# Patent

## Title
Modified galectin-9 protein

## Abstract

The present invention provides galectin-9 variant having substantially the same bioactivity as in wild-type galectin-9 and being superior in protease stability, solubility, and yield. The galectin-9 variant according to the present invention is a protein including the following NCRD and the following CCRD composed of an N-terminal region and a C-terminal region. The C terminus of the NCRD and the N terminus of the CCRD are directly or indirectly bound to each other. The NCRD is (N1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 1. The N-terminal region of the CCRD is (C-N1) a peptide composed of an amino acid sequence obtained by deletion of 1 to 17 amino acids in an amino acid sequence represented by SEQ ID NO: 3. The C-terminal region is (C-C1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 5.

## Background
TECHNICAL FIELD
The present invention relates to galectin-9 protein variant.
BACKGROUND ART
The wild-type galectin-9 has a function of suppressing excess immunoreaction or repairing breakdown of immune system such as activation of immunity to cancer, through differentiation or homeostasis of T cells. The wild-type galectin-9 is composed of two Carbohydrate Recognition Domains (CRDs) and a link peptide region linking them. It is suggested that the wild-type galectin-9 that is a recombinant generated using Escherichia coli as a host induces suppression of transfer of cancer and regression of cancer by a direct action on tumor cells (activity of inducing adhesion among tumor cells and apoptosis of tumor cells) and an action via an immune system. Moreover, it is identified that the wild-type galectin-9 does not act on non-activated lymphocytes and induces activated T cells, specifically apoptosis of CD4-positive T cells which cause an excess immunoreaction. Furthermore, it is also identified that the wild-type galectin-9 has potent apoptosis inducibility to synoviocytes involved in such conditions as deformation of joints in rheumatism.
The above-mentioned functions of the wild-type galectin-9 show that the wild-type galectin-9 is useful as therapeutic drugs for various diseases. However, in order to actually distribute the wild-type galectin-9 as a therapeutic drug, there are three problems of protease sensitivity, low solubility, and low yield of recombinant protein. Regarding the problem of protease sensitivity of the wild-type galectin-9 among these problems, the inventors of the present invention have reported the stabilized galectin-9 having a molecular structure more stable to protease (Patent Document 1). However, no effective solution to the problems of low solubility and low yield of recombinant protein has been reported. Therefore, it is strongly required to provide galectin-9 variant superior in solubility and yield in order to commercialize a pharmaceutical utilizing superior functions of the wild-type galectin-9.
PRIOR ART DOCUMENTS
Patent Document
Patent Document 1: WO 2005/093064
SUMMARY OF INVENTION
Problem to be Solved by the Invention
Hence, the present invention is intended to provide a galectin-9 variant having a bioactivity that is substantially the same as wild-type galectin-9 and being superior in protease stability and solubility.
Means for Solving Problem
In order to achieve the aforementioned objects, the present invention is a protein or a salt thereof, including: NCRD; and CCRD, a C terminus of the NCRD and an N terminus of the CCRD being directly or indirectly bound to each other. The NCRD is (N1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 1, (N2) a peptide which is composed of an amino acid sequence obtained by deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 and has a carbohydrate binding ability, or (N3) a peptide which is composed of an amino acid sequence exhibiting an 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 and has a carbohydrate binding ability. The CCRD is a peptide composed of an N-terminal region and a C-terminal region and has a carbohydrate binding ability. The N-terminal region is (C-N1) a peptide composed of an amino acid sequence obtained by deletion of 1 to 17 amino acids in an amino acid sequence represented by SEQ ID NO: 3, and the C-terminal region is (C-C1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 5, (C-C2) a peptide composed of an amino acid sequence obtained by deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5, or (C-C3) a peptide composed of an amino acid sequence exhibiting an 80% or more identity to the amino acid sequence represented by SEQ ID NO: 5.
The nucleic acid according to the present invention includes a base sequence that encodes the protein according to the present invention.
The expression vector according to the present invention includes the nucleic acid according to the present invention.
The transformant according to the present invention includes the nucleic acid or the expression vector according to the present invention.
The pharmaceutical according to the present invention includes at least one of the protein or a salt thereof, the nucleic acid, and the expression vector according to the present invention.
The protein or a salt thereof according to the present invention is a wild-type galectin-9 variant and hereinafter referred to as the galectin-9 variant according to the present invention.
Effects of the Invention
The galectin-9 variant according to the present invention has the same bioactivity as wild-type galectin-9 and is superior in protease stability (also referred to as resistance or sensitivity) and solubility and is also superior in yield at the time of, for example, production as a recombinant protein due to the solubility. As described above, the galectin-9 variant according to the present invention not only has bioactivity of wild-type galectin, but also is superior in stability, handleability, and yield in production. Thus, it can be said that the galectin-9 variant according to the present invention is really useful as a pharmaceutical material.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic view showing a sequence of stabilized galectin-9.
FIG. 2 is a photograph showing a result of SDS-PAGE of the galectin-9 variant in Example 1 of the present invention.
FIG. 3 shows graphs showing concentrations of the deletion-type galectin-9 variant in Example 1 of the present invention.
FIG. 4 shows graphs showing concentrations of the deletion and substitution-type galectin-9 variant in Example 1 of the present invention.
FIG. 5 shows graphs showing the protease stability in Example 1 of the present invention.



DESCRIPTION OF EMBODIMENTS
1. Modified Galectin-9
As mentioned above, the galectin-9 variant according to the present invention is a protein or a salt thereof, including: NCRD; and CCRD, a C terminus of the NCRD and an N terminus of the CCRD being directly or indirectly bound to each other. The NCRD is (N1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 1, (N2) a peptide which is composed of an amino acid sequence obtained by deletion, substitution, insertion and/or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 1 and has a carbohydrate binding ability, or (N3) a peptide which is composed of an amino acid sequence exhibiting an 80% or more identity to the amino acid sequence represented by SEQ ID NO: 1 and has a carbohydrate binding ability. The CCRD is a peptide composed of an N-terminal region and a C-terminal region and has a carbohydrate binding ability. The N-terminal region is (C-N1) a peptide composed of an amino acid sequence obtained by deletion of 1 to 17 amino acids in an amino acid sequence represented by SEQ ID NO: 3, and the C-terminal region is (C-C1) a peptide composed of an amino acid sequence represented by SEQ ID NO: 5, (C-C2) a peptide composed of an amino acid sequence obtained by deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 5, or (C-C3) a peptide composed of an amino acid sequence exhibiting an 80% or more identity to the amino acid sequence represented by SEQ ID NO: 5.
The wild-type galectin-9 is a protein having two carbohydrate recognition domains (CRDs) as mentioned above. The galectin-9 variant according to the present invention has substantially the same bioactivity as wild-type galectin-9 and exhibits superior protease stability and solubility than the wild-type galectin-9. The galectin-9 variant according to the present invention further exhibits superior yield at that time of production as a recombinant protein due to the solubility, for example. In the present invention, “substantially the same bioactivity” means that the bioactivity of the galectin-9 variant is the same kind of bioactivity as or a similar kind of bioactivity to the wild-type galectin-9, for example. Examples of the bioactivity of the wild-type galectin-9 include binding activity specific to a specific carbohydrate strand, cytotoxic activity, apoptosis induction activity, anti-inflammatory activity, anti-allergy activity, immunomodulatory activity, physiological activity, and biological activity. In the present invention, the “substantially the same bioactivity as wild-type galection-9” means bioactivity associated with treatments of diseases and the like and does not encompass the meaning of protease stability, solubility, and yield that are the objects of the present invention, for example.
The galectin-9 variant according to the present invention preferably has bioactivity substantially the same or more than the wild-type galectin-9, for example. The “substantially the same bioactivity as wild-type galectin-9” means the same extent of the substantially the same bioactivity as wild-type galectin-9. The substantially the same or more than can be, for example, about 0.001 to about 100 times, preferably about 0.01 to about 100 times, more preferably about 0.1 to about 100 times, yet more preferably about 0.5 to about 100 times the bioactivity of the wild-type galectin-9.
In the present invention, the “solubility” means, for example, solubility to an aqueous medium, and examples of the aqueous medium include water, a saline solution, various buffer solutions such as a phosphate buffer solution and the like, and mixtures thereof.
In the galectin-9 variant according to the present invention, the NCRD is a C-type carbohydrate recognition domain (CRD) on the N terminus side, and the CCRD is a C-type carbohydrate recognition domain (CRD) on the C terminus side.
In the galectin-9 variant according to the present invention, the NCRD is, as mentioned above, any of the peptides (N1), (N2), and (N3). The NCRD is, as mentioned above, a peptide having a carbohydrate binding ability.
The peptide (N1) is composed of an amino acid sequence represented by SEQ ID NO: 1.







