JPWO2019176866A1 - Bispecific polypeptide based on uteroglobin - Google Patents

Abstract

A low molecular weight bispecific polypeptide is provided. The Fc portion of a dual-character antibody prepared based on a natural antibody is changed to a low-molecular-weight uteroglobin, the uteroglobin monomer is mutated, a heterodimer uteroglobin is prepared, and the bispecificity is utilized. Create a polypeptide.

Description

The present invention relates to a polypeptide having bispecificity, specifically a bispecific polypeptide having a heterodimer uteroglobin as a structural basis, and a method for producing the same.

A naturally occurring antibody molecule is a molecule having a molecular weight of about 150 kDa, which usually exhibits a single binding property that recognizes one type of antigen and has a C-terminal region Fc region of an immunoglobulin heavy chain. The Fc region alone is considered to be a heavy chain homodimer containing the CH2 and CH3 regions.
On the other hand, by using the recombination technique, a bispecific antibody having two different binding properties can be created. Bispecific antibodies can simultaneously inhibit two receptors on the cell surface, can bind to two ligands at the same time, can signal by binding two different receptors, and can transfer cells to each other. It shows usefulness such as being able to act (Non-Patent Document 1).

In the production of bispecific antibodies, amino acid residues present in the CH3 region of one heavy chain are replaced with larger residues (knobs) and amino acid residues present in the CH3 region of the other heavy chain. Is replaced with a smaller residue (hole; void) so that the protrusion is placed in the void, thereby promoting hetero (heterogeneous) heavy chain formation and inhibiting homo (homo) heavy chain formation. The knob into hole method of causing is known (Patent Document 1). Further, the amino acid residue existing in the CH3 region of one heavy chain is replaced with an amino acid residue having a positive charge, and the amino acid residue existing in the CH3 region of the other heavy chain is replaced with an amino acid residue having a negative charge. It has also been reported that the substituted amino acid residues are electrostatically interacted with each other by substituting with, thereby promoting hetero-heavy chain formation and inhibiting homo-heavy chain formation (Patent Documents 2, 3 and 3). 4 and 5). The advantages of these methods are that the efficiency of producing bispecific antibody is relatively high, that the antigenicity is not easily impaired because the amino acid inside the CH3 region is modified, and that the Fc region has antibody-dependent cellular cytotoxicity. It is possible to produce an antibody having high in-vivo stability while maintaining the activity such as (ADCC) and complement-dependent cytotoxicity (CDC). On the other hand, it has the disadvantage that unexpected dimers may be formed, recombination may occur in the light chain, and the antigenicity may be changed. In addition, since the antibody has a large molecular weight of about 150 kDa, it is possible that the effect is diminished by reducing the permeability to tissues while maintaining high blood retention. Furthermore, since it is difficult to develop an ordinary antibody into an expression system that can be produced at a relatively low cost such as Escherichia coli due to its molecular weight and three-dimensional structure, its production is almost limited to an expensive mammalian expression system. is the current situation.

Uteroglobin is a relatively low molecular weight (15.8 kDa) homodimer polypeptide that does not interact with cells such as natural killer cells and macrophages that have a cell-killing 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 it has a great advantage in terms of protein production. In addition, uteroglobin is expected to have an anti-inflammatory effect (Patent Document 6), and is a highly safe polypeptide used in clinical trials. Uteroglobin is used as an antibody base for expressing a divalent antibody (Non-Patent Document 2), but this is a divalent antibody devised based on homodimer formation, which is a characteristic of wild-type uteroglobin. Therefore, there is no bispecificity, and the binding region with the antigen is also limited to those using scFv.

WO 1996/027011 Gazette WO2006 / 106905 Gazette Special Table 2011-508604 Gazette Special Table 2014-509857 Gazette WO 2015/046467 Gazette Special Table 2002-511856 Gazette

Biochem Pharmacol. 2012; 84 (9): 1105-12 PLoS One. 2013 Dec 19; 8 (12): e82878

Problems with antibody drugs containing bispecific antibodies generally include high drug prices, limited efficacy, and limited target antigens. High drug prices are common to biopharmacy, and the reason is that it often takes a development period to establish a manufacturing process compared to low molecular weight compounds, and in particular, antibodies use expensive animal cell expression systems due to their large molecular weight. There is no choice but to mention. Bispecific antibodies have been reported to have at least 50 or more various non-wild-type derivatives with extremely diverse structures and aims. On the other hand, the current situation is that trial and error is unavoidable in the creation of functional and effective bispecific antibodies.

Based on the above findings, a bispecific molecule having a lower molecular weight than that of a general antibody is obtained, and uteroglobin is used as a structural basis instead of the Fc region in a bispecific antibody prepared based on a natural antibody. 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 focused on the site where human uteroglobin is associated when forming a homodimer from the estimated structure. As a result, it was found that a heterodimer can be efficiently formed by adding a mutation of an amino acid residue to the associated site.

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

The present invention relates to a bispecific polypeptide based on heterodimer uteroglobin, a method for producing the same, and heterodimer uteroglobin itself and a method for producing the same. Therefore, the present invention includes the following aspects.
<Bispecific polypeptide defining heterodimer uteroglobin>
[1]
A bispecific polypeptide based on the heterodimer uteroglobin.
[2]
Heterodimer uteroglobin comprises chains A and B, where the A and B chains differ from each other and have mutations of one or several amino acid residues in the wild-type uteroglobin monomer, and their The bispecific polypeptide according to [1], wherein a heterodimer is formed by an association derived from a mutation.
[3]
The mutations in the A and B chains are pair substitutions of amino acid residues, which cause associations that promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin. The bispecific polypeptide according to [1] or [2].
[4]
The pair substitutions of the amino acid residues are the 5th serine (S) and the 68th leucine (L), the 27th leucine (L) and the 68th leucine, and the 28th in the amino acid sequence represented by SEQ ID NO: 1. A pair of phenylalanine (F) and serine (S) at position 66, aspartic acid (D) at position 33 and lysine (K) at position 51, and leucine (L) at position 44 and threonine (T) at position 47. The bispecific polypeptide according to [3], which is a substitution.
[5]
The pair substitution of the amino acid residue 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 according to the above.
[6]
The pair substitution replaces aspartic acid (D) at position 33 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 replaced with glutamic acid (E) or aspartic acid (D).
[7]
The 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 is glutamic acid (E). The bispecific polypeptide according to [6], which is substituted with).

