WO2019176866A1

WO2019176866A1 - Bispecific polypeptide structurally based on uteroglobin

Info

Publication number: WO2019176866A1
Application number: PCT/JP2019/009745
Authority: WO
Inventors: 真由美新山; 宏樹秋葉; 康弘阿部; 永田　諭志; 知子伊勢; 知生子長尾; 春彦鎌田; 浩平津本; 賢司水口; アファナシェヴァ・アリーナ; 井上　豪; 庸太福田
Original assignee: 国立研究開発法人医薬基盤・健康・栄養研究所; 国立大学法人大阪大学
Priority date: 2018-03-12
Filing date: 2019-03-11
Publication date: 2019-09-19
Also published as: JPWO2019176866A1; JP7398677B2

Abstract

Provided is a low-molecular weight bispecific polypeptide. A heterodimer uteroglobin is prepared by modifying, to a low-molecular weight uteroglobin, an Fc region in a bispecific antibody constructed from a natural antibody, and causing a uteroglobin monomer to be mutated, and a bispecific polypeptide is constructed by using the same.

Description

Bispecific polypeptides based on uteroglobin

The present invention relates to a polypeptide having bispecificity, in particular, a bispecific polypeptide based on a heterodimeric uteroglobin and a method for producing the same.

A natural antibody molecule is usually a molecule with a molecular weight of about 150 kDa, which exhibits a single binding property that recognizes one type of antigen and has the C-terminal region Fc region of an immunoglobulin heavy chain. The Fc region alone is thought to be a heavy chain homodimer containing CH2 and CH3 regions.
On the other hand, bispecific antibodies with two different binding properties can be created by using recombinant techniques. Bispecific antibodies can simultaneously inhibit two receptors on the cell surface, bind simultaneously with two ligands, allow signal transduction by binding two different receptors, and allow cells to interact with each other. The usefulness of being able to act is shown (nonpatent literature 1).

For the production of a bispecific antibody, an amino acid residue present in the CH3 region of one heavy chain is replaced with a larger residue (knob), and an amino acid residue present in the CH3 region of the other heavy chain Is replaced with a smaller residue so that the protrusion is located within the gap, thereby promoting heterogeneous heavy chain formation and inhibiting homogeneous heavy chain formation. A knob-in-to-hole method is known (Patent Document 1). In addition, an amino acid residue present in the CH3 region of one heavy chain is replaced with an amino acid residue having a positive charge, and an amino acid residue present in the CH3 region of the other heavy chain is negatively charged. There is also reported a method in which the substituted amino acid residues are electrostatically interacted with each other, thereby promoting hetero heavy chain formation and inhibiting homo heavy chain formation (

Patent Documents

2, 3, 4 and 5). Advantages of these methods are that the production efficiency of the bispecific antibody is relatively high, that the amino acid in the CH3 region is modified, so that antigenicity is not easily impaired, and antibody-dependent cellular cytotoxicity of the Fc region. Examples thereof include the ability to produce antibodies with high in vivo stability while maintaining activities such as (ADCC) and complement-dependent cytotoxicity (CDC). On the other hand, an unexpected dimer may be formed, and there is a drawback that recombination occurs in the light chain and antigenicity may change. In addition, since the antibody has a large molecular weight of about 150 kDa, the retention in blood is maintained at a high level, but the effect may be reduced by lowering the tissue permeability. Furthermore, normal antibodies are difficult to develop into expression systems that can be produced at a relatively low cost such as Escherichia coli due to the characteristics of molecular weight and three-dimensional structure, and their production is almost limited to expensive mammalian expression systems. is the current situation.

Uteroglobin is a relatively low molecular weight (15.8 kDa) homodimeric polypeptide and does not interact with cells such as natural killer cells and macrophages that have a cytocidal effect. In addition, since it is a low molecular weight polypeptide, it is expected that it can be developed into various expression systems without depending only on the expression system in mammalian cells, and has a great advantage from the viewpoint of protein production. In addition, uteroglobin is a highly safe polypeptide that is expected to have an anti-inflammatory effect (Patent Document 6) and is used in clinical trials. Uteroglobin is used as an antibody base for expressing a bivalent antibody (Non-Patent Document 2). This is a bivalent antibody devised based on the formation of a homodimer that is characteristic of wild-type uteroglobin. For this reason, there is no bispecificity, and the binding region with the antigen is limited to those using scFv.

WO1996 / 027011 WO2006 / 106905 Publication Special table 2011-508604 Special Table 2014-509857 WO2015 / 046467 Special Table 2002-511856

Problems with antibody drugs including bispecific antibodies generally include high drug prices, limited efficacy, and limited target antigens. High drug prices are common to biopharmaceuticals, and the cause of this is that development time is often required to establish a manufacturing process compared to low molecular weight compounds. Particularly, antibodies use expensive animal cell expression systems due to their large molecular weight. It must be unavoidable. Bispecific antibodies have so far been reported with at least 50 various non-wild-type derivatives with a wide variety of structures and targets. On the other hand, the creation of functional and effective bispecific antibodies is still inevitable through trial and error.

Based on the above knowledge, bispecific molecules that have a low molecular weight compared to general antibodies are sought, and uteroglobin is used as the structural basis instead of the Fc region in bispecific antibodies created based on natural antibodies. Diligent studies were conducted on polypeptides having bispecificity.
The present inventors estimated the three-dimensional structure of human uteroglobin from the structural analysis of rabbit uteroglobin, and paid attention to the site where human uteroglobin associates when forming a homodimer from the estimated structure. As a result, it was found that heterodimers can be efficiently formed by adding amino acid residue mutations to the sites where they are associated.

Surprisingly, according to the present invention, it is possible to efficiently form a bispecific polypeptide simply by substituting at least one amino acid residue in each monomer of wild-type uteroglobin.

The present invention relates to bispecific polypeptides based on heterodimeric uteroglobin, methods for producing the same, and heterodimeric uteroglobin itself and methods for producing the same. Accordingly, the present invention includes the following aspects.
<Bispecific polypeptide defining heterodimeric uteroglobin>
[1]
A bispecific polypeptide based on a heterodimeric uteroglobin.
[2]
The heterodimeric uteroglobin comprises an A chain and a B chain, wherein the A chain and the B chain are different from each other and have one or several amino acid residue mutations in the wild type uteroglobin monomer, and The bispecific polypeptide according to [1], wherein a heterodimer is formed by association derived from a mutation.
[3]
The mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin. The bispecific polypeptide according to [1] or [2].
[4]
The paired substitution of amino acid residues is the 5th serine (S) and 68th leucine (L), 27th leucine (L) and 68th leucine, 28th in the amino acid sequence represented by SEQ ID NO: 1. Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) The bispecific polypeptide according to [3], which is a substitution.
[5]
The pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain, [ 4] The bispecific polypeptide of description.
[6]
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain. The bispecific polypeptide according to [4], wherein the 51st lysine (K) is substituted with glutamic acid (E) or aspartic acid (D).
[7]
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E The bispecific polypeptide according to [6], wherein

<Bispecific Polypeptide Having Additional Mutation in Heterodimeric Uteroglobin>
[8]
The bispecific polypeptide according to any one of [1] to [7], wherein the uteroglobin A chain and B chain are disulfide bonded.
[9]
Both the uteroglobin A chain and the B chain are at least one selected from the 44th leucine (L), 34th methionine (M) and 59th leucine (L) of the amino acid sequence represented by SEQ ID NO: 1. The bispecific polypeptide according to any one of [1] to [8], which has a mutation that substitutes for cysteine (C).
[10]
The bispecific polypeptide according to any one of [1] to [9], wherein the uteroglobin is human uteroglobin.
[10-2]
The amino acid residue pair substitution is that the 29th serine (S) in the amino acid sequence represented by SEQ ID NO: 1 in the A chain is substituted with lysine (K), and the sequence shown in SEQ ID NO: 1 in the B chain. The bispecificity according to [3], wherein the lysine (K) at position 62 is substituted with aspartic acid (D) and the serine (S) at position 29 is substituted with aspartic acid (D). Sex polypeptide.
[10-3]
The pair substitution of amino acid residues is a pair of substitutions in the A chain with the 28th phenylalanine (F) of the amino acid sequence represented by SEQ ID NO: 1 and the 66th serine (S) in the B chain [3 ] The bispecific polypeptide of description.