SEQ ID NO: 1: 148aa


MAFSGSQAPYLSPAVPFSGTIQGGLQDGLQITVNGTVLSSSGTRFAVNFQ


 


TGFSGNDIAFHFNPRFEDGGYVVCNTRQNGSWGPEERKTHMPFQKGMPFD


 


LCFLVQSSDFKVMVNGILFVQYFHRVPFHRVDTISVNGSVQLSYISFQ






The peptide (N2) is a peptide composed of an amino acid sequence obtained by deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of the peptide (N1). The peptide (N2) is only required to have the same function as the peptide (N1) and specifically have a carbohydrate binding ability and is not limited to particular peptides. One or more is, for example, 1 to 45, preferably 1 to 30, more preferably 1 to 15, yet more preferably 1 to 7, particularly preferably 1, 2, or 3.
The peptide (N3) is a peptide composed of an amino acid sequence exhibiting 70% or more identity to the amino acid sequence of the peptide (N1). The peptide (N3) is only required to have the same function as the peptide (N1) and specifically have a carbohydrate binding ability and is not limited to particular peptides. The identity is, for example, preferably 75% or more, more preferably 80% or more, yet more preferably 85% or more, yet more preferably 90% or more, particularly preferably 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
As mentioned above, the CCRD is a peptide composed an N-terminal region and a C-terminal region and has a carbohydrate binding ability. The N-terminal region and the C-terminal region are shown below and are only required to have a carbohydrate binding ability as the CCRD in the state of linking to each other.
The N-terminal region in the CCRD is the peptide (C-N1), i.e., a peptide composed of an amino acid sequence obtained by deletion of 1 to 17 amino acids in an amino acid sequence represented by SEQ ID NO: 3. The galectin-9 variant exhibiting the above-mentioned effects of the present invention is composed by the N-terminal region having such deletion of amino acids. Each of the 10th and 11th amino acids (X) in SEQ ID NO: 3 is proline or histidine, one of them is preferably proline, and XX is, for example, preferably Pro-Pro (PP), Pro-His (PH), or His-Pro (HP). Each of 12th and 13th amino acid residues (X) in SEQ ID NO: 3 is proline or alanine, one of them is preferably proline, and XX is, for example, preferably Pro-Pro (PP), Pro-Ala (PA), or Ala-Pro (AP).









SEQ ID NO: 3: 17aa



TPAIPPMMYXXXXYPMP






As to the number of amino acid residues to be deleted in the peptide (C-N1), the lower limit is 1, preferably 6, more preferably 8, the upper limit is 17, preferably 14, more preferably 13, yet more preferably 12, and the range is, for example, preferably 6 to 14, more preferably 8 to 14 or 9 to 14, yet more preferably 8 to 13 or 9 to 13, particularly preferably 10 to 12. In the present invention, the description of the numerical range means the disclosure of values included in the range. That is, for example, the description of 6 to 14 means the disclosure of 6, 7, 8, 9, 10, 11, 12, 13, and 14.
The deletion of amino acids in the peptide (C-N1) may be any of the deletion of consecutive amino acids and the deletion of non-consecutive amino acids, for example. The deletion is preferably the deletion of consecutive amino acids, particularly preferably the deletion of consecutive amino acid from the N terminus. Both of the deletion of consecutive amino acids and the deletion of non-consecutive amino acids may be present in the N terminal region. As the deletion of consecutive amino acids, the above-mentioned number of amino acids is preferably deleted, and as a specific example, for example, preferably 6 to 14 amino acids, more preferably 8 to 14 amino acids or 9 to 14 amino acids, yet more preferably 8 to 13 amino acids or 9 to 13 amino acids, particularly preferably 10 to 12 amino acids are deleted based on the amino acid at the N terminus as a first amino acid.
It is preferred that a proline residue is conserved in the peptide (C-N1). Specifically, it is preferred that, in the amino acid sequence represented by SEQ ID NO: 3, at least one amino acid residue selected from the group consisting of 10th, 11th, 12th, 13th, 15th, and 17th amino acid residues is proline Moreover, as mentioned above, it is preferred that, in the amino acid sequence represented by SEQ ID NO: 3, at least one of the 10th and the 11th amino acid residues is proline, and they are, for example, Pro-Pro (PP), Pro-His (PH), or His-Pro (HP). As mentioned above, it is preferred that, in the amino acid sequence represented by SEQ ID NO: 3, at least one of the 12th and the 13th amino acid residues is proline, and they are, for example, Pro-Pro (PP), Pro-Ala (PA), or Ala-Pro (AP).
Specific examples of the peptide (C-N1) include amino acid sequences represented by SEQ ID NOs: 7 to 20. In the present invention, the peptide (C-N1) is not limited to the following amino acid sequences.










TABLE 1









N-terminal region




in CCRD












Amino acid sequence
SEQ ID NO















mC8
YPHPAYPMP
7



 



mC9
PHPAYPMP
8



 



mC9-HP
HPPAYPMP
9



 



mC10
HPAYPMP
10



 



mC10-1P1A1
HAAYPMP
11



 



mC10-1P1A2
HPAYAMP
12



 



mC10-HPAP
HPAPPMP
13



 



mC10-HPPY
HPPYPMP
14



 



mC11
PAYPMP
15



 



mC11-AP
APYPMP
16



 



mC11-PP
PPYPMP
17



 



mC12
AYPMP
18



 



mC13
YPMP
19



 



mC14
PMP
20










The peptide of the C-terminal region in the CCRD is, as mentioned above, any of the peptides (C-C1), (C-C2), and (C-C3).
The peptide (C-C1) is composed of an amino acid sequence represented by SEQ ID NO: 5.







SEQ ID NO: 5: 129aa


FITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNPRFDENA


 


VVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCLKVAVDGQHL


 


FEYYHRLRNLPTINRLEVGGDIQLTHVQT






The peptide (C-C2) is composed of an amino acid sequence obtained by deletion, substitution, insertion, and/or addition of one or more amino acids in the amino acid sequence of the peptide (C-C1). One or more is, for example, 1 to 39, preferably 1 to 26, more preferably 1 to 13, yet more preferably 1 to 6, particularly preferably 1, 2, or 3.
The peptide (C-C3) is composed of an amino acid sequence exhibiting 70% or more identity to the amino acid sequence in the peptide (C-C1). The identity is, for example, preferably 75% or more, more preferably 80% or more, yet more preferably 85% or more, yet more preferably 90% or more, particularly preferably, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
Specific examples of the CCRD composed of the N-terminal region and the C-terminal region include amino acid sequences represented by SEQ ID NOs: 21 to 34. In the present invention, the peptide of the CCDR is not limited to the following sequences.









TABLE 2









CCRD












Amino acid
Base



Sequence
SEQ ID NO
SEQ ID NO














mC8
YPHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHI
21
63



AFHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWIL



CEAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC9
PHPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIA
22
64



FHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILC



EAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC9-HP
HPPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIA
23
65



FHLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILC



EAHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC10
HPAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAF
24
66



HLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCE



AHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC10-1P1A1
HAAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAF
25
67



HLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCE



AHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC10-1P1A2
HPAYAMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAF
26
68



HLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCE



AHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC10-HPAP
HPAPPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAF
27
69



HLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCE



AHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC10-HPPY
HPPYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAF
28
70



HLNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCE



AHCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC11
PAYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFH
29
71



LNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEA



HCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC11-AP
APYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFH
30
72



LNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEA



HCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC11-PP
PPYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFH
31
73



LNPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEA



HCLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC12
AYPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHL
32
74



NPRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAH



CLKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC13
YPMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLN
33
75



PRFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHC



LKVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT


 


mC14
PMPFITTILGGLYPSKSILLSGTVLPSAQRFHINLCSGNHIAFHLNP
34
76



RFDENAVVRNTQIDNSWGSEERSLPRKMPFVRGQSFSVWILCEAHCL



KVAVDGQHLFEYYHRLRNLPTINRLEVGGDIQLTHVQT









In the galectin-9 variant according to the present invention, the NCRD and the CCRD may be, as mentioned above, proteins which are directly or indirectly bound to each other and are preferably the former.
In the latter case, the C terminus of the NCRD and the N terminus of the CCRD may be indirectly bound to each other via a linker, for example. Examples of the linker include an amino acid and a peptide. The number of amino acid residues in the peptide is not limited to particular numbers, preferably is low, and is, for example, 2 to 5, preferably 4 or less, 3 or less, 2 or less, 1.
The sequence of the peptide is not limited to particular sequences and can be set appropriately. Specific examples of the peptide includes peptides including or composed of sequences such as His-Met (HM), Arg-Ile-Pro (RIP), Asn-Leu (NL), Asp-Phe-Val (DFV), and Gly-Ser-Ala (GSA).
A specific example of the galectin-9 variant according to the present invention can be a peptide composed of any one of amino acid sequences of SEQ ID NOs: 35 to 48 shown in Table 3. The galectin-9 variant according to the present invention is not limited to these examples.










