WO2019176866A1 - Polypeptide bispécifique structurellement basé sur une utéroglobine - Google Patents
Polypeptide bispécifique structurellement basé sur une utéroglobine Download PDFInfo
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- WO2019176866A1 WO2019176866A1 PCT/JP2019/009745 JP2019009745W WO2019176866A1 WO 2019176866 A1 WO2019176866 A1 WO 2019176866A1 JP 2019009745 W JP2019009745 W JP 2019009745W WO 2019176866 A1 WO2019176866 A1 WO 2019176866A1
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- uteroglobin
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- polypeptide
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- amino acid
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
Definitions
- the present invention relates to a polypeptide having bispecificity, in particular, a bispecific polypeptide based on a heterodimeric uteroglobin and a method for producing the same.
- a natural antibody molecule is usually a molecule with a molecular weight of about 150 kDa, which exhibits a single binding property that recognizes one type of antigen and has the C-terminal region Fc region of an immunoglobulin heavy chain.
- the Fc region alone is thought to be a heavy chain homodimer containing CH2 and CH3 regions.
- bispecific antibodies with two different binding properties can be created by using recombinant techniques. Bispecific antibodies can simultaneously inhibit two receptors on the cell surface, bind simultaneously with two ligands, allow signal transduction by binding two different receptors, and allow cells to interact with each other. The usefulness of being able to act is shown (nonpatent literature 1).
- an amino acid residue present in the CH3 region of one heavy chain is replaced with a larger residue (knob), and an amino acid residue present in the CH3 region of the other heavy chain Is replaced with a smaller residue so that the protrusion is located within the gap, thereby promoting heterogeneous heavy chain formation and inhibiting homogeneous heavy chain formation.
- a knob-in-to-hole method is known (Patent Document 1).
- an amino acid residue present in the CH3 region of one heavy chain is replaced with an amino acid residue having a positive charge, and an amino acid residue present in the CH3 region of the other heavy chain is negatively charged.
- Patent Documents 2, 3, 4 and 5 There is also reported a method in which the substituted amino acid residues are electrostatically interacted with each other, thereby promoting hetero heavy chain formation and inhibiting homo heavy chain formation.
- Advantages of these methods are that the production efficiency of the bispecific antibody is relatively high, that the amino acid in the CH3 region is modified, so that antigenicity is not easily impaired, and antibody-dependent cellular cytotoxicity of the Fc region. Examples thereof include the ability to produce antibodies with high in vivo stability while maintaining activities such as (ADCC) and complement-dependent cytotoxicity (CDC).
- ADCC antibody-dependent cytotoxicity
- CDC complement-dependent cytotoxicity
- the antibody since the antibody has a large molecular weight of about 150 kDa, the retention in blood is maintained at a high level, but the effect may be reduced by lowering the tissue permeability. Furthermore, normal antibodies are difficult to develop into expression systems that can be produced at a relatively low cost such as Escherichia coli due to the characteristics of molecular weight and three-dimensional structure, and their production is almost limited to expensive mammalian expression systems. is the current situation.
- Uteroglobin is a relatively low molecular weight (15.8 kDa) homodimeric polypeptide and does not interact with cells such as natural killer cells and macrophages that have a cytocidal effect.
- it since it is a low molecular weight polypeptide, it is expected that it can be developed into various expression systems without depending only on the expression system in mammalian cells, and has a great advantage from the viewpoint of protein production.
- uteroglobin is a highly safe polypeptide that is expected to have an anti-inflammatory effect (Patent Document 6) and is used in clinical trials.
- Uteroglobin is used as an antibody base for expressing a bivalent antibody (Non-Patent Document 2).
- bispecific antibodies generally include high drug prices, limited efficacy, and limited target antigens.
- High drug prices are common to biopharmaceuticals, and the cause of this is that development time is often required to establish a manufacturing process compared to low molecular weight compounds.
- antibodies use expensive animal cell expression systems due to their large molecular weight. It must be unavoidable.
- Bispecific antibodies have so far been reported with at least 50 various non-wild-type derivatives with a wide variety of structures and targets. On the other hand, the creation of functional and effective bispecific antibodies is still inevitable through trial and error.
- bispecific molecules that have a low molecular weight compared to general antibodies are sought, and uteroglobin is used as the structural basis instead of the Fc region in bispecific antibodies created based on natural antibodies.
- Diligent studies were conducted on polypeptides having bispecificity.
- the present inventors estimated the three-dimensional structure of human uteroglobin from the structural analysis of rabbit uteroglobin, and paid attention to the site where human uteroglobin associates when forming a homodimer from the estimated structure. As a result, it was found that heterodimers can be efficiently formed by adding amino acid residue mutations to the sites where they are associated.
- the present invention relates to bispecific polypeptides based on heterodimeric uteroglobin, methods for producing the same, and heterodimeric uteroglobin itself and methods for producing the same. Accordingly, the present invention includes the following aspects. ⁇ Bispecific polypeptide defining heterodimeric uteroglobin> [1] A bispecific polypeptide based on a heterodimeric uteroglobin.
- the heterodimeric uteroglobin comprises an A chain and a B chain, wherein the A chain and the B chain are different from each other and have one or several amino acid residue mutations in the wild type uteroglobin monomer, and
- the mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin.
- the paired substitution of amino acid residues is the 5th serine (S) and 68th leucine (L), 27th leucine (L) and 68th leucine, 28th in the amino acid sequence represented by SEQ ID NO: 1. Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) The bispecific polypeptide according to [3], which is a substitution.
- the pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain, [ 4] The bispecific polypeptide of description. [6] Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain.
- the bispecific polypeptide according to any one of [1] to [9], wherein the uteroglobin is human uteroglobin.
- the amino acid residue pair substitution is that the 29th serine (S) in the amino acid sequence represented by SEQ ID NO: 1 in the A chain is substituted with lysine (K), and the sequence shown in SEQ ID NO: 1 in the B chain.
- the pair substitution of amino acid residues is a pair of substitutions in the A chain with the 28th phenylalanine (F) of the amino acid sequence represented by SEQ ID NO: 1 and the 66th serine (S) in the B chain [3 ]
- the bispecific polypeptide of description is a pair of substitutions in the A chain with the 28th phenylalanine (F) of the amino acid sequence represented by SEQ ID NO: 1 and the 66th serine (S) in the B chain [3 ] The bispecific polypeptide of description.
- ⁇ Bispecific polypeptide that defines the binding region> [11] A first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region
- the bispecific polypeptide according to any one of [1] to [10], [10-2] and [10-3], which comprises a second polypeptide.