<Bispecific polypeptide with further mutation in heterodimer uteroglobin>
[8]
The bispecific polypeptide according to any one of [1] to [7], wherein the uteroglobin A chain and the B chain are disulfide-bonded.
[9]
Both the uteroglobin A chain and the B chain are selected from at least one of the 44th leucine (L), the 34th methionine (M) and the 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 replaces cysteine (C).
[10]
The bispecific polypeptide according to any one of [1] to [9], wherein the uteroglobin is human uteroglobin.
[10-2]
As for the pair substitution of amino acid residues, the 29th serine (S) in the amino acid sequence represented by SEQ ID NO: 1 in the A chain is replaced with lysine (K), and the pair substitution is represented by SEQ ID NO: 1 in the B chain. [3] The double specificity according to [3], wherein the 62nd lysine (K) in the amino acid sequence is replaced with aspartic acid (D) and the 29th serine (S) is replaced with aspartic acid (D). Sex polypeptide.
[10-3]
The pair substitution of the amino acid residue 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 described.

<Bispecific polypeptide that defines the binding region>
[11]
The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer 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 according to 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 the target molecule of the 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 do not destroy the specificity of the region. [11] The bispecific polypeptide according to the above.
[16]
The bispecific polypeptide according to [15], wherein the link between the A chain and the first binding region and the link between the B chain and the second binding region are mediated by a linker.
[17]
The bispecific polypeptide of [16], wherein the linker has the amino acid sequence set forth in SEQ ID NO: 2.
[18]
The bispecific polypeptide according to any one of [1] to [17], wherein the first binding region and the second binding region each have monovalent specificity.

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

<Method for producing bispecific polypeptide>
[23]
The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer uteroglobin B chain is linked to a second binding region that is different from the first binding region. The method for producing a bispecific polypeptide according to any one of [1] to [14], which comprises a second polypeptide.
a) Prepare a first vector containing a nucleic acid encoding the first polypeptide.
b) Prepare a second vector containing the nucleic acid encoding the second polypeptide.
c) the cells are co-transfected with the first and second vectors, the resulting transfects are cultured, and d) the heterodimer containing the first and second polypeptides is recovered.
A method for producing the bispecific polypeptide.
[24]
The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer uteroglobin B chain is linked to a second binding region that is different from the first binding region. The method for producing a bispecific polypeptide according to any one of [1] to [14], which comprises a second polypeptide.
a) Prepare a first cell expressing the first polypeptide,
b) Prepare a second cell expressing the second polypeptide,
c) co-culture the first and second cells, and d) recover the heterodimer containing the first and second polypeptides.
A method for producing the bispecific polypeptide.

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

<Heterodimer uteroglobin>
[32]
Containing A and B chains, the A and B chains differ from each other and have mutations in one or several amino acid residues in the wild-type uteroglobin monomer, and heterodimers by associations derived from the mutations. Heterodimer uteroglobin that forms the body.
[33]
The mutations in the A and B chains are pair substitutions of amino acid residues, which cause associations that promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin. The heterodimer uteroglobin according to [32].
[34]
Affinity and repulsive electrostatic interactions derived from pair substitutions result in associations that promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin, [33]. The heterodimer uteroglobin described.
[35]
The pair substitutions of the amino acid residues are the 5th serine (S) and the 68th leucine (L), the 27th leucine (L) and the 68th leucine, and the 28th in the amino acid sequence represented by SEQ ID NO: 1. A pair of phenylalanine (F) and serine (S) at position 66, aspartic acid (D) at position 33 and lysine (K) at position 51, and leucine (L) at position 44 and threonine (T) at position 47. The heterodimeric uteroglobin according to [34], which is a substitution.
[36]
The pair substitution of the amino acid residue 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 heterodimer uteroglobin according to the above.
[37]
The pair substitution replaces aspartic acid (D) at position 33 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 [36], wherein the 51st lysine (K) is replaced with glutamic acid (E) or aspartic acid (D).
[38]
The 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 is glutamic acid (E). ), The heterodimeric uteroglobin according to [35].

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

The advantages of bispecific antibodies are that they can recognize two targets at the same time, that they can obtain synergistic effects similar to co-administration, and that the cost of developing two types of antibodies can be reduced. In addition to the advantages of these bispecific antibodies, since the present invention is based on uteroglobin, it is a small molecule compared to general antibodies, high tissue permeability can be expected, and there is no Fc region, so the Fc region. It is highly safe because it can suppress unexpected effects caused by the presence of. Since the polypeptide of the present invention can be synthesized using various expression systems such as Escherichia coli, it is expected that low cost can be realized and the problem of high drug price common to biopharmacy can be avoided.

It shows one aspect of the heterodimer uteroglobin. FIG. 1-1 shows an embodiment in which the formation of the heterodimer uteroglobin is promoted. Heterodimeric uteroglobin has A and B chains, and the A chain is a mutation from aspartic acid (D) to lysine (K) at position 33 of the amino acid sequence represented by SEQ ID NO: 1 for wild-type uteroglobin. This is an example in which (D33K mutation) and the B chain have a mutation from the 51st lysine (K) to glutamic acid (E) (K51E mutation), respectively. 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 electrostatically interact with each other, resulting in heterogeneity. It shows how the formation of dimeric uteroglobin is promoted. In FIG. 1-2, due to the pair substitution, 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 electrostatically interact with each other to form a homodimer. It shows how the formation of uteroglobin is inhibited. It is a schematic diagram which shows the structure of wild-type uteroglobin and the electrostatic interaction site. Both the X chain in the light black box and the Y chain in the white box interact electrostatically at the 33rd aspartic acid (D) on one side and the 51st lysine (K) on the other side to form a homodimer. The state of formation is shown. It is a structural schematic diagram of wild-type human uteroglobin estimated from the structural analysis of rabbit uteroglobin. A- and B- indicate A chain and B chain, respectively. For example, A-S5 indicates S (serine) at the 5th position of the A chain. Dotted lines indicate possible pairs. The pairing criterion is the result of a comprehensive search for visually close pairs of amino acids exposed to the outside. The distance between β carbons is calculated in Pymol (the number written as 5.4 etc. corresponds to it: unit Å), and it is in the range of 5 to 10 Å, and it is clear from the angle between the α carbon and β carbon bonds. Those that cannot be paired are excluded. FIG. 3-1 is a view of the model structure from the N-terminal side of the A chain. FIG. 3-2 is a view seen from the opposite side. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing 2-mercaptoethanol are shown. [1] is a wild-type uteroglobin expression product, [2] is a mutant uteroglobin (K51E) expression product, [3] is a ROBO4 scFv expression product, and [4] is a ROBO4 scFv-variant uteroglobin (D33K) (ROBO4 scFv-D33K). ) Expression product, [5] is an expression product obtained by double transfection with a plasmid encoding mutant uteroglobin (K51E) and ROBO4 scFv-variant uteroglobin (ROBO4 scFv-D33K), respectively, and [6] is a mutation. Electrofection of each expression product 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 addition, [7] is a mixture of ROBO4 scFv-mutant uteroglobin (D33K) [4] expression product and mutant uteroglobin (K51E) expression product [2] in a molar ratio of 1: 1 and 1 on ice. It is the result of electrophoresis after allowing for a long time. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing 2-mercaptoethanol 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, and [9] is a ROBO4 scFv-variant uteroglobin (F28S) (ROBO4 scFv-F28S). ) Expression product, [10] is an expression product obtained by double transfection with a plasmid encoding mutant uteroglobin (S66F) and ROBO4 scFv-variant uteroglobin (ROBO4 scFv-F28S), respectively, and [11] is a mutation. Electrofection of each expression product 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. In addition, [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 allowing for a long time. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing 2-mercaptoethanol 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, and [14] is a ROBO4 scFv-variant 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), respectively. And [16] were 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 plasmid. In addition, [17] is a mixture of ROBO4 scFv-mutant uteroglobin (S29D, K62D) [14] expression product and mutant uteroglobin (S29K) expression product [13] in a molar ratio of 1: 1 and on ice. It is the result of electrophoresis after standing for 1 hour in.