TABLE 3









Full length











Amino acid
Base



SEQ ID NO
SEQ ID NO















mC8
35
77



mC9
36
78



mC9-HP
37
79



mC10
38
80



mC10-1P1A1
39
81



mC10-1P1A2
40
82



mC10-HPAP
41
83



mC10-HPPY
42
84



mC11
43
85



mC11-AP
44
86



mC11-PP
45
87



mC12
46
88



mC13
47
89



mC14
48
90










In the amino acid sequence of the galectin-9 variant according to the present invention, each of the amino acids may be substituted with another amino acid or amino acid analogue in the class to which the each amino acid belongs, for example. The class can be, for example, a class defined by chemical characteristics and/or physical characteristics. Examples of the characteristics includes hydrophobicity, hydrophilicity, electric charge, and size, and specific examples thereof include non-polar amino acid (hydrophobic amino acid), polar amino acid (neutral amino acid), positive charge amino acid (acidic amino acid), and negative charge amino acid (acidic amino acid). Examples of the non-polar amino acid include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, and methionine. Examples of the polar amino acid include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Examples of the positive charge amino acid include arginine, lysine, and histidine. Examples of the negative charge amino acid include aspartic acid and glutamic acid.
In the galectin-9 variant according to the present invention, the salt of the protein is not limited to particular salts and may be in the form of a salt of any of the above-mentioned proteins.
The galectin-9 variant according to the present invention is only required to exert the effects of the present invention, and an amino acid residue(s) may be further varied, for example. Examples of the variation include partial dissociation of amino acid residues, variation to a derivative, binding of a protecting group, and binding of a carbohydrate strand, and specific examples thereof include amidation and esterification of a carboxyl group such as the C terminus.
A method for producing the galectin-9 variant according to the present invention is not limited to particular methods, and the galectin-9 variant may be produced by gene-engineering techniques or synthesis techniques, based on the amino acid sequence, for example. The former may be a cell system using a host or an acellular protein synthesis system, for example. In the case of the gene-engineering techniques, for example, a nucleic acid having a base sequence corresponding to the amino acid sequence, an expression vector including the nucleic acid, and the like can be used. The method for producing the galectin-9 variant, the nucleic acid which encodes the galectin-9 variant, and the expression vector are described below.
2. Nucleic Acid, Expression Vector, and Transformant
(1) Nucleic Acid
The nucleic acid according to the present invention includes a base sequence which encodes the galectin-9 variant according to the present invention. A protein which encodes the nucleic acid is caused to express in a host or an acellular protein synthesis system by gene-engineering techniques using the nucleic acid according to the present invention, for example. Thus, the galectin-9 variant according to the present invention can be produced.
The base sequence of the nucleic acid according to the present invention is not limited to particular base sequences and can be designed by replacement with a codon based on the amino acid sequence of the galectin-9 variant according to the present invention. The nucleic acid according to the present invention can be synthesized by gene-engineering techniques or organic synthesis techniques and can be referred to as synthesized DNA such as cDNA or synthesized RNA. The nucleic acid according to the present invention may be, for example, any of a sense strand and an antisense strand and may be a hybrid thereof.
The nucleic acid according to the present invention can be, for example, a polynucleotide including a polynucleotide that encodes the NCRD (NCRD polynucleotide) and a polynucleotide that encodes the CCRD (CCRD polynucleotide), wherein the 5′ end of the CCRD polynucleotide is directly or indirectly bound to the 3′ end of the NCRD polynucleotide. A reading frame of the NCRD polynucleotide and a reading frame of the CCRD polynucleotide are bound to each other so as to correspond to the amino acid of the NCRD and the amino acid of the CCRD, for example. In the case of the indirect binding, a linker may be present between the NCRD polynucleotide and the CCRD polynucleotide. In this case, it is preferred that the sequence of the linker is designed so that the reading frame of the CCRD polynucleotide does not change. The linker is, for example, a polynucleotide.
In the NCRD, the polynucleotide (n1) that encodes the peptide (N1) is represented by a base sequence represented by SEQ ID NO: 2, for example.







SEQ ID NO: 2







atggccttcagcggttcccaggctccctacctgagtccagctgtcccctt


 


ttctgggactattcaaggaggtctccaggacggacttcagatcactgtca


 


atgggaccgttctcagctccagtggaaccaggtttgctgtgaactttcag


 


actggcttcagtggaaatgacattgccttccacttcaaccctcggtttga


 


agatggagggtacgtggtgtgcaacacgaggcagaacggaagctgggggc


 


ccgaggagaggaagacacacatgcctttccagaaggggatgccctttgac


 


ctctgcttcctggtgcagagctcagatttcaaggtgatggtgaacggtat


 


cctcttcgtgcagtacttccaccgcgtgcccttccaccgtgtggacacca


 


tctccgtcaatggctctgtgcagctgtcctacatcagcttccag






The polynucleotide (c-n1) that encodes the peptide (C-N1) that is the N-terminal region of the CCRD is, for example, represented by a base sequence obtained by deletion of 1 to 17 sets of codon consisting of three consecutive bases as one set in a base sequence represented by SEQ ID NO: 4. The number of sets of codon to be deleted and the position(s) of the codon(s) to be deleted correspond to the number of amino acid residues to be deleted and the position(s) of the amino acid(s) to be deleted described for the peptide (C-N1), for example. In SEQ ID NO: 4, the underlined bases represent a sequence that encodes the 10th to 13th (four) amino acids in SEQ ID NO: 3 and can be set appropriately depending on the amino acids.







SEQ ID NO: 4







actcccgccatcccacctatgatgtacnnnnnnnnnnnntatccgatgc


 


ct






Specific examples of the polynucleotide (c-n1) include the following base sequences, for example. In the present invention, the polynucleotide (c-n1) is not limited to the following sequences.









TABLE 4









N-terminal region in CCRD










Base sequence
SEQ ID NO













mC8
tacccccaccccgcctatccgatgcct
49


 


mC9
ccccaccccgcctatccgatgcct
50


 


mC9-HP
cacccgcccgcctatccgatgcct
51


 


mC10
caccccgcctatccgatgcct
52


 


mC10-1P1A1
cacgccgcctatccgatgcct
53


 


mC10-1P1A2
caccccgcctatgcgatgcct
54


 


mC10-HPAP
caccccgccccgccgatgcct
55


 


mC10-HPPY
caccccccctatccgatgcct
56


 


mC11
cccgcctatccgatgcct
57


 


mC11-AP
gccccctatccgatgcct
58


 


mC11-PP
cccccctatccgatgcct
59


 


mC12
gcctatccgatgcct
60


 


mC13
tatccgatgcct
61


 


mC14
ccgatgcct
62









In the C-terminal region of the CCRD, the polynucleotide (c-c1) that encodes the peptide (C-C1) is, for example, represented by the base sequence of SEQ ID NO: 6.







SEQ ID NO: 6







ttcatcaccaccattctgggagggctgtacccatccaagtccatcctcct


 


gtcaggcactgtcctgcccagtgctcagaggttccacatcaacctgtgct


 


ctgggaaccacatcgccttccacctgaacccccgttttgatgagaatgct


 


gtggtccgcaacacccagatcgacaactcctgggggtctgaggagcgaag


 


tctgccccgaaaaatgcccttcgtccgtggccagagettctcagtgtgga


 


tcttgtgtgaagctcactgcctcaaggtggccgtggatggtcagcacctg


 


tttgaatactaccatcgcctgaggaacctgcccaccatcaacagactgga


 


