therefore, it is necessary to overcome such a problem to produce an efficient Fc fusion protein.
Another limitation of the Fc fusion technique is an unintended immune response. The Fc domain of an immunoglobulin also has effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In particular, four human IgG isoforms bind, with different affinity, to an activating Fcγ receptor (FcγR including FcγRI, FcγRIIa, and FcγRIIIa), an inhibitory FcγRIIb receptor, and a first factor of a complement (C1q) to cause very different effector functions (Bruhns, P. et al., Blood 113(16): 3716-3725, 2009). The binding of IgG to FcγR or C1q depends on residues located in a hinge region and a CH2 domain. However, the effector function of the Fc domain causes apoptosis, cytokine secretion, or inflammation in a subject when a therapeutic protein fused to the Fc domain is administered to the subject in vivo, and thus the effector function is needed to be suppressed to reduce unwanted reactions, unless absolutely necessary.
In order to overcome the above-identified problems, some modified Fc domain proteins have been developed, including those described in US2006/0074225A1, Armour, K. L. et al. (Eur. J. Immunol., 29(8): 2613-2624, 1999), Shields, R. L. et al. (J. Biol. Chem. 276(9): 6591-6604, 2001), and Idusogie, E E. et al. (J. Immunol. 164(8): 4178-4184, 2000).
In addition, despite the improvement in a half-life, there is still a need to further improve the half-life of a therapeutic protein fused to an Fc domain.
However, the abovementioned prior arts have the disadvantage of not achieving a satisfactory prolongation level of a half-life or having other side effects.
Therefore, the present invention is to solve the abovementioned various problems, and an object of the present invention is to provide a novel modified Fc domain protein which does not affect the production and the activity of a bioactive protein while a therapeutic protein fused without effector functions such as ADCC and CDC exhibits a longer half-life. However, such an object is exemplary and does not limit the scope of the present invention.
In accordance with an aspect of the present invention, provided is an immunoglobulin Fc domain variant protein in which 18th and 196th amino acids of a modified IgG4 Fc domain protein including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 are mutated to other amino acids.
In accordance with another aspect of the present invention, provided is a fusion protein in which at least one biologically active protein (API, active pharmaceutical ingredient) is linked to an N-terminus and/or a C-terminus of the immunoglobulin Fc domain variant protein.
In accordance with still another aspect of the present invention, provided is a polynucleotide which encodes the immunoglobulin Fc domain variant protein or the fusion protein.
In accordance with still another aspect of the present invention, provided is a homo- or hetero-dimer which contains the fusion protein.
In accordance with still another aspect of the present invention, provided is a composition which contains, as an active ingredient, the fusion protein, a polynucleotide encoding the fusion protein, or the homo- or hetero-dimer.
In accordance with still another aspect of the present invention, provided is a method for prolonging the half-life in vivo of a biologically active protein, the method including a step for administering, to a subject, a fusion protein obtained by fusing the biologically active protein to the immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.
By the fusion to another biologically active protein, the immunoglobulin Fc domain variant protein according to one exemplary embodiment of the present invention can increase the half-life of the biologically active protein and inactivate antibody Fc domain-dependent effector functions, such as ADCC and CDC which are side effects caused when using an antibody-based protein therapeutic agent. However, the effects of the present invention are not limited thereto.
According to an aspect of the present invention, provided is an immunoglobulin Fc domain variant protein in which 18th and 196th amino acids of a modified IgG4 Fc domain protein including an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 are mutated to other amino acids.
As used herein, the term “modified IgG4 immunoglobulin Fc domain protein” or “modified IgG4 Fc domain protein” refers to a recombinant antibody Fc domain protein prepared by combining all or part of a hinge, CH2, and CH3, which are constituent elements of an Fc domain of an immunoglobulin, with those derived from different types of antibody molecules, that is, IgG, IgD, IgE, and IgM. As a representative modified IgG4 immunoglobulin Fc domain protein, those disclosed in Korean Patent No. 897938 are mentioned.
As used herein, the term “immunoglobulin Fc domain variant protein” or “Fc domain variant protein” refers to a mutant protein in which at least one amino acid in the modified IgG4 Fc domain protein is substituted, deleted, or added.
In the immunoglobulin Fc domain variant protein, the mutation of the 18th amino acid may be a mutation selected from the group consisting of T18N, T18K, and T18Q, the mutation of the 196th amino acid may be a mutation selected from the group consisting of M196A, M196F, M196I, M196L, M196V, M196P, and M196W, the mutation of the 18th amino acid is more preferably T18Q, and the mutation of the 196th amino acid is preferably M196L.
Lysine (K) at a C-terminus of the immunoglobulin Fc domain variant protein may or may not be removed. Whether lysine is removed can be determined according to whether another API is linked to the C-terminus. For example, when API is linked to the C-terminus of the immunoglobulin Fc domain variant protein of the present invention, lysine removal may be advantageous in terms of protein production, and when API is not linked to the C-terminus, it is not necessary to remove lysine.
The immunoglobulin Fc domain variant protein may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 9.
According to another aspect of the present invention, provided is a fusion protein in which at least one biologically active protein (API, active pharmaceutical ingredient) is linked to an N-terminus and/or the C-terminus of the immunoglobulin Fc domain variant protein (
As used herein, the term “biologically active protein” is a concept distinguished from structural proteins, which are proteins constituting tissues and organs of a human body, and refers to a protein which performs specific functions such as metabolism and intercellular or intracellular signal transmission that occur in the human body, and can be used for diagnosis and/or treatment of diseases caused by deficiency or excess of the protein.
As shown in
The fusion protein according to one exemplary embodiment of the present invention may contain one or two biologically active proteins. When the fusion protein contains two biologically active proteins, the fusion protein itself exhibits bispecificity (
In the fusion protein, the biologically active protein may be a cytokine, an enzyme, a blood coagulation factor, an extracellular domain of a membrane receptor, a growth factor, a peptide hormone, or an antibody mimetic.
In the fusion protein, the length of the linker peptide may be 2 to 60 a.a., 4 to 55 a.a., 5 to 50 a.a., 5 to 46 a.a., 5 to 45 a.a., or 5 to 30 a.a. Among the linker peptides, a linker peptide, which links API to the N-terminus of the immunoglobulin Fc domain variant protein, may include a part or all of a hinge region of a heavy chain of an IgG1 antibody, and a case where the linker peptide includes a part of the hinge region may be a case where an artificial linker peptide is added to an N-terminus or a C-terminus of a part of the hinge region. Furthermore, the hinge region may be a hybrid-type hinge region in which parts of hinge regions of two or more heavy chains of the antibody are mixed. Specifically, the linker peptide may be a combination of one or more selected from the group consisting of GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTC PPCP (SEQ ID NO: 21), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 22), (G4S)n (unit sequence: SEQ ID NO: 23, n is an integer of 1 to 10), (GSSGGS)n (unit: SEQ ID NO: 24, n is an integer of 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 29), (EAAAK)n (unit: SEQ ID NO: 30, n is an integer of 1 to 10), CRRRRRREAEAC (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), GGGGGG (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), (Ala-Pro)n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 35), PLGLWA (SEQ ID NO: 36), TRHRQPRGWE (SEQ ID NO: 37), AGNRVRRSVG (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), GFLG (SEQ ID NO: 43), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GGSGGSGGS (SEQ ID NO: 46), GGGSGG (SEQ ID NO: 47), and GSTSGSGKPGSGEGS (SEQ ID NO: 48).
In this case, the cytokine may be a chemokine, an interferon, an interleukin, a colony-stimulating factor, or a tumor necrosis factor, the chemokine may be CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, XCL1, XCL2, or CX3CL1, the interferon may be interferon-alpha, interferon-beta, interferon-epsilon, interferon-kappa, interferon-delta, interferon-gamma, interferon-tau, interferon-omega, interferon-nu, or interferon-zeta, the interleukin may be IL-2, IL-4, IL-7, IL-10, IL-12α, IL-12β, IL-13, IL-15 or IL-21, the colony-stimulating factor may be a macrophage colony-stimulating factor (M-CSF), a granulocyte macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), or promegapoietin, and the tumor necrosis factor may be TNFα, TNFβ, a CD40 ligand (CD40L) a Fas ligand (FasL), a TNF-related apoptosis inducing ligand (TRAIL), or LIGHT (tumor necrosis factor tumor necrosis factor superfamily member 14, TNFSF14).
In the fusion protein, the enzyme may be a tissue-type plasminogen activator (tPA), urokinase, alteplase, reteplase, tenecteplase, streptokinase, anlsteplase, taaphylokinase, nattokinase, limbrokinase, collagenase, glutenase, alglucerase, velaglucerase, imiglucerase, taliglucerase-α, β-glucocerebrosidase, α-galactosiase A, α-glucosiase, α-L-iduroniase, arylsulphatase B, agalsidase, adenosine deaminase, phenylalanine ammonia lyase, dornase, rasburicase, pegloticase, glucarpidase, or ocriplasmin, but is not limited thereto.