<Bispecific polypeptide that defines the binding region>
[11]
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region The bispecific polypeptide according to any one of [1] to [10], [10-2] and [10-3], which comprises a second polypeptide.
[12]
The bispecific poly of any one of [1] to [11], which binds to one or more target molecules selected from the group consisting of a receptor or a fragment thereof, a cancer antigen, an MHC antigen, and a differentiation antigen. peptide.
[13]
The bispecific polypeptide according to any one of [1] to [12], wherein the target molecule binds to a target molecule of a cancer antigen.
[14]
The bispecific polypeptide according to any one of [1] to [13], wherein either the first or second polypeptide binds to a cancer antigen.
[15]
In the first polypeptide and the second polypeptide, the connection between the A chain and the first binding region and the connection between the B chain and the second binding region are such that the specificity of the region is not destroyed. [11] The bispecific polypeptide according to [11].
[16]
The bispecific polypeptide according to [15], wherein the linkage between the A chain and the first binding region and the linkage between the B chain and the second binding region are via a linker.
[17]
The bispecific polypeptide according to [16], wherein the linker has the amino acid sequence represented by SEQ ID NO: 2.
[18]
The bispecific polypeptide according to any one of [1] to [17], wherein each of the first binding region and the second binding region has monovalent specificity.

<Multispecific polypeptide>
[19]
Any one of [1] to [18], wherein one or both of the first binding region and the second binding region links at least one additional binding region in a manner that does not destroy the specificity of the region. Bispecific polypeptide.
<Antibody-drug complex>
[20]
[1] A complex comprising the bispecific polypeptide according to any one of [19] and a drug.
<Pharmaceutical composition>
[21]
[1] A pharmaceutical composition comprising the bispecific polypeptide according to any one of [19] to [19].
[22]
[20] A pharmaceutical composition comprising the complex according to [20].

<Method for producing bispecific polypeptide>
[23]
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing the bispecific polypeptide according to any one of [1] to [14], comprising a second polypeptide,
a) providing a first vector comprising a nucleic acid encoding the first polypeptide;
b) providing a second vector comprising a nucleic acid encoding a second polypeptide;
c) co-transfecting the cells with the first and second vectors, culturing the resulting transfectants, and d) recovering the heterodimer comprising the first and second polypeptides,
A method for producing the bispecific polypeptide.
[24]
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing the bispecific polypeptide according to any one of [1] to [14], comprising a second polypeptide,
a) providing a first cell expressing a first polypeptide;
b) providing a second cell expressing the second polypeptide;
c) co-culturing the first and second cells; and d) recovering the heterodimer comprising the first and second polypeptides.
A method for producing the bispecific polypeptide.

<Nucleic acids, vectors, etc.>
[25]
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region The bispecific polypeptide according to any one of [1] to [14], wherein the nucleic acid encodes the first polypeptide or the second polypeptide.
[26]
[25] A vector comprising the nucleic acid according to [25].
[27]
[26] A cell comprising the vector according to [26].
[28]
The cell according to [26], including both the vector according to [25], which includes a nucleic acid encoding the first polypeptide and the second polypeptide.
[29]
The cell according to [27] or [28], wherein the cell is Escherichia coli.
[30]
A co-culture containing a cell comprising a vector comprising a nucleic acid encoding a first polypeptide and a cell comprising a vector comprising a nucleic acid encoding a second polypeptide.
[31]
The co-culture according to [30], wherein the cell is Escherichia coli.

<Heterodimeric uteroglobin>
[32]
A and B chains, which are different from each other, have one or several amino acid residue mutations in the wild-type uteroglobin monomer, and are heterodimeric due to associations derived from that mutation Heterodimeric uteroglobin that forms the body.
[33]
The mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin. The heterodimeric uteroglobin according to [32].
[34]
Affinity and repulsive electrostatic interactions from pair substitution result in association, which promotes the formation of heterodimeric uteroglobin and inhibits the formation of homodimeric uteroglobin [33] The described heterodimeric uteroglobin.
[35]
The paired substitution of amino acid residues is the 5th serine (S) and 68th leucine (L), 27th leucine (L) and 68th leucine, 28th in the amino acid sequence represented by SEQ ID NO: 1. Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) The heterodimeric uteroglobin according to [34], which is a substitution.
[36]
The pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain, [ 35] The heterodimeric uteroglobin according to [35].
[37]
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain. The heterodimer uteroglobin according to [36], wherein the 51st lysine (K) is substituted with glutamic acid (E) or aspartic acid (D).
[38]
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E ) Is a heterodimeric uteroglobin according to [35].

<Heterodimeric Uteroglobin with Additional Mutations>
[39]
The heterodimeric uteroglobin according to any one of [32] to [38], wherein the uteroglobin A chain and the B chain are disulfide bonded.
[40]
Both the uteroglobin A chain and the B chain are at least one selected from the 44th leucine (L), 34th methionine (M) and 59th leucine (L) of the amino acid sequence represented by SEQ ID NO: 1. The heterodimeric uteroglobin according to any of [32] to 39], which has a mutation substituting for cysteine (C).
[41] The heterodimeric uteroglobin according to any one of [32 to 40], which is human uteroglobin.

The advantages of the bispecific antibody include that it can recognize two targets at the same time, can obtain a synergistic effect similar to co-administration, and can reduce the cost of developing two types of antibodies. In addition to the advantages of these bispecific antibodies, since the present invention is based on uteroglobin, it is a low molecular weight compared to general antibodies, can be expected to have high tissue permeability, and has no Fc region. It is possible to suppress unexpected effects that occur due to the presence of this, and safety is high. And since the polypeptide of this invention can be synthesize | combined using various expression systems, such as colon_bacillus | E._coli, it can implement | achieve low cost and is anticipated that the problem of the high drug price common to biopharmaceuticals can be avoided.

1 illustrates one embodiment of a heterodimeric uteroglobin. FIG. 1-1 is an embodiment in which the formation of heterodimeric uteroglobin is promoted. Heterodimeric uteroglobin has an A chain and a B chain, and is a mutation from the 33rd aspartic acid (D) to lysine (K) amino acid sequence represented by SEQ ID NO: 1 with respect to wild-type uteroglobin. (D33K mutation), and the B chain has a mutation from the 51st lysine (K) to glutamic acid (E) (K51E mutation). Here, due to the pair substitution, the D33K site of the A chain and the K51E site of the B chain, and the 51st K site of the A chain and the 33rd D site of the B chain interact electrostatically, Fig. 2 shows the formation of dimeric uteroglobin promoted. FIG. 1-2 shows that the pair substitution results in electrostatic interaction between the D33K site of the A chain and the K51 site of the A chain, and the K51E site of the B chain and the D33 site of the B chain. It shows how the formation of uteroglobin is inhibited. It is a schematic diagram which shows the structure and electrostatic interaction site | part of wild type uteroglobin. Both the light black boxed X chain and the white boxed Y chain interact electrostatically in one 33rd aspartic acid (D) and the other 51st lysine (K) to form a homodimer. The state of forming is shown. It is a structural schematic diagram of wild-type human uteroglobin deduced from the structural analysis of rabbit uteroglobin. A- and B- represent A chain and B chain, respectively. For example, A-S5 indicates S (serine) at the 5-position of the A chain. Dotted lines indicate possible pairs. The criteria for pairing are the results of exhaustive searches for pairs that are visually adjacent to the exposed amino acids. The distance between β carbons is calculated in Pymol (the number written as 5.4 corresponds to the unit: Å), which falls within the range of 5 to 10 mm, and is apparent from the angle between the α carbon-β carbon bonds. Excludes those that cannot be paired. FIG. 3-1 shows the model structure as viewed from the N-terminal side of the A chain. FIG. 3-2 is a view from the opposite side. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing are shown. [1] is wild-type uteroglobin expression product, [2] is mutant uteroglobin (K51E) expression product, [3] is ROBO4 scFv expression product, [4] is ROBO4 scFv-mutant uteroglobin (D33K) (ROBO4 scFv-D33K ) Expression product, [5] is an expression product obtained by double transfection with plasmids encoding mutant uteroglobin (K51E) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-D33K), respectively, and [6] is mutation Electrophoresis of expression products obtained by co-culturing cells transfected with a plasmid encoding type uteroglobin (K51E) and cells transfected with a plasmid encoding ROBO4 scFv-mutant uteroglobin (D33K) Is the result of In [7], ROBO4 scFv-mutant uteroglobin (D33K) [4] and mutant uteroglobin (K51E) expression product [2] were mixed at a molar ratio of 1: 1, and 1 on ice. It is the result of electrophoresis after leaving still for time. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing are shown. [1] is a wild-type uteroglobin expression product, [8] is a mutant uteroglobin (S66F) expression product, [3] is a ROBO4 scFv expression product, [9] is a ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S ) Expression product, [10] is the double transfection with plasmids encoding mutant uteroglobin (S66F) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-F28S), respectively, and [11] is the mutation Of the expression products obtained by co-culturing cells transfected with a plasmid encoding type uteroglobin (S66F) and cells transfected with a plasmid encoding ROBO4 scFv-mutant uteroglobin (F28S) Is the result of [12] is a mixture of ROBO4 scFv-mutant uteroglobin (F28S) [9] expression product and mutant uteroglobin (S66F) expression product [8] in a molar ratio of 1: 1, and 1 on ice. It is the result of electrophoresis after leaving still for time. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing are shown. [1] is a wild-type uteroglobin expression product, [13] is a mutant uteroglobin (S29K) expression product, [3] is a ROBO4 scFv expression product, [14] is a ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv -S29D, K62D) expression product, [15] is an expression product obtained by double transfection with plasmids encoding mutant uteroglobin (S29K) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-S29D, K62D), And [16] are obtained by co-culturing cells transfected with a plasmid encoding mutant uteroglobin (S29K) and cells transfected with a plasmid encoding ROBO4 scFv-mutant uteroglobin (S29D, K62D) It is the result of each electrophoresis of the expressed product. In [17], ROBO4 scFv-mutated uteroglobin (S29D, K62D) [14] and mutant uteroglobin (S29K) expression product [13] were mixed at a molar ratio of 1: 1 and on ice. It is the result of electrophoresis after leaving still for 1 hour.

