Modified antibodies and radioactive metal labeled antibodies

Abstract

The present invention relates to a labeling technology that is easily metabolized in the liver after being administered to a patient without reducing the function of the antibody, and reduces the accumulation of radionuclides in organs such as the liver, and provides a modified antibody comprising an IgG antibody and an IgG binding peptide bound thereto. The IgG binding peptide comprises an amino acid sequence consisting of 13 to 17 amino acid residues such as GPDCAYH(Xaa1)GELVWCTFH (wherein Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid), and is connected at the N-terminus of the IgG binding peptide via a modified linker to a compound represented by the following formula (II-1) at the position of its lysine residue.

Description

Technical Field

[0001] The present invention relates to the modification and radioactive metal labeling of IgG antibodies. Background Art

[0002] Antibodies are used for the detection of target molecules in various research and development because of their specificity for target molecules. In the medical field, antibodies that can specifically bind to affected areas such as cancer are labeled with radionuclides and administered into the body to perform diagnosis using single photon emission computed tomography (SPECT) and positron emission tomography (PET). In addition, antibodies labeled with radionuclides are also attracting attention as pharmaceuticals for treating diseases.

[0003] As a method for labeling antibodies with radionuclides, a method in which a chelating compound is combined with an antibody and the chelating compound is loaded with a radioactive metal element is being used (Non-Patent Document 1). The combination of the above-mentioned chelating compound is mainly carried out via the amino group of lysine contained in the antibody, the sulfhydryl group of cysteine, and the activated carboxyl group, etc., but even if it has specificity for the functional group, there is no site specificity, so there is a problem of reducing the antibody activity due to modification of the antigen binding site of the antibody, and it is difficult to control the number of the combined compound.

[0004] In order to overcome the above problems, antibodies with specific functional groups introduced in a site-specific manner have been used to modify the antibodies. For example, non-natural amino acids (non-patent documents 2 to 4) and free cysteine ​​(non-patent documents 5 to 6) are introduced into specific sites by genetic engineering modification, so that modification at specific sites can be achieved. Although such site-specific antibody modification techniques are being continuously developed, in most cases, it is necessary to modify the antibody itself in the form of antibody engineering. Considering that the antibody function is reduced and the development cost is high due to the modification, it cannot necessarily be considered a favorable method.

[0005] Therefore, as a method capable of modifying antibodies specifically and simply, the following method has been proposed: by linking a ligand to an IgG-binding peptide that has binding properties to a specific site of the Fc domain of an IgG antibody, the IgG-binding peptide is allowed to bind to the antibody, thereby eliminating the need to change the sequence of the antibody molecule and making it possible to label IgG with a radioactive metal nuclide without causing a decrease in function associated with genetic modification of the antibody molecule (Patent Documents 1 and 2).

[0006] On the other hand, when low-molecular-weight polypeptides labeled with radioactive metal nuclides are administered to a living body, radioactivity is observed in the kidneys for a long period of time from the early stage of administration. Therefore, when radioactive metal-labeled antibodies are administered to a living body, there is a possibility of radiation exposure and renal damage to the kidneys. However, radioactive-labeled compounds that reduce nonspecific renal accumulation by using specific low-molecular-weight polypeptides have been proposed (Patent Documents 3 and 4, Non-Patent Documents 7 and 8).

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2017 / 217347 Pamphlet

[0010] Patent Document 2: International Publication No. WO2016 / 186206 Pamphlet

[0011] Patent Document 3: International Publication No. WO2013 / 081091 Pamphlet

[0012] Patent Document 4: International Publication No. 2017 / 150549 Pamphlet

[0013] Non-patent literature

[0014] Non-patent document 1: Rodwell, JD et al., Proceedings of the National Academy of Sciences of the United States of America, 1986, 83, pp. 2632-2636

[0015] Non-patent document 2: Axup, JY et al., Proceedings of the National Academy of Sciences of the United States of America, 2012, 109, pp. 16101-16106

[0016] Non-patent document 3: Tian, ​​F. et al., Proceedings of the National Academy of Sciences of the United States of America, 2014, 111, pp. 1766-1771

[0017] Non-patent document 4: Zimmerman, ES et al., Bioconjugate chemistry, 2014, 25, pp. 351-361

[0018] Non-patent document 5: Shen, BQ et al., Nature biotechnology, 2012, 30, pp.184-189

[0019] Non-patent literature 6: Bernardes, GJ et al., Nature protocols, 2013, 8, pp. 2079-2089

[0020] Non-patent document 7: Arano, Yasushi, et al., "Chemical design of radiolabeled antibody fragments for low renal radioactivity levels." Cancer research 59.1 (1999): 128-134.

