PTP sigma-Fc fusion protein and pharmaceutical composition containing the same

Abstract

The present invention relates to a PTPsigma-Fc fusion protein that contains a protein derived from PTPsigma and an Fc domain derived from immunoglobulin and that can inhibit PTPsigma signaling by blocking the interaction between the extracellular domain of PTPsigma and its ligand. The PTPsigma-Fc fusion protein of the present invention inhibits PTPsigma-mediated signaling, promotes the growth of hematopoietic stem cells, and can be used to prevent or treat PTPsigma-mediated diseases. The PTPsigma-Fc fusion protein of the present invention regulates PTPsigma signaling using protein-protein interactions, thereby overcoming the problems associated with conventional inhibitors that target the active site of PTP. Furthermore, mutations introduced into the fusion protein improve its aqueous solubility, allowing for high-volume administration during human and animal therapy.

Description

[Technical Field]

[0001] The present invention relates to a PTPsigma-Fc fusion protein and a pharmaceutical composition containing the same, and more particularly to a PTPsigma-Fc fusion protein that can be used to promote the growth of blood stem cells and prevent or treat PTPsigma-mediated diseases by interfering with the interaction between the extracellular domain of PTPsigma and its ligand, and a pharmaceutical composition containing the same. [Background technology]

[0002] Proteins play a crucial role in signal transduction for regulating intracellular functions. Protein signaling occurs through the regulation of protein amount or activity, and various modifications, such as phosphorylation, glycosylation, methylation, and acetylation, occur to induce protein activation or inactivation.

[0003] Among these, protein phosphorylation plays an important role in regulating various cellular phenomena such as cell growth, differentiation, immune response, and brain function. Inappropriate control of protein phosphorylation can cause many diseases, including cancer, diabetes, immune disorders, and nervous system disorders.

[0004] Therefore, protein kinases and protein phosphatases, which regulate protein phosphorylation and dephosphorylation, are important target proteins for the development of therapeutic agents. While the efficacy of protein kinase inhibitors as therapeutic agents has already been proven in clinical trials, research into therapeutic agents targeting protein phosphatases has been actively conducted recently, but no therapeutic agents have yet been reported that have been clinically successful.

[0005] Protein tyrosine phosphatase (PTP) is an enzyme that hydrolyzes the phosphate group on phosphorylated tyrosine residues. The tyrosine phosphorylation-dephosphorylation process is a very important tool in intracellular signal transduction systems, particularly in cell responses to extracellular stimuli, cell growth, differentiation, and death. Therefore, PTPs occupy an important position as therapeutic targets for diseases such as cancer, vascular diseases, immune diseases, and neurological diseases that are accompanied by such changes in cellular states. As examples of PTP inhibitors, Korean Patent Publication No. 10-1194968 describes a technique for preventing or treating PTP1B-mediated diseases such as diabetes by inhibiting the activity of PTP1B using 1,1'-(1,2-ethynediyl)bis-benzene derivatives, and Korean Patent Publication No. 10-1826690 describes a therapeutic agent for neuroinflammatory diseases using PTP inhibitors that target PTP1B, TC-PTP, SHP2, LYP, and RPTPβ.

[0006] The development of conventional PTP inhibitors has primarily targeted the active site of PTP enzymes. However, the PTP active site pocket is relatively shallow, limiting the area where strong interactions with inhibitor compounds can occur. Furthermore, the similarity of the enzyme reaction domains between PTPs makes it difficult for inhibitor compounds to have selectivity. Furthermore, because the PTP active site pocket is positively charged, compounds that bind to it are negatively charged to mimic the phosphate group, which is the substrate of the PTP enzyme. However, such charged compounds have difficulty passing through the cell membrane.

[0007] In other words, conventional PTP inhibitors that target the PTP active site have limitations in that they are difficult to optimize in terms of enzyme function inhibitory potency, selectivity, cell membrane permeability, etc. Therefore, there is a need to develop therapeutic agents that can overcome these limitations.

[0008] On the other hand, receptor-type PTPs such as PTPsigma transmit signals through receptor-ligand interactions between the extracellular domain (ectodomain) and the ligand in addition to the active site. The receptor-ligand interaction of PTPsigma is known to be involved in signal transduction such as the inhibition of neuronal regeneration, and Parkinson's disease is known to be a PTPsigma-mediated disease.

[0009] In this situation, the inventors of the present invention have conducted research to develop a PTPsigma inhibitor that can overcome the limitations of conventional PTP inhibitor compounds, such as enzyme function inhibitory potency, selectivity, cell membrane permeability, etc. As a result, they have developed a PTPsigma-Fc fusion protein that can suppress PTPsigma signaling by interfering with receptor-ligand interaction that occurs in the extracellular domain of PTPsigma using protein-protein interactions, while overcoming the limitations of conventional PTP inhibitor compounds. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a PTPsigma-Fc fusion protein that can inhibit PTPsigma signal transduction by blocking the interaction between the extracellular domain of PTPsigma and its ligand.

[0011] Another object of the present invention is to provide a composition for promoting the growth of blood stem cells, which comprises the PTP sigma-Fc fusion protein.

[0012] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a PTP sigma-mediated disease, which comprises the PTP sigma-Fc fusion protein. [Means for solving the problem]

[0013] To achieve the above object, the present invention provides a PTPsigma-Fc fusion protein in which a protein derived from PTPsigma (Protein Tyrosine Phosphatase sigma) is linked to at least one of the two chains constituting the Fc domain derived from immunoglobulin (Ig).

[0014] In the present invention, the PTPsigma-derived protein may be a protein derived from the extracellular domain (ectodomain) of PTPsigma.