- the bispecific poly of any one of [1] to [11] which binds to one or more target molecules selected from the group consisting of a receptor or a fragment thereof, a cancer antigen, an MHC antigen, and a differentiation antigen. peptide.
- a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing the bispecific polypeptide according to any one of [1] to [14], comprising a second polypeptide, a) providing a first vector comprising a nucleic acid encoding the first polypeptide; b) providing a second vector comprising a nucleic acid encoding a second polypeptide; c) co-transfecting the cells with the first and second vectors, culturing the resulting transfectants, and d) recovering the heterodimer comprising the first and second polypeptides, A method for producing the bispecific polypeptide.
- a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing the bispecific polypeptide according to any one of [1] to [14], comprising a second polypeptide, a) providing a first cell expressing a first polypeptide; b) providing a second cell expressing the second polypeptide; c) co-culturing the first and second cells; and d) recovering the heterodimer comprising the first and second polypeptides.
- a method for producing the bispecific polypeptide comprising the first and second polypeptides.
- a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region The bispecific polypeptide according to any one of [1] to [14], wherein the nucleic acid encodes the first polypeptide or the second polypeptide.
- a vector comprising the nucleic acid according to [25].
- a cell comprising the vector according to [26].
- a and B chains which are different from each other, have one or several amino acid residue mutations in the wild-type uteroglobin monomer, and are heterodimeric due to associations derived from that mutation Heterodimeric uteroglobin that forms the body.
- the mutation of the A chain and the B chain is a pair substitution of amino acid residues, and the pair substitution results in association, thereby promoting the formation of heterodimeric uteroglobin and inhibiting the formation of homodimeric uteroglobin.
- the heterodimeric uteroglobin according to [32].
- Pairs of phenylalanine (F) and 66th serine (S), 33rd aspartic acid (D) and 51st lysine (K), and 44th leucine (L) and 47th threonine (T) The heterodimeric uteroglobin according to [34], which is a substitution.
- the pair substitution of amino acid residues is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain, [ 35] The heterodimeric uteroglobin according to [35].
- Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 in the A chain with lysine (K), arginine (R) or histidine (H), and in the B chain.
- lysine K
- arginine R
- histidine H
- the heterodimer uteroglobin according to [36], wherein the 51st lysine (K) is substituted with glutamic acid (E) or aspartic acid (D).
- Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E ) Is a heterodimeric uteroglobin according to [35].
- the advantages of the bispecific antibody include that it can recognize two targets at the same time, can obtain a synergistic effect similar to co-administration, and can reduce the cost of developing two types of antibodies.
- the present invention since the present invention is based on uteroglobin, it is a low molecular weight compared to general antibodies, can be expected to have high tissue permeability, and has no Fc region. It is possible to suppress unexpected effects that occur due to the presence of this, and safety is high.
- the polypeptide of this invention can be synthesize
- FIG. 1-1 illustrates one embodiment of a heterodimeric uteroglobin.
- FIG. 1-1 is an embodiment in which the formation of heterodimeric uteroglobin is promoted.
- Heterodimeric uteroglobin has an A chain and a B chain, and is a mutation from the 33rd aspartic acid (D) to lysine (K) amino acid sequence represented by SEQ ID NO: 1 with respect to wild-type uteroglobin. (D33K mutation), and the B chain has a mutation from the 51st lysine (K) to glutamic acid (E) (K51E mutation).
- FIG. 2 shows the formation of dimeric uteroglobin promoted.
- FIG. 1-2 shows that the pair substitution results in electrostatic interaction between the D33K site of the A chain and the K51 site of the A chain, and the K51E site of the B chain and the D33 site of the B chain. It shows how the formation of uteroglobin is inhibited.
- It is a schematic diagram which shows the structure and electrostatic interaction site
- Both the light black boxed X chain and the white boxed Y chain interact electrostatically in one 33rd aspartic acid (D) and the other 51st lysine (K) to form a homodimer.
- D 33rd aspartic acid
- K 51st lysine
- It is a structural schematic diagram of wild-type human uteroglobin deduced from the structural analysis of rabbit uteroglobin.
- A- and B- represent A chain and B chain, respectively.
- A-S5 indicates S (serine) at the 5-position of the A chain.
- Dotted lines indicate possible pairs.
- the criteria for pairing are the results of exhaustive searches for pairs that are visually adjacent to the exposed amino acids.
- FIG. 3-1 shows the model structure as viewed from the N-terminal side of the A chain.
- FIG. 3-2 is a view from the opposite side. The results of electrophoresis of various expression products under reducing conditions containing 2-mercaptoethanol and non-reducing conditions not containing are shown.
- [1] is wild-type uteroglobin expression product
- [2] is mutant uteroglobin (K51E) expression product
- [3] is ROBO4 scFv expression product
- [4] is ROBO4 scFv-mutant uteroglobin (D33K) (ROBO4 scFv-D33K )
- Expression product [5] is an expression product obtained by double transfection with plasmids encoding mutant uteroglobin (K51E) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-D33K), respectively
- [6] is mutation Electrophoresis of expression products obtained by co-culturing cells transfected with a plasmid encoding type uteroglobin (K51E) and cells transfected with a plasmid encoding ROBO4 scFv-mutant uteroglobin (D33K) Is the result of In [7], ROBO4
- [1] is a wild-type uteroglobin expression product
- [8] is a mutant uteroglobin (S66F) expression product
- [3] is a ROBO4 scFv expression product
- [9] is a ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S ) Expression product
- [10] is the double transfection with plasmids encoding mutant uteroglobin (S66F) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-F28S), respectively
- [11] is the mutation Of the expression products obtained by co-culturing cells transfected with a plasmid encoding type uteroglobin (S66F) and cells
- [1] is a wild-type uteroglobin expression product
- [13] is a mutant uteroglobin (S29K) expression product
- [3] is a ROBO4 scFv expression product
- [14] is a ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv -S29D, K62D) expression product
- [15] is an expression product obtained by double transfection with plasmids encoding mutant uteroglobin (S29K) and ROBO4 scFv-mutant uteroglobin (ROBO4 scFv-S29D, K62D)
- [16] are obtained by co-culturing cells transfected with a plasmid encoding mutant uteroglobin
- Uteroglobin is a homodimeric polypeptide in which two structurally identical monomers are electrostatically associated (Nature structural biology 1994, vol. 1, No. 8, 538).
- the amino acid sequence of human wild-type uteroglobin monomer is shown in SEQ ID NO: 1.