In the fusion protein, the blood coagulation factor may be a factor VIII or factor IX.
In the fusion protein, the membrane receptor may be a receptor tyrosine kinase (RTK) superfamily, a T cell receptor, an Fc receptor, a chemokine receptor, a Toll-like receptor family, a guanylyl cyclase-coupled receptor (GCCR), or a receptor serine/threonine kinase, the receptor tyrosine kinase may be an EGFR family, an insulin receptor family, a platelet-derived growth factor receptor (PDGFR) family, a vascular endothelial growth factor receptor (VEGFR) family, a fibroblast growth factor receptor (FGFR) family, a colon cancer kinase (CCK) family, a nerve growth factor receptor (NGFR) family, a hepatocyte growth factor receptor (HGFR) family, an erythropoietin-producing human hepatocyte receptor (EphR) family, a tyrosine-protein kinase receptor (AXL) family, an angiopoietin receptor (TIER) family, a receptor tyrosine kinase-related receptor (RYKR) family, a discoidin domain receptor (DDR) family, a rearranged during transfection (RET) family, a leukocyte receptor tyrosine kinase (LTK) family, a receptor tyrosine kinase-like orphan receptors (ROR) family, a muscle-specific kinase (MuSK) family, CD80 (B7.1), or CD83, the Fc receptor may be an Fc gamma receptor, an Fc alpha receptor, or an Fc epsilon receptor, the cytokine receptor may be a type 1 cytokine receptor, a type 2 cytokine receptor, a tumor necrosis factor receptor family, a chemokine receptor, or a TGF-beta receptor family, the Toll-like receptor family may be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13, the guanylyl cyclase-coupled receptor may be natriuretic factor receptor 1 (NPR1), NPR2, or NPR3, and the receptor serine/threonine kinase may be a TGF-β superfamily receptor, a bone morphogenetic protein (BMP) receptor, or an activin receptor-like kinase (ALK).
In the fusion protein, the growth factor may be adrenomedullin, angiopoietin, a bone morphogenetic protein, a ciliary neurotrophic factor, a leukemia inhibitory factor, an epidermal growth factor (EGF), ephrin, erythropoietin (EPO), fibroblast growth factor (FGF), a glial cell line-derived neurotrophic factor (GDNF) family, growth differentiation factor-9 (GDF9), a hepatocyte growth factor (HGF), a hepatoma-derived growth factor (HDGF), insulin, an insulin-like growth factor (IGF), a keratinocyte growth factor (KGF), a migration-stimulating factor (MSF), a macrophage-stimulating factor, neuregulin, neurotrophin, a placental growth factor (PGF), a platelet-derived growth factor (PDGF), a T-cell growth factor (TCGF), thrombopoietin (TPO), a transforming growth factor (TGF) family, or a vascular endothelial growth factor (VEGF).
In the fusion protein, the peptide hormone may be a human growth hormone (hGH), a GLP-1 analog, a GLP-2 analog, insulin, an adrenocorticotropic hormone, amylin, angiotensin, neuropeptide Y, enkephalin, neurotensin, an atrial natriuretic peptide, calcitonin, choloecystokinin, gastrin, ghelin, glucagon, a follicle-stimulating hormone, leptin, a melanocyte-stimulating hormone (MSH), oxytocin, a parathyroid hormone, prolactin, renin, somatostatin, a thyroid-stimulating hormone (TSH), a thyrotropin-releasing hormone (TRH), vasopressin, or a vasoactive intestinal peptide.
In the fusion protein, the antibody mimetic may be scFv, VNAR, VHH, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, a Kunitz domain peptide, a monobody, a repebody, VLR, or nanoCLAMP.
In the fusion protein, the IL-10 protein may be an IL 10 variant protein in which isoleucine, which is the 87th amino acid, is substituted with alanine, or a monomeric IL-10 variant protein in which a linker peptide having a length of 6 to 10 a.a. is inserted between asparagine, which is the 134th amino acid, and lysine, which is the 135th amino acid, and may include the amino acid sequence set forth in SEQ ID NO: 54 or 56.
As used herein, the term “fusion protein” refers to a recombinant protein in which two or more proteins or domains responsible for specific functions in a protein are linked such that each protein or domain is responsible for an original function thereof. A linker peptide having a flexible structure can be generally inserted between the two or more proteins or domains. As the linker peptide, GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTC PPCP (SEQ ID NO: 21), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 22), (G4S)n (unit sequence: SEQ ID NO: 23, n is an integer of 1 to 10), (GSSGGS)n (unit: SEQ ID NO: 24, n is an integer of 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 29), (EAAAK)n (unit: SEQ ID NO: 30, n is an integer of 1 to 10), CRRRRRREAEAC (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), GGGGGG (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), (Ala-Pro)n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 35), PLGLWA (SEQ ID NO: 36), TRHRQPRGWE (SEQ ID NO: 37), AGNRVRRSVG (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 40), GGGGSGGGGSGGGGS (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), GFLG (SEQ ID NO: 43), AKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GGSGGSGGS (SEQ ID NO: 46), GGGSGG (SEQ ID NO: 47), and GSTSGSGKPGSGEGS (SEQ ID NO: 48) may be memtioned.
As used herein, the term “antibody” refers to an immunoglobulin molecule which is a heterotetrameric protein produced by binding two identical heavy chains and two identical light chains, and performs antigen-specific binding through an antigen-binding site composed of a variable region (VL) of the light chain and a variable region (VH) of the heavy chain, thereby causing an antigen-specific humoral immune response. As the antibody, depending on the origin thereof, IgA, IgG, IgD, IgE, IgM, IgY, or the like is mentioned.
As used herein, the term “antigen-binding fragment of an antibody” refers to a fragment which has antigen-binding ability derived from an antibody and includes both a fragment produced by cleaving an antibody with a protein cleaving enzyme as well as a single-chain fragment produced in a recombinant manner, and examples thereof include Fab, F(ab′)2, scFv, diabody, triabody, sdAb, or VHH.
As used herein, the term “Fab” refers to an antigen-binding antibody fragment (fragment antigen-binding) which is produced by cleaving an antibody molecule with papain, which is a proteinase and is a dimer of two peptides of VH-CH1 and VL-CL, and the other fragment produced by the papain is referred to as Fc (fragment crystallizable).
As used herein, the term “F(ab′)2” refers to a fragment that includes an antigen-binding site among fragments produced by cleaving an antibody with pepsin, which is a proteinase, and is in a form of a tetramer in which the two Fab's are linked by a disulfide bond. The other fragment produced by the pepsin is referred to as pFc′.
As used herein, the term “Fab” refers to a molecule having a similar structure to that of Fab produced by separating the abovementioned F(ab′)2 under weak reducing conditions.
As used herein, the term “scFv” is an abbreviation for a “single chain variable fragment”, and refers to a fragment which is not a fragment of an actual antibody, but is a kind of fusion protein prepared by linking the heavy-chain variable region (VH) to the light-chain variable region (VL) of the antibody through a linker peptide having a size of about 25 a.a., and is known to have antigen-binding ability even though the fragment is not a unique antibody fragment (Glockshuber et al., Biochem. 29(6): 1362-1367, 1990).
As used herein, the terms “diabody” and “triabody” refer to antibody fragments in a form of two and three scFv's linked by a linker, respectively.
As used herein, the term “single domain antibody (sdAb)” refers to an antibody fragment which is also referred to as a nanobody and consists of a single variable region fragment of an antibody. The sdAb derived from the heavy chain is mainly used, but a single variable region fragment derived from the light chain is also reported to specifically bind to an antigen. VNAR composed of variable region fragments of a shark antibody and VHH composed of variable region fragments of a camelid antibody, which consist only of dimers of single chains unlike conventional antibodies composed of a heavy chain and a light chain, are also included in sdAb.