[0021] Non-patent literature 8: Uehara, Tomoya, et al., "Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels." Bioconjugate chemistry 18.1 (2007): 190-198. Summary of the invention

[0022] However, when radioactive metal-labeled antibodies are administered into the body, high accumulation in organs with a well-developed reticuloendothelial system, such as the liver and spleen, has been confirmed, and there is concern that unnecessary radiation exposure may be caused to normal organs of the patient. Although the techniques of Patent Documents 3 and 4 reduce kidney accumulation, there is concern that antibody function may be reduced due to binding to Fab fragments.

[0023] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a labeling technology that can reduce the accumulation of radionuclides in organs such as the liver after administration to a patient and is easily metabolized in the liver without reducing the function of the antibody.

[0024] One embodiment of the present invention provides a modified antibody comprising an IgG antibody and an IgG-binding peptide that binds to the IgG antibody, wherein the IgG-binding peptide comprises an amino acid sequence represented by the following formula (I) and consisting of 13 to 17 amino acid residues, and a compound represented by the following formula (II-1) is linked to the N-terminus of the IgG-binding peptide via a modified linker at the position of its lysine residue.

[0025] (X 1-3 )-C-(X2)-H-(Xaa1)-G-(Xaa2)-LVWC-(X 1-3 ) (I)

[0026] In formula (I),

[0027] X is each independently any amino acid residue other than cysteine,

[0028] C is a cysteine ​​residue,

[0029] H is a histidine residue,

[0030] Xaa1 is a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid,

[0031] G is a glycine residue,

[0032] Xaa2 is a glutamic acid residue, a glutamine residue or an asparagine residue,

[0033] L is a leucine residue,

[0034] V is a valine residue, and

[0035] W is a tryptophan residue,

[0036] The two cysteine ​​residues may be bound to each other via a disulfide bond or a linker, and the C-terminus may be amidated.

[0037] [Chemical formula 1]

[0038]

[0039] In formula (II-1), n ​​is 0 or 1.

[0040] Another aspect of the present invention provides a radioactive metal-labeled antibody comprising the above-mentioned modified antibody coordinated with a radioactive metal, and a radiopharmaceutical containing the same.

[0041] Another embodiment of the present invention provides a modified IgG binding peptide and an IgG antibody bound to the modified IgG binding peptide, wherein the modified IgG binding peptide comprises: an IgG binding peptide comprising an amino acid sequence consisting of 13 to 17 amino acid residues represented by the above formula (I); and a modified linker represented by the following formula (III) bound to the N-terminus of the IgG binding peptide.

[0042] L1-L2-L3(III)

[0043] In formula (III),

[0044] L1 is a polyethylene glycol linker bound to the N-terminus of the IgG binding peptide,

[0045] L2 is an amino acid sequence consisting of 0 or more and 5 or less amino acid residues,

[0046] L3 is a group having a DBCO (dibenzocyclooctyne) group at the terminal.

[0047] By using the modified IgG-binding peptide and the IgG antibody bound thereto, and preparing a ligand having an azide group capable of binding to DBCO by a click reaction, the ligand can be linked to the modified IgG-binding peptide or the IgG antibody by a click reaction.

[0048] Another aspect of the present invention provides a compound represented by the following formula (II) or a salt thereof.

[0049] [Chemical formula 2]

[0050]

[0051] In formula (II), n is 0 or 1, R 1 and R 2 Both are hydrogen atoms, or one is a hydrogen atom and the other is CO(CH2) m N3 (m is an integer of 1 to 10), or both of them together with the nitrogen atom form a maleimide group or an isothiocyanate group.

[0052] The compound of formula (II) can be ligated via the NR 1 R 2 The group represented by the present invention is combined with the functional group of proteins such as IgG antibodies and IgG binding peptides, and therefore, can be used for radioactive metal labeling of various target molecules including IgG antibodies. Therefore, another embodiment of the present invention provides a compound or a salt thereof formed by combining a polypeptide that can bind to a target molecule with a compound of the above formula (II).

[0053] According to the present invention, since the excretion-promoting linker having a phenylalanine residue and a lysine residue is provided in the Fc region of the antibody, it is possible to obtain a radioactive metal-labeled antibody which promotes the excretion of the radioactive metal from the body through liver metabolism and has excellent in vivo kinetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] [ Figure 1A ] to show [ 111 In]H-FGK-DOTA and 111 This graph shows the mean and standard deviation of the radioactivity distribution (injected dose%, % injected dose (ID)) of In]H-DOTA at 1 hour after administration.

[0055] [ Figure 1B ] to show [ 111 In]H-FGK-DOTA and 111 The graph shows the mean value and standard deviation of the radioactivity distribution (injected amount % (ID)) of In]H-DOTA at 6 hours after administration.

[0056] [ Figure 1C ] to show [ 111 In]H-FGK-DOTA and 111 The figure shows the mean value and standard deviation of the radioactivity distribution (injected amount % (ID)) of In]H-DOTA at 24 hours after administration.

[0057] [ Figure 2A ] to show [ 111 In]H-FGK-DOTA and 111 A graph showing the time course of the radioactivity distribution (%ID) of In]H-DOTA in the liver.