[0015] In the present invention, the PTPsigma-derived protein may be a protein derived from the first to second Ig domains (Ig1-Ig2) or the first to third Ig domains (Ig1-Ig3) of the extracellular domain of PTPsigma.

[0016] In the present invention, the PTPsigma-derived protein can contain amino acid residues 30 to 231 of the PTPsigma protein.

[0017] In the present invention, at least one residue of isoleucine (I), valine (V), leucine (L), and tyrosine (Y) in the PTP sigma-derived protein can be substituted with alanine (A), serine (S), threonine (T), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), or histidine (H).

[0018] In the present invention, based on the amino acid sequence numbers of the PTP sigma protein, I36, V104, V108, L125, L143, V At least one of residues 145, L155, L187, Y224, and V227 can be substituted with A, S, T, N, Q, D, E, K, R, or H.

[0019] In the present invention, the PTP sigma-derived protein may be in the form of a homodimer or a heterodimer.

[0020] In the present invention, the chain constituting the Fc domain can contain amino acid residues 221 to 447 of the IgG1 heavy chain (according to Kabat EU numbering).

[0021] In the present invention, at least one amino acid in one chain of the Fc domain is substituted with an amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F), and tyrosine (Y), and at least one amino acid in the other chain is substituted with an amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0022] In the present invention, one chain of the Fc domain may comprise a T366W substitution (according to Kabat EU numbering), and the other chain may comprise T366S, L368A, and Y407V substitutions (according to Kabat EU numbering).

[0023] The present invention also provides a composition for promoting the growth of blood stem cells, comprising the PTP sigma-Fc fusion protein.

[0024] In the present invention, the composition for promoting the growth of blood stem cells may be used to regenerate blood stem cells damaged by radiation or anti-cancer drug administration.

[0025] The present invention also provides a method for promoting the growth of blood stem cells, comprising the step of administering a pharmaceutical composition containing a PTP sigma-Fc fusion protein.

[0026] The present invention also provides a pharmaceutical composition comprising the PTP sigma-Fc fusion protein.

[0027] In the present invention, the pharmaceutical composition may be for preventing or treating a PTP sigma-mediated disease.

[0028] In the present invention, the PTP sigma-mediated disease may be a neurological disorder selected from the group consisting of traumatic brain injury, Alzheimer's disease, and Parkinson's disease.

[0029] In the present invention, the PTP sigma-mediated disease can be selected from the group consisting of multiple sclerosis and rheumatoid arthritis.

[0030] In the present invention, the PTP sigma-mediated disease may be a synaptic plasticity-related disease selected from the group consisting of mania, depression, bipolar disorder, and memory impairment.

[0031] The present invention also provides a method for preventing or treating a PTPsigma-mediated disease, comprising administering a pharmaceutical composition containing a PTPsigma-Fc fusion protein. [Effects of the Invention]

[0032] In the present invention, the reaction between the extracellular domain of PTPsigma and its ligand can be blocked by using a fusion protein in which a protein derived from PTPsigma is bound to the Fc region of immunoglobulin. The present invention uses a modulator that utilizes protein-protein interactions, thereby overcoming limitations of conventional inhibitors that target the active site of PTPs, such as enzyme function inhibitory potency, selectivity, and cell membrane permeability. Furthermore, by introducing mutations into the fusion protein to improve its aqueous solubility, it can be administered in high volumes during human and animal therapy.

[0033] The PTPsigma-Fc fusion protein of the present invention can inhibit PTPsigma-mediated signal transduction and promote the growth of blood stem cells, and can be used to prevent or treat PTPsigma-mediated diseases. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 shows the schematic design of the homodimer (a) and heterodimer (b) of the PTP sigma-Fc fusion protein of the present invention. [Figure 2] FIG. 2 shows an amino acid sequence map of a PTP sigma-Fc fusion protein according to one embodiment of the present invention. [Figure 3] FIG. 3 shows an amino acid sequence map of a solubility-enhancing mutant of a PTP sigma-Fc fusion protein according to one embodiment of the present invention. [Figure 4] FIG. 4 shows the amino acid sequence encoded by an expression vector for producing a PTP sigma-Fc homodimer according to one embodiment of the present invention. [Figure 5] FIG. 5 shows the amino acid sequence encoded by an expression vector for producing a PTP sigma-Fc heterodimer according to one embodiment of the present invention. [Figure 6] FIG. 6 shows the results of non-reducing SDS-PAGE after preparation of a PTPsigma-Fc fusion protein according to one embodiment of the present invention. [Figure 7] FIG. 7 is a graph showing the regenerative effect of hematopoietic stem cells by a PTP sigma-Fc fusion protein according to one embodiment of the present invention. [Figure 8] FIG. 8 shows the locations of residues to be mutated in the tertiary structure of PTP sigma Ig1-Ig2. [Figure 9] FIG. 9 shows the structure of PTP-lar in complex with Ig1-Ig2 and sucrose octasulfate, superimposed with the tertiary structure of PTPsigma Ig1-Ig2. [Figure 10] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 11]10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 12] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 13] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 14] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 15] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 16] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 17] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 18] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 19] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 20] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 21] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 22]10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 23] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 24] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 25] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 26] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 27] 10 to 27 show amino acid sequences encoded by PTP sigma mutant-Fc expression vectors according to embodiments of the present invention. [Figure 28] FIG. 28 is a graph showing the yield and solubility of a PTP sigma-Fc fusion protein according to one embodiment of the present invention. [Figure 29] FIG. 29 is a graph showing the regenerative effect of hematopoietic stem cells by a PTP sigma-Fc fusion protein according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0036] The present invention relates to a PTPsigma-Fc fusion protein that can inhibit PTPsigma signal transduction by blocking the receptor-ligand interaction between the extracellular domain of PTPsigma and its ligand.