As used herein, the term “antibody mimetic” is a concept including a protein having similar functions to those of antibodies prepared from non-antibody-derived protein scaffolds such as a monobody and a variable lymphocyte receptor (VLR), that is, having antigen-binding ability, unlike a normal full-length antibody in which two heavy chains and two light chains form a quaternary structure of a heterotetramer to exhibit functions. Examples of such an antibody mimetic include Affibody derived from a Z domain of protein A (Nygren, P. A., FEBS J. 275(11): 2668 to 2676, 2008), Affilin derived from Gamma-B crystallin or Ubiquitin (Ebersbach et al., J. Mol. Biol. 372(1): 172-185, 2007), Affimer derived from Cystatin (Johnson et al., Anal. Chem. 84(15): 6553-6560, 2012), Affitin derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383 (5): 1058-1068, 2008), Alphabody derived from a triple helix coiled coil protein (Desmet et al., Nat. Commun. 5: 5237, 2014), Anticalin derived from lipocalin (Skerra et al., FEBS J. 275(11): 2677-2683, 2008), Avimer derived from domains of various membrane receptors (Silverman et al., Nat. Biotechnol. 23(12): 1556-1561, 2005), DARPin derived from Ankyrin repeat motif (Stumpp et al., Drug Discov. Today. 13(15-16): 695-701, 2008), Fynomer derived from a SH3 domain of a Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5): 3196-3204, 2007), Kunitz domain peptides derived from Kunitz domains of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug Discov. Dev. 9(2): 261-268, 2006), a monobody derived from the 10th type 3 domain of fibronectin (Koide and Koide, Methods Mol. Biol. 352: 95-109, 2007), nanoCLAMP derived from carbohydrate-binding module 32-2 (Suderman et al., Protein Exp. Purif 134: 114-124, 2017), a variable lymphocyte receptor (VLR) derived from a hagfish (Boehm et al., Ann. Rev. Immunol. 30: 203-220, 2012), and a repebody engineered to enhance antigen affinity based on the VLR (Lee et al., Proc. Natl. Acad. Sci. USA, 109: 3299-3304, 2012).
According to still another aspect of the present invention, provided is a polynucleotide which encodes the immunoglobulin Fc domain variant protein or the fusion protein.
The polynucleotide may be any polynucleotide as long as the polynucleotide is a polynucleotide encoding a protein containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 9, and may have a codon optimized for the type of host cell used to produce the protein. Such a technique for optimizing a codon according to a host cell is well known in the art (Fuglsang et al., Protein Expr. Purif 31(2): 247-249, 2003). The polynucleotide more preferably contains a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13.
According to still another aspect of the present invention, provided is a recombinant vector containing the polynucleotide.
In the recombinant vector, the polynucleotide may be contained in a form of a gene construct operably linked to a regulatory sequence.
As used herein, the term “operably linked to” means that a target nucleic acid sequence (for example, in vitro transcription/translation system or in a host cell) is linked to the regulatory sequence in such a way that the target nucleic acid sequence can be expressed.
As used herein, the term “regulatory sequence” is meant to include a promoter, an enhancer, and other regulatory elements (for example, polyadenylation signal). Examples of the regulatory sequence include a sequence which directs such that a target nucleic acid is constantly expressed in many host cells, a sequence (for example, a tissue-specific regulatory sequence) which directs such that a target nucleic acid is expressed only in a specific tissue cell, and a sequence (for example, an inducible regulatory sequence) which directs such that expression is induced by a specific signal. Those skilled in the art could understand that the design of an expression vector may vary depending on factors such as the selection of a host cell to be transformed and the desired level of protein expression. The expression vector of the present invention can be introduced into a host cell to express the fusion protein. Regulatory sequences which enable expression in the eukaryotic cell and the prokaryotic cell are well known to those skilled in the art. As described above, these regulatory sequences generally include regulatory sequences responsible for transcription initiation, and optionally, a poly-A signal responsible for transcription termination and stabilization of a transcript. Additional regulatory sequences may include a translation enhancing factor and/or a naturally-combined or heterologous promoter region, in addition to the transcription regulatory factor. For example, possible regulatory sequences which enable expression in a mammalian host cell include a CMV-HSV thymidine kinase promoter, SV40, an RSV-promoter (Rous sarcoma virus), a human kidney urea 1α-promoter, a glucocorticoid-inducing MMTV-promoter (Moloney mouse tumor virus), a metallothionein- or tetracycline-inducible promoter, or an amplifying agent such as a CMV amplifying agent and an SV40 amplifying agent. It is considered that for expression in a nerve cell, a neurofilament-promoter, a PGDF-promoter, an NSE-promoter, a PrP-promoter, or a thy-1-promoter can be used. The abovementioned promoters are known in the art, and are described in the literature (Charron, J. Biol. Chem. 270: 25739 to 25745, 1995). For the expression in the prokaryotic cell, a number of promoters, including a lac-promoter, a tac-promoter, or a trp promoter, have been disclosed. In addition to the factors capable of initiating transcription, the regulatory sequences may include a transcription termination signal, such as an SV40-poly-A site and a TK-poly-A site, on the downstream of the polynucleotide according to one exemplary embodiment of the present invention. In the present specification, suitable expression vectors are known in the art, and examples thereof include Okayama-Berg cDNA expression vector pcDV1 (Parmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27 (Korean Patent No. 1442254), pX (Pagano et al., Science 255: 1144-1147, 1992), a yeast two-hybrid vector such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75: 791-803, 1995), and a prokaryotic expression vector such as lambda gt11 and pGEX (Amersham Pharmacia). The vector may further include a polynucleotide encoding a secretion signal, in addition to the nucleic acid molecules of the present invention. The secretion signals are well known to those skilled in the art. Moreover, depending on the used expression system, a leader sequence which can lead the fusion protein according to one exemplary embodiment of the present invention to a cellular compartment is combined with a coding sequence of the polynucleotide according to one exemplary embodiment of the present invention, and is preferably a leader sequence capable of directly secreting a decoded protein or the protein thereof into a pericytoplasmic or extracellular medium.
In addition, the vector of the present invention can be prepared, for example, by a standard recombinant DNA technique, and examples of the standard recombinant DNA technique include ligation of a smooth terminus and an adhesion terminus, a restriction enzyme treatment to provide a proper terminus, removal of a phosphate group by an alkaline phosphatase treatment to prevent inappropriate binding, and enzymatic linkage by T4 DNA ligase. The vector of the present invention can be prepared by recombining DNA encoding a signal peptide obtained by chemical synthesis or a genetic recombination technique, the immunoglobulin Fc domain variant protein according to one exemplary embodiment of the present invention, or DNA encoding a fusion protein containing the same with a vector containing an appropriate regulatory sequence. The vector containing a regulatory sequence can be commercially purchased or prepared, and in one exemplary embodiment of the present invention, a pBispecific backbone vector (Genexine, Inc., Korea), a pAD15 vector, pGP30 (Genexine, Inc. Korea), or a pN293F vector (Y-Biologics, Inc., Korea) was used as a skeleton vector.
The expression vector may further include a polynucleotide encoding a secretion signal sequence, and the secretion signal sequence induces the extracellular secretion of the recombinant protein expressed in the cell, and may be a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus glycoprotein Ds (HSV gDs) signal sequence, or a growth hormone signal sequence.
The expression vector according to one exemplary embodiment of the present invention may be an expression vector capable of expressing the protein in a host cell, and the expression vector may be in any form such as a plasmid vector, a viral vector, a cosmid vector, a phagemid vector, or an artificial human chromosome.
According to still another aspect of the present invention, provided is a homo- or hetero-dimer which contains the fusion protein.
In the homodimer, as shown in
Optionally, the homo- or hetero-dimers (500, 600, 700, and 800) according to one exemplary embodiment of the present invention may contain one or more linker peptides (14) between the biologically active proteins (11, 11a, 11b, 11c, and 11d) and the hinge region (13) or between the region of the immunoglobulin Fc domain variant protein (12) and the biologically active proteins. These fusion partners may be linked to the N-terminus or the C-terminus of the immunoglobulin Fc domain variant protein through the linker peptides (14) (
In particular, the biologically active protein (11a) linked to the N-terminus of the fusion protein may be a ligand which acts as a target moiety, and the other biologically active protein (11b) linked to the C-terminus of the same fusion protein may be a functional protein having a desired biological activity (
When the heterodimer according to one exemplary embodiment of the present invention is intended to be prepared, in order to minimize the formation of the homodimer, a knobs-into-holes (KIH) technique, which is well known in the art, can be used.
As used herein, the term “knobs-into-holes (KIH)” refers to one design strategy in antibody engineering used for heterodimerization of heavy chains in the production of the bispecific IgG antibody. In the KIH technique, when two fusion proteins forming a heterodimer are divided into a first fusion protein and a second fusion protein, in the modified IgG4 Fc region of the first fusion protein, serine, which is the 10th amino acid of a CH3 domain, may be substituted with cysteine (C), and threonine (T), which is the 22nd amino acid, may be substituted with tryptophan (W) (knobs structure), and in the Fc region of the second fusion protein, tyrosine (Y), which is the 5th amino acid of a CH3 domain, may be substituted with cysteine (C), threonine (T), which is the 22nd amino acid, may be substituted with serine (S), leucine (L), which is the 24th amino acid, may be substituted with alanine (A), and tyrosine (Y), which is the 63rd amino acid, may be substituted with valine (V) (holes structure). Conversely, in the Fc region of the first fusion protein, tyrosine (Y), which is the 5th amino acid of the CH3 domain, may be substituted with cysteine (C), threonine (T), which is the 22nd amino acid, may be substituted with serine (S), leucine (L), which is the 24th amino acid, may be substituted with alanine (A), and tyrosine (Y), which is the 63rd amino acid, may be substituted with valine (V) (holes structure), and in the modified IgG4 Fc region of the second fusion protein, serine (S), which is the 10th amino acid of the CH3 domain, may be substituted with cysteine (C), and threonine (T), which is the 22nd amino acid, may be substituted with tryptophan (W) (knobs structure).