<Heterodimeric uteroglobin and bispecific polypeptide based on it>
Uteroglobin is a homodimeric polypeptide in which two structurally identical monomers are electrostatically associated (Nature structural biology 1994, vol. 1, No. 8, 538). The amino acid sequence of human wild-type uteroglobin monomer is shown in SEQ ID NO: 1. Wild-type uteroglobin is a protein with no relatively low molecular weight (15.8 kDa) sugar chain, and its properties combine high solubility and pH stability, and is resistant to proteolytic enzymes (JBC 2009, vol.284, No.39, 26646.).

Uteroglobin is also called CC10, and a paper on crystallization of human uteroglobin has been reported (1.9 Å diffractivity, Nat Struct Biol vol. 1 (8) 1994 538-545). Here, uteroglobin is a kind of protein secreted into the bronchus, and is described as having phospholipase A2 (PLA2) inhibition and PCB binding activity. Crystallization was performed by obtaining and purifying the wild-type protein from individuals under the conditions: 100 μug / mL protein, 70% ammonium sulfate, 20 mM Tris (pH 7.5), 16-18% glycerol. Structural analysis with a resolution of 1.9 angstrom 、, human uteroglobin has a structure that can bind phospholipids such as phosphatidylcholine and phosphatidylinositol, and has a similar structure with 62% amino acid homology compared to rabbit uteroglobin Are listed.

Human uteroglobin is not registered in the protein data bank (PDB), but structural similarity to rabbit uteroglobin that is registered in the PDB is presumed. Therefore, when human uteroglobin forms a homodimer, a region where amino acids interact is presumed to some extent. Thus, as part of the structural analysis of human uteroglobin, the present inventors estimated the identification of amino acids to be mutated to form a heterodimer from the structural analysis of rabbit uteroglobin. The estimation of the structural analysis is described in detail below.

As a result, in the amino acid sequence of the human uteroglobin monomer represented by SEQ ID NO: 1, the 33rd aspartic acid (D) in one monomer and the 51st lysine (K) in the other monomer are associated with each other. The possibility was speculated. The association is presumed to be an affinity electrostatic interaction between amino acid residues having opposite charges. A schematic three-dimensional structure of wild-type uteroglobin is shown in FIG.
In addition, the fact that 33D of one monomer and 51K of the other monomer in wild-type human uteroglobin are associated with each other means that a heterodimer is formed by pair-substituting them with a certain amino acid residue. Was proved experimentally in the examples of the present specification, so it became clear.

Furthermore, based on the structural analysis of human uteroglobin, the amino acid residues whose side chains extend outside as uteroglobin dimers are identified, and other amino acids that may form heterodimer uteroglobin. Residue guessed.
Based on these facts and assumptions, the present invention provides various inventions based on heterodimeric uteroglobin.

Here, as part of the structural analysis of human uteroglobin, the identification of amino acids that should be mutated to form a heterodimer will be described in detail from the structural analysis of rabbit uteroglobin.
In Swiss-model (swissmodel.expasy.org), the human uteroglobin sequence is input and a model is constructed from the homology of amino acid sequences. The one with the best score is selected and the obtained PDB file is displayed on Pymol (pymol.org).
• The interatomic distance is calculated using the measurement tab. The displayed models are shown in FIGS. 3-1 and 3-2.

Table 1 shows pairs of amino acid residues that can be associated with each other to form a heterodimeric uteroglobin estimated as described above.
In Table 1, the first line means a pair of substitutions in the A chain with the fifth cysteine (S) of the amino acid sequence represented by SEQ ID NO: 1 and the 68th leucine (L) in the B chain. The same applies to the following lines. The A chain and the B chain can be converted to each other.

In other tables, the numbers are numbers from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1.

A preferred example of a pair of substitutions is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain.

In the present invention, the association may include an affinity electrostatic interaction, a repulsive interaction between amino acid residues having the same type of charge, and an interaction between hydrophobic amino acids.
In one aspect, the present invention promotes heterodimer formation by mutating amino acid residues that have an affinity electrostatic interaction in each monomer of wild-type uteroglobin, and promotes homodimerization. Inhibits the formation of mers. In the present invention, “pair substitution” is established by mutating one of the amino acid residues in each monomer having an affinity interaction with each other in wild-type uteroglobin to an amino acid residue having a repulsive interaction. That is, a heterodimeric uteroglobin with one paired substitution contains one amino acid mutation in the A chain and another amino acid mutation in the B chain. This embodiment is called “wild substitution type”.

Specific examples of pair substitution in the wild substitution type are shown in Table 2. The A chain and the B chain can be converted to each other.

Table 2 shows that aspartic substitution in this embodiment, the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 is changed to lysine (K), arginine (R) or histidine (H) in the A chain. A total of six examples are shown in which substitution is performed and the 51st lysine (K) in the B chain is substituted with glutamic acid (E) or aspartic acid (D). For example, in a heterodimeric uteroglobin having a D33K mutation in the A chain and a K51E mutation in the B chain relative to wild-type uteroglobin, the pair substitution results in D33K on the A chain and K51E on the B chain and The 51st K of the chain and the 33rd D of the B chain interact electrostatically to promote the formation of heterodimeric uteroglobin. This embodiment is shown in FIG.
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E ) Are preferred.

As another aspect of the present invention, a heterodimer is formed by newly generating a pair substitution at a portion where the amino acid residues of wild-type uteroglobin do not have a clear affinity interaction with each other. In this embodiment, it is preferred to generate an affinity electrostatic interaction with one pair substitution and add a homodimeric repulsion with another pair substitution, in which case the heterodimeric uteroglobin is Has two pair substitutions. This mode is called “charge pair formation type”.

Specific examples of pair substitution in the charge pair formation type are shown in Table 3. The A chain and the B chain can be converted to each other.

Table 3 shows the following example as a pair substitution in this embodiment.
An example with substitution site 5-68 will be described. From the first line, S68D is introduced into the A chain and L68R is introduced into the B chain (first pair substitution). From the second line, L68D is introduced into the A chain and S5K is introduced into the B chain (second pair substitution). ). In this case, the A chain becomes a mutant having two mutations of S5D and L68D, and the B chain becomes a mutant having two mutations of S5K and L68R. This establishes a pair of pair substitutions.
More specifically, as an example at substitution site 5-68, pair substitution of A and B chains is
First line: Six S5D-L68R, S5D-L68K, S5D-L68H, S5E-L68R, S5E-L68K, S5E-L68H,
2nd line: L68D-S5K, L68D-S5R, L68D-S5H, L68E-S5K, L68E-S5R, L68E-S5H,
There are 6 + 6 = 12 ways. In addition, the heterodimer which introduce | transduced each one pair substitution is also within the scope of the present invention.
There are 6 × 6 = 36 combinations of pair replacement in which one is selected from each of the first and second rows. Therefore, the total number of heterodimeric uteroglobins at the substitution site 5-68 is 12 + 36 = 48.
The same is true for the other pair of substitutions 27-68, 44-47, and 29-62.

The present invention includes, as yet another embodiment, a mode in which heterodimeric uteroglobin is formed as a pair substitution through an association in which hydrophobic amino acids interact with each other instead of electrostatic interaction. This embodiment is called “hydrophobic pocket inversion type”.

Specific examples of pair substitution in the hydrophobic pocket inverted type are shown in Table 4. The A chain and the B chain can be converted to each other.

Table 4 shows a total of nine examples of pair substitution in this aspect. Preferably, it has a F28S substitution in the A chain and S66F in the B chain.

Among amino acid residues, charged residues are known. Generally, lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids). As the negatively charged amino acid (negatively charged amino acid), aspartic acid (D), glutamic acid (E) and the like are known. Therefore, the amino acid residue having the same kind of charge in the present invention preferably means an amino acid residue having positive charges or an amino acid residue having negative charges.

Here, in the present invention, “mutation” of an amino acid residue specifically refers to substitution of the original amino acid residue with another amino acid residue, deletion of the original amino acid residue, This refers to addition of an amino acid residue, etc., but preferably means to substitute the original amino acid residue with another amino acid residue.