<Heterodimer uteroglobin and bispecific polypeptide based on it>
Uteroglobin is a homodimer polypeptide in which two structurally identical monomers are electrostatically associated (Nature structural biology 1994, vol.1, No.8,538). The amino acid sequence of the human wild-type uteroglobin monomer is shown in SEQ ID NO: 1. Wild-type uteroglobin is a relatively low molecular weight (15.8 kDa) sugar chain-free protein that combines high solubility and pH stability, and is also 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 (angstrom) diffractivity, Nat Struct Biol vol.1 (8) 1994 538-545). Here, uteroglobin is a type of protein secreted into the bronchi and is described as having phospholipase A2 (PLA2) inhibition and PCB binding activity. Crystallization was performed by obtaining and purifying wild-type protein from individuals under the conditions: 100 ug / mL protein, 70% ammonium sulfate, 20 mM tris (pH 7.5), 16-18% glycerol. Structural analysis with a resolution of 1.9 angstrom Å shows that human uteroglobin has a structure to which phospholipids such as phosphatidylcholine and phosphatidylinositol can bind, and has a similar structure with 62% amino acid homology compared to rabbit uteroglobin. Have been described.

Human uteroglobin is not registered in the Protein Data Bank (PDB), but structural similarity with rabbit uteroglobin registered in PDB is presumed. Therefore, when human uteroglobin forms a homodimer, the region where amino acids interact is presumed to some extent. Therefore, as part of the structural analysis of human uteroglobin, the present inventors estimated the identification of amino acids to be mutated in order to form a heterodimer from the structural analysis of rabbit uteroglobin. Structural analysis estimates are detailed 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 electrostatic interaction of affinity between amino acid residues having opposite charges. The schematic three-dimensional structure of the wild-type uteroglobin is shown in FIG.
The fact that 33D of one monomer and 51K of the other monomer are mutually associated in wild-type human uteroglobin means that a heterodimer is formed by substituting them with a certain amino acid residue. This was proved experimentally in the examples of the present specification, and thus became clear as a fact.

Furthermore, based on the structural analysis of human uteroglobin, amino acid residues with side chains extending outward as uteroglobin dimers were identified, and other amino acids with possible associations that could form heterodimer uteroglobin. The residue was inferred.
Based on these facts and speculations, the present invention provides various inventions based on the heterodimer uteroglobin.

Here, as part of the structural analysis of human uteroglobin, the identification of amino acids to be mutated to form a heterodimer will be described in detail with respect to the estimation from the structural analysis of rabbit uteroglobin.
Enter the sequence of human uteroglobin in Swiss-model (swissmodel.expasy.org) and build a model from the homology of amino acid sequences. Select the one with the best score and display the obtained PDB file on Pymol (pymol.org).
-Interatomic distance is calculated by the measurement tab. The displayed model is shown in FIGS. 3-1 and 3-2.

Table 1 shows the pairs of amino acid residues at which the associations that form the heterodimer uteroglobin estimated above are possible.
In Table 1, the first row means a pair of substitutions in the A chain with the 5th cysteine (S) of the amino acid sequence represented by SEQ ID NO: 1 and the 68th leucine (L) in the B chain. The same is true on the following lines. The A chain and the B chain can be converted to each other.

なお、他の表においても、数字は配列番号１で表されるアミノ酸配列のＮ末からの番号である。

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

A preferred example of the 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 similar charges, and an interaction between hydrophobic amino acids.
In one embodiment, the present invention promotes the formation of heterodimers by mutating amino acid residues that have an affinity electrostatic interaction with each monomer of wild-type uteroglobin, and homonidies. Inhibits the formation of monomers. 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, the heterodimer uteroglobin with one pair substitution contains one amino acid mutation in the A chain and another amino acid mutation in the B chain. This aspect is called "wild substitution type".

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

Table 2 shows, as a pair substitution in this embodiment, in the A chain, aspartic acid (D) at position 33 of the amino acid sequence represented by SEQ ID NO: 1 is replaced with lysine (K), arginine (R) or histidine (H). A total of six examples are shown in which the 51st lysine (K) in the B chain is replaced with glutamic acid (E) or aspartic acid (D). For example, in a heterodimer uteroglobin having a D33K mutation in the A chain and a K51E mutation in the B chain with respect to wild-type uteroglobin, the pair substitution results in D33K in the A chain and K51E in the B chain, respectively. The 51st K of the chain and the 33rd D of the B chain electrostatically interact with each other to promote the formation of the heterodimer uteroglobin. This aspect is shown in FIG.
The 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 is glutamic acid (E). ) Is preferred.

In another aspect of the present invention, in wild-type uteroglobin, a heterodimer is formed by causing a new pair substitution in a portion where amino acid residues do not have a clear affinity interaction with each other. In this embodiment, it is preferred that one pair substitution causes an affinity electrostatic interaction and another one pair substitution adds homodimer repulsion, in which case the heterodimer uteroglobin It has two pair substitutions. This aspect is called "charge pair forming type".

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

Table 3 shows the following examples as pair substitutions in this embodiment.
The example of substitution site 5-68 will be described. From the first row, S5D is introduced into the A chain and L68R is introduced into the B chain (first pair substitution), and from the second row, 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. As a result, a pair of pair substitutions is established.
More specifically, as an example at substitution site 5-68, the pair substitution of chain A and chain B
1st line: 6 of S5D-L68R, S5D-L68K, S5D-L68H, S5E-L68R, S5E-L68K, S5E-L68H,
2nd line: 6 of L68D-S5K, L68D-S5R, L68D-S5H, L68E-S5K, L68E-S5R, L68E-S5H,
So, there are 6 + 6 = 12 ways. It should be noted that the heterodimer into which each one pair substitution is introduced is also within the scope of the present invention.
There are 6 × 6 = 36 combinations of pair substitutions in which one is selected from each of the first and second rows. Therefore, the total number of heterodimer uteroglobins at the substitution site 5-68 is 12 + 36 = 48 ways.
The same is true for the other pair of substitutions 27-68, 44-47, and 29-62.