[0037] PTPsigma (Protein Tyrosine Phosphatase sigma) is a transmembrane receptor-type PTP that consists of three regions: the extracellular domain (ectodomain), the transmembrane region, and the cytoplasmic region. The extracellular domain begins at the N-terminal and consists of three Ig-like domains followed by multiple fibronectin domains. The extracellular domain mediates receptor-ligand interactions, and this interaction is transmitted to the cytoplasmic region, regulating the enzymatic activity of the PTP active domain.

[0038] Based on the assumption that the Ig-like domain of the extracellular domain of PTPsigma is involved in receptor-ligand interaction, the inventors of the present invention fused the Ig-like domain to the Fc region of immunoglobulin to prepare a PTPsigma-Fc fusion protein and tested the PTPsigma inhibitory ability of the fusion protein. As a result, it was confirmed that when irradiated hematopoietic stem cells were treated with the fusion protein, hematopoietic stem cells were effectively regenerated. This demonstrated that the PTPsigma-Fc fusion protein of the present invention can promote the regeneration of hematopoietic stem cells through PTPsigma.

[0039] Therefore, the PTPsigma-Fc fusion protein of the present invention can be used in compositions for promoting the growth of blood stem cells, and can also be used as a therapeutic agent for PTPsigma-mediated diseases such as neuronal damage diseases, autoimmune diseases, and synaptic plasticity-related diseases, like conventional PTPsigma inhibitors.

[0040] Furthermore, to improve the solubility of the fusion protein, amino acid residues that do not change the structure were selected from the hydrophobic amino acids exposed on the surface of the Ig-like domain of the PTP sigma and replaced with hydrophilic amino acids. As a result, it was confirmed that the solubility of the fusion protein was increased while maintaining the blood stem cell regeneration effect.

[0041] The PTPsigma-Fc fusion protein of the present invention comprises a PTPsigma-derived protein and an Fc domain.

[0042] Figure 1 shows a schematic design of a PTPsigma-Fc fusion protein according to the present invention, in which (a) shows a homodimer structure in which a PTPsigma-derived protein is linked to both chains of the Fc domain, and (b) shows a heterodimer structure in which a PTPsigma-derived protein is linked to only one chain of the Fc domain.

[0043] In the present invention, the base sequence of the PTPsigma-derived protein is interpreted as including not only the standard amino acid sequence of the PTPsigma protein but also its isoforms. The standard sequence of the PTPsigma protein refers to Isoform 1 (Q13332-1) based on Unitprot. Other isoforms include Isoform PTPS-MEA (Q13332-2), Isoform PTPS-MEB (Q13332-3), Isoform PTPS-MEC (Q13332-4), Isoform PTPS-F4-7 (Q13332-5), Isoform 2 (Q13332-6), and Isoform 3 (Q13332-7). The preferred base sequence may be the sequence of PTPsigma Isoform 3.

[0044] In the present invention, a protein derived from PTPsigma can be a protein derived from the extracellular domain (ectodomain) of PTPsigma. In this case, a protein derived from the extracellular domain of PTPsigma means that it contains all or part of the sequence of the extracellular domain of PTPsigma.

[0045] In particular, in the present invention, the protein derived from the extracellular domain of PTPsigma is preferably a protein derived from the Ig-like domain of the extracellular domain, and more preferably a protein derived from the first and second Ig-like domains (Ig1-Ig2) or the first and third Ig-like domains (Ig1-Ig3).

[0046] In one embodiment of the present invention, the PTPsigma-derived protein has an amino acid sequence derived from Ig1-Ig2 of the PTPsigma extracellular domain, and may comprise the sequence of PTPsigma protein from positions 30 to 231. In this case, the amino acid sequence number is based on isoform 3 (Q13332-7) of the PTPsigma protein.

[0047] Specifically, the PTPsigma-derived protein is a sequence of positions 30 to 231 of the PTPsigma protein, and can include the amino acid sequence of SEQ ID NO: 1 below.

[0048] [SEQ ID NO: 1] EEPPRFIKEPKDQIGVSGGVASFVCQATGDPKPRVTWNKKGKKVNSQRFETIEFDESAGAVLRIQPLRTPRDENVYECVAQNSVGEITVHAKLTVLREDQLPSGFPNIDMGPQLKVVERTRTATMLCAASGNPDPPEITWFKDFLPVDPSASNGRIKQLRSGALQIESSEETDQGKYECVATNSAGVRYSSPANLYVRVRRVA

[0049] In one embodiment of the present invention, the PTPsigma-derived protein has an amino acid sequence derived from Ig1-Ig3 of the extracellular domain of PTPsigma, and can include the sequence of positions 30 to 321 of the PTPsigma protein.

[0050] The sequence of positions 30 to 321 of the PTPsigma protein is the amino acid sequence of SEQ ID NO: 1 followed by the sequence of positions 232 to 321 of the PTPsigma protein, and can be represented as follows:

[0051] [The sequence from 30 to 321 of the PTP sigma protein] EEPPRFIKEPKDQIGVSGGVASFVCQATGDPKPRVTWNKKGKKVNSQRFETIEFDESAGAVLRIQPLRTPRDENVYECVAQNSVGEITVHAKLTVLREDQLPSGFPNIDMGPQLKVVERTRTATMLCAASGNPDPEITWFKDFLPV DPSASNGRIKQLRSGALQIESSEETDQGKYECVATNSAGVRYSSPANLYVRVRRVAPRFSILPMSHEIMPGGNVNITCVAVGSPMPYVKWMQGAEDLTPEDDMPVGRNVLELTDVKDSANYTCVAMSSLGVIEAVAQITVKSLPKA

[0052] In the PTPsigma-Fc fusion protein of the present invention, a PTPsigma mutant is formed in the PTPsigma-derived protein to improve the aqueous solubility of the fusion protein.

