KR20100043593A - Biotinylated biologically active molecule-antibody covalent complex in vitro - Google Patents

Abstract

PURPOSE: A complex in which biologically active molecule is bound to an antibody through biotin derivative is provided to improve stability of molecule. CONSTITUTION: A biotinylated biologically active molecule-antibody complex contains a biotin derivative, a biologically active molecule conjugated to the biotin derivative, an antibody bound to the biotin derivative, or fragment thereof. The biotin derivative contains two or more carboxyl group or carboxy group and amine group at veleric acid site. A method for preparing biotinylated biologically active molecule-antibody complex comprises: a step of preparing antibody or fragment connected to a linker peptide; a step of connecting biologically active molecule to biotin derivative; and a step of conneting the linker peptide with biotin derivative.

Description

Biotinylated biologically active molecule-antibody covalent complex in vitro}

The field of technology described herein relates to constructs in which a biologically active molecule is covalently bound to an antibody in vitro .

Recently, antibodies have been used a lot for the purpose of treating diseases. Antibodies are advantageous for mass expression and production because they are very stable both in vitro and in vivo and have long half-lives. In addition, since the antibody has a dimer structure inherently, the adhesion to the antigen is very high.

On the other hand, peptides having a high binding specificity for the antigen is excellent in therapeutic efficacy and can be usefully used in therapy with antibodies. In addition to the peptides, there are also substances useful for physiological activity that can be used for therapeutic purposes. For example, sugars and lipids exhibit inherent biological activity in vivo.

However, since peptides and bioactive substances of relatively small size are very unstable in vivo and have very short half-lives, a separate means for increasing stability and half-life is essential for use in medicine.

The content described herein is for the simple covalent attachment of biologically active molecules such as peptides to proteins such as antibodies and the like in vitro .

In addition, it is to stabilize the biologically active molecules such as peptides through this method so that the function can be stably exhibited in the body.

Complexes disclosed herein, biotin derivatives; Biologically active molecules bound to the biotin derivatives; And a biotinylated biologically active molecule-antibody complex comprising an antibody or fragment thereof bound to the biotin derivative.

Another complex disclosed herein is a biotin derivative; Biologically active molecules bound to the biotin derivatives; Linker peptides bound to the biotin derivatives; And a biotinylated biologically active molecule-antibody complex comprising an antibody or fragment thereof bound to the linker peptide.

The method disclosed herein is a method of preparing the biotinylated biologically active molecule-antibody complex as described above, which comprises obtaining an antibody or fragment thereof to which a linker peptide is linked, linking the biologically active molecule to a biotin derivative, and And covalently binding the linker peptide in vitro .

Also described herein are biotin derivatives; Biologically active molecules bound to the biotin derivatives; And a biotinylated biologically active molecule-antibody complex comprising an antibody or fragment thereof bound to the biotin derivative is disclosed.

Through the biotinylated biologically active molecule-antibody complex disclosed herein and a method for producing the same, it is possible to stabilize a biologically active but unstable molecule such as a peptide, and to simultaneously perform the biological activity of an antibody and the activity of various biologically active molecules such as peptides. Can enjoy.

The biotinylated biologically active molecule-antibody complexes disclosed herein may have the structure of FIG. 1. 1 shows that the biologically active molecule and the antibody are each bound to a biotin derivative. Another biotinylated biologically active molecule-antibody complex disclosed herein may have the form of FIG. 2. Referring to FIG. 2, it can be seen that the biologically active molecule and the antibody are respectively bound to the biotin derivative, but the antibody is not directly bound to the biotin derivative but is indirectly bound by the linker peptide.

As used herein, "biotin" refers to a compound that is a type of vitamin. Biotin can occur in eight different stereoisomeric forms. For example, D-(+)-biotin of formula (1), that is, (3aS, 4S, 6aR) -2-oxohexahydrocyeno [3,4-d] -imidazole-4- valeric acid may be mentioned. .