As yet another aspect of the present invention, as a pair substitution, an embodiment in which the heterodimer uteroglobin is formed through an association in which hydrophobic amino acids interact with each other rather than an electrostatic interaction is also included. This aspect is called "hydrophobic pocket reversal type".

Table 4 shows specific examples of pair substitution in the hydrophobic pocket reversal type. The A chain and the B chain can be converted to each other.

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

Among the 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). Aspartic acid (D), glutamic acid (E) and the like are known as negatively charged amino acids (negatively charged amino acids). Therefore, preferably, in the present invention, the amino acid residues having the same kind of charges mean amino acid residues between positive charges or amino acid residues between negative charges.

Here, in the present invention, "mutation" of an amino acid residue specifically means replacing the original amino acid residue with another amino acid residue, deleting the original amino acid residue, and newly. It refers to the addition of an amino acid residue, etc., but preferably means that the original amino acid residue is replaced with another amino acid residue.

In the present invention, the "homodimer" refers to a state in which polypeptides having the same amino acid sequence are associated with each other. The "heterodimer" refers to a state in which polypeptides having at least one amino acid residue different in the amino acid sequence are associated with each other.

In the present invention, the term "heterodimer uteroglobin" means that wild-type uteroglobin is a homodimer, whereas each monomer is heterodimer 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, in the "heterodimer uteroglobin", the heterodimer uteroglobin contains A chain and B chain, and the A chain and B chain are different from each other, and one or several of them in the wild type uteroglobin monomer, respectively. Has a mutation in the amino acid residue of, and both are associated by the mutation. Here, the A and B chains do not represent specific monomers in the heterodimer uteroglobin and are interchangeable. That is, the reference to each mutation in the A chain and the B chain should be understood as the reference to each mutation in the B chain and the A chain, respectively.

<Bispecific polypeptide with further mutation in heterodimer uteroglobin>
The heterodimer uteroglobin as described above can have yet another mutation. For example, in the heterodimer 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 contains leucine (L) at position 44, methionine (M) at position 34, and the amino acid sequence represented by SEQ ID NO: 1 in which both uteroglobin A and B chains are present. At least one selected from the 59th leucine (L) can have a mutation that replaces cysteine (C). As a result, the heterodimer uteroglobin and the bispecific polypeptide of the present invention have improved stability, and the amount of protein produced is significantly increased.

<Bispecific polypeptide that defines the binding region>
In the present invention, the "bispecific polypeptide" is a molecule containing at least a first polypeptide and a second polypeptide. That is, in the "bispecific polypeptide" of the present invention, the first polypeptide in which the heterodimer uteroglobin A chain is linked to the first binding region, and the heterodimer uteroglobin B chain are the first. Contains a second polypeptide that is linked to a second binding region that is different from the binding region of. 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. Is.

The bispecific polypeptides of the invention have binding specificity 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, for example, 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 conjugated receptor family, GPI. Examples thereof include a receptor family such as 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 Mouth 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 inhibitor (LIF) receptor, human or mouse hairy neurotrophic factor (CNTF) receptor and the like can be exemplified.

A cancer antigen is an antigen that is expressed as cells become malignant, or an antigen that is highly expressed in peripheral cells other than cancer cells due to the characteristics of cancer. In addition, abnormal sugar chains that appear on the cell surface or 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, roundabout homolog 4 (ROBO4) and the like.
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, and -H. MHC class II antigens include HLA-DR, -DQ, -DP.
Differentiation antigens include a group of differentiation antigens such as CD1, CD2, CD3, CD4, CD5, and CD6.

As mentioned above, the bispecific polypeptides of the invention have binding specificity or binding sites for two different ligands. That is, the first binding region and the second binding region each have monovalent specificity. One or both of the first binding region and the second binding region can connect at least one additional binding region in a manner that does not disrupt the specificity of the region. In that case, the polypeptide of the invention can be a "multispecific polypeptide". Here, the bispecific polypeptide of the present invention includes a "heterodimer uteroglobin" formed by the A and B chains of the heterodimer uteroglobin in the first and second polypeptides. ..

In the present invention, the "first polypeptide" and the "second polypeptide" respectively bind to "binding regions", such as antibody variable regions, receptor binding regions, ligand binding regions or enzyme active regions, and other molecules. It can include an active affinity polypeptide. In a preferred embodiment, the binding region comprises the heavy and light chains of immunoglobulin. Usually, the first polypeptide and the second polypeptide each contain at least one region derived from the primary sequence of uteroglobin. This region is where the first and second polypeptides meet.

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, diabody, single domain antibodies (VHH), chimeric antibodies, and immunoadhesion factors. Is included. "Antibody fragments" include fragments of Fv, Fv', Fab, Fab'and F (ab') 2. "Antibody", when it relates to the present invention, means a polypeptide containing one or more regions that bind to an epitope of an antigen of interest.

An "antibody fragment" can be obtained by treating an antibody with an enzyme, such as a protease such as papain or pepsin (Morimoto et al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al. ., 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 modified structure of an "antibody fragment" can be constructed by utilizing 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 a suitable host cell (eg, 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 See 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 linked via a linker or the like, if necessary. The two variable regions contained 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. Generally, scFv polypeptides contain a linker between the VH and VL regions, which forms the VH and VL pairing required for antigen binding. Usually, the linker linking VH and VL is a peptide linker having a length of 10 amino acids or more in order to form a pair portion between VH and VL in the same molecule. However, the scFv linker in the present invention is not limited to such peptide linkers as long as it does not interfere with the formation of scFv. As a review of scFv, Pluckthun, The Pharmacology of Monoclonal Antibody, Vol.113 (Rosenburg and Moore ed., Springer Verlag, NY, pp.269-315 (1994)) can be referred to.

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 dimer is a dimer composed of two polypeptide chains, in which the light chain variable region (VL) and the heavy chain variable region (VH) cannot bind to each other in the same chain, respectively. It is bound by a short, eg, 5 residue linker. Since VL and VH encoded on the same polypeptide chain cannot form a single chain V region fragment due to the short linker between them and form a dimer, the diabody has two antigen binding sites. Will have. At this time, when VL and VH for two different epitopes (a, b) are simultaneously expressed by a combination of VLa-VHb and VLb-VHa with a linker of about 5 residues, it is secreted as bispecific Db. To. At this time, the two different epitopes may be two different epitopes on the same antigen, or two different epitopes on each of the two different antigens. Since the "diabody" contains two molecules of scFv, it contains four variable regions, resulting in two antigen binding sites. Unlike the case of scFv that does not form a dimer, when the purpose is to form a dimer, the linker that connects VH and VL in each scFv molecule is usually about 5 amino acids when it is a peptide linker. To do. However, the scFv linkers that form the diabody are not limited to such peptide linkers as long as they do not interfere with the expression of scFv and do not interfere with the formation of the diabody.