- Wild-type uteroglobin is a protein with no relatively low molecular weight (15.8 kDa) sugar chain, and its properties combine high solubility and pH stability, and is resistant to proteolytic enzymes (JBC 2009, vol.284, No.39, 26646.).
- Uteroglobin is also called CC10, and a paper on crystallization of human uteroglobin has been reported (1.9 ⁇ diffractivity, Nat Struct Biol vol. 1 (8) 1994 538-545).
- uteroglobin is a kind of protein secreted into the bronchus, and is described as having phospholipase A2 (PLA2) inhibition and PCB binding activity. Crystallization was performed by obtaining and purifying the wild-type protein from individuals under the conditions: 100 ⁇ ug / mL protein, 70% ammonium sulfate, 20 mM Tris (pH 7.5), 16-18% glycerol.
- human uteroglobin has a structure that can bind phospholipids such as phosphatidylcholine and phosphatidylinositol, and has a similar structure with 62% amino acid homology compared to rabbit uteroglobin Are listed.
- Human uteroglobin is not registered in the protein data bank (PDB), but structural similarity to rabbit uteroglobin that is registered in the PDB is presumed. Therefore, when human uteroglobin forms a homodimer, a region where amino acids interact is presumed to some extent.
- the present inventors estimated the identification of amino acids to be mutated to form a heterodimer from the structural analysis of rabbit uteroglobin. The estimation of the structural analysis is described in detail below.
- the present invention provides various inventions based on heterodimeric uteroglobin.
- Table 1 shows pairs of amino acid residues that can be associated with each other to form a heterodimeric uteroglobin estimated as described above.
- the first line means a pair of substitutions in the A chain with the fifth cysteine (S) of the amino acid sequence represented by SEQ ID NO: 1 and the 68th leucine (L) in the B chain. The same applies to the following lines.
- the A chain and the B chain can be converted to each other.
- the numbers are numbers from the N-terminal of the amino acid sequence represented by SEQ ID NO: 1.
- a preferred example of a pair of substitutions is a pair of substitutions in the A chain with the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 and the 51st lysine (K) in the B chain.
- the association may include an affinity electrostatic interaction, a repulsive interaction between amino acid residues having the same type of charge, and an interaction between hydrophobic amino acids.
- the present invention promotes heterodimer formation by mutating amino acid residues that have an affinity electrostatic interaction in each monomer of wild-type uteroglobin, and promotes homodimerization. Inhibits the formation of mers.
- “pair substitution” is established by mutating one of the amino acid residues in each monomer having an affinity interaction with each other in wild-type uteroglobin to an amino acid residue having a repulsive interaction. That is, a heterodimeric uteroglobin with one paired substitution contains one amino acid mutation in the A chain and another amino acid mutation in the B chain. This embodiment is called “wild substitution type”.
- pair substitution in the wild substitution type are shown in Table 2.
- the A chain and the B chain can be converted to each other.
- Table 2 shows that aspartic substitution in this embodiment, the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 is changed to lysine (K), arginine (R) or histidine (H) in the A chain.
- lysine (K), arginine (R) or histidine (H) in the A chain A total of six examples are shown in which substitution is performed and the 51st lysine (K) in the B chain is substituted with glutamic acid (E) or aspartic acid (D).
- the pair substitution results in D33K on the A chain and K51E on the B chain and
- the 51st K of the chain and the 33rd D of the B chain interact electrostatically to promote the formation of heterodimeric uteroglobin.
- Pair substitution replaces the 33rd aspartic acid (D) of the amino acid sequence represented by SEQ ID NO: 1 with lysine (K) in the A chain, and the 51st lysine (K) in the B chain with glutamic acid (E ) Are preferred.
- a heterodimer is formed by newly generating a pair substitution at a portion where the amino acid residues of wild-type uteroglobin do not have a clear affinity interaction with each other.
- Table 3 shows the following example as a pair substitution in this embodiment.
- An example with substitution site 5-68 will be described. From the first line, S68D is introduced into the A chain and L68R is introduced into the B chain (first pair substitution). From the second line, L68D is introduced into the A chain and S5K is introduced into the B chain (second pair substitution). ). In this case, the A chain becomes a mutant having two mutations of S5D and L68D, and the B chain becomes a mutant having two mutations of S5K and L68R. This establishes a pair of pair substitutions.
- the present invention includes, as yet another embodiment, a mode in which heterodimeric uteroglobin is formed as a pair substitution through an association in which hydrophobic amino acids interact with each other instead of electrostatic interaction.
- This embodiment is called “hydrophobic pocket inversion type”.
- Table 4 shows a total of nine examples of pair substitution in this aspect. Preferably, it has a F28S substitution in the A chain and S66F in the B chain.
- amino acid residues charged residues are known.
- lysine (K), arginine (R), and histidine (H) are known as positively charged amino acids (positively charged amino acids).
- negatively charged amino acid (negatively charged amino acid) aspartic acid (D), glutamic acid (E) and the like are known. Therefore, the amino acid residue having the same kind of charge in the present invention preferably means an amino acid residue having positive charges or an amino acid residue having negative charges.
- mutants specifically refers to substitution of the original amino acid residue with another amino acid residue, deletion of the original amino acid residue, This refers to addition of an amino acid residue, etc., but preferably means to substitute the original amino acid residue with another amino acid residue.
- homodimer refers to a state in which polypeptides having the same amino acid sequence are associated with each other.
- Heterodimer refers to a state in which polypeptides having at least one amino acid residue in an amino acid sequence are associated with each other.
- heterodimeric uteroglobin means that wild-type uteroglobin is a homodimer, whereas each monomer is heterodimeric due to mutation of one or several amino acid residues in each monomer. It means a heterodimer of mutant uteroglobin that constitutes the body.
- heterodimeric uteroglobin is a heterodimeric uteroglobin comprising an A chain and a B chain, wherein the A chain and the B chain are different from each other, one or several in the wild type uteroglobin monomer, respectively. Of amino acid residues, and both are associated by that mutation.
- the A chain and the B chain do not represent specific monomers in the heterodimeric uteroglobin, and are interchangeable. That is, a reference to each mutation in the A chain and the B chain should be understood as a reference to each mutation in the B chain and the A chain, respectively.
- the heterodimeric uteroglobin as described above can further have another mutation.
- 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—”.
- the heterodimer uteroglobin of the present invention includes the leucine (L) at the 44th amino acid sequence, the methionine (M) at the 34th amino acid sequence represented by SEQ ID NO: 1, and both the uteroglobin A chain and the B chain.