In the present invention, “homodimer” refers to a state in which polypeptides having the same amino acid sequence are associated with each other. “Heterodimer” refers to a state in which polypeptides having at least one amino acid residue in an amino acid sequence are associated with each other.

In the present invention, “heterodimeric uteroglobin” means that wild-type uteroglobin is a homodimer, whereas each monomer is heterodimeric due to mutation of one or several amino acid residues in each monomer. It means a heterodimer of mutant uteroglobin that constitutes the body. In the present invention, “heterodimeric uteroglobin” is a heterodimeric uteroglobin comprising an A chain and a B chain, wherein the A chain and the B chain are different from each other, one or several in the wild type uteroglobin monomer, respectively. Of amino acid residues, and both are associated by that mutation. Here, the A chain and the B chain do not represent specific monomers in the heterodimeric uteroglobin, and are interchangeable. That is, a reference to each mutation in the A chain and the B chain should be understood as a reference to each mutation in the B chain and the A chain, respectively.

<Bispecific Polypeptide Having Additional Mutation in Heterodimeric Uteroglobin>
The heterodimeric uteroglobin as described above can further have another mutation. For example, in the heterodimeric uteroglobin of the present invention, the uteroglobin A chain and the B chain can form a disulfide bond. Disulfide bond formation refers to the process of forming a covalent bond between two cysteines present in one or two polypeptides, and this bond is schematized as “—S—S—”. Specifically, the heterodimer uteroglobin of the present invention includes the leucine (L) at the 44th amino acid sequence, the methionine (M) at the 34th amino acid sequence represented by SEQ ID NO: 1, and both the uteroglobin A chain and the B chain. At least one selected from the 59th leucine (L) can have a mutation replacing cysteine (C). Thereby, the stability of the heterodimeric uteroglobin and bispecific polypeptide of the present invention is improved, and the amount of protein produced is significantly increased.

<Bispecific polypeptide that defines the binding region>
In the present invention, a “bispecific polypeptide” is a molecule comprising at least a first polypeptide and a second polypeptide. That is, the “bispecific polypeptide” of the present invention comprises a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain in the first. A second polypeptide linked to a second binding region different from the binding region. Preferably, in the first polypeptide and the second polypeptide, the connection between the A chain and the first binding region and the connection between the B chain and the second binding region do not destroy the specificity of the region. It is.

The bispecific polypeptide of the present invention has binding specificities or binding sites for two different ligands. The ligand is not particularly limited, and any ligand may be used. Examples of the ligand include, but are not limited to, a receptor or a fragment thereof, a cancer antigen, an MHC antigen, a differentiation antigen, and the like.

Examples of receptors include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein coupled receptor family, GPI Examples of the receptor family include an anchor type receptor family, a tyrosine phosphatase type receptor family, an adhesion factor family, and a hormone receptor family. Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptor, human or mouse granulocyte colony stimulating factor (G-CSF) receptor, human or mouse thrombopoietin (TPO). ) Receptor, human or mouse insulin receptor, human or mouse Flt-3 ligand receptor, human or mouse platelet derived growth factor (PDGF) receptor, human or mouse interferon (IFN) -α, β receptor, human or Mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitory factor Examples include (LIF) receptors, human or mouse ciliary neurotrophic factor (CNTF) receptors, and the like.

Cancer antigens are antigens that are expressed as cells become malignant or antigens that are highly expressed in peripheral cells other than cancer cells due to the characteristics of cancer. In addition, abnormal sugar chains appearing on the cell surface and protein molecules when cells become cancerous also become cancer antigens, and are particularly called cancer sugar chain antigens. Examples of cancer antigens include CA19-9, CA15-3, and roundabout homolog 4 (ROBO4).
MHC antigens are roughly classified into MHC class I antigens and MHC class II antigens, and MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, -H, MHC class II antigens include HLA-DR, -DQ, and -DP.
Differentiation antigens include differentiation antigen groups such as CD1, CD2, CD3, CD4, CD5, and CD6.

As described above, the bispecific polypeptides of the present invention have binding specificities or binding sites for two different ligands. That is, each of the first binding region and the second binding region has a monovalent specificity. One or both of the first binding region and the second binding region can link at least one additional binding region in a manner that does not destroy the specificity of the region. In that case, the polypeptide of the present invention may be a “multispecific polypeptide”. Here, the bispecific polypeptide of the present invention includes a “heterodimeric uteroglobin” formed by the A and B chains of the heterodimeric uteroglobin in the first polypeptide and the second polypeptide. .

In the present invention, a “first polypeptide” and a “second polypeptide” each bind to a “binding region”, eg, an antibody variable region, a receptor binding region, a ligand binding region or an enzyme active region, or another molecule. An affinity polypeptide with activity can be included. In a preferred embodiment, the binding region comprises immunoglobulin heavy and light chains. Usually, the first polypeptide and the second polypeptide each comprise at least one region derived from the primary sequence of uteroglobin. This region is the portion where the first and second polypeptides associate.

The binding region is derived from the variable region of the antibody or has sequence identity with the variable region of the antibody. Examples of antibodies include full length antibodies, antibody fragments, single chain molecules, bispecific or bifunctional molecules, scFv, diabodies, single domain antibodies (VHH), chimeric antibodies, and immunoadhesive factors Is included. “Antibody fragments” include fragments of Fv, Fv ′, Fab, Fab ′ and F (ab ′) 2. “Antibody”, as it relates to the present invention, means a polypeptide that contains one or more regions that bind to an epitope of an antigen of interest.

“Antibody fragments” can be obtained by treating an antibody with an enzyme, for example, a protease such as papain, pepsin (MorimotoMet al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al ., See Science (1985) 229: 81). It can also be produced by gene recombination based on the amino acid sequence of the antibody fragment.

A low molecular weight antibody having a structure obtained by modifying an “antibody fragment” can be constructed using an antibody fragment obtained by enzyme treatment or gene recombination. Alternatively, a gene encoding the entire low molecular weight antibody can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co et al., J. Immunol. (1994) 152). : 2968-76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63 Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).

“ScFv” is a single-chain polypeptide in which two variable regions are bound via a linker or the like, if necessary. The two variable regions included in scFv are usually one heavy chain variable region (VH) and one light chain variable region (VL), but may be two VHs or two VLs. In general, scFv polypeptides include a linker between the VH and VL regions, thereby forming the VH and VL paired portions necessary for antigen binding. In general, in order to form a pair part between VH and VL in the same molecule, generally, the linker connecting VH and VL is a peptide linker having a length of 10 amino acids or more. However, the scFv linker in the present invention is not limited to such a peptide linker as long as it does not interfere with the formation of scFv. As a review of scFv, reference can be made to Pluckthun, The Pharmacology of Monoclonal Antibody, Vol.113 (Rosenburg and Moore ed., Springer Verlag, NY, pp.269-315 (1994)).

In addition, “diabody” refers to a divalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097). No., WO93 / 11161 etc.). A diabody is a dimer composed of two polypeptide chains, each of which is in a position where the light chain variable region (VL) and the heavy chain variable region (VH) cannot bind to each other in the same chain. They are linked by a short linker, for example, about 5 residues. Since VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to a short linker between them, a diabody forms two antigen-binding sites. Will have. At this time, if VL and VH for two different epitopes (a and b) are combined with VLa-VHb and VLb-VHa and linked together by a linker of about 5 residues, they are secreted as bispecific Db. The In this case, the two different epitopes may be two different epitopes on the same antigen, or may be two epitopes on each of the two different antigens. A “diabody” contains two molecules of scFv, so it contains four variable regions, resulting in two antigen-binding sites. Unlike the case of scFv that does not form a dimer, when aiming at the formation of a diabody, the linker that connects between VH and VL in each scFv molecule is usually about 5 amino acids when used as a peptide linker. To do. However, the scFv linker that forms a diabody is not limited to such peptide linkers as long as it does not interfere with scFv expression and does not interfere with diabody formation.

A “single domain antibody” is a molecule generally having a high affinity and specificity for a specific antigen, and is an antibody variable region consisting only of a heavy chain found in camelids or imitating this structure. This refers to an antibody variable region consisting of a single domain obtained by modifying human or other VH. These include those that have undergone stabilization and affinity improvement measures through humanization and amino acid optimization.

“Affinity polypeptide” refers to a peptide or partial structure consisting of several amino acids contained in the adhesion site of protein-protein interaction, or a peptide showing binding affinity for a specific molecule screened from a random peptide library. It refers to a polypeptide that is different from a structure, or a partial structure thereof. In a preferred embodiment, an adhesion molecule active center such as Arg-Gly-Asp-Ser, a cyclic polypeptide and the like are included. In addition, peptides having specific binding affinity found by methods such as phage surface display, yeast display, and ribosomal display, such as Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD-4C) is also preferred (Arap, W .; Pasqualini, R .; Ruoslahti, E. Science 1998, 279, 377.).