A "single domain antibody" is a molecule that generally has high affinity and specificity for a specific antigen, and is an antibody variable region consisting only of heavy chains found in camelids, or imitating this structure. It refers to an antibody variable region consisting of a single domain obtained by modifying VH such as humans. These include those that have undergone stabilization and affinity improvement measures through humanization and amino acid optimization.

"Affinity polypeptide" refers to a peptide consisting of several amino acids or a partial structure contained in the adhesion site of protein-protein interactions, or a peptide showing binding affinity for a specific molecule screened from a random peptide library. Refers to a polypeptide that is different in structure or its partial structure. In a preferred embodiment, an adhesion molecule active center such as Arg-Gly-Asp-Ser, a cyclic polypeptide or the like is included. In addition, peptides showing specific binding affinity found by methods such as phage surface presentation method, 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 connect 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 disrupt 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 and second binding regions are linked with scFv that binds to one or more ligands. Further, a polypeptide in which the above-mentioned affinity polypeptide is linked with a linker or the like can also be used as a multispecific polypeptide.

In the present invention, the "polypeptide" usually refers to a peptide and a protein having a length of about 10 amino acids or more. Further, it is usually a polypeptide of biological origin, but is not particularly limited, and may be, for example, a polypeptide consisting of an artificially designed sequence. Further, it may be any of a natural polypeptide, a synthetic polypeptide, a recombinant polypeptide and the like. Further, fragments of the above-mentioned polypeptides are also included in the polypeptides of the present invention.

The link between the uteroglobin A chain and the first binding region and the second binding different from the uteroglobin B chain and the first binding region in the first polypeptide and the second polypeptide of the bispecific polypeptide of the present invention. The connection with the region can be via a linker if necessary. Generally, the linker linking the A chain and the 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 it does not interfere with the specificity of the binding region and the binding characteristics. Preferred linkers include, for example, the GS4 (Gly + Ser x4, GSSSSGSSSS) (Non-Patent Document 2) linker shown in SEQ ID NO: 2.

In the present invention, the "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. Specific regions of each other are usually one or more amino acid residues that are subjected to association, preferably amino acid residues that approach and engage in interaction during association. Specific examples of the interaction include a case where amino acid residues approaching each other at the time of association form a hydrogen bond, an electrostatic interaction, a salt bridge, and the like.

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

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 plant toxins such as ricin can be mentioned.

<Nucleic acid, vector, etc.>
As another aspect of the present invention, a first polypeptide in which the heterodimer uteroglobin A chain is linked to the first binding region, and a second polypeptide in which the heterodimer 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 the binding region of the first polypeptide or a nucleic acid encoding the second polypeptide. ..

Numerous nucleic acid sequences encoding immunoglobulin regions, including VH, VL, hinges, 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 guidelines herein, one of ordinary skill in the art will combine such nucleic acid sequences and / or other nucleic acid sequences known in the art and base the structure on the heterodimer uteroglobin described herein. A nucleic acid sequence encoding a bispecific polypeptide can be made.

In addition, the nucleic acid sequence encoding the bispecific polypeptide of the invention can be determined by one of ordinary skill in the art based on the amino acid sequence provided herein and knowledge in the art. Besides the traditional method of producing a cloned DNA segment encoding a particular amino acid sequence, DNA of any desired sequence, which is now readily chemically synthesized, is routinely produced on order and of the DNA. The production process has been streamlined (GenScript, Eurofin Genomics, etc.).

<Method for producing bispecific polypeptide>
In a preferred embodiment, the present invention presents a first polypeptide in which the heterodimer uteroglobin A chain is linked to a first binding region, and a second in which the heterodimer uteroglobin B chain is different from the first binding region. Provided is a method for producing a bispecific polypeptide of the present invention, which comprises a second polypeptide linked to the binding region of. In another aspect of the production method of the invention, the invention presents a pair of amino acid residues of one mutation in the A chain and another mutation in the B chain such that associations between polypeptides are controlled. Provided is a method for producing a bispecific polypeptide based on the heterodimer uteroglobin of the present invention, which comprises pair substitution. Examples of such pair substitutions are shown in Tables 1-4.

In the above method of the present invention, the A chain and the B chain containing the pair substitution are prepared by modifying the nucleic acid encoding the wild-type uteroglobin.
The present invention relates to a first polypeptide in which the heterodimer uteroglobin A chain is linked to a first binding region, and a second binding region in which the heterodimer uteroglobin B chain is different from the first binding region. Provided are nucleic acids encoding a first or second polypeptide in a bispecific polypeptide of the invention, including a second polypeptide that is linked.
"Modifying a nucleic acid" means modifying a nucleic acid to correspond to an amino acid residue introduced by the "modification" in the present invention. More specifically, it refers to modifying a nucleic acid encoding the original (before modification) amino acid residue into a nucleic acid encoding the amino acid residue introduced by the modification. Usually, it means that the original nucleic acid is genetically manipulated or mutated so as to be a codon encoding a desired amino acid residue by inserting, deleting or substituting at least one base. That is, the codon encoding the original amino acid residue is replaced by the codon encoding the amino acid residue introduced by the modification. Such modification of nucleic acid can be appropriately carried out by using a technique known to those skilled in the art, for example, a site-specific mutagenesis method, a PCR mutagenesis method, or the like.

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 containing the nucleic acid of the present invention and a cell containing the vector of the present invention.
The vector is not particularly limited as long as it stably holds the inserted nucleic acid. For example, when Escherichia coli is used as the host, a pBluescript vector (manufactured by Stratagene) is preferable as the cloning vector, but it is commercially available. Various vectors of the above can be used. When a vector is used for the purpose of producing the polypeptide of the present invention, an expression vector is particularly useful. The expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in Escherichia coli, in cultured cells, or in an individual organism. For example, in vitro expression is pBEST vector (manufactured by Promega), Escherichia coli. If it is a pET vector (manufactured by Invitrogen), if it is a cultured cell, pME18S-FL3 vector (GenBank Accession No. AB009864), if it is an individual organism, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)), etc. Is preferable. Insertion of the DNA of the present invention into a vector can be carried out by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biologyedit. Ausubel et al. (1987) Publish. John Wiley & Sons . Section 11.4-11.11).

The host cell is not particularly limited, and various host cells are used depending on the purpose. Examples of cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), and insect cells (eg, Drosophylla S2). , Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells can be exemplified. Vector introduction into host cells includes, 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). It can be performed by a known method such as (manufactured by the company) or the microinjection method.
Furthermore, the present invention comprises a cell containing both a vector containing a nucleic acid encoding a first polypeptide and a nucleic acid encoding a second polypeptide, a cell containing a vector containing a nucleic acid encoding the first polypeptide, and a second. Provided are a cell mixture and co-culture of cells containing a vector containing a nucleic acid encoding a polypeptide. These cells and co-cultures are suitable for suitably preparing the bispecific polypeptide of the present invention.