[0053] In the present invention, the term "mutation" includes substitution, insertion, and / or deletion of amino acid residues, and preferably, the mutants of the present invention include substitution of amino acid residues. The substitution of amino acid residues can be represented by the residue in the parent amino acid sequence, the number of the amino acid residue, and the order of the substituted amino acid residues.

[0054] Fusion proteins containing Ig-like domains, as in the present invention, have poor aqueous solubility, making it difficult to administer them in large volumes to animals or humans. To solve this problem, in the present invention, amino acids that do not alter the structure were selected from the hydrophobic amino acids exposed on the surface of the Ig-like domain of PTP sigma and replaced with hydrophilic amino acids to improve solubility.

[0055] Specifically, a PTPsigma mutant for improving the solubility of a fusion protein may be a protein derived from the PTPsigma in which at least one residue of isoleucine (I), valine (V), leucine (L), and tyrosine (Y) is substituted with alanine (A), serine (S), threonine (T), asparagine (N), glutamine (Q), aspartic acid (D), glutamic acid (E), lysine (K), arginine (R), or histidine (H).

[0056] Among them, I36, V104, V108, L125, L143, and V108 are the sequences 30 to 231 of PTPsigma that do not affect the structure of PTPsigma. V It is preferable to substitute at least one residue among L145, L155, L187, Y224, and V227 with A, S, T, N, Q, D, E, K, R, or H, and from the standpoint of improving solubility, it is even more preferable to substitute at least one residue among L143, L155, L187, Y224, and V227 with A or N.

[0057] That is, when the amino acid sequence of SEQ ID NO: 1 of the present invention is used as a reference, residue 30 of the PTP sigma sequence corresponds to residue 1 of SEQ ID NO: 1, and therefore at least one residue from I7, V75, V79, L96, L114, L116, L126, L158, Y195, and V198 in SEQ ID NO: 1 can be substituted with A, S, T, N, Q, D, E, K, R, or H, and more preferably at least one residue from L114, L126, L158, Y195, and V198 in SEQ ID NO: 1 can be substituted with A or N.

[0058] In the experimental examples of the present invention, it was confirmed that the aqueous solubility of the fusion protein increased by up to six times due to the above mutation, and that it had an excellent effect in promoting the regeneration of blood stem cells. Therefore, it is expected that the PTPsigma-Fc fusion protein into which the above mutation has been introduced can be used as a composition for promoting the regeneration of blood stem cells, and that high-volume treatment is possible in the treatment of humans and animals.

[0059] In the present invention, the PTPsigma-derived protein is linked to an Fc domain derived from an immunoglobulin to form a PTPsigma-Fc fusion protein.

[0060] In the present invention, the Fc domain derived from an immunoglobulin refers to the C-terminal region of the heavy chain constant region of an immunoglobulin, which contains the CH2 and CH3 domains (or the CH2, CH3, and CH4 domains), and is used to encompass wild-type Fc domains and mutants thereof. The immunoglobulin from which the Fc domain is derived may be IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0061] In the present invention, each chain of the Fc domain may comprise a region from residue 221 of the human IgG1 heavy chain to the C-terminus, or a region including a hinge in the region. The numbering of amino acid residues in the Fc region is according to the Kabat EU numbering system, which defines the numbering of residues in a human immunoglobulin heavy chain.

[0062] In the present invention, each chain of the Fc domain can comprise an IgG1 heavy chain sequence of positions 221 to 447. The IgG1 heavy chain sequence of positions 221 to 447 can be represented by the amino acid sequence of SEQ ID NO: 2 below.

[0063] [SEQ ID NO: 2] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0064] In the present invention, the PTP sigma-derived protein may be in a form linked to at least one of the two chains that constitute the Fc domain.

[0065] In this case, the PTPsigma-derived protein and the Fc domain can be linked via 0 to 20 amino acid residues. That is, the PTPsigma-derived protein and the Fc domain can be linked directly or via a linker consisting of 1 to 20 amino acids. In this case, the PTPsigma-Fc-derived protein can be linked to the N-terminus or C-terminus of the Fc domain.

[0066] The PTPsigma-Fc fusion protein of the present invention may be a homodimer in which a PTPsigma-derived protein is linked to both chains of the Fc domain, or a heterodimer in which a PTPsigma-derived protein is linked to only one chain.

[0067] In one embodiment of the present invention, when the PTPsigma-Fc fusion protein is a heterodimer, recombinant variants can be made to form heterodimers in the Fc domain.

[0068] For example, the recombinant mutants for forming the dimers can be prepared using the knobs-into-hole technique.

[0069] The knob-into-hole technology is a technique designed to ensure that only heterodimers form between the heavy chains of antibody fragments. Here, a knob is designed with a protruding side chain on the opposite chain and is inserted into a hole in the opposite domain. This prevents homodimerization between the heavy chains due to side chain clashes, allowing only heterodimerization. In the present invention, the structure in which a knob or hole is formed on each chain of the Fc domain can be referred to as an Fc-knob or Fc-hole, respectively.

[0070] The Fc knob can be formed by substituting at least one amino acid in the chain constituting the Fc domain with a large amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F), and tyrosine (Y). For example, the Fc knob can be formed by substituting T366W mutation at positions 221 to 447 of the IgG1-Fc heavy chain.

[0071] The Fc-hole can be formed by substituting at least one amino acid in the chain constituting the Fc domain with a small amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). For example, the Fc-hole can be formed by T366S, L368A, and Y407V mutations in the sequence of positions 221 to 447 of the IgG1-Fc heavy chain.

[0072] FIG. 2 shows an amino acid sequence map of the polypeptide chains that make up the PTP sigma-Fc fusion protein according to one embodiment of the present invention.