As described above, the present invention can link at least one additional binding region in one or both of the first binding region and the second binding region in a manner that does not destroy the specificity of the region, The polypeptide can be a “multispecific polypeptide”. Examples of multispecific polypeptides include those in which one or both of the first binding region and the second binding region are linked to an scFv that binds to one or more ligands. Moreover, what connected above affinity polypeptide with the linker etc. can be utilized as multispecific polypeptide.

In the present invention, “polypeptide” generally refers to peptides and proteins having a length of about 10 amino acids or more. Moreover, although it is normally a polypeptide derived from a living organism | raw_food, it will not specifically limit, For example, the polypeptide which consists of a sequence designed artificially may be sufficient. Moreover, any of natural polypeptide, synthetic polypeptide, recombinant polypeptide, etc. may be sufficient. Furthermore, fragments of the above polypeptides are also included in the polypeptides of the present invention.

Linkage between uteroglobin A chain and first binding region and second binding different from uteroglobin B chain and first binding region in the first polypeptide and the second polypeptide of the bispecific polypeptide of the present invention Linkage with a region can be performed through a linker as necessary. In general, the linker that links the A chain and B chain to the binding region is a peptide linker having a length of 10 amino acids or more. However, the linker in the present invention is not particularly limited as long as the specificity and binding characteristics of the binding region are not hindered. As a preferable linker, for example, a GS4 (Gly + Ser x4, GSSSSGSSSS) linker represented by SEQ ID NO: 2 can be mentioned.

In the present invention, “association” of the A chain and the B chain means a state in which the A chain and the B chain interact in a specific region of each other. The specific regions of each other are usually one or a plurality of amino acid residues that are subjected to association, and are preferably amino acid residues that approach and participate in the interaction during the association. Specifically, the interaction includes a case where amino acid residues approaching at the time of association form a hydrogen bond, an electrostatic interaction, a salt bridge, and the like.

<Antibody-drug complex>
As another embodiment, the present invention includes a complex formed by covalently binding the bispecific polypeptide of the present invention and a drug, an ADC (antibody-drug conjugate) complex.
In general, systemic administration of drugs can cause unacceptable levels of toxicity not only to tumor cells to be eliminated but also to normal cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986): 603 -05). Thus, antibody-drug conjugates are used to locally deliver drugs to pursue maximum efficacy while minimizing toxicity and to kill or inhibit tumor cells in cancer therapy (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; U.S. Pat. No. 4,975,278). Similarly, the bispecific polypeptide of the present invention has a binding property to the corresponding antigen or ligand, while many drug molecules do not selectively kill cancer cells, and thus are difficult to use for cancer treatment. There is. Therefore, a highly selective and specific drug complex can be obtained by binding of the bispecific polypeptide of the present invention to a highly toxic drug such as a toxin.

Drugs used in the complex of the present invention include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., “Cancer” Immunol. “Immunother.” 21: 183-87 (1986)). In addition, bacterial toxins such as diphtheria toxin and phytotoxins such as ricin are included.

<Nucleic acids, vectors, etc.>
In another aspect, the present invention provides a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region. The bispecific polypeptide according to any one of claims 10 to 13, which comprises a second polypeptide linked to a binding region of the nucleic acid encoding the first polypeptide or the second polypeptide. .

Numerous nucleic acid sequences encoding immunoglobulin regions including VH, VL, hinge, CH1, CH2, CH3 and CH4 regions are known in the art (eg, Kabat et al. In SEQUENCES OFIMMUNOLOGICAL INTEREST, Public Health Service NIH , Bethesda, MD, 1991). Using the guidance herein, one of ordinary skill in the art can combine such nucleic acid sequences and / or other nucleic acid sequences known in the art to build on the heterodimeric uteroglobin described herein. Nucleic acid sequences encoding bispecific polypeptides can be made.

In addition, the nucleic acid sequence encoding the bispecific polypeptide of the present invention can be determined by one skilled in the art based on the amino acid sequence provided herein and knowledge in the art. In addition to the traditional method of producing cloned DNA segments encoding specific amino acid sequences, it is now readily chemically synthesized and DNA of any desired sequence is produced routinely upon order, The production process is streamlined (GenScript, Eurofin Genomics, etc.).

<Method for producing bispecific polypeptide>
In preferred embodiments, the present invention provides a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region. A method for producing a bispecific polypeptide of the present invention comprising a second polypeptide linked to a binding region of In another aspect of the production method of the present invention, the present invention relates to the pair of amino acid residues of one mutation in the A chain and another mutation in the B chain so that the association between the polypeptides is controlled. Methods of producing bispecific polypeptides based on the heterodimeric uteroglobin of the present invention comprising pair substitutions are provided. Examples of such pair substitutions are shown in Tables 1 to 4.

In the above method of the present invention, the A chain and B chain containing pair substitutions are made by modifying a nucleic acid encoding wild-type uteroglobin.
The present invention relates to a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second binding region in which the heterodimeric uteroglobin B chain is different from the first binding region. In the bispecific polypeptide of the invention, comprising a linked second polypeptide, a nucleic acid encoding the first polypeptide or the second polypeptide is provided.
“Modifying a nucleic acid” refers to modifying a nucleic acid so as to correspond to an amino acid residue introduced by “modification” in the present invention. More specifically, it means that a nucleic acid encoding an original (before modification) amino acid residue is modified to a nucleic acid encoding an amino acid residue introduced by the modification. Usually, it means that genetic manipulation or mutation treatment is carried out such that at least one base is inserted, deleted or substituted into the original nucleic acid so as to be a codon encoding the target amino acid residue. That is, the codon encoding the original amino acid residue is replaced by the codon encoding the amino acid residue introduced by modification. Such nucleic acid modification can be appropriately performed by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis.

In addition, the nucleic acid in the present invention is usually carried (inserted) into an appropriate vector and introduced into a host cell. The present invention provides a vector comprising the nucleic acid of the present invention and a cell comprising the vector of the present invention.
The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, if E. coli is used as the host, the cloning vector is preferably a pBluescript vector (Stratagene), but is commercially available. Various vectors can be used. An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E. coli, in cultured cells, or in an individual organism. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli PET vector (manufactured by Invitrogen), pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for organisms, etc. Is preferred. The insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons). Section 11.4-11.11).

There is no restriction | limiting in particular as said host cell, According to the objective, various host cells are used. Examples of cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2). , Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofectamine method (GIBCOBRL And a known method such as a microinjection method.
Furthermore, the present invention provides a cell comprising both a vector comprising a nucleic acid encoding each of the first polypeptide and the second polypeptide, and a cell comprising a vector comprising a nucleic acid encoding the first polypeptide, and a second A cell mixture or co-culture of cells comprising a vector comprising a nucleic acid encoding a polypeptide is provided. These cells and co-cultures are suitable for suitably preparing the bispecific polypeptide of the present invention.

In order to secrete the polypeptide expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be incorporated into the polypeptide of interest. These signals may be endogenous to the polypeptide of interest or may be heterologous signals.

In the above production method, the polypeptide is collected when the polypeptide of the present invention is secreted into the medium. When the polypeptide of the present invention is produced intracellularly, the cell is first lysed, and then the polypeptide is recovered.

To recover and purify the polypeptides of the invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxylapatite chromatography and lectin chromatography can be used.

In a preferred embodiment of the invention, a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region In the method for producing the bispecific polypeptide of the present invention comprising the second polypeptide linked to the binding region of the present invention, the purified polypeptide may not be a heterodimer simply by mixing together. It was found. See Example 3. Therefore, in the present invention, the first polypeptide and the second polypeptide are obtained by co-transfection of a vector encoding them into a cell (double transfection) or by including each of the vectors encoding them. Cells need to be co-cultured.

The present invention, as one aspect,
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing a bispecific polypeptide according to any of claims 1 to 13, comprising a second polypeptide comprising:
a) providing a first vector comprising a nucleic acid encoding the first polypeptide;
b) providing a second vector comprising a nucleic acid encoding a second polypeptide;
c) co-transfecting the cells with the first and second vectors, culturing the resulting transfectants, and d) recovering the heterodimer comprising the first and second polypeptides,
A method of producing the bispecific polypeptide is provided.

In addition, h, as another aspect, the present invention provides a method for producing the bispecific polypeptide of the present invention,
a) providing a first cell expressing a first polypeptide;
b) providing a second cell expressing the second polypeptide;
c) co-culturing the first and second cells; and d) recovering the heterodimer comprising the first and second polypeptides.
A method of producing the bispecific polypeptide is provided.