"Biotin derivative" means a compound that is partially modified with biotin. Biotin derivatives contain two or more carboxyl groups (-COOH) that can be used for amide bond reactions by modifications through specific chemical reactions to valeric acid moieties, or simultaneously include carboxyl and amine groups (-NH ₂ ). Can be used. One of the carboxyl groups or amine groups may be bound to a linker peptide in which a biologically active molecule is bound and the other carboxyl group is bound to an antibody or fragment thereof. As a biotin derivative, a biotin in which aspartate or glutamate is bonded to a carboxyl group of biotin, and sulfo-N-hydroxysuccimido (sulfo-NHS) further added to sulfo- NHS-Aspartateyl Biotin (Sulfo-NHS-Aspartatyl Biotin) and Sulfo-NHS-Glutamatyl Biotin are mentioned. Among them, the chemical formula of aspartatel biotin is as follows.

As another example of a biotin derivative, a biotin in which a lysin is bound to a carboxyl group of the biotin, and 6-Ahx-lysinyl biotin (6-Ahx-lysinyl biotin further added to 6-aminohexanoic acid (6-Ahx) -Ahx-Lysinyl Biotin). Among them, the chemical formula of 6-Ahx-lysinyl biotin is as follows.

In addition, biotin derivatives comprising two or more carboxyl groups or a carboxyl group and an amine group simultaneously by modification through a specific chemical reaction in the valeric acid site of biotin may be used as biotin derivatives for complexes described herein. Can be used.

As used herein, "biologically active molecule" is a generic concept that refers to a molecule that exhibits useful activity to a living body when administered in vivo. This includes, but is not limited to, peptides, sugars, lipids, and the like. Biologically active peptides can be peptides that affect the progression of various diseases. For example, it may be a peptide that inhibits factors that promote angiogenesis of cancer tissue. If the antibody is also an antibody that inhibits neovascularization of the same cancer tissue, each activity of the antibody and the peptide may be simultaneously exerted, thereby producing a synergistic effect.

When the biologically active molecule is a peptide, the biotin derivative by an amide bond between an amide bond between the terminal -COOH groups of the terminal -NH ₂ group and a biotin derivative of a peptide, or a -COOH group and the terminal ends of the peptide derivative of biotin -NH ₂ group And a complex between a biologically active peptide can be formed. The complex between the biotin derivative and the biologically active peptide is named "Kaleido" for convenience.

A "biologically active sugar" is a sugar having biological activity, which may be a monosaccharide or a polysaccharide. The monosaccharide may be pentose sugar or hexose sugar. Monosaccharides may include, but are not limited to, D or L hexose, allose, altrose, glucose, mannose, gallos, idose, galactose, or talos. It may be, but is not limited to, D or L pentose, ribose, arabinose, xylose or lyxose. It may also be a modified monosaccharide. For example, it may be a deoxy sugar, an alkylated sugar, or a di- or trisulfated monosaccharide. Various polysaccharides using the above-mentioned monosaccharides as binding units can be used. Biologically active sugars may, for example, prevent or treat antithrombotic activity, unstable angina, stenosis after angioplasty or insertion of vascular prostheses, or thromboembolic pathologies associated with diabetes. You may have

When the biologically active molecule is a sugar, hydrogen or a hydroxyl group (-OH) at any one or more carbon positions of the sugar carbons may be bonded to the biotin derivative by being substituted with a biotin derivative via a -NH ₂ group. As an example, pentasaccharides of the following general formula (4) may be mentioned.

In the above formula, R ₁ , R ₂ , R ₃ , R ₄ mean "-NH-biotin derivative".

The -COOH group of the biotin derivative may be linked through an amide bond between the -NH ₂ groups of the R ₁ , R ₂ , R ₃ , and R ₄ .

A "biologically active lipid" is a lipid that possesses biological activity, and examples of the representative lipids include, but are not limited to, phospholipids, sphingomyelin, galactolipids, and the like. The lipids exhibit various activities such as anticancer and anti aging.