agtggggggcgacatccagctgacccatgtgcagacatag






A specific example of the polynucleotide that encodes the CCRD (CCRD polynucleotide) composed of the N-terminal region and the C-terminal region can be a base sequence in which the 5′ end of the base sequence represented by SEQ ID NO: 6 is linked to the 3′ end of any of the base sequences represented by the sequence numbers shown in Table 2 (SEQ ID NOs: 63 to 76 shown in Table 2). In the present invention, the CCRD polynucleotide is not limited to these sequences.
A specific example of the nucleic acid that encodes the galectin-9 variant according to the present invention can be a polynucleotide composed of any of base sequences represented by SEQ ID NOs: 77 to 90 shown in Table 3. The nucleic acid according to the present invention is not limited to these examples.
In the nucleic acid, the polynucleotide may be DNA or RNA, for example. RNA can be, for example, a base sequence obtained by substitution of T in the base sequence of DNA shown as an example with U. DNA can be, for example, a sequence including or composed of deoxyribonucleotide, and RNA can be, for example, a sequence including or being composed of ribonucleotide. Moreover, the nucleic acid may be, for example, a sequence composed of artificial nucleic acids or a sequence in which the DNA or RNA further includes artificial nucleic acids. Examples of the artificial nucleic acids include LNA, PNA, and BNA.
(2) Expression Vector
The expression vector according to the present invention includes the nucleic acid according to the present invention. The expression vector according to the present invention is introduced into a host, and the protein that encodes the nucleic acid is expressed by the resultant transformant, for example. Thus, the galectin-9 variant according to the present invention can be produced. The expression vector according to the present invention is only required to functionally include the nucleic acid according to the present invention so that the galectin-9 variant according to the present invention can be expressed, and the other configuration is not limited to particular configurations.
The expression vector is only required to express the galectin-9 variant according to the present invention by introducing into a non-human host, for example. The non-human host is not limited to particular hosts and can be selected appropriately. Examples of the non-human host include microorganisms, animal cells, insect cells, plant cells, and cultured cells thereof. Examples of the microorganisms include prokaryotes and eukaryotes. Examples of the prokaryotes include bacteria belonging to Escherichia such as Escherichia coli, bacteria belonging to Bacillus such as Bacillus subtilis, bacteria belonging to Pseudomonas such as Pseudomonas putida, and bacteria belonging to Rhizobium such as Rhizobium Examples of the eukaryote include yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe. Examples of the animal cells include COS cells and CHO cells. Examples of the insect cells include Sf9 cells and Sf21 cells.
The expression vector can be produced by inserting the nucleic acid into a vector which becomes a skeleton (hereinafter also referred to as a “basic vector”), for example. The kind of the basic vector is not limited to particular kinds and can be determined appropriately depending on the kind of the host into which the expression vector is introduced, for example. In the case where bacteria such as Escherichia coli are transformed, examples of the basic vector include a pET vector (Merck), a pCold vector (TAKARA BIO INC.), and a PQE vector (QIAGEN). In the case where eukaryotes such as yeast are transformed, the basic vector can be, for example, pYE22m, and a commercially available vector for yeast expression such as pYES (Invitrogen) or pESC (Stratagene) can also be used. In the case where the transformation is performed using an Agrobacterium method, the basic vector is, for example, preferably a binary vector, and examples thereof include pBI121, pPZP202, pBINPLUS, and pBIN19.
The expression vector preferably includes a regulatory sequence which regulates expression of nucleic acid and protein, for example. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an ori sequence (ori). The derivation of the promoter is not limited to particular derivations, and examples thereof include cytomegalovirus (CMV), Rous sarcoma virus (RSV), simian virus-40 (SV-40), a muscle β actin promoter, and herpes simplex virus. Examples of the promoter further includes, besides these, a tissue-specific promoter such as a thymidine kinase promoter, a regulatory promoter such as a growth hormone regulatory promoter, a promoter under control of lac operon sequence, and an inducible promoter such as a zinc inducible metallothionein promoter. In the expression vector, the arrangement of the regulatory sequence is not limited to particular arrangements. In the expression vector, for example, the regulatory sequence is only required to be arranged so that the expression of nucleic acid and protein can be functionally regulated and can be arranged based on a known method. As the regulatory sequence, for example, a sequence including a basic vector in advance may be utilized, or the regulatory sequence may be further inserted into the basic vector. The regulatory sequence including the basic vector may be replaced with another regulatory sequence.
The expression vector may further have a coding sequence of a selection marker, for example. Examples of the selection marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.
(3) Transformant
The transformant according to the present invention includes the nucleic acid according to the present invention or the expression vector according to the present invention. The transformant according to the present invention is only required to functionally include the nucleic acid according to the present invention so that the galectin-9 variant according to the present invention can be expressed, and the other configurations are not limited to particular configurations.
The transformant can be obtained by introducing the nucleic acid according to the present invention into a host, for example. The host is not limited to particular hosts, and examples thereof include the above-mentioned non-human hosts. The transformant according to the present invention may include the nucleic acid according to the present invention as the expression vector according to the present invention, for example, and in this case, the transformant can be obtained by introducing the expression vector according to the present invention into a host, for example.
The method for introducing the nucleic acid or the expression vector into a host is not limited to particular methods, and the introduction can be performed by a known method. The method for the introduction can be set appropriately depending on the kind of the host.
The method for the introduction includes an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethyleneglycol method, a lipofection method using liposome, an electroporation method, an ultrasound nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction adjuvant, and a method via Agrobacterium. Examples of the lipsome include lipofectamine and cationic liposome. Examples of the introduction adjuvant include atelocollagen, nanoparticles, and a polymer.
3. Method for Producing Galectin-9 Variant
The method for producing the galectin-9 variant according to the present invention (hereinafter also referred to as “the production method according to the present invention”) includes the step of expressing the nucleic acid according to the present invention, for example. The production method according to the present invention allows the galectin-9 variant to be obtained with a superior yield compared with the case of producing a wild-type galectin-9 as a recombinant protein, for example.
The production method according to the present invention is characterized in that the nucleic acid according to the present invention is transcribed and translated to a protein that is encoded by the nucleic acid by the expression of the nucleic acid to synthesize the galectin-9 variant according to the present invention, and the other steps and conditions are not limited to particular steps and conditions.
In the production method according to the present invention, the expression of the nucleic acid according to the present invention may be, for example, as mentioned above, performed in a cell system using a host or an acellular protein synthesis system.
In the case of a cell system, for example, the galectin-9 variant according to the present invention can be synthesized by introducing the nucleic acid according to the present invention into a host. The host is, for example, as mentioned above, and the introduction of the nucleic acid according to the present invention into a host may be an introduction of the expression vector according to the present invention.
The production method according to the present invention preferably further include the step of cultivating a transformant obtained by the introduction of the nucleic acid according to the present invention into a host. The conditions of the step of cultivating are not limited to particular conditions and can be determined appropriately depending on the kind of the host.
The expressed galectin-9 variant according to the present invention may be used as it is after the expression or may be used after purification, for example. The method for the purification is not limited to particular methods, and examples thereof include salting out, electrophoresis, and various kinds of chromatography.
4. Uses of Galectin-9 Variant
The galectin-9 variant according to the present invention can be used as a pharmaceutical. Specifically, the galectin-9 variant conserves, for example, activity of wild-type galectin 9, such as, e.g., cytotoxic activity against malignant tumors, apoptosis induction activity against malignant tumors, anti-tumor activity against malignant tumors, apoptosis induction activity against activated T cells (e.g., CD4-positive T cells) or activated B cells, immunomodulatory activity, anti-inflammatory activity, and/or anti-allergy activity. Thus, the galectin-9 variant according to the present invention is applicable to the same pharmaceuticals as those to which the wild-type galectin 9 is applicable, for example.
That is, the pharmaceutical according to the present invention contains at least one of the galectin-9 variant (a protein or a salt thereof) according to the present invention, the nucleic acid according to the present invention, and the expression vector according to the present invention, and is characterized in that it is applicable to at least one use selected from the group consisting of immunomodulatory agents (which also encompass immunosuppressive agents), anti-tumor agents, tumor metastasis inhibitors, analgesic agents, anti-inflammatory agents, and antiphlogistics. When the pharmaceutical according to the present invention contains the nucleic acid according to the present invention or the expression vector according to the present invention, it is only required that, for example, the galectin-9 variant according to the present invention can be expressed by the nucleic acid in the body of a patient to which the pharmaceutical according to the present invention has been administered.
The pharmaceutical according to the present invention can be used for treatment or prevention of diseases, for example. The treatment may be either causal treatment or symptomatic treatment. Also, the treatment may be any of disappearance of symptoms, alleviation (improvement) of symptoms, and inhibition of the progression of symptoms, for example.
A subject to which the pharmaceutical according to the present invention is administered is not limited to particular subjects, and examples thereof include humans and non-human animals excluding humans. Examples of the non-human animals include mammals such as mice, rats, rabbits, horses, sheep, cows, pigs, dogs, and cats; fishes; birds such as chickens; helminths; insects; reptiles; and amphibians.
The administration method of the pharmaceutical according to the present invention is not limited to particular methods and can be determined appropriately depending on the type of disease. The administration method may be, for example, either oral administration or parenteral administration, and also, may be either direct administration or indirect administration. Examples of the parenteral administration include local, transdermal, intravenous, intramuscular, subcutaneous, intradermal, and intraperitoneal administrations.
When the pharmaceutical according to the present invention contains the galectin-9 variant according to the present invention, the dose of the pharmaceutical is not limited to particular doses and can be determined appropriately depending on a subject to which the pharmaceutical is administered, the type of disease, the severity of the disease, etc. A specific example is as follows: the daily dose per a kilogram body weight of a subject is, for example, 5 μg to 5 mg, preferably 50 μg to 500 μg, more preferably 100 μg to 500 μg, and yet more preferably 200 μg to 250 μg. The frequency of administration per day is, for example, 1 to 3 times.
When the pharmaceutical according to the present invention contains the nucleic acid according to the present invention, the administration method thereof may be either in vivo administration or ex vivo administration, for example. In the former case, the administration may be achieved by, for example, administering the nucleic acid according to the present invention to the living body of a patient. In the latter case, the administration may be achieved by, for example, transfecting an isolated tissue or isolated cells with the nucleic acid according to the present invention and then introducing the tissue or cells to the living organism of a patient.
When the pharmaceutical according to the present invention contains the nucleic acid according to the present invention, the dose of the pharmaceutical is not limited to particular doses and can be determined appropriately depending on a subject to which the pharmaceutical is administered, the type of disease, the severity of the disease, etc. The dose preferably is such that, for example, it allows the galectin-9 variant according to the present invention in the above-described amount to be expressed. A specific example is as follows: when the nucleic acid is administered locally, the daily dose per human tissue is, for example, 100 ng to 200 mg, preferably 500 ng to 50 mg, more preferably 1 μg to 2 mg, and still more preferably 5 μg to 500 μg. The frequency of administration per day is, for example, 1 to 3 times.
The form of the pharmaceutical according to the present invention can be determined appropriately depending on the administration method, examples of which are as described above, for example. Examples of the form of the pharmaceutical include solution formulations, dispersion formulations, semisolid formulations, powder and granular formulations, molded formulations, and extract formulations. Specific examples thereof include tablets, coated tablets, sugar-coated tablets, pills, troches, hard capsules, soft capsules, microcapsules, implants, powders, micro powders, granules, microgranules, injections, pharmaceutical solutions, elixirs, emulsions, irrigations, syrups, liquid medicines, emulsions, suspensions, liniments, lotions, aerosols, sprays, inhalants, liquid medicines for use in atomizers, ointment formulations, plaster formulations, patches, pastes, cataplasms, creams, oil-based medicines, suppositories (e.g., rectal suppositories), tinctures, liquid medicines for skin, eye drops, nasal drops, ear drops, embrocations, infusions, pharmaceutical solutions for injections, powders for use in preparation of liquid formulations, freeze-dried formulations, and gel preparations.
The pharmaceutical according to the present invention may contain a pharmaceutically acceptable additive(s), in addition to the galectin-9 variant according to the present invention, the nucleic acid according to the present invention, or the expression vector according to the present invention. The additive is not limited to particular additives, and can be selected appropriately depending on the form of the pharmaceutical. Any known substances for use in preparation of pharmaceuticals can be used as the additive. The amount of the additive contained in the pharmaceutical is only required to not interfere with the properties of the galectin-9 variant according to the present invention contained in the pharmaceutical as an active ingredient and is not limited to particular amounts. Examples of the additive include carriers, adjuvants, excipients, bulking agents, diluents, flavoring agents, fragrances, sweetening agents, vehicles, antiseptic agents, stabilizers, binding agents, pH adjusters, buffers, surfactants, bases, solvents, fillers, expanders, dissolution assisting agents, solubilizers, tonicity agents, emulsifying agents, suspending agents, dispersants, thickening agents, gelling agents, curing agents, absorbents, adhesives, elastic agents, plasticizers, disintegrants, propellants, preservatives, antioxidants, sunproofing agents, moisturizing agents, palliatives, antistatic agents, and soothing agents. One kind of additive may be used, or two or more kinds of additives may be used in combination.
EXAMPLES
The present invention will be described in detail with reference to examples below. However, the present invention is not limited to the aspects described in the examples.
Example 1
As mentioned above, the stabilized galectin-9 composed of the amino acid sequence obtained by deletion of the region from proline at position 149 to serine at position 177 in wild-type galectin-9M has been reported in WO 2005/093064. The amino acid sequence of the stabilized galectin-9 is shown in FIG. 1 (SEQ ID NO: 91). It has been demonstrated that the stabilized galectin-9 maintains the bioactivity of wild-type galectin-9M and has superior protease stability than the wild-type galectin-9M. Hence, the solubility, bioactivity, and protease stability of galectin-9 variants obtained by varying the stabilized galectin-9 were examined.
(1) Construction of Expression Vector
(1-1) Deletion Type Galectin-9 Variant
As galectin-9 variants, proteins each composed of the amino acid sequence in which NCRD and CCRD are directly linked were used. Among the variants, the amino acid sequence (SEQ ID NO: 1) of the NCRD and the amino acid sequence (SEQ ID NO: 5) of the C-terminal region of the CCRD were common, and only the N-terminal region of the CCRD was set to an amino acid sequence obtained by deletion of 4, 6, 8, 9, 10, 11, 12, 13, 14, or 16 amino acid residue from the N-terminus in the amino acid sequence (17 amino acid residues) of SEQ ID NO: 3. The respective galectin-9 variants are referred to as galectin-9 variants mC4, mC6, mC8, mC9, mC10, mC11, mC12, mC13, mC14, and mC16 according to the number of amino acids deleted in the N-terminal region of the CCRD. The amino acid sequences and the base sequences of the N-terminal region of the CCRD of and the full-length amino acid sequences and the full-length base sequences of the stabilized galectin-9 and the galectin-9 variants are summarized below.