An example of producing wild-type human uteroglobin as a basis for producing the bispecific polypeptide of the present invention is as follows. A gene encoding a wild-type human uteroglobin monomer and His tag, FLAG tag, GST tag, etc. is introduced into Escherichia coli, yeast, Brevibacillus, insect cells, mammalian cells and the like. A culture solution or cell disruption solution containing uteroglobin is passed through a resin that captures a His tag, and a fraction containing uteroglobin is eluted with a solution containing imidazole. The eluate is subjected to solution exchange by dialysis to lower the salt concentration, and then purified by anion exchange chromatography. As the resin, an anion exchange resin is used, and uteroglobin is separated and purified by a salt concentration gradient. After collecting the eluted fraction, it is further purified by gel filtration chromatography to collect highly pure uteroglobin. The eluted fraction is collected and concentrated using an ultrafiltration membrane to obtain a uteroglobin solution. The wild-type uteroglobin obtained here is a dimer.
The same applies to the production of mutant uteroglobin.

<Pharmaceutical composition>
The present invention, in another aspect, relates to a composition comprising a bispecific polypeptide of the present invention and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutical composition usually means a drug for treatment or prevention of a disease, or examination / diagnosis.

The pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection in suspension. For example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to prepare a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of the active ingredient in these preparations is set so as to obtain an appropriate volume within the indicated range.

A sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.

Examples of aqueous solutions for injection include isotonic solutions containing, for example, physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride). A suitable solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.), nonionic surfactant (polysorbate 80 (TM), HCO-50 etc.) may be used in combination.

Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent. Moreover, you may mix | blend with a buffer (for example, phosphate buffer and sodium acetate buffer), a soothing agent (for example, procaine hydrochloride), a stabilizer (for example, benzyl alcohol and phenol), and an antioxidant. The prepared injection solution is usually filled in a suitable ampoule.

The pharmaceutical composition of the present invention is preferably administered by parenteral administration. For example, the composition can be an injection, nasal, pulmonary, or transdermal composition. For example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.

The administration method can be appropriately selected depending on the age and symptoms of the patient. The dosage of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg body weight at a time. Alternatively, for example, the dose may be 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values. The dose and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.

In the present invention, the polypeptide or complex of the present invention is particularly useful as an active ingredient of a therapeutic or prophylactic agent for cancer, blood vessel-related diseases, and inflammatory diseases. Cancers include, but are not limited to: lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma), colon cancer, rectal cancer, colon cancer, breast cancer, Liver cancer, stomach cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, Nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms tumor, acute myeloid leukemia (acute myelocytic leukemia, acute myeloblastic leukemia) , Including acute promyelocytic leukemia, acute myelomonocytic leukemia and acute monocytic leukemia), chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma (Burkitt lymphoma, Chronic lymphocytic leukemia, mycosis fungoides, Center cell lymphoma, follicular lymphoma, diffuse large cell lymphoma, marginal zone lymphoma, hairy cell leukemia plasmacytoma, peripheral T cell lymphoma and adult T cell leukemia / lymphoma), Langerhans cell histiocytosis, multiple Myeloma, myelodysplastic syndrome, brain tumor (including glioma, astrocytoma, glioblastoma, meningioma and ependymoma), neuroblastoma, retinoblastoma, osteosarcoma, Kaposi sarcoma, Ewing Sarcoma, hemangiosarcoma, vascular epithelioma. Blood vessel-related diseases include arteriosclerosis and vasculitis. Inflammatory diseases include acute or chronic inflammatory diseases, specifically including but not limited to: alcoholic hepatitis. Viral hepatitis. Nonalcoholic steatohepatitis. Interstitial pneumonia. Ulcerative colitis. Crohn's disease.

The present invention also provides a kit for use in the therapeutic or prophylactic method of the present invention, comprising at least the polypeptide produced by the production method of the present invention or the pharmaceutical composition of the present invention. In addition, the kit may be packaged with a pharmaceutically acceptable carrier, a medium, instructions describing the method of use, and the like. The present invention also relates to the use of the polypeptide of the present invention or the polypeptide produced by the production method of the present invention in the manufacture of a therapeutic or prophylactic agent for immunoinflammatory diseases. The present invention also relates to the polypeptide of the present invention or the polypeptide produced by the production method of the present invention for use in the therapeutic or prophylactic method of the present invention.

The correspondence between the three-letter code and the one-character code of amino acids used in this specification is as follows.
Alanine: Ala: A
Arginine: Arg: R
Asparagine: Asn: N
Aspartic acid: Asp: D
Cysteine: Cys: C
Glutamine: Gln: Q
Glutamate: Glu: E
Glycine: Gly: G
Histidine: His: H
Isoleucine: Ile: I
Leucine: Leu: L
Lysine: Lys: K
Methionine: Met: M
Phenylalanine: Phe: F
Proline: Pro: P
Serine: Ser: S
Threonine: Thr: T
Tryptophan: Trp: W
Tyrosine: Tyr: Y
Valine: Val: V

Hereinafter, the present invention will be described in detail by way of examples. However, it should be noted that these are merely examples, not limiting the scope of the present invention.

Example 1: Construction of expression plasmid EcoRI cleavage sequence, DNA sequence encoding mouse IgGκ signal peptide, DNA sequence encoding full-length wild type human uteroglobin monomer (NCBI), linker sequence GSSSSGSSSS, His tag and stop codon sequence, XhoI A gene containing a cleavage sequence was synthesized. The synthesized gene and the mammalian expression vector pcDNA6.0 mycHisB (Invitrogen) were cleaved with EcoRI (NEB) and XhoI (NEB). The two cleaved products were ligated using a DNA ligation kit mighty mix (TAKARA) to prepare a wild-type uteroglobin mammalian expression plasmid.

The mutant was prepared as follows. Primers containing a sequence in which the codon aag encoding lysine (K), the 51st amino acid of the uteroglobin monomer, was converted to the codon gaa of glutamic acid (E) were synthesized. A PCR reaction was performed using the synthesized primer and a wild-type uteroglobin mammalian expression plasmid as a template to prepare a mutant uteroglobin (K51E) mammalian expression plasmid.

In the same manner, a mutant uteroglobin (D33K) mammalian expression plasmid was prepared by converting aspartate gac, the 33rd amino acid, into lysine aag. Subsequently, ROBO4 scFv-mutant uteroglobin (D33K) for mammalian expression, in which the mouse IgGκ signal peptide of the mutant uteroglobin (D33K) mammalian expression plasmid is inserted with a single chain antibody scFv sequence against ROBO4 and linker sequence GSSSSGSSSS. A plasmid was prepared.

In this way, wild-type uteroglobin mammalian expression plasmid [1], mutant uteroglobin (K51E) mammalian expression plasmid [2], ROBO4 scFv-mutant uteroglobin (D33K) (ROBO4 scFv-D33K) mammalian expression plasmid [ 4] was prepared.

A mutant uteroglobin (S66F) mammalian expression plasmid was prepared by converting serine (S) agc, which is the 66th amino acid, into phenylalanine (F) ttc in the same manner. Subsequently, a single chain antibody scFv sequence and a linker sequence GSSSSGSSSS were inserted into the 3 ′ end of the mouse IgGκ signal peptide of the mutant uteroglobin (F28S) mammalian expression plasmid in which the 28th amino acid phenylalanine ttc was converted to serine agc. ROBO4 scFv-mutant uteroglobin (F28S) mammalian expression plasmid was prepared.

Thus, a mutant uteroglobin (S66F) mammalian expression plasmid [8] and a ROBO4BOscFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [9] were prepared.

In the same manner, a mutant uteroglobin (S29K) mammalian expression plasmid was prepared by converting serine (S) agc, the 29th amino acid, into lysine (K) aag. Subsequently, the same method was used to convert the 29th amino acid serine (S) agc to aspartic acid (D) gac and the 62nd amino acid lysine (K) aaa to aspartic acid (D) gac. (S29D, K62D) ROBO4 scFv-mutant uteroglobin (S29D, K62D) mammalian expression plasmid in which the mouse IgGκ signal peptide of the mammalian expression plasmid inserts a single-chain antibody scFv sequence against ROBO4 and the linker sequence GSSSSGSSSS at the 3 ′ end Produced.

Thus, a mutant uteroglobin (S29K) mammalian expression plasmid [13] and a ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [14] were prepared.

Example 2: Preparation of expression product 2-1: Expression Transfection (transduction) was performed using ExpiFectamine293 Transfection kit (Thermo Fisher Scentific). The procedure is as follows.
Expi293F cells (Invitrogen) were cultured in 100 mL of Expi293 expression medium (Thermo Fisher Scentific) under conditions of 37 ° C., 125 rpm, and 8% CO ₂ at 3.0 × 10 ⁶ cells / mL. 100 μg of plasmid [1], [2], [4], [8], [9], [13] and [14] prepared in Example 1 and Opti-MEM I reduced serum medium (Thermo Fisher Scentific) 5 mL was mixed gently and left at room temperature for 5 minutes. 270 μL of ExpiFectamine293 reagent (Thermo Fisher Scentific) and 5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, each plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes. Gently add the mixed solution to 100 mL of Expi293F cell solution, and incubate at 37 ° C, 125 rpm, 8% CO ₂ for 20 hours. enhancer 2 (Thermo Fisher Scentific) 5mL were added 37 ° C., and cultured for one week at 125rpm, 8% CO ₂ conditions.