The lipid can be linked to the biotin derivative by an amide bond between the -NH ₂ residue of the lipid and the -COOH residue of the biotin derivative, as in the above peptide. In addition, as in the case of the sugar, it is possible to form an amide bond with the -COOH group of the biotin derivative through the mediation of -NH ₂ instead of hydrogen of any one of several carbon atoms of the lipid.

As described above, the complex of the biologically active molecule and the biotin derivative can be prepared in vitro . In other words, since no in vivo synthesis is required at all, the entire complex preparation process can be performed simply and easily.

As used herein, "antibody" refers to a specific antibody that exhibits specificity for a particular antigen. For example, it includes various antibodies administered to a living body for the treatment of various diseases. It may also be an antibody administered to a living body for diagnosis of various diseases. If the antibody and the biologically active molecule act on the same disease course, they can have a synergistic effect in the treatment or diagnosis of the disease. It may also be an antibody administered to a living body for the sole purpose of enhancing the stability of the biologically active molecules bound together.

As used herein, the term "antibody or fragment thereof" refers to an antibody or fragment thereof in the form of a full body. For example, fragments of antibodies include Fab, F (ab '), F (ab') 2, Fv, scFv, diabody, and the like. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage of intact antibodies can result in Fab or F (ab ′) 2 fragments, respectively. Fab has one antigen binding site as a structure having the variable region of the light and heavy chains, the constant region of the light chain and the first constant region of the light chain (CH1). F (ab ') differs from Fab in that it has a hinge region that includes one or more Cysteine residues at the C terminus of the heavy chain CH1 domain. In the F (ab ') 2 antibody, cysteine residues in the hinge region of Fab' form a disulfied bond. Fv is a minimal antibody having only the variable region of the heavy chain and the variable region of the light chain.

In the present specification, the "linker peptide" is a peptide that connects a biotin derivative and an antibody, and the sequence thereof is not limited. Examples of linker peptides include carboxylase enzymes or fragments thereof. The carboxylase enzyme is acetyl CoA carboxylase-1, Acetyl CoA carboxylase-2, Pyruvate carboxylase, Propionyl coei carboxyl. At least one selected from the group consisting of Propylyl CoA carboxylase and 3-Methylcrotonyl CoA carboxylase. For example, the linker peptide may be composed of about 20 to 70 amino acids. If more than 70 amino acids can interfere with the original function of the antibody and kaleido to be linked, because less than 20 amino acids because the mutual interference between the antibody and the kaleido appears. Amide linkage between -NH ₂ residue of lysine and -COOH group of biotin derivative in amino acid sequence consisting of conserved -Alanin-Methionin-Lysin-Methionin-conserved of linker peptide Is connected. On the other hand, enzymes can be used for the amide bond. Examples of enzymes include Biotin protein ligase. The linkage between the linker peptide and the antibody or fragment thereof is obtained using a gene recombination technique to obtain a sequence linking the DNA sequence encoding the linker peptide with the cDNA sequence encoding the antibody or fragment thereof and inserting the same into a vector and expressing in a host cell. It may be obtained by, but not limited to. Proteins in the form of fused linker peptides with antibodies or fragments thereof are referred to as "D-body" for convenience.

As used herein, the term "biotinylation" means a combination of biotin or a derivative thereof. The so-called "Kaleido", a biotinylated biologically active molecule, and the antibody or fragment thereof, called "D-body", to which a linker peptide is bound, are linked to each other by amide bonds. 3 and 4 show the aspect in which the kaleido and D-body are bonded via amide bonds. The amide bond may be performed by an enzyme such as, for example, biotin protein ligase, but is not limited thereto. As such, since Kaleido and D-body can be easily bound in vitro and no in vivo expression process is required, the process for preparing an active molecule-antibody complex is easy and simple. This can save significant time and money.

Hereinafter, a method for preparing a biotinylated biologically active molecule-antibody complex will be described in detail.