[0073] As shown in Figure 2, the PTPsigma-Fc fusion protein of the present invention can comprise a polypeptide consisting of a heavy chain signal peptide, the sequence of positions 30 to 231 of PTPsigma, and the sequence of positions 221 to 447 of the IgG1 heavy chain, thereby forming a homodimeric fusion protein. Alternatively, the PTPsigma-Fc fusion protein of the present invention can comprise a polypeptide consisting of a heavy chain signal peptide, the sequence of positions 30 to 231 of PTPsigma, and a knob mutant of the IgG1 heavy chain sequence of positions 221 to 447, and a hole mutant of the heavy chain signal peptide and the sequence of positions 221 to 447 of the IgG1 heavy chain, in which case a heterodimeric fusion protein can be formed.

[0074] FIG. 3 shows an amino acid sequence map of a solubility-enhancing mutant of a PTPsigma-Fc fusion protein according to one embodiment of the present invention, i.e., a PTPsigma mutant-Fc fusion protein.

[0075] As shown in Figure 3, the PTPsigma mutant-Fc fusion protein of the present invention can comprise a polypeptide consisting of a heavy chain signal peptide, a solubility-enhancing mutant of the PTPsigma sequence from positions 30 to 231, and an IgG1 heavy chain sequence from positions 221 to 447, thereby forming a homodimeric fusion protein. Alternatively, the PTPsigma mutant-Fc fusion protein of the present invention can comprise a polypeptide consisting of a heavy chain signal peptide, a solubility-enhancing mutant of the PTPsigma sequence from positions 30 to 231, and a knob mutant of the IgG1 heavy chain sequence from positions 221 to 447, as well as a heavy chain signal peptide and a hole mutant of the IgG1 heavy chain sequence from positions 221 to 447, thereby forming a heterodimeric fusion protein.

[0076] The PTPsigma-Fc fusion protein formed by the present invention can block the reaction between the extracellular domain of PTPsigma and its ligand, and can therefore be used as an inhibitor that suppresses signal transduction mediated by PTPsigma.

[0077] Accordingly, the present invention also provides a composition comprising said PTPsigma-Fc fusion protein.

[0078] In the present invention, we have confirmed that the PTPsigma-Fc fusion protein can block the receptor-ligand interaction between the extracellular domain of PTPsigma and its ligand, thereby suppressing the PTPsigma-mediated signal transduction that inhibits the growth of hematopoietic stem cells. Therefore, the PTPsigma-Fc fusion protein of the present invention can promote the growth of hematopoietic stem cells, and specifically can be used to promote the regeneration of hematopoietic stem cells whose growth has been inhibited by radiation or the use of anticancer drugs.

[0079] Therefore, the PTP sigma-Fc fusion protein of the present invention is expected to be effective in helping cancer patients who require radiation therapy and chemotherapy to recover. For example, when blood stem cells of patients suffering from cancers such as pancreatic cancer, liver cancer, breast cancer, brain cancer, prostate cancer, colorectal cancer, lung cancer, renal cancer, neuroblastoma, ovarian cancer, endometrial cancer, germ cell tumor, eye cancer, multiple myeloma, and gastric cancer are damaged by radiation therapy and / or chemotherapy, the PTP sigma-Fc fusion protein can be used as a therapeutic agent to help regenerate damaged blood stem cells and promote rapid recovery of cancer patients.

[0080] Furthermore, the extracellular domain of PTPsigma is known to be involved in signal transduction that inhibits neuronal regeneration and signal transduction that activates immune responses through interaction with chondroitin sulfate proteoglycan (CSPG). The PTPsigma-Fc fusion protein of the present invention can be used in pharmaceutical compositions for the prevention or treatment of PTPsigma-mediated diseases.

[0081] Specifically, the PTPsigma-Fc fusion proteins of the present invention can be used to prevent or treat PTPsigma-mediated neuronal damage diseases such as traumatic brain injury, Alzheimer's disease, and Parkinson's disease, and can be used to prevent or treat PTPsigma-mediated synaptic plasticity-related diseases selected from the group consisting of mania, depression, bipolar disorder, and memory disorders. Furthermore, the PTPsigma-Fc fusion proteins of the present invention can be used to prevent or treat PTPsigma-mediated autoimmune diseases such as rheumatoid arthritis or multiple sclerosis.

[0082] The PTPsigma-Fc fusion protein of the present invention is used in protein-protein interactions, and therefore overcomes the inherent problems of small molecule inhibitors that target the active site of PTP, such as enzyme inhibitory potency, selectivity, and cell membrane permeability. Furthermore, the present invention makes it possible to introduce mutations into the PTPsigma-Fc fusion protein to improve its aqueous solubility, which is expected to enable the administration of high volumes in human and animal therapy.

[0083] The composition of the present invention may contain a pharmaceutically acceptable carrier in addition to the PTPsigma-Fc fusion protein.

[0084] The pharmaceutically acceptable carriers are those commonly used in pharmaceutical formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0085] Compositions containing the PTP sigma-Fc fusion proteins of the present invention can be administered orally or parenterally, and in the case of parenteral administration, administration can be by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0086] Compositions containing the PTP sigma-Fc fusion proteins of the present invention can be formulated according to conventional methods into the form of a sterile injectable solution, a lyophilized preparation, a solution for a pre-filled syringe, an oral preparation, an external preparation, or a suppository. Because proteins or peptides are digested upon oral administration, oral compositions can be formulated to coat the active agent or to protect it from degradation in the stomach.

[0087] The appropriate dosage of the PTPsigma-Fc fusion protein of the present invention can be variously formulated depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity, etc. For example, the daily dosage of the PTPsigma-Fc fusion protein may be, for example, 0.001 to 100 mg / kg.