In this case, the position of the amino acid where the mutation occurred is based on a reference sequence (amino acid sequence of the human IgG1 CH3 domain of SEQ ID NO: 77). Even when additional mutations such as addition, deletion, or substitution of amino acids occur at a site independent of the knobs-into-holes structure on the CH domain, a fusion protein in which an amino acid corresponding to the position of the site is mutated based on the reference sequence may be used. Optionally, the knobs-into-holes structure can be introduced through other amino acid mutations well known in the art. Such a mutation is specifically described in the prior literature (Wei et al., Oncotarget 8(31): 51037-51049, 2017; Ridgway et al., Protein Eng. 9(7): 617-621, 1996; Carter, P., J. Immunol. Methods 48(1-2): 7-15, 2001; Merchant et al., Nat. Biotechnol. 16(7): 677-681, 1998). A bispecific dimer fusion protein can be produced through such a selective mutation, for example, a combination of a knob structure in which threonine, which is the 22nd amino acid of the CH3 domain of the first fusion protein, is substituted with tyrosine and a hole structure in which tyrosine, which is the 63rd amino acid of the CH3 domain of the second fusion protein, is substituted with threonine. Conversely, the knobs-into-holes structure may be formed by introducing a hole structure to the first fusion protein and introducing a knob structure to the second fusion protein.
According to still another aspect of the present invention, provided is a composition which contains, as an active ingredient, the fusion protein, a polynucleotide encoding the fusion protein, or the homo- or hetero-dimer.
According to still another aspect of the present invention, provided is a method for prolonging the half-life in vivo of a biologically active protein, the method including a step for administering, to a subject, a fusion protein obtained by fusing the biologically active protein to the immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.
According to still another aspect of the present invention, provided is a composition which contains, as an active ingredient, a fusion protein obtained by fusing the biologically active protein to the immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein.
The composition may contain a pharmaceutically acceptable carrier, and may further include a pharmaceutically acceptable adjuvant, excipient, or diluent in addition to the carrier.
As used herein, the term “pharmaceutically acceptable” refers to a composition which is physiologically acceptable and generally does not cause an allergic reaction, such as a gastrointestinal disorder and dizziness, or a similar reaction when administered to a human. Examples of the carrier, the excipient, and the diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Moreover, a filler, an anti-aggregation agent, a lubricant, a wetting agent, a fragrance, an emulsifier, and an antiseptic agent may be further contained.
Furthermore, when the pharmaceutical composition according to one exemplary embodiment of the present invention is administered to a mammal, the pharmaceutical composition can be formulated using methods known in the art to allow rapid, sustained, or delayed release of the active ingredient. Examples of the formulation include powder, a granule, a tablet, an emulsion, a syrup, an aerosol, a soft or hard gelatin capsule, a sterile injectable solution, and a sterile powder form.
The composition according to one exemplary embodiment of the present invention can be administered by various routes such as oral administration and parenteral administration, for example, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, or intravertebral administration, and can be administered using an implantation device for sustained release or continuous or repeated release. The administration can be performed once or several times a day within a desired range, and can be performed at an interval such as once a week, twice a week, and once a month, and the duration of administration is also not particularly limited.
The composition according to one exemplary embodiment of the present invention can be formulated in a suitable form with a pharmaceutically acceptable carrier that is commonly used. Examples of the pharmaceutically acceptable carrier include carriers for parenteral administration such as water, suitable oil, a saline solution, aqueous glucose, and glycol, and a stabilizer and a preservative may be further contained. Examples of the suitable stabilizer include antioxidants such as sodium hydrogen sulfite, sodium sulfite, or ascorbic acid. Examples of the suitable preservative include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. Moreover, the composition according to the present invention may suitably contain a suspending agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer agent, an antioxidant, or the like if necessary, depending on the administration method or the formulation. The pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].
The dosage of the composition to a patient depends on many factors including a height of the patient, a body surface area, an age, a specific compound administered, a gender, a time and a route of administration, general health, and other drugs administered simultaneously. The pharmaceutically active protein can be administered in an amount of 100 ng/body weight (kg) to 10 mg/body weight (kg), more preferably 1 to 500 μg/kg (body weight), and most preferably 5 to 50 μg/kg (body weight), but the dosage can be adjusted in consideration of the abovementioned factors.
According to still another aspect of the present invention, provided is a method for treating a disease of a subject, the method including a step for administering, to the subject in a therapeutically effective amount, a fusion protein obtained by fusing a biologically active protein to the immunoglobulin Fc domain variant protein, or a homo- or a hetero-dimer which contains the fusion protein.
As used herein, the term “therapeutically effective amount” refers to an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dose level can be determined according to factors including the type of subject, severity, an age, a gender, a drug activity, drug sensitivity, an administration time, an administration route, a rate of excretion, a treatment duration, drugs used simultaneously, and other factors well known in the medical field. The therapeutically effective amount of the composition according to the present invention is 0.1 mg/kg to 1 g/kg and more preferably 1 mg/kg to 500 mg/kg, but the effective dosage can be appropriately adjusted according to the age, the gender, and the condition of the patient.
The abovementioned disease is a disease requiring administration of the biologically active protein, for example, when the biologically active protein is a GLP-1 analog, the disease to be treated may be diabetes or metabolic syndrome, when the biologically active protein is a GLP-2 analog, the disease to be treated may be short bowel syndrome or an inflammatory bowel disease, when the biologically active protein is IL-2, the disease to be treated may be a malignant tumor, when the biologically active protein is FcεRIa, the disease to be treated may be IgE-mediated allergic diseases such as asthma and atopic dermatitis, and when the biologically active protein is an anti-CD40L antibody mimetic, the disease to be treated may be an autoimmune disease or an organ transplant rejection reaction.
Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, the present invention is not limited to Examples and Experimental Examples described below, and may be implemented in various other forms, and Examples and Experimental Examples described below are provided to enable the disclosure of the present invention to be complete and to fully convey the scope of the invention to those skilled in the art to which the present invention belongs.
A technique for linking an Fc domain of an antibody to a biologically active protein or an active pharmaceutical ingredient (API) to increase a half-life of API is a widely used technique. However, when in the Fc domain moiety of the antibody, an N-linked sugar chain is added to asparagine, which is the 297th amino acid, a sugar chain pattern is different from that of the human antibody or is not uniform and is produced in a heterogenous form depending on the characteristics of the host cell for antibody production. Therefore, the activity of a fusion protein to which API is linked may be not constant or an effect unnecessary in some cases, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), is exhibited to cause side effects. Moreover, it is known that the reason for the increase in half-life after administration of the antibody in vivo is that the antibody is absorbed due to endocytosis by vascular endothelial cells in vivo, then binds to a neonatal Fc receptor (hereinafter, referred to as “FcRn”) due to a low pH in the endosomal state to avoid degradation by lysosomes, and is separated from FcRn under extracellular physiological pH conditions in a process in which an endosome is recycled to a cell membrane (
However, since existing antibody Fc domains have different affinity with FcRn depending on the type of IgG, there is a limit to improving the half-life of API.
Accordingly, the present inventors designed an Fc domain protein variant which does not have activities of ADCC and CDC while increasing affinity of the existing Fc domain protein with FcRn.
Therefore, the present inventors designed a variant (SEQ ID NOs: 6 to 9) in which threonine (T), which is the 18th amino acid of the modified IgG4 Fc protein having the amino acid sequences set forth in SEQ ID NOs: 1 to 4, was substituted with glutamine (Q), and methionine (M), which is the 196th amino acid, was substituted with leucine (L), and the variant was named “NTIG (SEQ ID NO: 5)”.
Subsequently, the present inventors prepared various fusion proteins in which various biologically active proteins were linked to the N-terminus and/or the C-terminus of the immunoglobulin Fc domain variant protein (NTIG) prepared in Example 1.