First we look at the manufacturing process of D-body. Genetic recombination techniques can be used to obtain an antibody or fragment thereof to which a linker peptide is linked. For example, a DNA sequence encoding a linker peptide can be linked to the 3 'end of the antibody heavy chain cDNA sequence using genetic recombination techniques. Of course, instead of the antibody heavy chain cDNA sequence, a gene corresponding to the Fc region including the antibody heavy chain dimer construct may be coupled. The recombinant DNA sequence thus prepared is inserted into a vector. The vector can then be introduced into a host cell and then the cells can be cultured to obtain a fusion protein.

As used herein, a "vector" is a means for expressing a gene of interest in a host cell, including but not limited to plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Examples of vectors include expression vectors comprising membrane targeting or secretory signal sequences or leader sequences in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers. Vectors can be prepared in various ways according to methods known in the art for that purpose. The expression vector may comprise a selection marker for selecting a host cell containing the vector. In addition, replicable expression vectors may include the origin of replication.

The host cell of the vector can be substantially any cell useful for expression vectors. For example, it may be a higher eukaryotic host cell, such as a mammalian cell, or a lower eukaryotic host cell, such as a yeast cell. It may also be a prokaryotic cell, such as a bacterial cell.

Kaleido, on the other hand, is obtained by combining either -COOH or -NH ₂ of a biotin derivative with -NH ₂ or -COOH of a biologically active molecule. It can be bound by simple chemical reactions.

There is then a need for a process of combining the prepared D-body with the prepared kaleido. In Kaleido, a complex is formed by forming an amide bond with the -NH ₂ residue of lysine in the conserved -alanine-methionine-lysine-methionine- amino acid sequence of the D-body that does not bind a biologically active molecule. For convenience, the complex is referred to as "Kaleidobody". The amide bond can be carried out by enzymatic treatment. Examples of the enzymes include biotin protein ligase.

The pharmaceutical compositions disclosed herein are prepared by mixing a biotinylated biologically active molecule-antibody complex with a desired degree of purity with a physiologically acceptable carrier, diluent or stabilizer, and the like to provide a shelf life of an aqueous solution, lyophilized or other dry preparation. (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Physiologically acceptable carriers, diluents or stabilizers should be safe at the dosages and concentrations applied, phosphate, citrate And buffers such as other organic acids; antioxidants including ascorbic acid and methionine; (octadecyldimethylbenzyl ammonium chloride; alkyl parabens such as methyl or propyl parabens; catechol; nesorcinol; cyclohexanol; 3-penta Preservatives such as knoll; and m-cresol); low molecular weight polypeptides; serum albumin, gelatin or immuno- Proteins such as globulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, polysaccharides and other carbohydrates; chelating agents such as EDTA.

The compositions referred to herein may comprise one or more active ingredients when necessary for treatment, and it is preferred that the active ingredients do not counteract the activity of each other. The auxiliary active ingredient may be present in the pharmaceutical composition in an amount effective for the desired purpose.

The active ingredient is for example in a colloidal drug delivery system (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example coacervation techniques or interfacial polymerization For example, it can be enclosed in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules. This technique is disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980). Drugs used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. On the other hand, sustained release formulations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US Pat. No. 3,773,919), L Injection of a copolymer of glutamic acid with ethyl-L-glutamate, a non-degradable ethylene-vinyl acetate, a degradable lactic acid-glycolic acid copolymer, for example LUPRON DEPOT ™ (lactic acid-glycolic acid copolymer and leuprolide acetate) Possible microspheres), and poly-D-(-)-3-hydroxybutyric acid.

The pharmaceutical composition according to the invention can be administered by intravenous administration as a bolus, or by prolonged sequencing by intramuscular, intraperitoneal, intramedullary, subcutaneous, intraarticular, synovial, intramedullary, oral, topical or inhalation routes. It can be administered to a mammal, preferably a human, by known methods such as irrigation. Intravenous administration is preferred as the antibody type preparation.