ROBO4 scFv-D33K and K51E double transfection was performed as follows. Mix 50 μg of uteroglobin (K51E) mammalian expression plasmid [2] with 50 μg of ROBO4 scFv-mutated uteroglobin (D33K) mammalian expression plasmid [4] and add to 5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific). After gently mixing, the mixture was allowed to stand at room temperature for 5 minutes. 270 μL of ExpiFectamine293 reagent (Thermo Fisher Scentific) and 5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes. Gently add the mixed solution to Expi293F cell solution 3.0 × 10 ⁶ cells / mL, 100 mL, and incubate at 37 ° C, 125 rpm, 8% CO ₂ for 20 hours, then ExpiFectamine 293 Transfection Enhancer 1 (Thermo Fisher Scentific) 500 μL and ExpiFectamine 293 transfection enhancer 2 (Thermo Fisher Scentific) 5mL were added 37 ° C., and cultured for one week at 125rpm, 8% CO ₂ conditions.

In a similar manner, ROBO4 scFv-F28S and S66F double transfection using mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [10] And ROBO4 scFv-S29D, K62D and S29K double transfection using mutant uteroglobin (S29K) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (S29D, DK62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [15] was performed.

ROBO4 scFv-D33K_K51E co-culture was performed as follows. Expi293F cells (Invitrogen) were cultured in 37 mL of Expi293 expression medium (Thermo Fisher Scentific) at 37 ° C., 125 rpm, and 8% CO ₂ at 3.0 × 10 ⁶ cells / mL. 50 μg of uteroglobin (K51E) mammalian expression plasmid [2] and 2.5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes. ExpiFectamine293 reagent (Thermo Fisher Scentific) 135 μL and Opti-MEM I reducing serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes. Gently add the mixed solution to 50 mL of Expi293F cell solution and incubate at 37 ° C, 125 rpm, 8% CO ₂ for 20 hours. Then, 250 µL of ExpiFectamine 293 Transfection Enhancer 1 (Thermo Fisher Scentific) and ExpiFectamine 293 Transfection Enhancer 2 ( Thermo Fisher Scentific) 2.5 mL was added and cultured under conditions of 37 ° C., 125 rpm, 8% CO ₂ for 3 hours. Expi293F cells (Invitrogen) were cultured in 50 mL of Expi293 Expression medium (Thermo Fisher Scentific) under conditions of 37 ° C., 125 rpm, and 8% CO 2 at 3.0 × 10 ⁶ cells / mL. ROBO4 scFv-mutated uteroglobin (D33K) mammalian expression plasmid [4] 50 μg and Opti-MEM I reducing serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. ExpiFectamine293 reagent (Thermo Fisher Scentific) 135 μL and Opti-MEM I reducing serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes. Gently add the mixed solution to 50 mL of Expi293F cell solution, and incubate at 37 ° C, 125 rpm, 8% CO ₂ for 20 hours. Fisher Scentific) 2.5 mL was added and cultured under conditions of 37 ° C., 125 rpm, 8% CO 2 for 3 hours. Thereafter, 50 mL of the K51E cell solution and 50 mL of the ROBO4 scFv-D33K cell solution were mixed and cultured at 37 ° C., 125 rpm, 8% CO ₂ for 1 week.

In a similar manner, ROBO4FscFv-F28S and S66F co-culture using a mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [11], And ROBO4 scFv-S29D, K62D and S29K co-cultures using mutant uteroglobin (S29K) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [16 ] Went.

2-2: Purification [1], [2], [4], [5], [6], [8], [9], [10], [11] obtained by expression in 1-1 ], [13], [14], [15] and [16] were all purified as follows.
The culture solution was centrifuged and the supernatant was collected, and then filtered through a 0.45 um filter. The filtered sample was purified by affinity chromatography. The column was HisTrap Excel 5 mL (GE healthcare), and the column was equilibrated and washed with 50 mM Hepes-NaOH pH 7.5, 500 mM NaCl, and eluted with 50 mM Hepes-NaOH pH 7.5, 500 mM NaCl, 500 mM imidazole. The eluted fractions were collected and dialyzed overnight at 50 mM Hepes-NaOH pH 7.5, 4 ° C. The dialyzed solution was collected and subjected to anion exchange chromatography. The column was 5 mL of HiTrapQ HP (GE healthcare) and eluted with a concentration gradient of 50 mM Hepes-NaOH pH7.5, 0-1M NaCl. The eluted fraction was collected and subjected to gel filtration chromatography. The column used was HiLoad16 / 600 Superdex75 (GE healthcare), and the mobile phase was 50 mM Hepes-NaOH pH 7.5, 300 mM NaCl. The eluted fraction was collected and concentrated with Amicon Ultra15.

ROBO4 scFv expression product was also prepared [3].
Moreover, the sample preparation before electrophoresis shown by lane [7] in FIG. 4 was performed as follows. ROBO4 scFv-mutant uteroglobin (D33K) [4] expression product and mutant uteroglobin (K51E) expression product [2] were mixed at a molar ratio of 1: 1, and allowed to stand on ice for 1 hour.
By the same method, scFv-F28S and S66F mixed at a molar ratio of 1: 1 [12] and scFv-S29D, K62D and S29K mixed [17] were prepared.

Example 3 Electrophoresis Each solution of expression products [1]-[6], [8]-[11], [13]-[16] obtained in Example 2 was adjusted to 0.2 mg / mL. Dilute with 50 mM Hepes-NaOH pH 7.5, 300 mM NaCl.
For [1]-[17], a sample for electrophoresis under reducing conditions was prepared as follows.
To 10 μL of the diluted protein solution, 0.5 μL of 2-mercaptoethanol and 9.5 μL of 2 × Laemmli sample buffer (BIO-RAD) were added and heated at 95 ° C. for 5 minutes. For the sample under non-reducing conditions, 10 μL of 2 × Laemmli sample buffer (BIO-RAD) was added to 10 μL of the diluted protein solution and heated at 95 ° C. for 5 minutes. Apply each sample under reducing and non-reducing conditions to a 10-20% gradient gel (ato), 20 μL per lane, and conduct electrophoresis at 25 mA in 25 mM Tris, 192 mM glycine, 3.5 mM SDS solution for 60 minutes. It was. The gel after electrophoresis was stained with 25% ethanol, 10% acetonitrile, 0.1% Coomassie brilliant blue 250 staining solution, and decolorized with hot water.

The obtained results are shown in FIG. 4, FIG. 5, and FIG.
In FIG. 4, the molecular weights of the monomers of wild type uteroglobin and mutant uteroglobin (K51E) are 9.6 kDa, ROBO4 scFv is 25 kDa, and ROBO4 scFv-mutant uteroglobin (D33K) (ROBO4 scFv-D33K) is 36 kDa. From the results of structural analysis, it is known that wild-type uteroglobin has an intermolecular disulfide bond and forms a homodimer. From the results of electrophoresis under reducing conditions, bands were observed for [1], [2], [3] and [4] in the molecular weight of the monomer. This is because the disulfide bond is cleaved under reducing conditions. In [5], [6], and [7], bands existed in the molecular weights of the K51E monomer and the ROBO4 scFv-D33K monomer. On the other hand, since disulfide bonds are retained under non-reducing conditions, uteroglobin forms a homodimer, and in [1] and [2], a band is observed on the higher molecular weight side compared to reducing conditions. It was. Since the molecular weight of the homodimer is 19.2 kDa, the band position should be confirmed around 20 kDa, but it was observed on the lower molecular weight side. Since the disulfide bond is retained under non-reducing conditions, it cannot be separated purely by molecular weight, and is considered to have shifted to a lower molecular weight side than the original molecular weight position.

In [4] under non-reducing conditions, a main band (main band) was present at 72 kDa, a homodimer, and a minor band was present at 36 kDa, a monomer. In electrophoresis, the amount ratio can be determined depending on the degree of staining, so it was found that ROBO4 scFv-D33K mainly contains homodimers and contains a small amount of monomers.

Under non-reducing conditions, [5] and [6] have bands around 17, 36, 45 and 72 kDa, and ROBO4 scFv-D33K and K51E mixed have bands around 17, 36 and 72 kD Was. Based on the results thus far, 17 kDa is a K51E homodimer, 36 kDa is a ROBO4 scFv-D33K monomer, and 72 kDa is a ROBO4 scFv-D33K homodimer. When the ROBO4 scFv-D33K monomer 36 kDa and the K51E monomer 9.6 kDa form a heterodimer, it becomes 45 kDa, so the band around 45 kDa is considered to be a ROBO4 scFv-D33K_K51E heterodimer.