[0088] The present invention also relates to a method for preventing or treating a PTPsigma-mediated disease, which may comprise administering a pharmaceutical composition comprising a PTPsigma-Fc fusion protein.

[0089] The present invention further relates to a method for promoting the regeneration of blood stem cells, the method comprising administering a pharmaceutical composition containing a PTP sigma-Fc fusion protein, and the method may be for promoting the regeneration of blood stem cells damaged by radiation or anticancer drug administration in a cancer patient.

[0090] In the present invention, the subject of administration may be an individual, specifically an individual in need of administration of a PTPsigma-Fc fusion protein, and the individual may be an animal, typically a mammal.

[0091] <Example> The present invention will be described in more detail through the following examples, but these examples are intended to illustrate some experimental methods and compositions for the purpose of illustratively explaining the present invention, and the scope of the present invention is not limited to these examples.

[0092] <Production Example 1: Production of PTP sigma-Fc fusion protein> 1-1. Expression vector construction The expression vector was constructed based on pcDNA 3.1 / Myc His-A (Invitrogen) so as to have a polyhistidine tag at the C-terminus of the protein.

[0093] A polynucleotide encoding the amino acid sequence of the target protein was amplified by PCR and then cloned using restriction enzymes and ligases. This resulted in the construction of an Fc expression vector expressing the sequence from positions 221 to 447 of the human immunoglobulin G1 (IgG1) heavy chain, including the signal peptide of the human immunoglobulin G1 heavy chain. The amino acid sequence number of the Fc protein is based on "EU numbering."

[0094] A restriction enzyme recognition sequence was inserted into the vector through mutagenesis, and the nucleotides encoding the sequence from 30 to 231 of Uniprot Q133332-7 PTPsigma were inserted, thereby constructing a PTPsigma-Fc expression vector.

[0095] The amino acid sequence encoded by the PTPsigma-Fc expression vector is the same as that shown in Figure 4. In Figure 4, the portion underlined with dotted lines corresponds to the PTPsigma-derived protein (SEQ ID NO: 1), and the portion underlined with straight lines corresponds to IgG1 221 to 447 of the Fc domain (SEQ ID NO: 2).

[0096] Based on the vector constructed above, mutations were introduced to form knobs and holes that can produce heterodimeric proteins.

[0097] The nucleotide sequence of the PTPsigma-Fc expression vector was altered so that a knob was formed by the T366W mutation, thereby producing a PTPsigma-Fc-knob expression vector.

[0098] The nucleotide sequence of the Fc expression vector was altered so that holes were formed in the IgG1 heavy chain at positions 221 to 447 due to T366S, L368A, and Y407V mutations, thereby creating an Fc-hole expression vector encoding the amino acid sequence shown below.

[0099] Figure 5 shows the amino acid sequences encoded by the PTPsigma-Fc-knob expression vector (a) and the Fc-hole expression vector (b). In Figure 5, the dotted underlined portion corresponds to the PTPsigma-derived protein (SEQ ID NO: 1), and the straight underlined portion corresponds to the knob or hole mutant of IgG1 221-447 (SEQ ID NO: 2) in the Fc domain. Mutated residues are indicated with an asterisk.

[0100] 1-2. Protein expression ExpiCHO® cells were transfected with the expression vectors to transiently express each protein.

[0101] The transfection was performed at an initial cell density of 6 x 10 6 The cells / mL and expression vector concentration was 0.8 μg DNA / mL, and ExpiCHO® Expression Medium, OptiPRO® SFM, and ExpiFectamine® transfection kit (Gibco) were used in this process according to the manufacturer's instructions.

[0102] The transfected cells were cultured in a carbon dioxide incubator for 8 days, and then separated into cell culture medium containing each protein and cells by centrifugation.

[0103] 1-3. Purification To separate the target protein from the culture medium containing each recombinant protein, affinity chromatography was performed using a Ni-NTA (Qiagen) column.

[0104] Specifically, a Ni-NTA column was filled with a buffer solution containing 50 mM Tris-HCl (pH 7.5) and 200 mM NaCl, and the resulting culture medium was centrifuged and filtered through a 0.45 μm filter. After reacting with the culture medium, the column was washed sequentially with a buffer solution containing 50 mM Tris-HCl (pH 7.5) and 500 mM NaCl, and a buffer solution containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 30 mM imidazole. Finally, the protein bound to the column was eluted using a buffer solution containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, and 500 mM imidazole. After elution, the protein-containing buffer solution was exchanged for phosphate-buffered saline (PBS) using a Hitrap® Desalting (GE Healthcare) column.

[0105] 1-4. Combination of heterodimeric proteins The purified PTP sigma-Fc-knob protein and Fc-hole protein were quantified and mixed at the same molecular ratio. The mixed protein solution was added to a final concentration of 0.1 M arginine and 5 mM L-glutathione (reduced), and the mixture was incubated at 37°C for 2 hours to form heterodimeric proteins. After the reaction, the proteins were subjected to SDS-PAGE analysis.

[0106] Figure 6 shows the results of SDS-PAGE of the homodimeric and heterodimeric proteins formed in Preparation Example 1. In Figure 6, A shows the results for the PTPsigma-Fc homodimer, and B shows the results for the PTPsigma-Fc-knob:Fc-hole heterodimer. In the case of the PTPsigma-Fc homodimer, the yield was approximately 6 mg / L, and the PTPsigma-Fc-knob:Fc-hole heterodimer was confirmed to be formed with a purity of approximately 85%.

[0107] <Experimental Example 1: Measurement of the blood stem cell regeneration effect of PTP sigma-Fc fusion protein by colony-forming cell assay> Commercially available human CD34+ hematopoietic stem cells (STEMCELL Technologies) were used to measure the colony-forming ability of hematopoietic stem cells.