Appropriate secretory signals can be incorporated into the polypeptide of interest in order for the polypeptide expressed in the host cell to be secreted into the endoplasmic reticulum lumen, the pericellular cavity, or the extracellular environment. These signals may be endogenous or heterologous to the polypeptide of interest.

In the recovery of the polypeptide in the above production method, when the polypeptide of the present invention is secreted into the medium, the medium is recovered. When the polypeptide of the 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 cultures, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods can be used, including hydroxylapatite chromatography and lectin chromatography.

As a preferred embodiment of the present invention, a first polypeptide in which the heterodimer uteroglobin A chain is linked to the first binding region, and a second polypeptide in which the heterodimer uteroglobin B chain is different from the first binding region. In the method for producing a bispecific polypeptide of the present invention, which comprises a second polypeptide linked to the binding region of, simply mixing the purified polypeptides may not result in a heterodimer. Found. See Example 3. Therefore, in the present invention, the first polypeptide and the second polypeptide are each co-transfected (double-transfected) cells with a vector encoding them, or contain a vector encoding them. The cells need to be co-cultured.

The present invention, as one aspect,
The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer uteroglobin B chain is linked to a second binding region that is different from the first binding region. The method for producing a bispecific polypeptide according to any one of claims 1 to 13, which comprises a second polypeptide.
a) Prepare a first vector containing a nucleic acid encoding the first polypeptide.
b) Prepare a second vector containing the nucleic acid encoding the second polypeptide.
c) the cells are co-transfected with the first and second vectors, the resulting transfects are cultured, and d) the heterodimer containing the first and second polypeptides is recovered.
A method for producing the bispecific polypeptide is provided.

Further, the present invention is, as another aspect, a method for producing the bispecific polypeptide of the present invention.
a) Prepare a first cell expressing the first polypeptide,
b) Prepare a second cell expressing the second polypeptide,
c) co-culture the first and second cells, and d) recover the heterodimer containing the first and second polypeptides.
A method for producing the bispecific polypeptide is provided.

An example of producing wild-type human uteroglobin, which is the basis for producing the bispecific polypeptide of the present invention, is as follows. A wild-type human uteroglobin monomer and a gene encoding a His tag, FLAG tag, GST tag, etc. are introduced into Escherichia coli, yeast, Brevibacillus, insect cells, mammalian cells, and the like. A culture solution containing uteroglobin or a cell disruption solution is passed through a resin that captures His tags, and a fraction containing uteroglobin is eluted with a solution containing imidazole. The eluate is exchanged by a dialysis method to reduce the salt concentration, and then purified by anion exchange chromatography. An anion exchange resin is used as the resin, and uteroglobin is separated and purified according to the salt concentration gradient. After collecting the eluted fraction, it is further purified by gel filtration chromatography to collect high-purity 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>
In another aspect, the invention relates to a composition comprising the bispecific polypeptide of the invention and a pharmaceutically acceptable carrier.

In the present invention, the pharmaceutical composition usually means a drug for treating or preventing a disease, or for testing / diagnosis.

The pharmaceutical composition of the present invention can be formulated by a method 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 injectable suspension. For example, pharmacologically acceptable carriers or vehicles, specifically sterile water or saline, vegetable oils, emulsifiers, suspensions, surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives. , It is conceivable to formulate by appropriately combining with a binder and the like and mixing in a unit dose form required for generally accepted pharmaceutical practice. The amount of the active ingredient in these preparations is set so as to obtain an appropriate volume in the specified range.

Aseptic compositions for injection can be formulated according to routine formulation practices using vehicles such as distilled water for injection.

Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannose, D-mannitol, sodium chloride). Appropriate solubilizers such as alcohol (ethanol, etc.), polyalcohol (propylene glycol, polyethylene glycol, etc.), and nonionic surfactants (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. It may also be blended with a buffer (eg, phosphate buffer and sodium acetate buffer), a soothing agent (eg, procaine hydrochloride), a stabilizer (eg, benzyl alcohol and phenol), 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, it can be an injection type, a nasal administration type, a pulmonary administration type, or a transdermal administration type 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 dose of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of 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 weight, age, symptom and the like of the patient, but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.

In the present invention, the above-mentioned 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 cancers (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), colon cancer, rectal cancer, colon cancer, breast cancer, Liver cancer, gastric cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesenteric tumor, squamous epithelial cancer, cervical cancer, uterine body cancer, bladder cancer, esophageal cancer, head and neck cancer, Nasopharyngeal cancer, salivary gland tumor, thoracic adenoma, skin cancer, basal cell tumor, malignant melanoma, anal cancer, penis cancer, testicular cancer, Wilms tumor, acute myeloid leukemia (acute myelocytic leukemia, acute myeloblastic leukemia) , Acute premyelocytic leukemia, including acute myeloid monocytic leukemia and acute monocytic leukemia), chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma (Berkit lymphoma, Chronic lymphocytic leukemia, fungal sarcoma, mantle 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, myelodystrophy syndrome, brain tumors (including glioma, stellate cell tumor, glial blastoma, meningeal tumor and coat tumor), neuroblastoma, retina Sprout sarcoma, osteosarcoma, capos sarcoma, Ewing sarcoma, angiosarcoma, vascular epidermoid cell tumor. Vasculitis-related diseases include arteriosclerosis and vasculitis. Inflammatory diseases include, but are not limited to, acute or chronic inflammatory diseases, specifically: alcoholic hepatitis. Viral hepatitis. Non-alcoholic 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, which comprises at least the polypeptide produced by the production method of the present invention or the pharmaceutical composition of the present invention. The kit may also be packaged with a pharmaceutically acceptable carrier, medium, instructions describing how to use it, 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 production of a therapeutic or prophylactic agent for an immunoinflammatory disease. The present invention also relates to a polypeptide of the present invention or a 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 notation and the one-letter notation 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
Glutamic acid: 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, but it should be noted that these do not limit the scope of the present invention and are merely examples.

Example 1: Construction of expression plasmid
Genes containing EcoRI cleavage sequence, DNA sequence encoding mouse IgGκ signal peptide, DNA sequence encoding wild-type human uteroglobin monomer full length (NCBI), linker sequence GSSSSGSSSS, His tag and stop codon sequence, and XhoI cleavage sequence were synthesized. .. The synthesized gene and 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 plasmid for wild-type uteroglobin mammal expression.

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

In the same manner, a plasmid for expressing mutant uteroglobin (D33K) in mammals was prepared by converting the 33rd amino acid, aspartate gac, into lysine aag. Subsequently, the single-chain antibody scFv sequence against ROBO4 and the linker sequence GSSSSGSSSS were inserted at the 3'end of the mouse IgGκ signal peptide of the mutant uteroglobin (D33K) mammalian expression plasmid for ROBO4 scFv-mutant uteroglobin (D33K) mammalian expression. A plasmid was prepared.