In FIG. 5, the molecular weights of the monomers of wild type uteroglobin and mutant uteroglobin (S66F) are 9.6 kDa, ROBO4 scFv is 25 kDa, and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) is 36 kDa. From the results of structural analysis, it is known that wild-type uteroglobin has an intermolecular disulfide bond and forms a homodimer. From the results of electrophoresis under reducing conditions, [1], [3], [8], and [9] bands were observed in the molecular weight of the monomer. This is because the disulfide bond is cleaved under reducing conditions. In [10], [11], and [12], the bands were thin, but there were bands in the molecular weights of S66F monomer and ROBO4 scFv-F28S monomer. On the other hand, since disulfide bonds are retained under non-reducing conditions, uteroglobin forms a homodimer, and in [8] and [12], a band is observed on the higher molecular weight side compared with reducing conditions. It was.

In [9] under non-reducing conditions, a main band was present at 36 kDa as a monomer, a minor band was observed at 72 kDa as a homodimer, and a minor band was also observed near 80 kDa, which is considered to be a contaminant.

Similarly, bands considered to be heterodimers were observed in [10] and [11] under non-reducing conditions.

In FIG. 6, the molecular weights of the wild-type and mutant uteroglobin (S29K) monomers are 9.6 kDa, ROBO4 scFv is 25 kDa, ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) is 36 kDa. . From the results of structural analysis, it is known that wild-type uteroglobin has an intermolecular disulfide bond and forms a homodimer. From the results of electrophoresis under reducing conditions, [1], [3], [13], and [14] bands were observed in the molecular weight of the monomer. This is because the disulfide bond is cleaved under reducing conditions. Also in [15], [16], and [17], bands existed in the molecular weights of S29K monomer and ROBO4BOscFv-S29D, DK62D monomer.

In [14] under non-reducing conditions, a main band was present at 36 kDa as a monomer, a minor band was observed at 72 kDa as a homodimer, and a minor band was also observed near 80 kDa, which is considered to be a contaminant.

Under non-reducing conditions, bands considered to be heterodimers were also observed in [15] and [16].

Discussion From the results of electrophoresis, the scFv-D33K_K51E double transfection and co-culture formed a heterodimer of scFv-D33K_K51E. In addition, a 45 kDa band of heterodimer was not present in the results [7] and [12]. For this reason, it was found that a heterodimer was not formed by simply mixing scFv-D33K and K51E.

Since the bispecific polypeptide of the present invention is based on uteroglobin, it has a lower molecular weight than a general bispecific antibody. Therefore, high tissue permeability can be expected, and since there is no Fc region, an unexpected effect can be suppressed. Therefore, it is highly expected that the tool will be a tool for creating a highly safe pharmaceutical product.

Claims

Bispecific polypeptide based on the structure of heterodimer uteroglobin.
The heterodimeric uteroglobin comprises an A chain and a B chain, wherein the A chain and the B chain are different from each other and have one or several amino acid residue mutations in the wild type uteroglobin monomer, and The bispecific polypeptide according to claim 1, wherein heterodimer is formed by association derived from mutation.
The mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin. The bispecific polypeptide according to claim 1 or 2.
The paired substitution of amino acid residues is the 5th serine (S) and 68th leucine (L), 27th leucine (L) and 68th leucine, 28th in the amino acid sequence represented by SEQ ID NO: 1. Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) 4. The bispecific polypeptide according to claim 3, which is a substitution.
The pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain. Item 5. The bispecific polypeptide according to Item 4.
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain. The bispecific polypeptide according to claim 4, wherein the 51st lysine (K) is substituted with glutamic acid (E) or aspartic acid (D).
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E The bispecific polypeptide according to claim 6, which substitutes for
The bispecific polypeptide according to any one of claims 1 to 7, wherein the uteroglobin A chain and B chain are disulfide bonded.
Both the uteroglobin A chain and the B chain are at least one selected from the 44th leucine (L), 34th methionine (M) and 59th leucine (L) of the amino acid sequence represented by SEQ ID NO: 1. The bispecific polypeptide according to any one of claims 1 to 8, which has a mutation substituting for cysteine (C).
The bispecific polypeptide according to any one of claims 1 to 9, wherein the uteroglobin is human uteroglobin.
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region The bispecific polypeptide according to any of claims 1 to 10, comprising a second polypeptide.
The bispecific polypeptide according to any one of claims 1 to 11, which binds to one or more target molecules selected from the group consisting of a receptor or a fragment thereof, a cancer antigen, an MHC antigen, and a differentiation antigen.
The bispecific polypeptide according to any one of claims 1 to 12, wherein the target molecule binds to a target molecule of a cancer antigen.
The bispecific polypeptide according to any one of claims 1 to 13, wherein either the first polypeptide or the second polypeptide binds to a cancer antigen.
In the first polypeptide and the second polypeptide, the connection between the A chain and the first binding region and the connection between the B chain and the second binding region are such that the specificity of the region is not destroyed. 12. A bispecific polypeptide according to claim 11.
The bispecific polypeptide according to claim 15, wherein the linkage between the A chain and the first binding region and the linkage between the B chain and the second binding region are via a linker.
The bispecific polypeptide according to claim 16, wherein the linker has an amino acid sequence represented by SEQ ID NO: 2.
The bispecific polypeptide according to any one of claims 1 to 17, wherein each of the first binding region and the second binding region has monovalent specificity.
19. The two of claims 1 to 18, wherein one or both of the first binding region and the second binding region links at least one additional binding region in a manner that does not destroy the specificity of the region. Bispecific polypeptide.
A complex comprising the bispecific polypeptide according to any one of claims 1 to 19 and a drug.
A pharmaceutical composition comprising the bispecific polypeptide according to any one of claims 1 to 19.
A pharmaceutical composition comprising the complex according to claim 20.
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing a bispecific polypeptide according to any of claims 1 to 14, comprising a second polypeptide, comprising:
a) providing a first vector comprising a nucleic acid encoding the first polypeptide;
b) providing a second vector comprising a nucleic acid encoding a second polypeptide;
c) co-transfecting the cells with the first and second vectors, culturing the resulting transfectants, and d) recovering the heterodimer comprising the first and second polypeptides,
A method for producing the bispecific polypeptide.
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing a bispecific polypeptide according to any of claims 1 to 14, comprising a second polypeptide, comprising:
a) providing a first cell expressing a first polypeptide;
b) providing a second cell expressing the second polypeptide;
c) co-culturing the first and second cells; and d) recovering the heterodimer comprising the first and second polypeptides.
A method for producing the bispecific polypeptide.
A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region The bispecific polypeptide according to any of claims 1 to 14, comprising a second polypeptide, the nucleic acid encoding the first polypeptide or the second polypeptide.
A vector comprising the nucleic acid according to claim 25.
A cell comprising the vector according to claim 26.
27. The cell of claim 26, comprising both the vector of claim 25 comprising a nucleic acid encoding the first polypeptide and the second polypeptide.
The cell according to claim 27 or 28, wherein the cell is Escherichia coli.
A co-culture containing a cell containing a vector containing a nucleic acid encoding a first polypeptide and a cell containing a vector containing a nucleic acid encoding a second polypeptide.
The co-culture according to claim 30, wherein the cells are Escherichia coli.
A and B chains, which are different from each other, have one or several amino acid residue mutations in the wild-type uteroglobin monomer, and are heterodimeric due to associations derived from that mutation Heterodimeric uteroglobin that forms the body.
The mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin. 33. The heterodimeric uteroglobin of claim 32.
34. Affinity and repulsive electrostatic interactions from pair substitution result in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin. The described heterodimeric uteroglobin.
The paired substitution of amino acid residues is the 5th serine (S) and 68th leucine (L), 27th leucine (L) and 68th leucine, 28th in the amino acid sequence represented by SEQ ID NO: 1. Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) 35. The heterodimeric uteroglobin of claim 34 which is a substitution.
The pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain. Item 36. The heterodimeric uteroglobin according to Item 35.
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain. The heterodimeric uteroglobin according to claim 36, wherein the 51st lysine (K) is substituted with glutamic acid (E) or aspartic acid (D).
Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E 36) The heterodimeric uteroglobin of claim 35, wherein
The heterodimeric uteroglobin according to any one of claims 32 to 38, wherein the uteroglobin A chain and the B chain are disulfide bonded.
Both the uteroglobin A chain and the B chain are at least one selected from the 44th leucine (L), 34th methionine (M) and 59th leucine (L) of the amino acid sequence represented by SEQ ID NO: 1. 40. The heterodimeric uteroglobin according to any of claims 32 to 39, having a mutation that replaces cysteine (C).
The heterodimeric uteroglobin according to any one of claims 32 to 40, which is human uteroglobin.