TABLE 5









N-terminal region in CCRD
Full-length















Base

Base




Amino acid
SEQ ID
Amino acid
SEQ ID



Amino acid sequence
SEQ ID NO
NO
SEQ ID NO
NO
















G9Null
TPAIPPMMYXXXXYPMP
3
4 *1
91
92



(XXXX = PHPA)


 


mC8
YPHPAYPMP
7
49
35
77


 


mC9
PHPAYPMP
8
50
36
78


 


mC10
HPAYPMP
10
52
38
80


 


mC11
PAYPMP
15
57
43
85


 


mC12
AYPMP
18
60
46
88


 


mC13
YPMP
19
61
47
89


 


mC14
PMP
20
62
48
90





*1 In SEQ ID NO: 4, consecutive twelve bases are ccccaccccgcc (SEQ ID NO: 130) which encodes PHPA (SEQ ID NO: 132).






The expression vectors that express the galectin-9 variants were constructed by the following method. The amino acid sequence (SEQ ID NO: 91) of the stabilized galectin-9 (hereinafter, also referred to as G9Null) in Example 1 of WO 2005/093064 is shown in FIG. 1. First, the coding sequence (SEQ ID NO: 92) for the G9Null was inserted into the cloning site (BamHI site) of pET-11a, and an expression vector pET-G9Null of the G9Null was constructed in the usual manner. It has been already verified in WO 2005/093064 that the stabilized galectin-9 has the apoptosis induction activity of wild-type galectin-9 and has superior protease stability than the wild-type galectin-9.







SEQ ID NO: 92







ATGGCCTTCAGCGGTTCCCAGGCTCCCTACCTGAGTCCAGCTGTCCCCTT


 


TTCTGGGACTATTCAAGGAGGTCTCCAGGACGGACTTCAGATCACTGTCA


 


ATGGGACCGTTCTCAGCTCCAGTGGAACCAGGTTTGCTGTGAACTTTCAG


 


ACTGGCTTCAGTGGAAATGACATTGCCTTCCACTTCAACCCTCGGTTTGA


 


AGATGGAGGGTACGTGGTGTGCAACACGAGGCAGAACGGAAGCTGGGGGC


 


CCGAGGAGAGGAAGACACACATGCCTTTCCAGAAGGGGATGCCCTTTGAC


 


CTCTGCTTCCTGGTGCAGAGCTCAGATTTCAAGGTGATGGTGAACGGTAT


 


CCTCTTCGTGCAGTACTTCCACCGCGTGCCCTTCCACCGTGTGGACACCA


 


TCTCCGTCAATGGCTCTGTGCAGCTGTCCTACATCAGCTTCCAGCATATG


 


ACTCCCGCCATCCCACCTATGATGTACCCCCACCCCGCCTATCCGATGCC


 


TTTCATCACCACCATTCTGGGAGGGCTGTACCCATCCAAGTCCATCCTCC


 


TGTCAGGCACTGTCCTGCCCAGTGCTCAGAGGTTCCACATCAACCTGTGC


 


TCTGGGAACCACATCGCCTTCCACCTGAACCCCCGTTTTGATGAGAATGC


 


TGTGGTCCGCAACACCCAGATCGACAACTCCTGGGGGTCTGAGGAGCGAA


 


GTCTGCCCCGAAAAATGCCCTTCGTCCGTGGCCAGAGCTTCTCAGTGTGG


 


ATCTTGTGTGAAGCTCACTGCCTCAAGGTGGCCGTGGATGGTCAGCACCT


 


GTTTGAATACTACCATCGCCTGAGGAACCTGCCCACCATCAACAGACTGG


 


AAGTGGGGGGCGACATCCAGCTGACCCATGTGCAGACATAG






NCRD polynucleotide (SEQ ID NO: 2) which encodes NCRD peptide (SEQ ID NO: 1) was amplified by PCR using the following primers A1 and A2 with the pET-G9Null being used as a target sequence and purified by agarose gel electrophoresis.









(Primer)



A1:







(SEQ ID NO: 93)









5′-CGTCCTCGTCCTCATATGGCCTTCAGCGGTTCCCAGGCT-3′



 



A2:







(SEQ ID NO: 94)









5′-CTGGAAGCTGATGTAGGACAGCTG-3′






In the same manner, polynucleotides (CCRD1 to CCRD10, 10 types) which encode CCRDs of the respective variants were amplified by PCR using the following primer A4 and the following primers B1 to K1 corresponding to the respective variants with the pET-G9Null being used as a target sequence and purified by agarose gel electrophoresis.









(Primer)



B1:







(SEQ ID NO: 95)









5′-TACATCAGCTTCCAGCCACCTATGATGTACCCCCACCCC-3′



 



C1:







(SEQ ID NO: 96)









5′-TACATCAGCTTCCAGATGATGTACCCCCACCCCGCCTAT-3′



 



D1:







(SEQ ID NO: 97)









5′-TACATCAGCTTCCAGTACCCCCACCCCGCCTATCCGATG-3′



 



E1:







(SEQ ID NO: 98)









5′-TACATCAGCTTCCAGCCCCACCCCGCCTATCCGATGCCT-3′



 



F1:







(SEQ ID NO: 99)









5′-TACATCAGCTTCCAGCACCCCGCCTATCCGATGCCTTTC-3′



 



G1:







(SEQ ID NO: 100)









5′-TACATCAGCTTCCAGCCCGCCTATCCGATGCCTTTCATC-3′



 



H1:







(SEQ ID NO: 101)









5′-TACATCAGCTTCCAGGCCTATCCGATGCCTTTCATCACC-3′



 



I1:







(SEQ ID NO: 102)









5′-TACATCAGCTTCCAGTATCCGATGCCTTTCATCACCACC-3′



 



J1:







(SEQ ID NO: 103)









5′-TACATCAGCTTCCAGCCGATGCCTTTCATCACCACCATT-3′



 



K1:







(SEQ ID NO: 104)









5′-TACATCAGCTTCCAGCCTTTCATCACCACCATTCTGGGA-3′






Next, the polynucleotide (NCRD) which encodes the NCRD and the respective polynucleotides (CCRD 1 to CCRD 10) which encodes the CCRDs were mixed, these mixtures were subjected to second stage PCR using the primers A1 and A4, and the obtained PCR products were purified by agarose gel electrophoresis. The purified PCR products were cleaved by restriction enzymes NdeI and BamHI and purified, the resultants were then each ligated to a pET-11a vector that has been cleaved by the same restriction enzyme, and clones containing correct sequences were then selected in the usual manner. These clones were used as the expression vectors for the respective galectin-9 variants.









(Primer)



A4:







(SEQ ID NO: 105)









5′-CGACCGGGATCCCTATGTCTGCACATGGGTCAGCTG-3′






The clones were then each transfected into E. coli BL21(DE3) to produce transformants for recombinant protein expression, and the transformants were stored at −80° C. in the presence of about 15% glycerin.
(1-2) Deletion and Substitution Type Galectin-9 Variant
As galectin-9 variants, proteins were designed by substituting amino acids in the N-terminal region of the CCRD of the galectin-9 variants mC9 to mC12. The types of, the amino acid sequences and base sequences of the N-terminal regions of, and full-length amino acid sequences and full-length base sequences of the respective galectin-9 variants are summarized below. In the following sequences, the underlined portions indicate the amino acid residues substituted for the corresponding amino acid residues of the amino acid sequences of mC9 to mC12.










TABLE 6









N-terminal region in CCRD
Full-length













Amino acid
Amino acid
Base
Amino acid
Base



sequence
SEQ ID NO
SEQ ID NO
SEQ ID NO
SEQ ID NO
















mC9
PHPAYPMP
8
50
36
78


 


mC9-HP
HPPAYPMP
9
51
37
79


 


mC10
 HPAYPMP
10
52
38
80


 


mC10-1P1A1
HAAYPMP
11
53
39
81


 


mC10-1P1A2
HPAYAMP
12
54
40
82


 


mC10-HPAP
HPAPPMP
13
55
41
83


 


mC10-HPPY
HPPYPMP
14
56
42
84


 


mC11
PAYPMP
15
57
43
85


 


mC11-AP
APYPMP
16
58
44
86


 


mC11-PP
PPYPMP
17
59
45
87









The expression vectors that express the galectin-9 variants were constructed by the following method. First, with the expression vectors for mC9, mC10, mC11, and mC12 being used as target sequences, PCR using the primer A1 and the primers L2 to W2 each containing a mutant sequence corresponding to the substituted amino acid sequence was conducted, and the obtained PCR products were purified by agarose gel electrophoresis (muNCRD1 to muNCRD12, 12 types).