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

In the same manner, a mutant uteroglobin (S66F) mammalian expression plasmid was prepared by converting the 66th amino acid serine (S) agc into phenylalanine (F) ttc. 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) plasmid for expressing mammals in which the 28th amino acid phenylalanine ttc was converted to serine abc. A plasmid for expressing ROBO4 scFv-mutant uteroglobin (F28S) in mammals was prepared.

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

A mutant uteroglobin (S29K) mammalian expression plasmid was prepared by converting the 29th amino acid serine (S) ag into lysine (K) aag in the same manner. Subsequently, in the same manner, the 29th amino acid serine (S) agc was converted to aspartic acid (D) gac, and the 62nd amino acid lysine (K) aaa was converted to aspartic acid (D) gac. (S29D, K62D) ROBO4 scFv-mutant uteroglobin (S29D, K62D) mammalian expression plasmid with a single-chain antibody scFv sequence against ROBO4 and a linker sequence GSSSSGSSSS inserted at the 3'end of the mouse IgGκ signal peptide of the mammalian expression plasmid. Made.

In this way, a plasmid for expressing mutant uteroglobin (S29K) in mammals [13] and a plasmid for expressing ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) in mammals [14] were prepared.

Example 2: Preparation of expression product 2-1: Expression Transfection (transduction) was performed using the ExpiFectamine 293 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 37 ° C., 125 rpm, and 8% CO ₂ conditions 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 gently mixed and allowed to stand at room temperature for 5 minutes. 270 μL of ExpiFectamine293 reagent (Thermo Fisher Scentific) and 5 mL of Opti-MEM I reduced 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, then 500 μL of ExpiFectamine 293 Transfection Enhancer 1, Thermo Fisher Scentific and ExpiFectamine 293 Transfection. 5 mL of Enhancer 2 (Thermo Fisher Scentific) was added, and the cells were _{cultured under 37 ° C., 125 rpm, and 8% CO 2} conditions for 1 week.

ROBO4 scFv-D33K and K51E double transfection was performed as follows. 50 μg of uteroglobin (K51E) mammalian expression plasmid [2] and 50 μg of ROBO4 scFv-mutant uteroglobin (D33K) mammalian expression plasmid [4] were mixed and added to 5 mL of Opti-MEM I reduced serum medium (Thermo Fisher Scentific). After mixing gently, 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 reduced 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, 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) 5 mL was added, and the cells _{were cultured at 37 ° C., 125 rpm, and 8% CO 2} conditions for 1 week.

ROBO4 scFv-F28S and S66F double transfection with mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid in a similar manner [10] ROBO4 scFv-S29D, K62D and S29K double transfection using plasmids for mammalian expression of mutant uteroglobin (S29K) and ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) [15] was performed.

ROBO4 scFv-D33K_K51E co-culture was performed as follows. Expi293F cells (Invitrogen) were cultured in 50 mL of Expi293 expression medium (Thermo Fisher Scentific) under 37 ° C., 125 rpm, and 8% CO ₂ conditions at ^{3.0 × 10 6 cells / mL.} 50 μg of uteroglobin (K51E) mammalian expression plasmid [2] and 2.5 mL of Opti-MEM I reduced serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes. 135 μL of ExpiFectamine293 reagent (Thermo Fisher Scentific) and 2.5 mL of Opti-MEM I reduced 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 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 the cells were _{cultured at 37 ° C., 125 rpm, and 8% CO 2} conditions for 3 hours. Expi293F cells (Invitrogen) were cultured in 50 mL of Expi293 Expression medium (Thermo Fisher Scentific) at 37 ° C., 125 rpm, and 8% CO2 conditions at ^{3.0 × 10 6 cells / mL.} ROBO4 scFv-mutant uteroglobin (D33K) mammalian expression plasmid [4] 50 μg and Opti-MEM I reduced serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. 135 μL of ExpiFectamine293 reagent (Thermo Fisher Scentific) and 2.5 mL of Opti-MEM I reduced 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 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 the cells were cultured at 37 ° C., 125 rpm, and 8% CO2 conditions for 3 hours. Then, 50 mL of K51E cell solution and 50 mL of ROBO4 scFv-D33K cell solution were mixed and _{cultured at 37 ° C., 125 rpm and 8% CO 2} for 1 week.

ROBO4 scFv-F28S and S66F co-cultured with mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid in a similar manner [11], ROBO4 scFv-S29D, K62D and S29K co-cultures using the mutant uteroglobin (S29K) plasmid for mammalian expression and the ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) plasmid for mammalian expression [16] ] Was performed.

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

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

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

The obtained results are shown in FIGS. 4, 5, and 6.
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 in the molecular weight of the monomers in [1], [2], [3], and [4]. This is because the disulfide bond is cleaved under reducing conditions. Bands were also present in the molecular weights of the K51E monomer and ROBO4 scFv-D33K monomer in [5], [6], and [7]. On the other hand, since the disulfide bond is retained under non-reducing conditions, uteroglobin forms a homodimer, and in [1] and [2], a band is observed on the high molecular weight side as compared with the reducing conditions. It was. Since the molecular weight of the homodimer is 19.2 kDa, the position of the band should be confirmed near 20 kDa, but it was observed on the lower molecular weight side. Since the disulfide bond is retained under non-reducing conditions, it is considered that the disulfide bond cannot be separated purely by the molecular weight, and the disulfide bond is shifted to the lower molecular weight side from the position of the original molecular weight.

Under non-reducing conditions [4], the homodimer 72 kDa had a main band (main band) and the monomer 36 kDa had a minor band. Since the amount ratio can be determined by the degree of staining in electrophoresis, 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 the mixture of ROBO4 scFv-D33K and K51E has bands around 17, 36 and 72 kD. Was. From the results so 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 that 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, bands were observed in the molecular weight of the monomers in [1], [3], [8], and [9]. This is because the disulfide bond is cleaved under reducing conditions. The bands were also thin in [10], [11], and [12], but there were bands in the molecular weights of the S66F monomer and ROBO4 scFv-F28S monomer. On the other hand, since the disulfide bond is retained under non-reducing conditions, uteroglobin forms a homodimer, and in [8] and [12], a band is observed on the high molecular weight side as compared with the reducing conditions. It was.

Under non-reducing conditions [9], a main band was present at 36 kDa, which is a monomer, a minor band was observed at 72 kDa, which is 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 found in [10] and [11]].

In FIG. 6, the molecular weights of the monomers of wild-type uteroglobin and mutant uteroglobin (S29K) are 9.6 kDa, ROBO4 scFv is 25 kDa, and 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, bands were observed in the molecular weight of the monomers in [1], [3], [13], and [14]. This is because the disulfide bond is cleaved under reducing conditions. In addition, bands were also present in the molecular weights of the S29K monomer and ROBO4 scFv-S29D and K62D monomers in [15], [16], and [17].