- At least one selected from the 59th leucine (L) can have a mutation replacing cysteine (C).
- a “bispecific polypeptide” is a molecule comprising at least a first polypeptide and a second polypeptide. That is, the “bispecific polypeptide” of the present invention comprises a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain in the first. A second polypeptide linked to a second binding region different from the binding region.
- the connection between the A chain and the first binding region and the connection between the B chain and the second binding region do not destroy the specificity of the region. It is.
- the bispecific polypeptide of the present invention has binding specificities or binding sites for two different ligands.
- the ligand is not particularly limited, and any ligand may be used. Examples of the ligand include, but are not limited to, a receptor or a fragment thereof, a cancer antigen, an MHC antigen, a differentiation antigen, and the like.
- receptors include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein coupled receptor family, GPI
- the receptor family include an anchor type receptor family, a tyrosine phosphatase type receptor family, an adhesion factor family, and a hormone receptor family.
- Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptor, human or mouse granulocyte colony stimulating factor (G-CSF) receptor, human or mouse thrombopoietin (TPO).
- EPO erythropoietin
- G-CSF granulocyte colony stimulating factor
- 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) - ⁇ , ⁇ receptor, human or Mouse leptin receptor, human or mouse growth hormone (GH) receptor, human or mouse interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitory factor Examples include (LIF) receptors, human or mouse ciliary neurotrophic factor (CNTF) receptors, and the like.
- PDGF platelet derived growth factor
- IFN interferon
- GH growth hormone
- IL interleukin
- IGF insulin-like growth factor
- IGF insulin-like growth factor
- human or mouse leukemia inhibitory factor examples include (LIF) receptors, human or mouse ciliary neurotrophic factor (CNTF) receptors, and the like.
- Cancer antigens are antigens that are expressed as cells become malignant or antigens that are highly expressed in peripheral cells other than cancer cells due to the characteristics of cancer.
- abnormal sugar chains appearing on the cell surface and protein molecules when cells become cancerous also become cancer antigens, and are particularly called cancer sugar chain antigens.
- cancer antigens include CA19-9, CA15-3, and roundabout homolog 4 (ROBO4).
- MHC antigens are roughly classified into MHC class I antigens and MHC class II antigens, and MHC class I antigens include HLA-A, -B, -C, -E, -F, -G, -H, MHC class II antigens include HLA-DR, -DQ, and -DP.
- Differentiation antigens include differentiation antigen groups such as CD1, CD2, CD3, CD4, CD5, and CD6.
- the bispecific polypeptides of the present invention have binding specificities or binding sites for two different ligands. That is, each of the first binding region and the second binding region has a monovalent specificity. One or both of the first binding region and the second binding region can link at least one additional binding region in a manner that does not destroy the specificity of the region.
- the polypeptide of the present invention may be a “multispecific polypeptide”.
- the bispecific polypeptide of the present invention includes a “heterodimeric uteroglobin” formed by the A and B chains of the heterodimeric uteroglobin in the first polypeptide and the second polypeptide. .
- a “first polypeptide” and a “second polypeptide” each bind to a “binding region”, eg, an antibody variable region, a receptor binding region, a ligand binding region or an enzyme active region, or another molecule.
- a binding region eg, an antibody variable region, a receptor binding region, a ligand binding region or an enzyme active region, or another molecule.
- An affinity polypeptide with activity can be included.
- the binding region comprises immunoglobulin heavy and light chains.
- the first polypeptide and the second polypeptide each comprise at least one region derived from the primary sequence of uteroglobin. This region is the portion where the first and second polypeptides associate.
- the binding region is derived from the variable region of the antibody or has sequence identity with the variable region of the antibody.
- antibodies include full length antibodies, antibody fragments, single chain molecules, bispecific or bifunctional molecules, scFv, diabodies, single domain antibodies (VHH), chimeric antibodies, and immunoadhesive factors Is included.
- Antibody fragments include fragments of Fv, Fv ′, Fab, Fab ′ and F (ab ′) 2.
- “Antibody”, as it relates to the present invention, means a polypeptide that contains one or more regions that bind to an epitope of an antigen of interest.
- Antibody fragments can be obtained by treating an antibody with an enzyme, for example, a protease such as papain, pepsin (MorimotoMet al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al ., See Science (1985) 229: 81). It can also be produced by gene recombination based on the amino acid sequence of the antibody fragment.
- an enzyme for example, a protease such as papain, pepsin (MorimotoMet al., J. Biochem. Biophys. Methods (1992) 24: 107-17; Brennan et al ., See Science (1985) 229: 81). It can also be produced by gene recombination based on the amino acid sequence of the antibody fragment.
- a low molecular weight antibody having a structure obtained by modifying an “antibody fragment” can be constructed using an antibody fragment obtained by enzyme treatment or gene recombination.
- a gene encoding the entire low molecular weight antibody can be constructed, introduced into an expression vector, and then expressed in an appropriate host cell (for example, Co et al., J. Immunol. (1994) 152). : 2968-76; Better and Horwitz, Methods Enzymol. (1989) 178: 476-96; Pluckthun and Skerra, Methods Enzymol. (1989) 178: 497-515; Lamoyi, Methods Enzymol. (1986) 121: 652-63 Rousseaux et al., Methods Enzymol. (1986) 121: 663-9; Bird and Walker, Trends Biotechnol. (1991) 9: 132-7).
- “ScFv” is a single-chain polypeptide in which two variable regions are bound via a linker or the like, if necessary.
- the two variable regions included in scFv are usually one heavy chain variable region (VH) and one light chain variable region (VL), but may be two VHs or two VLs.
- scFv polypeptides include a linker between the VH and VL regions, thereby forming the VH and VL paired portions necessary for antigen binding.
- the linker connecting VH and VL is a peptide linker having a length of 10 amino acids or more.
- the scFv linker in the present invention is not limited to such a peptide linker as long as it does not interfere with the formation of scFv.
- scFv As a review of scFv, reference can be made to Pluckthun, The Pharmacology of Monoclonal Antibody, Vol.113 (Rosenburg and Moore ed., Springer Verlag, NY, pp.269-315 (1994)).
- diabody refers to a divalent antibody fragment constructed by gene fusion (P. Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404,097). No., WO93 / 11161 etc.).
- a diabody is a dimer composed of two polypeptide chains, each of which is in a position where the light chain variable region (VL) and the heavy chain variable region (VH) cannot bind to each other in the same chain. They are linked by a short linker, for example, about 5 residues.