(Primer)


L2:







(SEQ ID NO: 106)







5′-ATAGGCGGGCGGGTGCTGGAAGCTGATGTAGGA-3′


 


M2:







(SEQ ID NO: 107)







5′-GATAGGCGGCGTGCTGGAAGCTGATGTA-3′


 


N2:







(SEQ ID NO: 108)







5′-AAGGCATCGCATAGGCGGGGTGCTGGAA-3′


 


O2:







(SEQ ID NO: 109)







5′-TGATGAAAGCCATCGGATAGGCGGGGTG-3′


 


P2:







(SEQ ID NO: 110)







5′-TGATGAAAGCCATCGCATAGGCGGCGTGCTGGAAGCTGATG-3′


 


Q2:







(SEQ ID NO: 111)







5′-TGAAAGGCGGCGGATAGGGGGGGTGCTG-3′


 


R2:







(SEQ ID NO: 112)







5′-GGCATCGGCGGGGCGGGGTGCTGGAAGCT-3′


 


S2:







(SEQ ID NO: 113)







5′-TCGGATAGGGGGGGTGCTGGAAGCTGAT-3′


 


T2:







(SEQ ID NO: 114)







5′-CGGATAGGGGGCCTGGAAGCTGATGTAGGA-3′


 


U2:







(SEQ ID NO: 115)







5′-TCGGATAGGGGGGCTGGAAGCTGATGTA-3′


 


V2:







(SEQ ID NO: 116)







5′-TGGTGATGAAAGCCATCGCATAGGCCTGGAAGCTGAT-3′


 


W2:







(SEQ ID NO: 117)







5′-AGGCATCGGAGGGGCCTGGAAGCTGATGTA-3′






In the same manner, with the expression vectors for mC9, mC10, mC11, and mC12 being used as target sequences, PCR using the primer A4 and the primers L1 to W1 each containing a mutant sequence corresponding to the substituted amino acid sequence was conducted, and the obtained PCR products were purified by agarose gel electrophoresis (muCCRD1 to muCCRD12, 12 types).







(Primer)


L1:







(SEQ ID NO: 118)







5′-AGCTTCCAGCACCCGCCCGCCTATCCGATGCCT-3′


 


M1:







(SEQ ID NO: 119)







5′-TTCCAGCACGCCGCCTATCCGATGCCTT-3′


 


N1:







(SEQ ID NO: 120)







5′-CCCGCCTATGCGATGCCTTTCATCACCA-3′


 


O1:







(SEQ ID NO: 121)







5′-TATCCGATGGCTTTCATCACCACCATTC-3′


 


P1:







(SEQ ID NO: 122)







5′-TTCCAGCACGCCGCCTATGCGATGGCTTTCATCACCACCATTC-3′


 


Q1:







(SEQ ID NO: 123)







5′-CTATCCGCCGCCTTTCATCACCACCATT-3′


 


R1:







(SEQ ID NO: 124)







5′-CCCCGCCCCGCCGATGCCTTTCATCACC-3′


 


S1:







(SEQ ID NO: 125)







5′-CAGCACCCCCCCTATCCGATGCCTTTCA-3′


 


T1:







(SEQ ID NO: 126)







5′-TTCCAGGCCCCCTATCCGATGCCTTTCA-3′


 


U1:







(SEQ ID NO: 127)







5′-CCAGCCCCCCTATCCGATGCCTTTCATC-3′


 


V1:







(SEQ ID NO: 128)







5′-TTCCAGGCCTATGCGATGGCTTTCATCACCACCATTC-3′


 


W1:







(SEQ ID NO: 129)