Under non-reducing conditions [14], a main band was present at 36 kDa, which is a monomer, a minor band was observed at 72 kDa, which is 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 found in [15] and [16]].

Discussion From the results of electrophoresis, a heterodimer of scFv-D33K_K51E was formed by co-culture with scFv-D33K_K51E double transfection. Also, the 45 kDa band of the heterodimer was not present in the results [7] and [12]. Therefore, it was found that a heterodimer is 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, unexpected effects can be suppressed, and therefore, it is highly expected to be a tool for creating highly safe drugs.

Claims (41)

A bispecific polypeptide based on the heterodimer uteroglobin. Heterodimer uteroglobin comprises chains A and B, where the A and B chains differ from each other and have mutations of one or several amino acid residues in the wild-type uteroglobin monomer, and their The bispecific polypeptide according to claim 1, wherein a heterodimer is formed by an association derived from the mutation. The mutations in the A and B chains are pair substitutions of amino acid residues, which cause associations that promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin. The bispecific polypeptide according to claim 1 or 2. The pair substitutions of the amino acid residues are the 5th serine (S) and the 68th leucine (L), the 27th leucine (L) and the 68th leucine, and the 28th in the amino acid sequence represented by SEQ ID NO: 1. A pair of phenylalanine (F) and serine (S) at position 66, aspartic acid (D) at position 33 and lysine (K) at position 51, and leucine (L) at position 44 and threonine (T) at position 47. The bispecific polypeptide according to claim 3, which is a substitution. Claimed that the pair substitution of the amino acid residue 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 4. The bispecific polypeptide according to Item 4. The pair substitution replaces aspartic acid (D) at position 33 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 replaced with glutamic acid (E) or aspartic acid (D). The 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 is glutamic acid (E). ), The bispecific polypeptide according to claim 6. The bispecific polypeptide according to any one of claims 1 to 7, wherein the uteroglobin A chain and the B chain are disulfide-bonded. Both the uteroglobin A chain and the B chain are selected from at least one of the 44th leucine (L), the 34th methionine (M) and the 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 that replaces cysteine (C). The bispecific polypeptide according to any one of claims 1 to 9, wherein the uteroglobin is human uteroglobin. The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer 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 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 the target molecule of the cancer antigen. The bispecific polypeptide according to any one of claims 1 to 13, wherein either the first or 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 do not destroy the specificity of the region. The bispecific polypeptide according to claim 11. The bispecific polypeptide according to claim 15, wherein the link between the A chain and the first binding region and the link between the B chain and the second binding region are mediated by a linker. The bispecific polypeptide of claim 16, wherein the linker has the amino acid sequence set forth in SEQ ID NO: 2. The bispecific polypeptide according to any one of claims 1 to 17, wherein the first binding region and the second binding region each have monovalent specificity. 2. The second of claims 1 to 18, wherein one or both of the first binding region and the second binding region connect at least one additional binding region in a manner that does not disrupt the specificity of the region. Heavy-specific polypeptide. A complex comprising the bispecific polypeptide and drug according to any one of claims 1-19. A pharmaceutical composition containing the bispecific polypeptide according to any one of claims 1 to 19. A pharmaceutical composition containing the complex according to claim 20. The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer uteroglobin B chain is linked to a second binding region that is different from the first binding region. The method for producing a bispecific polypeptide according to any one of claims 1 to 14, which comprises a second polypeptide.
a) Prepare a first vector containing a nucleic acid encoding the first polypeptide.
b) Prepare a second vector containing the nucleic acid encoding the second polypeptide.
c) the cells are co-transfected with the first and second vectors, the resulting transfects are cultured, and d) the heterodimer containing the first and second polypeptides is recovered.
A method for producing the bispecific polypeptide. The heterodimer uteroglobin A chain is linked to the first polypeptide, and the heterodimer uteroglobin B chain is linked to a second binding region that is different from the first binding region. The method for producing a bispecific polypeptide according to any one of claims 1 to 14, which comprises a second polypeptide.
a) Prepare a first cell expressing the first polypeptide,
b) Prepare a second cell expressing the second polypeptide,
c) co-culture the first and second cells, and d) recover the heterodimer containing the first and second polypeptides.
A method for producing the bispecific polypeptide. The heterodimer uteroglobin A chain is linked to a first polypeptide linked to a first binding region, and the heterodimer uteroglobin B chain is linked to a second binding region different from the first binding region. The nucleic acid encoding the first or second polypeptide in the bispecific polypeptide according to any one of claims 1 to 14, comprising the second polypeptide. A vector comprising the nucleic acid of claim 25. A cell comprising the vector according to claim 26. The cell of claim 26, comprising both the vector of claim 25, which comprises a first polypeptide and a nucleic acid encoding a second polypeptide. The cell according to claim 27 or 28, wherein the cell is Escherichia coli. A co-culture containing cells containing a vector containing a nucleic acid encoding a first polypeptide and cells containing a vector containing a nucleic acid encoding a second polypeptide. The co-culture according to claim 30, wherein the cells are Escherichia coli. Containing A and B chains, the A and B chains differ from each other and have mutations in one or several amino acid residues in the wild-type uteroglobin monomer, and heterodimers by associations derived from the mutations. Heterodimer uteroglobin that forms the body. The mutations in the A and B chains are pair substitutions of amino acid residues, and the pair substitutions cause associations that promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin. The heterodimer uteroglobin according to claim 32. Affinity and repulsive electrostatic interactions resulting from pair substitutions cause associations, which promote the formation of heterodimer uteroglobin and inhibit the formation of homodimer uteroglobin, claim 33. The heterodimer uteroglobin described. The pair substitutions of the amino acid residues are the 5th serine (S) and the 68th leucine (L), the 27th leucine (L) and the 68th leucine, and the 28th in the amino acid sequence represented by SEQ ID NO: 1. A pair of phenylalanine (F) and serine (S) at position 66, aspartic acid (D) at position 33 and lysine (K) at position 51, and leucine (L) at position 44 and threonine (T) at position 47. The heterodimeric uteroglobin according to claim 34, which is a substitution. Claimed that the pair substitution of the amino acid residue 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 35. Heterodimer uteroglobin. The pair substitution replaces aspartic acid (D) at position 33 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 replaced with glutamic acid (E) or aspartic acid (D). The 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 is glutamic acid (E). ), The heterodimeric uteroglobin according to claim 35. Uteroglobin The heterodimer uteroglobin according to any one of claims 32 to 38, wherein the A chain and the B chain are disulfide-bonded. Both the uteroglobin A chain and the B chain are selected from at least one of the 44th leucine (L), the 34th methionine (M) and the 59th leucine (L) of the amino acid sequence represented by SEQ ID NO: 1. The heterodimer uteroglobin according to any one of claims 32 to 39, which has a mutation that replaces cysteine (C). The heterodimer uteroglobin according to any one of claims 32 to 40, which is a human uteroglobin.

Images