- VL and VH encoded on the same polypeptide chain cannot form a single-chain V region fragment due to a short linker between them, a diabody forms two antigen-binding sites. Will have.
- VL and VH for two different epitopes (a and b) are combined with VLa-VHb and VLb-VHa and linked together by a linker of about 5 residues, they are secreted as bispecific Db.
- the two different epitopes may be two different epitopes on the same antigen, or may be two epitopes on each of the two different antigens.
- a “diabody” contains two molecules of scFv, so it contains four variable regions, resulting in two antigen-binding sites. Unlike the case of scFv that does not form a dimer, when aiming at the formation of a diabody, the linker that connects between VH and VL in each scFv molecule is usually about 5 amino acids when used as a peptide linker. To do. However, the scFv linker that forms a diabody is not limited to such peptide linkers as long as it does not interfere with scFv expression and does not interfere with diabody formation.
- a “single domain antibody” is a molecule generally having a high affinity and specificity for a specific antigen, and is an antibody variable region consisting only of a heavy chain found in camelids or imitating this structure. This refers to an antibody variable region consisting of a single domain obtained by modifying human or other VH. These include those that have undergone stabilization and affinity improvement measures through humanization and amino acid optimization.
- Affinity polypeptide refers to a peptide or partial structure consisting of several amino acids contained in the adhesion site of protein-protein interaction, or a peptide showing binding affinity for a specific molecule screened from a random peptide library. It refers to a polypeptide that is different from a structure, or a partial structure thereof.
- an adhesion molecule active center such as Arg-Gly-Asp-Ser, a cyclic polypeptide and the like are included.
- peptides having specific binding affinity found by methods such as phage surface display, yeast display, and ribosomal display such as Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (RGD-4C) is also preferred (Arap, W .; Pasqualini, R .; Ruoslahti, E. Science 1998, 279, 377.).
- the present invention can link at least one additional binding region in one or both of the first binding region and the second binding region in a manner that does not destroy the specificity of the region
- the polypeptide can be a “multispecific polypeptide”.
- multispecific polypeptides include those in which one or both of the first binding region and the second binding region are linked to an scFv that binds to one or more ligands.
- affinity polypeptide with the linker etc. can be utilized as multispecific polypeptide.
- polypeptide generally refers to peptides and proteins having a length of about 10 amino acids or more. Moreover, although it is normally a polypeptide derived from a living organism
- Linkage between uteroglobin A chain and first binding region and second binding different from uteroglobin B chain and first binding region in the first polypeptide and the second polypeptide of the bispecific polypeptide of the present invention Linkage with a region can be performed through a linker as necessary.
- the linker that links the A chain and B chain to the binding region is a peptide linker having a length of 10 amino acids or more.
- the linker in the present invention is not particularly limited as long as the specificity and binding characteristics of the binding region are not hindered.
- a GS4 (Gly + Ser x4, GSSSSGSSSS) linker represented by SEQ ID NO: 2 can be mentioned.
- association of the A chain and the B chain means a state in which the A chain and the B chain interact in a specific region of each other.
- the specific regions of each other are usually one or a plurality of amino acid residues that are subjected to association, and are preferably amino acid residues that approach and participate in the interaction during the association.
- the interaction includes a case where amino acid residues approaching at the time of association form a hydrogen bond, an electrostatic interaction, a salt bridge, and the like.
- the present invention includes a complex formed by covalently binding the bispecific polypeptide of the present invention and a drug, an ADC (antibody-drug conjugate) complex.
- a drug an ADC (antibody-drug conjugate) complex.
- systemic administration of drugs can cause unacceptable levels of toxicity not only to tumor cells to be eliminated but also to normal cells (Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986): 603 -05).
- antibody-drug conjugates are used to locally deliver drugs to pursue maximum efficacy while minimizing toxicity and to kill or inhibit tumor cells in cancer therapy (Syrigos and Epenetos (1999) Anticancer Research 19: 605-614; U.S. Pat. No. 4,975,278).
- the bispecific polypeptide of the present invention has a binding property to the corresponding antigen or ligand, while many drug molecules do not selectively kill cancer cells, and thus are difficult to use for cancer treatment. There is. Therefore, a highly selective and specific drug complex can be obtained by binding of the bispecific polypeptide of the present invention to a highly toxic drug such as a toxin.
- Drugs used in the complex of the present invention include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., “Cancer” Immunol. “Immunother.” 21: 183-87 (1986)).
- bacterial toxins such as diphtheria toxin and phytotoxins such as ricin are included.
- the present invention provides a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region.
- the bispecific polypeptide according to any one of claims 10 to 13, which comprises a second polypeptide linked to a binding region of the nucleic acid encoding the first polypeptide or the second polypeptide. .
- nucleic acid sequences encoding immunoglobulin regions including VH, VL, hinge, CH1, CH2, CH3 and CH4 regions are known in the art (eg, Kabat et al. In SEQUENCES OFIMMUNOLOGICAL INTEREST, Public Health Service NIH , Bethesda, MD, 1991). Using the guidance herein, one of ordinary skill in the art can combine such nucleic acid sequences and / or other nucleic acid sequences known in the art to build on the heterodimeric uteroglobin described herein. Nucleic acid sequences encoding bispecific polypeptides can be made.
- nucleic acid sequence encoding the bispecific polypeptide of the present invention can be determined by one skilled in the art based on the amino acid sequence provided herein and knowledge in the art.
- the traditional method of producing cloned DNA segments encoding specific amino acid sequences it is now readily chemically synthesized and DNA of any desired sequence is produced routinely upon order, The production process is streamlined (GenScript, Eurofin Genomics, etc.).
- the present invention provides a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region.
- a method for producing a bispecific polypeptide of the present invention comprising a second polypeptide linked to a binding region of
- the present invention relates to the pair of amino acid residues of one mutation in the A chain and another mutation in the B chain so that the association between the polypeptides is controlled.
- Methods of producing bispecific polypeptides based on the heterodimeric uteroglobin of the present invention comprising pair substitutions are provided. Examples of such pair substitutions are shown in Tables 1 to 4.
- the A chain and B chain containing pair substitutions are made by modifying a nucleic acid encoding wild-type uteroglobin.
- the present invention relates to a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a second binding region in which the heterodimeric uteroglobin B chain is different from the first binding region.
- a nucleic acid encoding the first polypeptide or the second polypeptide is provided in the bispecific polypeptide of the invention.
- “Modifying a nucleic acid” refers to modifying a nucleic acid so as to correspond to an amino acid residue introduced by “modification” in the present invention.
- nucleic acid encoding an original (before modification) amino acid residue is modified to a nucleic acid encoding an amino acid residue introduced by the modification.