5′-TTCCAGGCCCCTCCGATGCCTTTCATCACC-3′






Next, the former PCR products (muNCRD1 to muNCRD12) and the latter PCR products (muCCRD1 to muCCRD12) were mixed, these mixtures were amplified by second stage PCR using the primers A1 and A4, and the obtained PCR products were purified by agarose gel electrophoresis. The purified PCR products were cleaved by restriction enzymes NdeI and BamHI and purified, the resultants were then each ligated to a pET-11a vector that has been cleaved by the same restriction enzyme, and clones containing correct sequences were then selected in the usual manner. These clones were used as the expression vectors for the respective galectin-9 variants.
The clones were then each transfected into E. coli BL21(DE3) to produce transformants, and the transformants were stored at −80° C. in the presence of about 15% glycerin.
(2) Measurement of Expression and Yield of Galectin-9 Variant
Galectin-9 variants were expressed by the following method using the transformants produced in the item (1) above, and the yields were measured with respect to test samples of recombinant proteins right after expression (also referred to as preparations right after expression) and test samples of recombinant proteins from which insoluble matters were removed after three months storage after expression (also referred to as preparations after three months storage).
The transformants were each added to LB-broth that contains 100 μg/mL ampicillin and cultured at 37° C. overnight. 400 mL of 2XYT, 4 mL of 10 mg/mL ampicillin, and 8 mL of each of E. coli culture solutions obtained by the culture were added to 1000 mL flask. Subsequently, the resultants were each cultured at 37° C. while shaking the flask until A600 nm becomes about 0.7. 0.4 mL of 0.1 mol/L isopropyl-β-D(−)thiogalactopyranoside (IPTG) was then added to each of the flasks and the resultants were cultured at 20° C. overnight (16 to 20 hours). Bacterial cells were then recovered from the culture solutions by centrifugation.
The recovered bacterial cells were suspended in the flasks each containing 80 mL of buffer solution for extraction. The buffer solution for extraction had the following composition: 10 mmol/L Tris-HCl (pH7.5), 0.5 mol/L NaCl, 1 mmol/L dithiothreitol (DTT), 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100. Each of the suspensions was then sonicated under the following conditions: output control=5 and % duty cycle=100. Thereafter, the resultants were stirred at 4° C. for 30 minutes. With regard to the sonication, one cycle of the treatment for 2 minutes and the pause for 1 minute was repeated for a total of four cycles. Subsequently, the suspensions were subjected to the centrifugation at 15,000×g for 30 minutes to remove insoluble matters, and thereby recovered supernatants.
3 mL of lactose-agarose suspension (50% [v/v] in PBS, 1.5 ml as a gel) was added to each of the obtained supernatants, and the resultants were stirred at 4° C. for 1 hour. The resultants were then each subjected to the centrifugation at 2,000×g for 5 minutes to recover lactose-agarose gel, and the recovered lactose-agarose gels were each suspended in TBS that contains 0.03% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS) and then charged in a minicolumn. Subsequently, after washing each gel with TBS that contains 0.03% CHAPS in an amount of 10 times the amount of the gel, protein was eluted with 3 mL of eluate. The elute had the following composition: 20 mmol/L Tris-HCl (pH7.5), 0.15 mol/L NaCl, and 0.2 mol/L lactose. The respective elutes were dialyzed against PBS, then subjected to the centrifugation at 25,000×g for 20 minutes to remove insoluble matters, and the supernatants thereby obtained were filter sterilized to obtain final samples. Hereinafter, these will be referred to as test samples right after preparation. 3 μg of each sample was subjected to SDS-PAGE and stained with coomassie brilliant blue R-250. In the usual manner, the purity test of protein and the concentration measurement of protein by SDS-PAGE were then performed, and the yield of each galectin-9 variant was calculated. This was considered as the yield of recombinant protein right after preparation.
Also the test samples right after preparation were stored at 4° C. for 3 months. After the storage, the samples were subjected to the centrifugation at 25,000×g for 20 minutes to remove insoluble matters, and the supernatants thereby obtained were filter sterilized to obtain test samples after 3 months storage. In the same manner as the test samples right after preparation, the concentration of each protein was then measured, and the yield of each galectin-9 variant was calculated in the usual manner. This was considered as the yield of recombinant protein after 3 months storage.
Also, as a comparative example, using the expression vector ET-G9Null for the stabilized galectin-9, the expression and evaluation of protein were performed in the same manner.
FIG. 2 shows the photograph of SDS-PAGE. In FIG. 2, the lane M indicates molecular weight markers, and each lane indicates the test sample right after preparation of each galectin-9 variant. As shown in FIGS. 2 and 3, it was verified that all galectin-9 variants can be expressed in Escherichia coli and can be highly purified by affinity chromatography using lactose-agarose.
FIG. 3 shows the concentrations of the deletion type galectin-9 variants prepared in the item (1-1) above and the stabilized galectin-9. In FIG. 3, A indicates the results of the test samples right after preparation and B indicates the results of the test samples after 3 months storage. In FIG. 3, each of the vertical axes indicates the concentration (μg/ml) of the galectin-9 variant or the stabilized galectin-9 in the sample; and the average value of two expression experiments is shown with respect to each galectin-9 variant and the average value and standard deviation of twelve expression experiments are shown with respect to the stabilized galectin-9.
As shown in FIG. 3, with respect to the test samples right after preparation, the galectin-9 variants mC8, mC9, mC10, mC11, mC12, mC13, and mC14 each showed higher protein concentration than the stabilized galectin-9 (G9Null). Among others, mC9, mC10, mC12 and mC14 each showed very high protein concentration. Also with respect to the test samples after 3 months storage, the galectin-9 variants mC10, mC11, mC12, and mC13 each showed significantly higher protein concentration than the stabilized galectin-9 (G9Null).
Since the stabilized galectin-9 (G9Null) exceeded its maximum solubility in the elution step from the column at the time of preparing a final sample after expression (test sample right after preparation), a part of the stabilized galectin-9 was insolubilized. This insoluble matter was removed by the centrifugation step and the filtration step after the elution step.
Therefore, as shown in FIG. 3, the stabilized galectin-9 showed a low protein concentration in the test sample as mentioned above. On the other hand, the insolubilization in the elution step of each galectin-9 variant was significantly suppressed as compared to the stabilized galectin-9. Therefore, as shown in FIG. 3, the galectin-9 variants showed significantly high protein concentrations in the test samples as compared to the stabilized galectin-9.
In the present example, with respect to all recombinant proteins, fixed-volume final samples (test samples right after preparation) were prepared under the same conditions. Therefore, the fact that the concentration of each galectin-9 variant in the test sample is significantly higher than that of the stabilized galectin-9 in the test sample means that the solubility of each galectin-9 variant is significantly higher than that of the stabilized galectin-9. Also the high concentration of each galectin-9 variant in the test sample means that each galectin-9 variant was recovered with high yield.
In the synthesis of a target protein, the purification of the protein involving centrifugal treatment, filtration treatment, and the like, and the pharmaceutical preparation of the protein, generally, the solubilization of the protein is utilized with respect to an aqueous solvent such as a buffer solution. Therefore, the higher the solubility of the protein is, the less the loss of the protein in recovery becomes, for example. Also the higher solubility of the protein results in superior handleability. Since the galectin-9 variants according to the present invention are superior in solubility as mentioned above, it can be said that they are superior in the production, purification, and pharmaceutical preparation of protein as compared to the stabilized galectin-9 having the same bioactivity.
FIG. 4 shows the concentrations of the deletion and substitution type galectin-9 variants prepared in the item (1-2) above and the deletion type galectin-9 variants prepared in the item (1-1) above. In FIG. 4, A indicates the results of the test samples right after preparation and B indicates the results of the test samples after 3 months storage. In FIG. 4, each of the vertical axes indicates the concentration (μg/ml) of the galectin-9 variant in the test sample; and the average value of two expression experiments is shown with respect to each galectin-9 variant and the average value and standard deviation of twelve expression experiments are shown with respect to the stabilized galectin-9.
As shown in FIG. 4, with respect to the test samples right after preparation, the deletion and substitution type galectin-9 variants mC9-HP, mC10-1P1A1, mC10-1P1A2, mC10-HPAP, mC10-HPPY, mC11-AP, and mC11-PP each showed higher protein concentration than the stabilized galectin-9. Also with respect to the test samples after 3 months storage, deletion and substitution type galectin-9 variants mC10-HPAP, mC10-HPPY, and mC11-AP each showed higher protein concentration than the stabilized galectin-9.
Furthermore, as shown in FIG. 4, with respect to the test samples right after preparation, the deletion and substitution type galectin-9 variants mC10-HPAP, mC10-HPPY, mC11-AP, and mC11-PP respectively showed higher protein concentrations than the corresponding deletion type galectin-9 variants. Also with respect to the test samples after 3 months storage, the deletion and substitution type galectin-9 variants mC10-HPPY and mC11-AP respectively showed higher protein concentrations than the corresponding galectin-9 variants. Among others, in the case of comparing the test samples after 3 months storage, the protein concentration of the deletion and substitution type mC10-HPPY in which proline residues were conserved and tyrosine residues were substituted with proline residues was increased about 3.3 times the protein concentration of the deletion type mC10. Also the protein concentration of the deletion and substitution type mC11-AP in which proline-alanine was substituted with alanine-proline was increased about 2.2 times the protein concentration of the deletion type mC11. From these results, it was found that the solubility of the galectin-9 variant can be further improved by conservation of proline residues at position 12 or 15 and position 17 of SEQ ID NO: 3 and further substitution for proline residues in the N-terminal region of the CCRD.
(3) Measurement of Apoptosis Induction Activity of Galectin-9 Variant to Jurkat Cell
Jurkat cells cultured in a RPMI1640-10% FBS culture medium were recovered by centrifugation and suspended in a new culture medium so as to achieve a concentration of 3×104 cells/90 μL. 90 μL of the obtained suspension was seeded into each well of a 96-well plate. After the cells were cultured for 3 hours in a CO2 incubator, 10 μL of each of the samples containing galectin-9 variants was then added to each well. The samples were prepared by diluting the respective galectin-9 variants of the item (1) above with PBS so as to achieve concentrations of 0.01, 0.03, 0.1, 0.3, and 1 μmol/L in the respective wells. Next, after the cells were cultured for 24 hours, 10 μL of WST-8 reagent was added to each well, and the cells were further cultured for 3 hours. 10 μL of 1.2% SDS was then added to each well, the absorbance of each well at the wavelengths of 450 nm and 620 nm was measured using a microplate reader, and the difference between the absorbance at 450 nm and the absorbance at 620 nm was calculated. This assay was performed with three wells as one set. Also, as a control, the same measurement was performed by adding 10 μl of PBS instead of the samples. With the calculated value of the control being considered as 100%, the relative values (%) with respect to the calculated values in the cases of adding the samples were then obtained. These relative values were considered as the values showing the number of cells.
Also, as a comparative example, the number of cells was measured in the same manner using the stabilized galectin-9 (G9Null) instead of the galectin-9 variants.
From these results, the concentrations (LD 50) of the galectin-9 variants and the stabilized galectin-9 which cause 50% decrease of the number of Jurkat cells were obtained. With the LD 50 of the stabilized galectin-9 being considered as 100%, the relative values (%) of the respective galectin-9 variants were obtained as specific activities. The results are shown in Table 7.









TABLE 7







Apoptosis induction activity



(%)


















Stabilized galectin-9
G9Null
100


Galectin-9 variant
mC6
170



mC8
130



mC9
190



mC10
190



mC11
210



mC12
230



mC13
120



mC10-HPPY
250



mC10-HPAP
120



mC11-AP
190









As can be seen from Table 7, the galectin-9 variants each showed significantly higher apoptosis induction activity to Jurkat cells than the stabilized galectin-9 (G9Null). From this result, it was found that the galectin-9 variants sufficiently maintain the bioactivity of the stabilized galectin-9 and are superior in solubility as mentioned above.
(4) Protease Stability
With respect to the galectin-9 variant mC10-HPPY prepared in the item (2) above, the stability to protease present in human tissues was examined.
The galectin-9 variant was dissolved in 100 mmol/L Tris-HCl (pH8.0) that contains 150 mmol/L NaCl and 1 mmol/L CaCl2 at a concentration of 0.06 mg/mL, and further, protease was mixed thereto, followed by incubation at 37° C. The proportion of protease (P) to be added to the galectin-9 variant (G) was G:P=100:1 in weight ratio. As the protease, elastase (trade name: Elastase (product of Elastin Products Company, Inc.)) or matrix metalloproteinase-3 (MMP-3, trade name: Matrix Metalloproteinase-3 (product of Biogenesis)) was used. MMP-3 was activated by activation treatment at 37° C. for 8 hours using 20 mmol/L Aminophenyl mercuric acetate before use. Sampling was performed over time during the incubation, and each sample was subjected to SDS-PAGE and stained with coomassie brilliant blue R-250. Also, as a comparative example, wild-type galectin-9 preparation (G9S, amino acid sequence of SEQ ID NO: 131) or the stabilized galectin-9 (G9Null) prepared in the item (2) above was used instead of the galectin-9 variant.
FIG. 5 shows the photographs of SDS-PAGE. In FIG. 5, A indicates the results obtained by using elastase and B indicates the results obtained by using MMP-3. In FIG. 5, the lane M indicates molecular weight markers. As shown in A and B of FIG. 5, degradation of wild-type G9S was recognized only 0.5 hours after the start of the incubation, and the wild-type G9S was mostly degraded in 2 hours. In contrast, degradation of the galectin-9 variant mC10-HPPY was not recognized even after 2 hours incubation as with the stabilized galectin-9 whose protease stability has been verified in WO2005/093064. From these results, it was confirmed that mC10-HPPY is also superior in protease stability. The same applies to other galectin-9 variants.
As described above, it was verified that each galectin-9 variant according to the present invention has superior solubility than the stabilized galectin-9, maintains the bioactivities of the wild-type galectin-9, and has superior protease stability than the wild-type galectin. Specifically, as described in the item (2) above, the solubility (i.e., yields) of all galectin-9 variants was significantly increased as compared to the stabilized galectin-9. Among others, the solubility (i.e., yields) of mC10, mC11, mC12, mC10-HPPY, mC10-HPAP, and mC11-AP was increased more than 5 times the solubility of the stabilized galectin-9. In particular, it was verified that mC10-HPPY could be present in a stable manner in PBS at least up to a concentration of 2.5 mg/mL, which is about 7 times the concentration of the stabilized galectin-9. Also, as described in the item (3) above, all galectin-9 variants showed the apoptosis induction activity to Jurkat cells, which is one of the bioactivities of the wild-type galectin-9. Among others, mC10, mC11, mC12, mC10-HPPY, mC10-HPAP, and mC11-AP each showed the activity 2 to 2.5 times the activity of the stabilized galectin-9. Furthermore, as described in the item (4) above, each of the galectin-9 variants showed significant protease stability as compared to the wild-type galectin-9. From these results, it can be said that the galectin-9 variants have excellent properties as medical materials of high productivity, and among others, mC10-HPPY has an excellent property.
Example 2
The present example examined the influence of long-term storage of the above galectin-9 variants on their solubility and the degranulation inhibitory activities of the galectin-9 variants against RBL-2H3 cells.
(1) Influence of Long-Term Storage on Solubility
Each of the galectin-9 variants was stored at 4° C. for about a year (344 days). Thereafter, the influence of this long-term storage on the solubility of the galectin-9 variant was examined.
The filter-sterilized test samples (the galectin-9 variants) prepared in the item (2) in Example 1 right after the preparation were stored at 4° C. for about a year (344 days). The protein concentration in each test sample was adjusted as follows: 1.88 mg/mL in mC10-HPPY: 1.16 mg/mL in mC10-HPAP, and 1.14 mg/mL in mC11-AP. Subsequently, the test sample after the storage was centrifuged at 25,000×g for 20 minutes. Insoluble matters were removed, and the supernatant was collected. The absorbance of each test sample after the one-year storage was measured at 280 nm, and the protein concentration was calculated. Assuming that the protein concentration of the galectin-9 variant in the test sample before the storage was 100%, the relative value (%) of the protein concentration of the galectin-9 variant in the test sample after the storage was then determined. The results thereof are shown in Table 8 below.