[0108] Immediately after thawing, the human CD34+ hematopoietic stem cells were cultured at 37°C and 5% carbon dioxide using StemSpan (registered trademark) SFEM and StemSpan (registered trademark) CD34+ Expansion Supplement (10X) (STEMCELL Technologies) according to the manufacturer's instructions.

[0109] Cultured human CD34+ hematopoietic stem cells were plated at 2 × 10 3 The cells were seeded at a cell density of 1000 cells / well. To measure colony formation ability at this time point, MethoCult® H4434 Classic (STEMCELL Technologies) culture medium was used. Immediately after seeding, the fusion protein from Preparation 1 and PBS were applied to each well. 24 hours after seeding, the hematopoietic stem cells were damaged by irradiating them with 300 cGy of radiation using a Cs-137 irradiator.

[0110] After irradiation, colony formation was measured using the CytoSelect® 96-Well Hematopoietic Colony Forming Cell Assay (Cell Biolabs) kit. Specifically, hematopoietic stem cells cultured at 37°C and 5% carbon dioxide for 9 days after irradiation were mixed with the reagents of the colony formation assay kit, and fluorescence was measured at 355 nm / 460 nm wavelengths using a Victor X2 (Perkin-Elmer) plate reader.

[0111] Figure 7 shows the results of measuring colony-forming ability. It can be seen that when hematopoietic stem cells are irradiated, their colony-forming ability is significantly reduced. However, when hematopoietic stem cells are treated with the PTPsigma-Fc homodimer of the present invention, their colony-forming ability is restored to almost the same level as that of the untreated group (control). Similar results were obtained when the heterodimer consisting of PTPsigma-Fc-knob:Fc-hole was treated.

[0112] This confirmed that the PTPsimga-Fc fusion protein of the present invention can be used to effectively repair hematopoietic stem cells damaged after radiation therapy in cancer patients.

[0113] <Production Example 2: Production of mutant PTP sigma-Fc fusion protein> To improve the solubility of the fusion protein, mutations were engineered into the PTPsigma-derived protein.

[0114] Specifically, mutations were designed based on the tertiary structure of PTPsigma, and were designed so that the mutations would not impair the CSPG-binding ability of PTPsigma and would not affect the PTPsigma inhibitory effect.

[0115] The residues in the Ig1-Ig2 tertiary structure of the PTPsigma extracellular domain were analyzed one by one using the PyMOL program, and residues with large solvent-accessible surface areas and hydrophobic properties were selected. Among these, residues that interact with surrounding residues structurally and may affect the structure when mutated were excluded.

[0116] Next, to avoid affecting CSPG binding, the complex structure of PTP-lar, which is homologous to PTPsigma, and sucrose octasulfate, a CSPG analog, was used as PTP-sigma interaction information. First, the PTPsigma Ig1-Ig2 structure was superimposed on the PTP-lar Ig1-Ig2 structure, and residues in PTPsigma Ig1-Ig2 at the sucrose octasulfate interaction site were excluded from the mutation targets.

[0117] As a result, the positions of the amino acids to be mutated were I36, V104, V108, L125, L143, V The positions of these residues in the tertiary structure of PTPsigma Ig1-Ig2 are shown in Figure 8. Figure 9 shows the superposition of the tertiary structure of PTPsigma Ig1-Ig2 and the complex structure of PTP-lar with sucrose octasulfate.

[0118] These amino acids were changed to hydrophilic amino acids to improve solubility, and mutants consisting of a combination of one or more of these amino acids were used. Mutations to hydrophilic amino acids were performed by selecting any of A, S, T, N, Q, D, E, K, R, and H amino acids, and the positions and residues of the mutations are shown in Table 1 below. In Table 1, the numbers of amino acid residues are based on the amino acid sequence numbers of PTPsigma.

[0119] [Table 1]

[0120] Based on the positions and residues of the mutations, representative sequences of amino acid mutations of PTP sigma Ig1-Ig2 containing mutations that improve aqueous solubility were designed as shown in Table 2 below.

[0121] [Table 2]

[0122] Figures 10 to 27 show the amino acid sequences encoded by vectors for forming fusion proteins of each PTPsigma mutant and Fc domain for the representative mutant sequences in Table 2. In each sequence, (a) shows the sequence for forming a PTPsigma mutant-Fc homodimer fusion protein, and (b) shows the PTPsigma mutant-Fc knob sequence for forming an asymmetric heterodimer.

[0123] In Figures 10 to 27, the dotted underlined portions correspond to mutant sequences of positions 30 to 231 of PTPsigma (SEQ ID NO: 1), a protein derived from PTPsigma, and the straight underlined portions correspond to positions 221 to 447 of the Fc domain of IgG1 (SEQ ID NO: 2) or its knob mutants. Mutated residues in the parent protein are indicated with an asterisk.

[0124] <Experimental Example 2: Measurement of yield and solubility of PTP sigma-Fc fusion protein mutants> The yield and solubility of mutant fusion proteins designed in the same manner as in Preparation Example 2 were measured and compared with those of the fusion protein (wild type) in Preparation Example 1.

[0125] To measure the yield of PTPsigma-Fc fusion proteins containing mutants, 8 ml of elution was concentrated to approximately 1 ml immediately after purification, and the insoluble contents were precipitated by centrifugation. The supernatant sample was then collected. The concentration of the supernatant sample was then measured using a detergent compatible assay (DC assay), and the amount of soluble protein was calculated based on the concentration and volume.