- genetic manipulation or mutation treatment is carried out such that at least one base is inserted, deleted or substituted into the original nucleic acid so as to be a codon encoding the target amino acid residue. That is, the codon encoding the original amino acid residue is replaced by the codon encoding the amino acid residue introduced by modification.
- nucleic acid modification can be appropriately performed by those skilled in the art using known techniques such as site-directed mutagenesis and PCR mutagenesis.
- the nucleic acid in the present invention is usually carried (inserted) into an appropriate vector and introduced into a host cell.
- the present invention provides a vector comprising the nucleic acid of the present invention and a cell comprising the vector of the present invention.
- the vector is not particularly limited as long as it stably holds the inserted nucleic acid.
- the cloning vector is preferably a pBluescript vector (Stratagene), but is commercially available.
- Various vectors can be used.
- An expression vector is particularly useful when a vector is used for the purpose of producing the polypeptide of the present invention.
- the expression vector is not particularly limited as long as it is a vector that expresses a polypeptide in vitro, in E.
- coli in cultured cells, or in an individual organism.
- pBEST vector manufactured by Promega
- E. coli PET vector manufactured by Invitrogen
- pME18S-FL3 vector GenBank Accession No. AB009864
- pME18S vector Mol Cell Biol. 8: 466-472 (1988)
- the insertion of the DNA of the present invention into a vector can be performed by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons). Section 11.4-11.11).
- host cell there is no restriction
- various host cells are used.
- cells for expressing the polypeptide include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), fungal cells (eg, yeast, Aspergillus), insect cells (eg, Drosophila S2). , Spodoptera SF9), animal cells (eg, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells.
- Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation method, electric pulse perforation method (Current protocols in Molecular Biology edit.
- the present invention provides a cell comprising both a vector comprising a nucleic acid encoding each of the first polypeptide and the second polypeptide, and a cell comprising a vector comprising a nucleic acid encoding the first polypeptide, and a second A cell mixture or co-culture of cells comprising a vector comprising a nucleic acid encoding a polypeptide is provided.
- These cells and co-cultures are suitable for suitably preparing the bispecific polypeptide of the present invention.
- an appropriate secretion signal can be incorporated into the polypeptide of interest.
- These signals may be endogenous to the polypeptide of interest or may be heterologous signals.
- the polypeptide is collected when the polypeptide of the present invention is secreted into the medium.
- the polypeptide of the present invention is produced intracellularly, the cell is first lysed, and then the polypeptide is recovered.
- ammonium sulfate or ethanol precipitation acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, Known methods including hydroxylapatite chromatography and lectin chromatography can be used.
- a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region and a second polypeptide in which the heterodimeric uteroglobin B chain is different from the first binding region
- the purified polypeptide may not be a heterodimer simply by mixing together. It was found. See Example 3. Therefore, in the present invention, the first polypeptide and the second polypeptide are obtained by co-transfection of a vector encoding them into a cell (double transfection) or by including each of the vectors encoding them. Cells need to be co-cultured.
- a first polypeptide in which a heterodimeric uteroglobin A chain is linked to a first binding region, and a heterodimeric uteroglobin B chain is linked to a second binding region that is different from the first binding region A method for producing a bispecific polypeptide according to any of claims 1 to 13, comprising a second polypeptide comprising: a) providing a first vector comprising a nucleic acid encoding the first polypeptide; b) providing a second vector comprising a nucleic acid encoding a second polypeptide; c) co-transfecting the cells with the first and second vectors, culturing the resulting transfectants, and d) recovering the heterodimer comprising the first and second polypeptides, A method of producing the bispecific polypeptide is provided.
- the present invention provides a method for producing the bispecific polypeptide of the present invention, a) providing a first cell expressing a first polypeptide; b) providing a second cell expressing the second polypeptide; c) co-culturing the first and second cells; and d) recovering the heterodimer comprising the first and second polypeptides.
- a method of producing the bispecific polypeptide is provided.
- An example of producing wild-type human uteroglobin as a basis for producing the bispecific polypeptide of the present invention is as follows.
- a gene encoding a wild-type human uteroglobin monomer and His tag, FLAG tag, GST tag, etc. is introduced into Escherichia coli, yeast, Brevibacillus, insect cells, mammalian cells and the like.
- a culture solution or cell disruption solution containing uteroglobin is passed through a resin that captures a His tag, and a fraction containing uteroglobin is eluted with a solution containing imidazole.
- the eluate is subjected to solution exchange by dialysis to lower the salt concentration, and then purified by anion exchange chromatography.
- an anion exchange resin is used, and uteroglobin is separated and purified by a salt concentration gradient. After collecting the eluted fraction, it is further purified by gel filtration chromatography to collect highly pure uteroglobin. The eluted fraction is collected and concentrated using an ultrafiltration membrane to obtain a uteroglobin solution.
- the wild-type uteroglobin obtained here is a dimer. The same applies to the production of mutant uteroglobin.
- the present invention in another aspect, relates to a composition comprising a bispecific polypeptide of the present invention and a pharmaceutically acceptable carrier.
- the pharmaceutical composition usually means a drug for treatment or prevention of a disease, or examination / diagnosis.
- the pharmaceutical composition of the present invention can be formulated by methods known to those skilled in the art. For example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection in suspension.
- a pharmacologically acceptable carrier or medium specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative
- a pharmaceutical preparation by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice. The amount of the active ingredient in these preparations is set so as to obtain an appropriate volume within the indicated range.
- a sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.
- aqueous solutions for injection examples include isotonic solutions containing, for example, physiological saline, glucose and other adjuvants (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride).
- a suitable solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.), nonionic surfactant (polysorbate 80 (TM), HCO-50 etc.) may be used in combination.
- oily liquid examples include sesame oil and soybean oil, and benzyl benzoate and / or benzyl alcohol may be used in combination as a solubilizing agent.
- a buffer for example, phosphate buffer and sodium acetate buffer
- a soothing agent for example, procaine hydrochloride
- a stabilizer for example, benzyl alcohol and phenol
- an antioxidant for example, benzyl alcohol and phenol
- composition of the present invention is preferably administered by parenteral administration.
- the composition can be an injection, nasal, pulmonary, or transdermal composition.
- it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dosage of the pharmaceutical composition containing the polypeptide can be set, for example, in the range of 0.0001 mg to 1000 mg per kg body weight at a time. Alternatively, for example, the dose may be 0.001 to 100,000 mg per patient, but the present invention is not necessarily limited to these values.