2-1: Design and Production of GLP-1E-NTIG
The present inventors designed a fusion protein (GLP-1E-NTIG, SEQ ID NO: 50) in which the NTIG protein (SEQ ID NO: 7) prepared in Example 1 was linked to the C-terminus of the GLP-1/Exendin-4 hybrid peptide (SEQ ID NO: 49), which is a hybrid peptide of GLP-1 and Exendin-4, through a linker peptide ((G4S)3-IgD/IgG1 hybrid hinge, SEQ ID NO: 14) containing a hinge region in a hybrid form, and the fusion protein was named “PG-11”. GLP-1E-NTIG was designed to add a tPA signal sequence (SEQ ID NO: 51) for secretion from a host cell. Meanwhile, a polynucleotide encoding PG-11 was synthesized using oligonucleotide synthesis, PCR amplification, and a site-directed mutagenesis technique, and then inserted into a pGP30 expression vector (Genexine, Inc., Korea). The prepared vector was transiently expressed using an ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector prepared as above and an ExpiFectamine reagent contained in the kit were mixed in an ExpiCHO-S cell, the resultant was cultured for 1 day in an incubator with conditions of 8% CO2 and 37° C., then the temperature was lowered to 32° C., and the culture was performed until the 7th day.
Thereafter, capturing and purification of protein A were performed, it was checked whether a candidate material was purified through SDS-PAGE analysis under non-reducing and reducing conditions, and formulation was performed with a formulation buffer solution according to the pI value of the candidate material. The formulated material was quantified using Nano drop, and the final purity was checked using SEC-HPLC (
2-2: Design and Production of GLP-2-2G-NTIG
The present inventors designed a fusion protein (GLP-2-2G-NTIG, SEQ ID NO: 53) in which NTIG (SEQ ID NO: 7) was linked, through the linker peptide (SEQ ID NO: 14), to the C-terminus of a GLP-2 analog (GLP-2-2G, SEQ ID NO: 52) that is a mutant in which alanine (A), which is the 2nd amino acid of a human GLP-2 peptide, is substituted with glycine (G), and the fusion protein was named “PG-22”. Similarly, the PG-22 was also designed to add the tPA signal sequence (SEQ ID NO: 51) for secretion. Similar to Example 2-1, a polynucleotide encoding PG-22 was prepared and inserted into the pGP30 expression vector (Genexine, Inc., Korea) to produce a protein using the ExpiCHO system, and then the final purity was checked (
2-3: Design and Production of NTIG-IL-10V
The present inventors designed a fusion protein (NTIG-IL-10V, SEQ ID NO: 55) in which an IL-10 variant (SEQ ID NO: 54) was linked to the C-terminus of NTIG through a linker peptide (SEQ ID NO: 26), and the fusion protein was named “PG-088”. Similar to Example 2-1, a polynucleotide encoding PG-088 was prepared and inserted into a pBispec expression vector (Genexine, Inc., Korea) to produce a protein using the ExpiCHO system, and then the final purity was checked (
2-4: Design and Production of NTIG-IL-10V Monomer
The present inventors designed a fusion protein (NTIG-IL-10Vm, SEQ ID NO: 57) in which an IL-10 monomeric variant (SEQ ID NO: 56) was linked to the C-terminus of NTIG through the linker peptide (SEQ ID NO: 26), and the fusion protein was named “PG-088m”. Similar to Example 2-1, a polynucleotide encoding PG-088m was prepared and inserted into the pBispec expression vector to produce a protein using the ExpiCHO system, and then the final purity was checked (
3-1: Preparation of Anti-PD-L1-NTIG-IL-2
A polynucleotide encoding each of a fusion protein (anti-PD-L1-NTIG-IL-2) in which scFv targeting PD-L1 was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-2 was linked to the C-terminus, and a fusion protein (anti-PD-L1-modified IgG4 Fc-IL-2) using a modified IgG4 Fc domain protein of SEQ ID NO: 2 instead of NTIG was inserted into the pBispec expression vector, and each protein was produced using the ExpiCHO system.
3-2: Design and Production of FcεRIα-NTIG-IL-10V
The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10V, SEQ ID NO: 59) in which an extracellular domain (SEQ ID NO: 58) of FcεRIα, which is a receptor specifically binding to IgE, was linked to the N-terminus of NTIG-IL-10V designed in Example 2-3 through a linker peptide (SEQ ID NO: 18), the bispecific fusion protein was named “PG-075”, and a polynucleotide encoding the fusion protein was prepared and inserted into the pBispec expression vector. The PG-075 construct was also designed so that the tPA signal sequence (SEQ ID NO: 51) was positioned at the N-terminus for secretion, was produced using the ExpiCHO system, and subjected to SDS-PAGE and western blotting (
3-3: Design and Production of FcεRIα-NTIG-IL-10Vm
The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10Vm, SEQ ID NO: 60) in which the extracellular domain (SEQ ID NO: 58) of FcεRIα, which is a receptor specifically binding to IgE, was linked to the N-terminus of NTIG-IL-10Vm designed in Example 2-4 through the linker peptide (SEQ ID NO: 18), the bispecific fusion protein was named “PG-110”, and a polynucleotide encoding the fusion protein was prepared and inserted into a pAD15 expression vector. The PG-110 construct was also designed so that the tPA signal sequence (SEQ ID NO: 51) was positioned at the N-terminus for secretion. A gene inserted into the pAD15 expression vector was transfected into a CHO DG44 cell, then subjected to methotrexate (MTX) amplification, and subjected to a single clone sorting operation to prepare a high-efficiency expressing cell line, and then a PG-110 protein was produced and purified from the high-efficiency expressing cell line (
4-1: Preparation of Chimeric Anti-CD40L Antibody
The present inventors prepared an anti-CD40L antibody obtained by introducing an FcγRIIα binding inhibitory variant into heavy-chain CH2 and CH3 domains of an antibody (C10) in which an Fc region of the mouse-derived anti-CD40L (anti-CD154) antibody described in WO2016/182335A1 was substituted with a human IgG1 Fc region, and the anti-CD40L antibody was named “C10M”.
4-2: Preparation of Chimeric Anti-CD40L-NTIG Hybrid Antibody
The present inventors designed a hybrid heavy chain protein in which a heavy chain moiety in a Fab moiety of the C10M antibody is linked to the N-terminus, does not bind to FcγR, of the NTIG protein prepared in Example 1, and the hybrid heavy chain protein was named “PG-129”.
A polynucleotide encoding a hybrid heavy chain of the hybrid heavy chain protein in which the heavy chain moiety (VH-CH1, SEQ ID NO: 61) in Fab of the C10M antibody is linked to NTIG (SEQ ID NO: 7) was prepared and inserted into a pAD15 expression vector (WO2015/009052A). Moreover, a polynucleotide encoding a light heavy moiety (VL—CL, SEQ ID NO: 62) in Fab of the C10M antibody was inserted into the pAD15 expression vector so that the polynucleotide can be regulated under a dual promoter. Thereafter, the vector was co-transfected into a CHO DG44 (from Dr. Chasin, Columbia University, USA) cell using a Neon-transfection system.
The expression vector completed as above was also transiently expressed using the ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector construct prepared as above and the ExpiFectamine reagent contained in the kit were mixed in the ExpiCHO-S cell, the resultant was cultured for 1 day in an incubator with conditions of 8% CO2 and 37° C., then the temperature was lowered to 32° C., and the culture was performed until the 7th day.
Thereafter, capturing and purification of protein A were performed, it was checked whether a candidate material was purified through PAGE analysis and western blot analysis under non-reducing and reducing conditions, and formulation was performed with a formulation buffer solution according to the pI value of the candidate material. The formulated material was quantified using Nano drop, and the final purity was checked using SEC-HPLC.
As a result, as shown in
4-3: Preparation of Humanized Anti-CD40L-NTIG Hybrid Antibody
The chimeric antibodies have sufficiently improved disadvantages of the conventional human antibodies by introducing the NTIG technique (immunoglobulin Fc domain variant protein) of the present invention, but the present inventors have determined that the antigen-binding site was still a mouse-derived antibody and thus unnecessary immune responses in the human body may occur, and, in order to prepare a safer hybrid antibody, designed a humanized antibody (anti-CD40L Fab-NTIG) in which the framework part except for an antigenic determinant of Fab, which is the antigen-binding fragment of the antibody that is the base of the present invention, was substituted with the sequence of the human antibody (Tables 1 and 2), and the humanized antibody was named “PG-400”.
Furthermore, the present inventors designed to prepare a bispecific hybrid antibody (anti-CD40L Fab-NTIG-IL-10Vm) in which the IL-10 monomeric variant protein (SEQ ID NO: 56), which is a cytokine having an immunosuppressive activity, was linked to the heavy chain C-terminus of the hybrid antibody (Table 3), and the bispecific hybrid antibody was named “PG-410”. The structure of the light chain of the bispecific hybrid antibody is the same as shown in Table 2.