TABLE 8








Protein concentration




(proportion relative to that in test



Galectin-9 variants
sample before storage, %)



















mC10-HPPY
100



mC10-HPAP
90










As can be seen from Table 8, the protein concentrations of mC10-HPPY and mC10-HPAP remained substantially the same even after the storage for about one year. It was thus found that mC10-HPPY and mC10-HPAP can maintain high solubility even after long-term storage.
(2) Measurement of Degranulation Inhibitory Activities of Galectin-9 Variants Against RBL-2H3 Cells
RBL-2H3 cells cultured in a RPMI 1640-10% FBS medium were collected by trypsinization. The collected RBL-2H3 cells were then suspended in a fresh medium so as to achieve a cell density of 2×104 cells/100 μL. The thus-obtained suspension was seeded into a 96-well plate so that each well contained 100 μL of the suspension. Thereafter, the cells were cultured for 24 hours in a CO2 incubator”.
Each well was washed once with an activity measurement buffer solution (Hanks' balanced salt solution containing 20 mmol/L HEPES-NaOH (pH 7.5) and 1 mg/mL bovine serum albumin). To each well, 90 μL of the activity measurement buffer solution was then added, and further, 10 μL of each of samples containing the above-described galectin-9 variants was added. The samples were prepared by diluting each galectin-9 variant used in the item (1) in Example 1 with PBS so that the concentration of the galectin-9 variant in each well was 0.1, 0.25, 0.5, 0.75, or 1 μmol/L. Subsequently, the 96-well plate was allowed to stand still for 10 minutes, and anti-2,4,6-trinitrophenyl (TNP) mouse monoclonal antibody (IgE) and TNP-labeled bovine serum albumin were then added so that their concentrations in each well were 0.3 μg/mL and 0.048 μg/mL, respectively. The resultant mixture was cultured for 1 hour. Thereafter, the medium was collected from each well, and the cultured cells were then lysed using 0.1% Triton X-100. The β-hexosaminidase (β-HEX) activity was measured in the collected medium and in the cell lysate. The total of the β-HEX activity in the medium and the β-HEX activity in the cell lysate was calculated as a total β-HEX activity. The proportion (%) of the β-HEX activity in the medium to the total β-HEX activity was then determined. The thus-determined proportion was used as an index of degranulation. This assay was performed with three wells as one set.
Furthermore, the measurement was performed in the same manner, except that: as a control, 10 μL of PBS was added instead of the sample; and as a comparative example, the stabilized galectin-9 (G9Null) in Example 1 was added instead of the galectin-9 variant.
From the results thereof, the concentration (LD50) at which each of the galectin-9 variants and the stabilized galectin-9 decreased the degranulation of the RBL-2H3 cells by 50% was determined. Assuming that LD50 of the stabilized galectin-9 was 100%, the relative values (%) of LD50 of the respective galectin-9 variants were then determined as the degranulation inhibitory activities (%). The results thereof are shown in Table 9 below.









TABLE 9







Degranulation inhibitory activity



(%)


















Stabilized galectin-9
G9Null
100


Galectin-9 variants
mC6
94



mC8
180



mC9
160



mC10
250



mC11
170



mC12
190



mC13
190



mC10-HPPY
150



mC10-HPAP
170



mC11-AP
170









As can be seen from Table 9, all the galectin-9 variants exhibited degranulation inhibitory activities against the RBL-2H3 cells, similarly to the stabilized galectin-9 (G9 Null). In particular, mC8, mC9, mC10, mC11, mC12, mC13, mC10-HPPY, mC10-HPAP, and mC11-AP exhibited significantly high degranulation inhibitory activities, as compared with the stabilized galectin-9 (G9Null). From these results, it was found that the galectin-9 variants sufficiently conserve the physiological activities of the stabilized galectin-9.
As specifically described above, it was confirmed that the galectin-9 variant according to the present invention maintains high solubility even after long-term storage. It was also confirmed that the galectin-9 variant according to the present invention conserves the physiological activities of the wild-type galectin 9. More specifically, as described in the item (1) above, all the galectin-9 variants exhibited substantially the same protein concentrations even after the storage for about a year, and maintained high solubility after long-term storage. Also, as described in the item (2) above, all the galectin-9 variants maintained degranulation inhibitory activity against RBL-2H3 cells, which is one of the physiological activities of the wild-type galectin 9. In particular, the degranulation inhibitory activities of mC8, mC9, mC10, mC11, mC12, mC13, mC10-HPPY, mC10-HPAP and mC11-AP were 1.5 to 2.5 times higher than that of the stabilized galectin-9. From these results, it can be said that the galectin-9 variant is stable and has excellent properties as a raw material of pharmaceuticals.
While the present invention has been described above with reference to illustrative embodiments and examples, the present invention is by no means limited thereto. Various changes and modifications that may become apparent to those skilled in the art may be made in the configuration and specifics of the present invention without departing from the scope of the present invention.
This application claims priority from Japanese Patent Application No. 2012-254349 filed on Nov. 20, 2012. The entire subject matter of the Japanese Patent Application is incorporated herein by reference.
INDUSTRIAL APPLICABILITY
As described above, the galectin-9 variant according to the present invention has the same bioactivity as wild-type galectin 9, is superior in protease stability and solubility, and is also superior in yield at the time of producing as a recombinant protein. As described above, the galectin-9 variant according to the present invention not only has bioactivity of wild-type galectin, but also is superior in stability, handleability, and yield in production. Thus, it can be said that the galectin-9 variant according to the present invention is really useful as a pharmaceutical material.
SEQUENCE LISTING
TF13029WO_ST25.txt

## Claims

1. A protein or a salt thereof, consisting of:
NCRD; and
CCRD, a C terminus of the NCRD and an N terminus of the CCRD being directly bound to each other, wherein:
the NCRD is
(N1) a peptide consisting of an amino acid sequence represented by SEQ ID NO: 1, or
(N2) a peptide consisting of an amino acid sequence obtained by deletion, substitution and/or insertion of between 1 and 15 amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 and has a carbohydrate binding ability, and
the CCRD is
a peptide consisting of an N-terminal region and a C-terminal region and having a carbohydrate binding ability, wherein:
the N-terminal region is (C-N1) a peptide consisting of an amino acid sequence obtained by deletion of between 8 and 14 amino acid residues in an amino acid sequence of SEQ ID NO: 3, and
the C-terminal region is
(C-C1) a peptide consisting of an amino acid sequence represented by SEQ ID NO: 5, or
(C-C2) a peptide consisting of an amino acid sequence obtained by deletion, substitution and/or insertion of between 1 and 13 amino acid residues in the amino acid sequence of SEQ ID NO: 5.

2. The protein or a salt thereof according to claim 1, wherein
the deletion of the amino acids in the N-terminal region of the CCRD is a deletion of consecutive amino acids or inconsecutive amino acids.

3. The protein or a salt thereof according to claim 1, wherein
the deletion of the amino acids in the N-terminal region of the CCRD is a deletion of consecutive amino acids.

4. The protein or a salt thereof according to claim 3, wherein
the deletion of the amino acids in the N-terminal region of the CCRD is a deletion of consecutive amino acids from the N terminus.

5. The protein or a salt thereof according claim 1, wherein
at least one amino acid residue selected from the group consisting of 10th, 11th, 12th, 13th, 15th, and 17th amino acids in the amino acid sequence of SEQ ID NO: 3 of the peptide (C-N1) is proline.

6. The protein or a salt thereof according to claim 1, wherein
10th and 11th amino acid residues in the amino acid sequence of SEQ ID NO: 3 of the peptide (C-N1) are Pro-Pro, Pro-His, or His-Pro.

7. The protein or a salt thereof according to claim 1, wherein
12th and 13th amino acid residues in the amino acid sequence of SEQ ID NO: 3 of the peptide (C-N1) are Pro-Pro, Pro-Ala, or Ala-Pro.

8. The protein or a salt thereof according to claim 1, wherein
the N-terminal region in the CCRD is a peptide composed of any one of amino acid sequences of SEQ ID NOs: 7 to 20.

9. The protein or a salt thereof according to claim 1, wherein
the CCRD is a peptide composed of any one of amino acid sequences of SEQ ID NOs: 21 to 34.

10. The protein or a salt thereof according to claim 1,
consisting of any one of amino acid sequences of SEQ ID NOs: 35 to 48.

11. A nucleic acid comprising a nucleotide sequence which encodes the protein according to claim 1.

12. An expression vector comprising the nucleic acid according to claim 11.

13. A transformant comprising a nucleic acid comprised of a base sequence which encodes the protein according to claim 1 or an expression vector comprising the nucleic acid comprised of a base sequence which encodes the protein according to claim 1.