- the dose and administration method vary depending on the patient's weight, age, symptoms, etc., but those skilled in the art can set an appropriate dose and administration method in consideration of these conditions.
- the polypeptide or complex of the present invention is particularly useful as an active ingredient of a therapeutic or prophylactic agent for cancer, blood vessel-related diseases, and inflammatory diseases.
- Cancers include, but are not limited to: lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma), colon cancer, rectal cancer, colon cancer, breast cancer, Liver cancer, stomach cancer, pancreatic cancer, renal cancer, prostate cancer, ovarian cancer, thyroid cancer, bile duct cancer, peritoneal cancer, mesothelioma, squamous cell carcinoma, cervical cancer, endometrial cancer, bladder cancer, esophageal cancer, head and neck cancer, Nasopharyngeal cancer, salivary gland tumor, thymoma, skin cancer, basal cell tumor, malignant melanoma, anal cancer, penile cancer, testicular cancer, Wilms tumor, acute myeloid leukemia (acute myelocytic leuk)
- Blood vessel-related diseases include arteriosclerosis and vasculitis.
- Inflammatory diseases include acute or chronic inflammatory diseases, specifically including but not limited to: alcoholic hepatitis. Viral hepatitis. Nonalcoholic steatohepatitis. Interstitial pneumonia. Ulcerative colitis. Crohn's disease.
- the present invention also provides a kit for use in the therapeutic or prophylactic method of the present invention, comprising at least the polypeptide produced by the production method of the present invention or the pharmaceutical composition of the present invention.
- the kit may be packaged with a pharmaceutically acceptable carrier, a medium, instructions describing the method of use, and the like.
- the present invention also relates to the use of the polypeptide of the present invention or the polypeptide produced by the production method of the present invention in the manufacture of a therapeutic or prophylactic agent for immunoinflammatory diseases.
- the present invention also relates to the polypeptide of the present invention or the polypeptide produced by the production method of the present invention for use in the therapeutic or prophylactic method of the present invention.
- Example 1 Construction of expression plasmid EcoRI cleavage sequence, DNA sequence encoding mouse IgG ⁇ signal peptide, DNA sequence encoding full-length wild type human uteroglobin monomer (NCBI), linker sequence GSSSSGSSSS, His tag and stop codon sequence, XhoI A gene containing a cleavage sequence was synthesized.
- the synthesized gene and the mammalian expression vector pcDNA6.0 mycHisB (Invitrogen) were cleaved with EcoRI (NEB) and XhoI (NEB).
- the two cleaved products were ligated using a DNA ligation kit mighty mix (TAKARA) to prepare a wild-type uteroglobin mammalian expression plasmid.
- TAKARA DNA ligation kit mighty mix
- the mutant was prepared as follows. Primers containing a sequence in which the codon aag encoding lysine (K), the 51st amino acid of the uteroglobin monomer, was converted to the codon gaa of glutamic acid (E) were synthesized. A PCR reaction was performed using the synthesized primer and a wild-type uteroglobin mammalian expression plasmid as a template to prepare a mutant uteroglobin (K51E) mammalian expression plasmid.
- a mutant uteroglobin (D33K) mammalian expression plasmid was prepared by converting aspartate gac, the 33rd amino acid, into lysine aag. Subsequently, ROBO4 scFv-mutant uteroglobin (D33K) for mammalian expression, in which the mouse IgG ⁇ signal peptide of the mutant uteroglobin (D33K) mammalian expression plasmid is inserted with a single chain antibody scFv sequence against ROBO4 and linker sequence GSSSSGSSSS. A plasmid was prepared.
- wild-type uteroglobin mammalian expression plasmid [1] mutant uteroglobin (K51E) mammalian expression plasmid [2]
- ROBO4 scFv-mutant uteroglobin (D33K) ROBO4 scFv-D33K mammalian expression plasmid [ 4] was prepared.
- a mutant uteroglobin (S66F) mammalian expression plasmid was prepared by converting serine (S) agc, which is the 66th amino acid, into phenylalanine (F) ttc in the same manner. Subsequently, a single chain antibody scFv sequence and a linker sequence GSSSSGSSSS were inserted into the 3 ′ end of the mouse IgG ⁇ signal peptide of the mutant uteroglobin (F28S) mammalian expression plasmid in which the 28th amino acid phenylalanine ttc was converted to serine agc. ROBO4 scFv-mutant uteroglobin (F28S) mammalian expression plasmid was prepared.
- mutant uteroglobin (S66F) mammalian expression plasmid [8] and a ROBO4BOscFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [9] were prepared.
- a mutant uteroglobin (S29K) mammalian expression plasmid was prepared by converting serine (S) agc, the 29th amino acid, into lysine (K) aag. Subsequently, the same method was used to convert the 29th amino acid serine (S) agc to aspartic acid (D) gac and the 62nd amino acid lysine (K) aaa to aspartic acid (D) gac.
- mutant uteroglobin (S29K) mammalian expression plasmid [13] and a ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [14] were prepared.
- Example 2 Preparation of expression product 2-1: Expression Transfection (transduction) was performed using ExpiFectamine293 Transfection kit (Thermo Fisher Scentific). The procedure is as follows. Expi293F cells (Invitrogen) were cultured in 100 mL of Expi293 expression medium (Thermo Fisher Scentific) under conditions of 37 ° C., 125 rpm, and 8% CO 2 at 3.0 ⁇ 10 6 cells / mL.
- ROBO4 scFv-D33K and K51E double transfection was performed as follows. Mix 50 ⁇ g of uteroglobin (K51E) mammalian expression plasmid [2] with 50 ⁇ g of ROBO4 scFv-mutated uteroglobin (D33K) mammalian expression plasmid [4] and add to 5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific). After gently mixing, the mixture was allowed to stand at room temperature for 5 minutes.
- ExpiFectamine293 reagent (Thermo Fisher Scentific) and 5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes.
- Expi293F cell solution 3.0 ⁇ 10 6 cells / mL, 100 mL, and incubate at 37 ° C, 125 rpm, 8% CO 2 for 20 hours, then ExpiFectamine 293 Transfection Enhancer 1 (Thermo Fisher Scentific) 500 ⁇ L and ExpiFectamine 293 transfection enhancer 2 (Thermo Fisher Scentific) 5mL were added 37 ° C., and cultured for one week at 125rpm, 8% CO 2 conditions.
- ROBO4 scFv-F28S and S66F double transfection using mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [10]
- ROBO4 scFv-S29D, K62D and S29K double transfection using mutant uteroglobin (S29K) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (S29D, DK62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [15] was performed.