In order to produce humanized anti-CD40L hybrid antibodies (PG-400 and PG-410), a polynucleotide encoding each of the heavy chain and the light chain of the antibody was prepared, and then for temporary expression in an animal cell, plasmid DNA was transfected into a HEK293F cell using the N293F vector system (Y-BIOLOGICS). In order to prepare a nucleic acid, 25 μg of plasmid DNA was added to and mixed with 3 ml of the medium, and then 25 μl of 2 mg/ml Polyethylenimine (PEI, PolyPlus, USA) was added thereto, followed by mixing. The reaction solution was allowed to stand at room temperature for 15 minutes, and then put into 40 ml of a culture solution cultured at 1×106 cells/ml, and the resultant was cultured for 24 hours under conditions of 120 rpm, 37° C., and 8% CO2. 24 hours after DNA transfection, a nutritional supplement medium component (Soytone, BD, USA) was added so that the final concentration was 10 g/L. After culturing for 7 days, the cell culture solution was centrifuged at 5000 rpm for 10 minutes to collect a supernatant.
Subsequently, a protein A resin filled a column and was washed with 1×DPBS, the collected supernatant was combined with the resin at 4° C. at a rate of 0.5 ml/min, and a protein was eluted with 0.1 M glycine. In order to replace a buffer solution, the eluted solution was put into a dialysis tube (GeBAflex tube, Geba, Israel) and dialyzed at 4° C. with 1×DPBS. The obtained substance was subjected to gel filtration using a Superdex 200 resin, and then formulated with a PBS (pH of 7.4) buffer solution.
For the purified hybrid antibody, the yield and the purity of the hybrid antibody were assayed through SDS-PAGE analysis, size-exclusion FPLC, and size-exclusion HPLC analysis.
As a result, as shown in
4-4: Design of Anti-CD40L scFv-NTIG-IL-10 Fusion Protein
The present inventors sorted out a variable region moiety in the Fab of the humanized anti-CD40L antibody, designed scFv using the sorted-out variable region moiety, designed a single chain-based antibody binding-fragment fusion protein by inserting scFv instead of the VH-CH1 moiety of the heavy chain construct of the anti-CD40L antibody used in Example 4-3, and designed a bispecific fusion protein in which the IL-10 monomeric variant (SEQ ID NO: 56) used in Example 4-3 was linked to the C-terminus of the anti-CD40L scFv-NTIG through the linker peptide (SEQ ID NO: 26) (Table 4). In the present Example, the anti-CD40L scFv is composed of the light-chain variable region (VL), the linker, the heavy-chain variable region (VH) in this order, but conversely, an anti-CD40L scFv, which is composed of VH, the linker, and VL in this order, may be used because the CD40L-binding ability is maintained even in a case of using the latter anti-CD40L scFv, and also regarding the type of linker, various types can be used.
The present inventors designed bispecific fusion proteins containing both the GLP-1 analog and the GLP-2 analog as shown in Table 5. In these bispecific fusion proteins, the knobs-into-holes (KIH) structure is introduced so as to form a heterodimer by an intermolecular disulfide bond and a hydrophobic interaction between the first fusion protein and the second fusion protein.
A difference between Examples 5-1 and 5-2 is whether an N-linked sugar chain attachment site is included so that an N-glycan sugar chain can be linked to the linker linking the GLP-1 analog of the first fusion protein to NTIG. This is because GLP-1 as well as Exendin 4 (Ex4), oxyntomodulin (OXM), GLP-2 all bind to GLP-1R which is a GLP-1 receptor, whereas only GLP-2 binds to GLP-2R, and GLP-1R is expressed in various organs such as a brain, a heart, a liver, muscle, and pancreas in addition to gastrointestinal tract (GI tract), and thus it is necessary to lower the affinity with GLP-1R somewhat in order to allow GLP-1 to function specifically in the gastrointestinal tract. For the reason, in order to lower the binding force of the GLP-1 analog to GLP-1R somewhat, the present inventors used a protein to which a glycan attachment domain was added so that glycan can be attached to the linker moiety (including the IgD/IgG1 hybrid hinge) of the GLP-1 analog (Example 5-1).
The vector constructs prepared as above were transiently expressed using the ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. Specifically, the vector constructs prepared as above and the ExpiFectamine reagent contained in the kit were mixed in the ExpiCHO-S cell, the resultant was cultured for 1 day in an incubator with conditions of 8% CO2 and 37° C., then the temperature was lowered to 32° C., and the culture was performed until the 7th day.
The supernatant obtained through the culturing and the heterodimer proteins (were named ‘MG12-4’ and ‘MG12-5’, respectively) of Examples 5-1 and 5-2 purified through a protein A column and a secondary column were appropriately diluted with a 4×LDS sample buffer solution and water for injection to prepare a sample so that the final concentration was 3 to 10 μg/20 μL. In a case of a sample under the reducing conditions, each substance to be analyzed, the 4×LDS sample buffer solution, a 10×reducing agent, and the water for injection were appropriately diluted to prepare a sample so that the final concentration was 3 to 10 μg/20 μL, and the sample was heated in a 70° C. heating block for 10 minutes. The prepared sample was loaded in an amount of 20 μL in each well of a gel fixed in an electrophoresis apparatus which was installed in advance. For a size marker, the sample was loaded at 3 to 5 μL/well. After a power supply device was set to 120 V and 90 minutes, electrophoresis was performed. The gel subjected to the electrophoresis was separated, and then stained using a staining solution and a destaining solution, and the results were analyzed. As a result, as shown in
The present inventors investigated how the production efficiency of the protein varies depending on the type of hinge when API is linked to the N-terminus of the NTIG protein, while linking various types of API to the N-terminus and/or the C-terminus of the NTIG protein of the present invention.
6-1: Preparation of FcεRIα-NTIG-IL-13a2
The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-13Rα2) in which FcεRIα (SEQ ID NO: 58) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-13Rα2 (SEQ ID NO: 74) was linked to the C-terminus, and the bispecific fusion protein was named “PG-50”. While producing PG-50, a hinge region linking FcεRIα to NTIG was investigated for protein productivity when using an IgD hinge (SEQ ID NO: 15) or a combination of a (G4S)3 linker and an IgG1 hinge (SEQ ID NO: 16). In this way, a fusion protein using the IgD hinge was named “PG-50-1”, and a fusion protein using the (G4S)3 linker/IgG1 hinge was named “PG-50-2”. In a case of the IgD hinge, one intermolecular disulfide bond was formed, and in a case of the (G4S)3 linker/IgG1 hinge, two intermolecular disulfide bonds were formed.
As a result, as shown in
6-2: Preparation of GLP-2-2G-NTIG-IL-10 wt
The present inventors analyzed the effects of various types of hinges on a protein production yield using different types of API. For the analysis, specifically, a construct was prepared in which GLP-2-2G (SEQ ID NO: 52) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-10 wt (SEQ ID NO: 75) was linked to the C-terminus of NTIG. Here, a bispecific fusion protein (GLP-2-2G-NTIG-IL-10 wt) was designed by linking GLP-2-2G to NTIG using a (G4S)3 linker+IgD/IgG1 hybrid hinge (SEQ ID NO: 14), and linking IL-10 wt to the C-terminus of NTIG using a linker peptide consists of the amino acid sequence set forth in SEQ ID NO: 26, and was named “PG-073”. A polynucleotide encoding the fusion protein designed as above was prepared and then inserted into the pBispec expression vector, a fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein expression level in the culture supernatant was checked by SDS-PAGE. The IgD/IgG1 hybrid hinge forms two intermolecular disulfide bonds.
As a result, as shown in
6-3 and 6-4: Preparation of GLP-2-2G (3 Point)-NTIG-IL-10Vm
Accordingly, the present inventors designed fusion proteins (GLP-2V (3 point)-NTIG-IL-10Vm) in which in the GLP-2-2G-NTIG-IL-10wt fusion protein, GLP-2-2G was changed to a mutant (SEQ ID NO: 76) containing a 3 point mutation, the hinge portion was replaced with a different type of linker or hinge (each SEQ ID NO: 17 or 18), and IL-10 wt was replaced with IL-10Vm prepared in Example 2-4, and the fusion proteins were named “PG210-5 (Example 6-3)” and “PG210-6 (Example 6-4), respectively.
After a polynucleotide encoding each of the fusion proteins was inserted into the pBispec expression vector, a fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein expression level in the culture supernatant was checked by SDS-PAGE.
As a result, as shown in
The present inventors compared the binding affinity of the NTIG protein of the present invention with FcRn to IgG4 Fc and IgG1 antibodies.
For the comparison, specifically, the recombinant FcRn (rhFcRn, R&D systems, USA), which is a ligand, was dissolved in an acetate buffer at a concentration of 5 μg/ml, and then fixed to a biosensor chip (Series S CM5 sensor chip, GE Healthcare, USA) using amine coupling.