For the prophylaxis or treatment of a disease, suitable dosages of the pharmaceutical compositions herein can be determined by the type, severity and course of the disease to be treated as defined above, whether the pharmaceutical composition administered is prophylactic or therapeutic, It will depend on the therapy, the patient's clinical history and response to the drug, and discretion and the attending physician. The drug is suitably administered to the patient at one time or through a series of treatments. For example, depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (eg 0.1 to 20 mg / kg) of the antibody is administered to a patient by one or more separate or continuous irrigation Initial candidate dose for. Typical daily dosages are about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days, depending on the condition, treatment continues until the desired signs of disease are suppressed. However, other dosages may be effective. The progress of this therapy can be easily monitored by conventional techniques and assays.

Hereinafter, one example of the complexes described herein will be described in more detail, but it will be apparent to those skilled in the art that this does not limit the scope of the present invention.

1. Preparation and Screening of Antibodies

 The antibody may be a monoclonal antibody. Antibodies can be prepared according to methods well known in the art. For example, mouse monoclonal antibodies can be prepared by the methods described in Schwaber et al., Which are hereby incorporated by reference in their entirety (Schwaber, J and Cohen, EP, "Human x Mouse Somatic Cell Hybrid Clones"). Secreting Immunoglobulins of Both Parental Types, "Nature, 244 (1973), 444-447).

On the other hand, individual monoclonal antibodies can be screened based on the desired binding capacity using a typical Enzyme-Linked ImmunoSorbent Assay (ELISA) format. Functional assays such as molecular interactions or cell-based assays for the conjugates can be screened for desired activity. The affinity (Kd values) is then tested for the selected members based on strong activity.

The final selected clones can be humanized. Humanization processes are well known in the art. For example, it may be humanized by a method disclosed in Almagro et al. (Almagro, J. C. and Fransson, J., “Humanization of antibodies,” Frontiers in Bioscience, 13 (2008), 1619-1633).

In addition to humanized antibodies, chimeric or fully human antibodies can be prepared and used. Methods of making chimeric antibodies and fully human antibodies are well known in the art.

2. Screening of Peptides

Peptides can be screened, for example, from known phage libraries.

3. Manufacture of D-body

Prepare a DNA sequence encoding the antibody prepared in 1. above. In addition, a gene corresponding to a site that binds to biotin is prepared from the cDNA sequence encoding acetyl coei carboxylase-1. The two genes prepared above are combined using genetic recombination techniques. Insert the recombinant DNA sequence into the vector. After introducing the vector into the host cell, the cells are cultured to prepare a fusion protein D-body.

4. Preparation of Biotin Derivatives

After biotin and lysine are combined, 6-Ahx is further added thereto to prepare 6-Ahx-lysinyl biotin of the following Chemical Formula 3.

 (Formula 3)

5. Kaleido Production

The biologically active peptide obtained in the above 2 is chemically attached in vitro to one amine group of the biotin derivative having one carboxyl group and one amine group on the valeric acid site of the original biotin prepared in the above 4.

6. Kaleido body production

The D-body prepared in 3. and the kaleido prepared in 5. are covalently bound through an enzymatic reaction using a biotin protein ligase.

1 schematically shows a complex in which a biologically active molecule and an antibody are bound to a biotin derivative.

FIG. 2: Schematic illustration of a complex in which an antibody is bound to a biotin derivative via a linker peptide, wherein the biologically active molecule and the antibody are bound to a biotin derivative.

3 shows the manner in which the biotin derivative to which the biologically active molecule is bound and the linker peptide to the bound antibody.

4 schematically shows the coupling between the Kaleido and D-bodies.

Figure 5 details the coupling between the Kaleido and D-bodies.