- ROBO4 scFv-D33K_K51E co-culture was performed as follows.
- Expi293F cells (Invitrogen) were cultured in 37 mL of Expi293 expression medium (Thermo Fisher Scentific) at 37 ° C., 125 rpm, and 8% CO 2 at 3.0 ⁇ 10 6 cells / mL.
- 50 ⁇ g of uteroglobin (K51E) mammalian expression plasmid [2] and 2.5 mL of Opti-MEM I reducing serum medium (Thermo Fisher Scentific) were gently mixed and allowed to stand at room temperature for 5 minutes.
- ExpiFectamine293 reagent (Thermo Fisher Scentific) 135 ⁇ L and Opti-MEM I reducing serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes. Gently add the mixed solution to 50 mL of Expi293F cell solution and incubate at 37 ° C, 125 rpm, 8% CO 2 for 20 hours.
- Expi293F cells (Invitrogen) were cultured in 50 mL of Expi293 Expression medium (Thermo Fisher Scentific) under conditions of 37 ° C., 125 rpm, and 8% CO 2 at 3.0 ⁇ 10 6 cells / mL.
- ExpiFectamine293 reagent (Thermo Fisher Scentific) 135 ⁇ L and Opti-MEM I reducing serum medium (Thermo Fisher Scentific) 2.5 mL were gently mixed and allowed to stand at room temperature for 5 minutes. After 5 minutes, the plasmid solution and ExpiFectamine solution were gently mixed and allowed to stand at room temperature for 20 minutes.
- ROBO4FscFv-F28S and S66F co-culture using a mutant uteroglobin (S66F) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (F28S) (ROBO4 scFv-F28S) mammalian expression plasmid [11]
- ROBO4 scFv-S29D, K62D and S29K co-cultures using mutant uteroglobin (S29K) mammalian expression plasmid and ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) mammalian expression plasmid [16 ] Went.
- the eluted fractions were collected and dialyzed overnight at 50 mM Hepes-NaOH pH 7.5, 4 ° C.
- the dialyzed solution was collected and subjected to anion exchange chromatography.
- the column was 5 mL of HiTrapQ HP (GE healthcare) and eluted with a concentration gradient of 50 mM Hepes-NaOH pH7.5, 0-1M NaCl.
- the eluted fraction was collected and subjected to gel filtration chromatography.
- the column used was HiLoad16 / 600 Superdex75 (GE healthcare), and the mobile phase was 50 mM Hepes-NaOH pH 7.5, 300 mM NaCl.
- the eluted fraction was collected and concentrated with Amicon Ultra15.
- ROBO4 scFv expression product was also prepared [3]. Moreover, the sample preparation before electrophoresis shown by lane [7] in FIG. 4 was performed as follows. ROBO4 scFv-mutant uteroglobin (D33K) [4] expression product and mutant uteroglobin (K51E) expression product [2] were mixed at a molar ratio of 1: 1, and allowed to stand on ice for 1 hour. By the same method, scFv-F28S and S66F mixed at a molar ratio of 1: 1 [12] and scFv-S29D, K62D and S29K mixed [17] were prepared.
- Example 3 Electrophoresis Each solution of expression products [1]-[6], [8]-[11], [13]-[16] obtained in Example 2 was adjusted to 0.2 mg / mL. Dilute with 50 mM Hepes-NaOH pH 7.5, 300 mM NaCl. For [1]-[17], a sample for electrophoresis under reducing conditions was prepared as follows. To 10 ⁇ L of the diluted protein solution, 0.5 ⁇ L of 2-mercaptoethanol and 9.5 ⁇ L of 2 ⁇ Laemmli sample buffer (BIO-RAD) were added and heated at 95 ° C. for 5 minutes.
- BIO-RAD Laemmli sample buffer
- the band position should be confirmed around 20 kDa, but it was observed on the lower molecular weight side. Since the disulfide bond is retained under non-reducing conditions, it cannot be separated purely by molecular weight, and is considered to have shifted to a lower molecular weight side than the original molecular weight position.
- a main band (main band) was present at 72 kDa, a homodimer, and a minor band was present at 36 kDa, a monomer.
- the amount ratio can be determined depending on the degree of staining, so it was found that ROBO4 scFv-D33K mainly contains homodimers and contains a small amount of monomers.
- [5] and [6] have bands around 17, 36, 45 and 72 kDa, and ROBO4 scFv-D33K and K51E mixed have bands around 17, 36 and 72 kD Was.
- 17 kDa is a K51E homodimer
- 36 kDa is a ROBO4 scFv-D33K monomer
- 72 kDa is a ROBO4 scFv-D33K homodimer.
- the molecular weights of the monomers of wild type uteroglobin and mutant uteroglobin are 9.6 kDa
- ROBO4 scFv is 25 kDa
- ROBO4 scFv-mutant uteroglobin F28S
- ROBO4 scFv-F28S is 36 kDa.
- the molecular weights of the wild-type and mutant uteroglobin (S29K) monomers are 9.6 kDa
- ROBO4 scFv is 25 kDa
- ROBO4 scFv-mutant uteroglobin (S29D, K62D) (ROBO4 scFv-S29D, K62D) is 36 kDa.
- wild-type uteroglobin has an intermolecular disulfide bond and forms a homodimer.
- [1], [3], [13], and [14] bands were observed in the molecular weight of the monomer.
- the bispecific polypeptide of the present invention is based on uteroglobin, it has a lower molecular weight than a general bispecific antibody. Therefore, high tissue permeability can be expected, and since there is no Fc region, an unexpected effect can be suppressed. Therefore, it is highly expected that the tool will be a tool for creating a highly safe pharmaceutical product.
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Abstract
L'invention concerne un polypeptide bispécifique de faible masse moléculaire. Une uteroglobine hétérodimère est préparée en modifiant, pour former une utéroglobine de faible masse moléculaire, une région Fc d'un anticorps bispécifique construit à partir d'un anticorps naturel, et en amenant un monomère d'utéroglobine à muter, et un polypeptide bispécifique est construit en l'utilisant.
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US20130189735A1 (en) * | 2009-01-19 | 2013-07-25 | Luciano Zardi | Process for engineering polyvalent, polyspecific fusion proteins using uteroglobin as skeleton and so obtained products. |
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US20130189735A1 (en) * | 2009-01-19 | 2013-07-25 | Luciano Zardi | Process for engineering polyvalent, polyspecific fusion proteins using uteroglobin as skeleton and so obtained products. |
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