Subsequently, in order to compare the FcRn-binding affinity of NTIG of the present invention and the FcRn-binding affinity of IgG4 Fc, the present inventors analyzed, through surface plasmon resonance (SPR), the binding affinity of, with FcRn, PG-11 prepared in Example 2-1 and dulaglutide (trade name Trulicity) which is a fusion protein in which commercial GLP-1 is linked to IgG4 Fc. As the analyzer, Biocore 8K (GE Healthcare, USA) was used, and all reagents used in the analysis were purchased from GE Healthcare. PG-11 and Trulicity were dissolved in a running buffer solution (PBST, pH of 6.0), then dilution was sequentially performed from 1,000 nM by ½, and multi-cycle kinetic analysis was performed. The experiment was performed at a flow rate of 30 μl/min at 25° C., an association of an analyte was performed for 60 seconds, and then dissociation was performed for 60 seconds. Based on the results of the experiment, the optimal antibody concentration was selected (Trulicity: 8,000 nM and PG-11: 2,000 nM), multi-cycle kinetic analysis was performed, and the equilibrium dissociation constant (KD) was measured using the Biacore Insight program (GE Healthcare, USA). At this time, the buffer solution was replaced with a PBS buffer solution having a pH of 7.4 during regeneration.
As shown in Table 6 and
In addition, in order to compare the FcRn-binding affinity of NTIG of the present invention and the FcRn-binding affinity of IgG4 Fc, the present inventors analyzed, through surface plasmon resonance (SPR), the binding affinity of, with FcRn, PG-11 and Rituximab which is a commercial recombinant chimeric anti-CD20 antibody of an IgG1 family. PG-11 and Rituximab were dissolved in a running PBST buffer solution (pH of 6.0) at various concentrations (2,000 nM to 1.96 nM for PG-11, and 4,000 nM to 3.9 nM for Rituximab), and then similar to the comparative analysis of PG-11 and Trulicity, association, dissociation, and regeneration were performed under the conditions of 60 seconds for the association, 60 seconds for the dissociation, and 30 seconds for the regeneration. During the regeneration, a PBS buffer solution having a pH of 7.4 was used.
As shown in Table 7 and
As described above, NTIG according to one exemplary embodiment of the present invention has high affinity with FcRn compared to the Fc region of conventional IgG1 or IgG4, and thus when NTIG is administered in vivo, the half-life is expected to be significantly improved.
Subsequently, the present inventors intended to compare the affinity of the modified IgG4 Fc domain protein of SEQ ID NO: 2 with FcRn, on the basis of NTIG (SEQ ID NO: 7) according to one exemplary embodiment of the present invention.
For the comparison, instead of calculating a simple KD value, the present inventors calculated a steady state affinity (KD) by associating the proteins at a pH of 6.0 and performing dissociation at a pH of 7.3 (Piche-Nicholas et al., MAbs. 10(1): 81-94, 2018). Specifically, for the binding affinity of anti-PD-L1-NTIG-IL-2 and anti-PD-L1-modified IgG4 Fc-IL-2 prepared in Example 3-1 with rhFcRn, SPR analysis was performed by changing the pH conditions during the association and the dissociation as described above.
As a result, as shown in Table 8 and
These results suggest that the application of NTIG exhibits an effect of enhancing the half-life in vivo through enhancement of the FcRn-binding force of the produced protein as well as a superior effect on the physical properties the and stability of the protein, compared to the previously known application of the modified IgG4 Fc of SEQ ID NO: 2.
As the buffer solution used for the analysis, the running buffer solution (1×PBST, pH of 7.3), the sample dilution buffer solution (1×PBST, pH of 6.0), the dissociation buffer solution (1×PBST, pH of 7.3), and the regeneration buffer solution (1×PBS, pH of 8.0) were used.
As a result of analysis, as shown in Table 9 and
The Fc region of IgG1 is known to have binding affinity with FcγRI and FcγRIIIa. These Fc receptors are associated with effector functions such as ADCC and CDC, are helpful in anticancer treatment when using antibodies or Fc fusion proteins for the anticancer treatment, but have disadvantages in which side effects are caused when using API for the purpose of treating other diseases other than anti-cancer treatment.
Accordingly, the present inventors analyzed the degree of the affinity of the NTIG fusion protein according to one exemplary embodiment of the present invention with FcγRI and FcγRIIIa compared to the IgG1 antibody through bio-layer interferometry analysis (BLI).
As a result, as shown in
Furthermore, the present inventors performed comparative analysis of the binding affinity of Rituximab and PG-11 according to one exemplary embodiment of the present invention with FcγRIIa, FcγRIIb, and FcγRIIIb.
As a result, as shown in
The antibody Fc region may induce complement-dependent cytotoxicity (CDC) by binding to the complement C1q, and thus may cause a serious problem in the clinical application of the Fc fusion protein for treating general diseases other than cancer treatment. Therefore, the degree of the binding affinity of the NTIG-containing fusion protein according to one exemplary embodiment of the present invention with the complement C1q was analyzed compared to Rituximab, which is an IgG1-based antibody.
For the comparison, specifically, PG-11 and Rituximab were each diluted with a coating buffer solution (0.1 M Na2HPO4, pH of 9.6) so that each concentration was 2 μg/ml. Subsequently, 100 μl of each diluted sample was loaded into each well and reacted overnight at 4° C. Next, washing was performed three times by adding 300 μl of a washing buffer solution to the plate each time, 200 μl of a blocking buffer solution (2% BSA/PBS) was added to each well and reacted at room temperature for 2 hours. 300 μl of the washing buffer solution was added to the plate, then washing was repeatedly performed three times, an anti-C1q antibody was diluted with an assay buffer solution to 10, 3, 1, 0.3, and 0.1 μg/ml by ⅓, and mixed (duplication) in an amount of 50 μl in each well, and then the resultant was reacted at room temperature for 2 hours. Then, the washing buffer solution was mixed in the plate by 300 μl each time and washing was repeatedly performed eight times. A C1q-HRP conjugate was diluted 400-fold, and 50 μl of the resultant was added to each well and reacted at room temperature for 1 hour. The washing buffer solution was added to the plate by 300 μl each time, washing was repeatedly performed eight times, 50 μl of TMB as a substrate was mixed, and the resultant was reacted at room temperature. When the color development reaction appeared in blue, 50 μl of a stop solution was added to each well to stop the reaction, and then optical density was measured using a microplate reader set at 450 nm.
As a result of the analysis, as shown in
Therefore, it was confirmed that NTIG according to one exemplary embodiment of the present invention is a safe protein carrier capable of minimizing side effects due to the Fc region such as ADCC or CDC while improving the half-life of API.
Whether the GLP-2 activity was properly exhibited with respect to a number of fusion proteins containing GLP-2 among Examples described above was investigated by cAMP Hunter™ eXpress GPCR analysis (Table 10).
As a result of analysis, as shown in
5-1: Analysis of Affinity with IL-10 Receptor
The present inventors analyzed the affinity of the fusion proteins of Examples 2-3 and 2-4 with the IL-10 receptor 1 (IL-10R1) through the bio-layer interferometry analysis (BLI).
For the analysis, the present inventors attached an IL-10R His-tag protein to a 96-well plate using a Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, 200 μl of D. W. was dispensed into a 96-well plate, and an amine biosensor included in the kit was inserted to perform hydration for 10 minutes. Subsequently, after 200 μl of D. W. was additionally dispensed into a new 96-well plate, EDC and NHS were mixed at 1:1 in an amount of 1/20 of the required sample amount and then diluted with D. W., and 200 μl of the resultant was dispensed into the 96-well plates. Next, the IL-10R His-tag protein was diluted to 10 μg/ml in a 10 mM acetate solution having a pH of 5.0, and then 200 μl of the resultant was added to the 96-well plates. After 200 μl of 1 M ethanolamine was added to the 96-well plates, the biosensor plate and the sample plate were placed in an Octet®-K2 instrument and measured. After the measurement was completed, 200 μl of a 1× kinetics buffer solution was added to the sample plate, and then a baseline was determined. Thereafter, the prepare NTIG-IL-10 fusion protein was diluted with the 1× kinetics buffer solution to various concentrations (0, 62.5, 125, 250, 500, and 1,000 nM), 200 μl of the resultant was dispensed to the sample plate, and then bio-layer interferometry (BLI) was measured using the Octet®-K2 instrument.
As a result, as shown in
5-2: Analysis of Immunosuppressive Activity of IL-10Vm Fusion Protein
The present inventors analyzed immunosuppressive activities in vitro of various fusion proteins (PG-088m, PG-110, PG-410, and PG-210-6) containing the monomeric IL-10 variant protein (IL-10Vm) according to one exemplary embodiment of the present invention.