Claims (17)

Biotin derivatives; Biologically active molecules bound to the biotin derivatives; And Biotinylated biologically active molecule-antibody complex comprising an antibody or fragment thereof bound to the biotin derivative. The method of claim 1, The biotin derivative is a biotinylated biologically active molecule-antibody complex, characterized in that it comprises two or more carboxyl groups at the valeric acid moiety of biotin, or a carboxyl group and an amine group at the same time. The biotin derivative according to claim 1, wherein the biotin derivative is a biotin derivative in which aspartate, glutamate, or lysine is bonded to a carboxyl group, or a biotin derivative in which sulfo-N-hydroxysuccimido or 6-aminohexanoic acid is further added thereto. A biotinylated biologically active molecule-antibody complex. The biotinylated biologically active molecule-antibody complex of claim 1, wherein the antibody or fragment thereof is mediated by a linker peptide rather than directly binding to the biotin derivative. The biotinylated biologically active molecule-antibody complex of claim 4, wherein the linker peptide consists of about 20 to about 70 amino acids. 6. The biotinylated biologically active molecule-antibody complex according to claim 5, wherein the linker peptide is a carboxylase enzyme or fragment thereof. 7. The carboxylase enzyme of claim 6, wherein the carboxylase enzyme is acetyl coa carboxylase-1, acetyl coa carboxylase-2, pyruvate carboxylase, propionyl coa carboxylase and 3-methylcrotonyl coa carboxylase. At least one selected from the group consisting of biotinylated biologically active molecule-antibody complexes. The biotin derivative of claim 4, wherein the linker peptide and the biotin derivative are amine residues and biotin of the conserved -alanine-methionine-lysine-methionine- amino acid sequence of the linker peptide. A biotinylated biologically active molecule-antibody complex, characterized by being linked by an amide bond between the terminal carboxyl groups of the derivative. The biotinylated biologically active molecule-antibody complex according to claim 4, wherein the linker peptide and the antibody are obtained in linkage with each other by expression of recombinant DNA obtained by gene recombination. The biotinylated biologically active molecule-antibody complex of claim 1, wherein the biologically active molecule is a biologically active peptide, a biologically active sugar, or a biologically active lipid. A method for preparing a biotinylated biologically active molecule-antibody complex according to claim 4, Obtaining an antibody or fragment thereof to which a linker peptide is linked, Linking the biologically active molecule to the biotin derivative, and A method for producing a biotinylated biologically active molecule-antibody complex comprising linking the biotin derivative with the linker peptide. 12. The method of claim 11, wherein the biotin derivative comprises at least two carboxyl groups at the valeric acid site of biotin, or simultaneously contains a carboxyl group and an amine group. The method of claim 11, wherein the obtaining of the antibody or fragment thereof to which the linker peptide is linked, A biotinylated biologically active molecule, characterized in that a fusion protein is obtained by inserting a DNA sequence linked with a DNA sequence encoding a linker peptide and a cDNA sequence encoding an antibody or fragment thereof into a vector and then introducing the vector into a host cell. Method for producing antibody complex. The method of claim 11, wherein linking the biologically active molecule to a biotin derivative, The biologically active molecule is a biologically active peptide, wherein the biotinylated biologically active molecule-antibody is characterized by being linked by an amide bond between the terminal amine group or carboxyl group of the peptide and the terminal carboxyl group or amine group of the biotin derivative. Method for preparing a composite. The method of claim 11, wherein the linking of the biotin derivative and the linker peptide, Characterized in that the terminal carboxyl group of the biotin derivative is linked by an amide bond with an amine residue in the conserved -alanine-methionine-lysine-methionine-amino acid sequence of the linker peptide. Method for preparing biotinylated biologically active molecule-antibody complex. 16. The biotinylated biological according to claim 15, wherein the amine group in the conserved -Alanin-Methionine-Lysin-Methionine- amino acid sequence of the linker peptide is an amine group of lysine. Method for preparing an active molecule-antibody complex. A pharmaceutical composition comprising the biotinylated biologically active molecule-antibody complex according to any one of claims 1 to 10 as an active ingredient.

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