[0126] To measure the aqueous solubility of PTPsigma-Fc fusion proteins containing mutants, 8 ml of protein was concentrated to 1 ml immediately after protein purification, and the supernatant concentration after centrifugation was measured using a DC assay. This was then concentrated to 400 μl, and the same process was repeated to measure the concentration of the supernatant. The amount of soluble protein was calculated by multiplying each concentration by the volume, and this was converted to a percentage.

[0127] The yield and solubility calculated by the above method are shown in Table 3 below and FIG.

[0128] [Table 3]

[0129] From the results of the yield and solubility measurements, I36, V104, V108, L125, L143, V Mutants in which at least one of residues 145, L155, L187, Y224, and V227 was substituted with A, S, T, N, Q, D, E, K, R, or H showed improved aqueous solubility compared to the wild type, and the yield also tended to improve.

[0130] In particular, the above experiments confirmed that mutants in which at least one residue among L143, L155, L187, Y224, and V227 was substituted with A or N showed excellent results in terms of yield and solubility.

[0131] This confirmed that the fusion protein of the present invention can be administered in high doses to animals and humans by improving its solubility through the introduction of specific natural mutations into the PTP sigma-derived protein.

[0132] <Experimental Example 3: Measurement of the blood stem cell regeneration effect of PTP sigma-Fc fusion protein mutants> Commercially available human CD34+ hematopoietic stem cells (STEMCELL Technologies) were used to measure the colony-forming ability of hematopoietic stem cells.

[0133] Immediately after thawing the human CD34+ hematopoietic stem cells, they were cultured at 37°C and 5% carbon dioxide using StemSpan® SFEM and StemSpan® CD34+ Expansion Supplement (10X) (STEMCELL Technologies) according to the manufacturer's instructions.

[0134] Cultured human CD34+ hematopoietic stem cells were plated at 1.4 × 10 3 Cells were seeded at a cell density of 1000 cells / well. MethoCult® H4434 Classic (STEMCELL Technologies) culture medium was used to measure colony formation. Immediately after seeding, PTP sigma-Fc fusion protein and PBS were added to each well. 24 hours after seeding, 5 μM 5-fluorouracil (5FU) was administered together with the fusion protein. 14 days after 5FU treatment, the number of colonies was measured by manual counting.

[0135] The effect of wild-type PTPsigma-Fc fusion protein and PTPsigma-Fc fusion protein containing the L155N and L187N mutations on hematopoietic stem cell damage induced by anticancer drugs was measured using the above method, and the results are shown in Figure 29. In Figure 29, 5FU- indicates a control without 5FU added, 5FU+ indicates a sample with 5FU added, 2N indicates a sample with 5FU added and treated with PTPsigma-Fc fusion protein mutants (L155N and L187N), and wt indicates a sample with 5FU added and treated with PTPsigma-Fc fusion protein (wild-type).

[0136] Referring to Figure 29, it was confirmed that hematopoietic stem cells were severely damaged when treated with an anticancer drug (5FU), but when treated with the anticancer drug and the PTP sigma mutant-Fc fusion protein together, the damaged hematopoietic stem cells recovered to the same extent as the control when no anticancer drug was treated.

[0137] This confirmed that the PTPsimga-Fc fusion protein of the present invention can be used to effectively regenerate hematopoietic stem cells damaged after anticancer drug treatment in cancer patients.

[0138] While the specific details of the present invention have been described above, it is obvious to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. One of the two chains that make up the Fc domain from immunoglobulin (Ig) a protein derived from PTPsigma (Protein Tyrosine Phosphatase sigma) linked to the N-terminus or C-terminus of at least one chain; the PTPsigma-derived protein has the amino acid sequence of SEQ ID NO: 1; A PTP sigma-Fc fusion protein in which one or more residues of L126, L158, and Y195 in SEQ ID NO: 1 are substituted with A or N, the chain constituting the Fc domain comprises amino acid residues 221 to 447 of the IgG1 heavy chain (according to the Kabat EU numbering system); The PTPsigma-Fc fusion protein inhibits PTPsigma signaling. The PTP sigma-Fc fusion protein.

2. The PTPsigma-derived protein contains amino acid residues 30 to 321 of the PTPsigma protein. The PTP sigma-Fc fusion protein of claim 1 .

3. 2. The PTP sigma-Fc fusion protein of claim 1, wherein the PTP sigma-Fc fusion protein is a homodimer or a heterodimer.

4. one chain of the Fc domain comprises a T366W substitution (Kabat EU numbering system); The other chain contains (Kabat EU numbering standard) T366S, L368A, and Y407V substitutions. The PTP sigma-Fc fusion protein of claim 1.

5. A blood sample containing the PTPsigma-Fc fusion protein according to any one of claims 1 to 4. Composition for promoting stem cell growth.

6. 6. The composition for promoting the growth of blood stem cells according to claim 5, wherein the composition is for regenerating blood stem cells damaged by radiation or administration of anti-cancer drugs.

7. A pharmaceutical composition for preventing or treating a PTPsigma-mediated disease, comprising the PTPsigma-Fc fusion protein according to any one of claims 1 to 4.

8. The pharmaceutical composition for preventing or treating a PTP sigma-mediated disease according to claim 7, wherein the PTP sigma-mediated disease is a neurological disorder selected from the group consisting of traumatic brain injury, Alzheimer's disease, and Parkinson's disease.

9. 8. The pharmaceutical composition for preventing or treating a PTP sigma-mediated disease according to claim 7, wherein the PTP sigma-mediated disease is selected from the group consisting of multiple sclerosis and rheumatoid arthritis.

10. 8. The pharmaceutical composition for preventing or treating a PTP sigma-mediated disease according to claim 7, wherein the PTP sigma-mediated disease is a synaptic plasticity-related disease selected from the group consisting of mania, depression, bipolar disorder, and memory impairment.

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