For the analysis, specifically, after a supernatant was removed from a T75 flask including a macrophage Raw264.7 in culture, washing was performed once with PBS, then 3 ml of TrypLE™ Select Enzyme was added, and the resultant was reacted at 37° C. for 3 to 5 minutes. Thereafter, 10 ml of a fresh medium was added to the T75 flask, and a cell suspension from the bottom of the flask was transferred to a 50 ml conical tube and then centrifuged at 1,500 rpm for 5 minutes. Next, the supernatant was removed and the tube was gently tapped to release cells. Then, 5 ml of a fresh medium were mixed and the cells were counted. The cell concentration was adjusted to be 1×105 cells/ml, and 100 μl of the resultant was dispensed into a 96-well flat-bottom plate.
Thereafter, PG-088m prepared in Example 2-4, PG-110 prepared in Example 3-3, PG-410 prepared in Example 4-3, and PG-210-6 prepared in Example 6-4 were sequentially diluted to various concentrations (0 to 1,000 nM for PG-210-6 and PG-410, and 0 to 1,507 nM for PG-110 and PG-088m), and then 50 μl of each protein was dispensed into the 96-well plate into which the macrophage Raw264.7 was dispensed. After 20 minutes, a lipopolysaccharide (LPS) was treated in an increment of 50 μl so that the final concentration was 400 ng/ml, and the resultant was cultured at 36° C. for 16 hours. The next day, the supernatant was collected, and then TNF-α ELISA (Biolegend, USA) analysis was performed according to the protocol of the manufacturer.
As a result, as shown in
5-3: Analysis of IgE-Binding Ability
The present inventors analyzed the affinity of the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 3-3 with mouse IgE and human IgE through the bio-layer interferometry analysis (BLI).
For the analysis, the present inventors first attached the FcεRIα-NTIG protein having no IL-10Vm as a control group and the fusion protein prepared in Example 3-3 to a 96-well plate using a Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, 200 μl of D. W. was dispensed into a 96-well plate, and then an amine biosensor included in the kit was inserted to perform hydration for 10 minutes. Subsequently, after 200 μl of D. W. was additionally dispensed, EDC and NHS were mixed at 1:1 in an amount of 1/20 of the required sample amount and then diluted with D. W., and 200 μl of the resultant was dispensed into the 96-well plate. Next, the FcεRIα-modified IgG4 Fc protein and the FcεRIα-NTIG-IL-10Vm fusion protein were diluted to 10 μg/ml in a 10 mM acetate solution having a pH of 5.0, and then 200 μl of the resultant was added to the 96-well plate. After 200 μl of 1 M ethanolamine was added to the 96-well plate, the biosensor plate and the sample plate were placed in an Octet®-K2 instrument and measured. After the measurement was completed, 200 μl of a 1× kinetics buffer solution was added to the sample plate, and then a baseline was determined. Thereafter, anti-DNP mouse IgE (Sigma) was diluted with the 1× kinetics buffer solution to various concentrations (50 pM to 3.125 nM), 200 μl of the resultant was dispensed to the sample plate, and then bio-layer interferometry (BLI) was measured using the Octet®-K2 instrument.
As a result, as shown in
In addition to the above results, in order to measure the binding degree of human FcεRIα and human IgE used in the present invention, the present inventors analyzed the affinity of the fusion protein (PG-110) of Example 3-3 with human IgE using the abovementioned BLI method.
As a result, as shown in
5-4: Analysis of Inhibitory Activity Against IgE
Subsequently, the present inventors investigated whether the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 3-3 exhibited inhibitory activity against IgE by an in vitro experiment.
For the investigation, specifically, FcεRIα-modified IgG4 Fc as a control group and the fusion protein (PG-110) of Example 3-3 as an experimental material were sequentially diluted to various concentrations to prepare samples, and the samples were mixed with 1 μl/ml of anti-DNP mouse IgE and reacted at room temperature for 30 minutes. Then, marrow-derived mast cells obtained from a mouse were washed with an HBSS buffer solution, adjusted so that the concentration was 5×105 cells/60 μl, and then dispensed into 96-well plate. 20 μl of the sample reacted with IgE and the test drug was added to previously prepared mast cells, and the resultant was cultured for 25 minutes in an incubator with conditions of 37° C. and 5% CO2. Next, 20 μl of DNP BSA (500 ng/ml) was added, the resultant was cultured again for 30 minutes in the incubator with conditions of 37° C. and 5% CO2 and centrifuged at 4° C. at a rate of 1,500 rpm, and 30 μl of a supernatant was separated. 30 μl of the separated supernatant and 30 μl of a substrate (4-nitrophenyl N-acetyl-p-glucosaminide, 5.84 mM) were mixed and cultured for 25 minutes in the incubator with conditions of 37° C. and 5% CO2. 400 μl of a 0.1 M sodium carbonate buffer solution (pH of 10) was mixed and the reaction was terminated. Thereafter, the relative amounts of β-hexominidase secreted from the mast cells were compared by measuring the absorbance at 405 nm, and an effect of suppressing mast cells according to the concentration of each drug was checked. The analysis was independently conducted three times and an average thereof was obtained.
As a result, as shown in
The present inventors investigated the binding affinity of PG-400 and PG-410 respectively prepared in Examples 4-2 and 4-3 of the present invention with human CD40L through the BLI analysis.
For the investigation, specifically, 200 μl of distilled water was dispensed onto a 96-well black flat-bottom plate, a biosensor tray was placed on the hydrated black plate, and then an amine biosensor was inserted to perform hydration for 10 minutes. Then, 200 μl of distilled water was dispensed onto a new 96-well black sample plate, EDC and NHS were mixed at 1:1 in an amount of 1/20 of the required amount and then diluted with distilled water, and the 200 μl of the resultant was dispensed into the 96-well black sample plate. Next, human CD40L (Peptrotech, Korea) was diluted to 5 μg/ml in a 10 mM acetate solution (pH of 6.0), and then 200 μl of the resultant was added to the 96-well black sample plate. 200 μl of 1 M ethanolamine was added to the plate, the biosensor plate and the sample plate were placed in an Octet®-K2 instrument, and the degree of binding of CD40L was measured on the amine biosensor. After the measurement was completed, the 96-well black sample plate was taken out from the measuring instrument, 200 μl of a 1× kinetics buffer solution was mixed, and a baseline was set. PG-410 and PG-400, which are analytes, were sequentially diluted with the 1× kinetics buffer solution so that the concentrations were 200 to 12.5 nM, and then 200 μl of the resultant was dispensed into the 96-well black sample plate. The sample plate was placed again in the Octet®-K2 instrument, and then the degree of binding to CD40L was measured by the BLI analysis.
As a result, as shown in
The present inventors checked the pharmacokinetic profile (PK) by comparing the half-life, the area under the curve (AUC), the highest concentration in the blood (Cmax), and the like of the bispecific fusion protein (PG-110), which was prepared above, according to Example 3-3.
Specifically, the fusion protein (PG-110) to be tested was administered to three male Sprague Dawley (SD) rats per group by subcutaneous (SC), intravenous (IV), intraperitoneal (IP), and intramuscular (IM) routes at a dose of 1 mg/kg. Blood was obtained prior to injection and after 0.5, 1, 5, 10, 24, 48, 72, 120, 168, 240, and 336 hours after injection, and then stored at room temperature for 30 minutes to cause aggregation. Aggregated blood was centrifuged at 3,000 rpm for 10 minutes, and then a serum of each sample was obtained and stored in a cryogenic freezer. The concentrations of the fusion proteins according to one exemplary embodiment of the present invention in sera were analyzed using an IgG-Fc Quantitation set (Bethyl, Cat #E80-104, Lot #E80-104-30), which can specifically detect Fc among the administered proteins.
As a result, as shown in
The present invention has been described with reference to the abovementioned Examples and Experimental Examples, but the description is merely exemplary, and those skilled in the art could understand that various modifications and other equivalent embodiments can be made therefrom. Therefore, the real technical protection scope of the present invention shall be determined by the technical spirit of the appended claims.
The NTIG protein according to one exemplary embodiment of the present invention is a fusion partner of various biologically active proteins, and increases the therapeutic effect of the biologically active protein by effectively increasing the half-life of the biologically active protein as well as enables safer drug delivery by minimizing effector functions such as ADCC and CDC, which are disadvantages of the Fc fusion protein. Therefore, the NTIG protein according to one exemplary embodiment of the present invention can be effectively used in the preparation of protein medicines.
This application claims priority under the Paris Convention for the Protection of Industrial Property Rights for U.S. Patent Application No. 62/847,470 filed on May 14, 2019, the contents of which are incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/006346 | 5/14/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62847470 | May 2019 | US |