[0058] [ Figure 2B ] to show [ 111 In]H-FGK-DOTA and 111 A graph showing the time course of the radioactivity distribution (%ID) of In]H-DOTA in the kidney.

[0059] [ Figure 2C ] to show [ 111 In]H-FGK-DOTA and 111 A graph showing the temporal changes in the radioactivity distribution (%ID) of In]H-DOTA in the excretion system. DETAILED DESCRIPTION

[0060] <Modified IgG Binding Peptide>

[0061] In one embodiment of the present invention, the modified IgG-binding peptide comprises an IgG-binding peptide consisting of 13 to 17 amino acid residues represented by the above formula (I), and the N-terminus of the IgG-binding peptide is modified with a DBCO linker represented by the above formula (III).

[0062] The IgG-binding peptide of formula (I) is not particularly limited as long as it can bind to IgG, but the IgG-binding peptide described in WO2017 / 217347 is preferred.

[0063] Hereinafter, the IgG-binding peptide used in the present invention will be described in detail.

[0064] "IgG" as used in this specification refers to IgG of mammals, such as primates such as humans and chimpanzees, experimental animals such as rats, mice, and rabbits, livestock animals such as pigs, cattle, horses, sheep, and goats, and pets such as dogs and cats, preferably human IgG (IgG1, IgG2, IgG3, or IgG4). IgG in this specification is more preferably human IgG1, IgG2, or IgG4, or rabbit IgG, and particularly preferably human IgG1, IgG2, or IgG4.

[0065] The IgG-binding peptide used in the present invention comprises an amino acid sequence represented by the above formula (I) and consisting of 13 to 17 amino acid residues, and is capable of binding to human IgG.

[0066] In the above formula (I), X at the N-terminus or C-terminus 1-3 This symbol indicates that 1 to 3 independent and arbitrary amino acid residues X other than cysteine ​​(C or Cys) are consecutive, and the amino acid residues constituting them are identical or different residues, but preferably consist of a sequence of 3 residues that are not all identical. Similarly, X2 indicates that 2 independent and arbitrary amino acid residues X other than cysteine ​​(C or Cys) are consecutive, and the amino acid residues constituting them are identical or different residues, but preferably consist of a sequence of the 2 consecutive amino acid residues being different residues.

[0067] The two cysteine ​​residues of formula (I) can be disulfide bonded to form a cyclic peptide. Generally, in the IgG binding peptide of formula (I), the two outer cysteine ​​residues (excluding Xaa1 when Xaa1 is a cysteine ​​residue) are disulfide bonded. Alternatively, in the peptide of formula (I), the thioether groups in the two outer cysteine ​​residues are connected via a linker represented by the following formula,

[0068] [Chemical formula 3]

[0069]

[0070] The dotted line portion in the above formula represents the portion bonded to the thioether group. This linker is more stable to reduction reactions, etc., than a normal disulfide bond. Therefore, this linker is preferably used when a radioactive metal nuclide such as zirconium that can destabilize a disulfide bond is used.

[0071] In the case of the amino acid sequence of the IgG-binding peptide of formula (I) above having 17 amino acid residues, the amino acid residue X at the 1st and 2nd and 16th and 17th positions from the N-terminus may be deleted, and such a peptide consists of 13 amino acids.

[0072] The term "in the case of 17 amino acid residues" used in this specification is a convenient term for numbering the IgG binding peptide of formula (I) from the 17th position in sequence starting from the N-terminus of the longest amino acid length of 17 residues when the amino acid residues of the peptide are referred to by amino acid numbers.

[0073] In addition, the amino acid residues other than cysteine ​​(C) in the amino acid sequence of the peptide of the above formula (I), that is, the amino acid residues at positions 1 to 3, 5, 6, and 15 to 17 from the N-terminus in the case of 17 amino acid residues, are preferably selected from the following amino acid residues. Here, each capital letter is a single letter description of the amino acid:

[0074] Amino acid residue at position 1 = S, G, F, R or none;

[0075] Amino acid residue at position 2 = D, G, A, S, P, homocysteine ​​or none;

[0076] Amino acid residue at position 3 = S, D, T, N, E or R;

[0077] Amino acid residue at position 5 = A or T;

[0078] Amino acid residue at position 6 = Y or W;

[0079] Amino acid residue at position 15 = S, T or D;

[0080] Amino acid residue at position 16 = H, G, Y, T, N, D, F, homocysteine ​​or none;

[0081] Amino acid residue at position 17 = Y, F, H, M or none.

[0082] Preferred specific examples of the IgG-binding peptide of formula (I) include:

[0083] 1) DCAYH(Xaa1)GELVWCT(sequence number 1);

[0084] 2) GPDCAYH(Xaa1)GELVWCTFH(sequence number 2);

[0085] 13)RGNCAYH(Xaa1)GQLVWCTYH(sequence number 3); and

[0086] 14)G(Xaa2)DCAYH(Xaa1)GELVWCT(Xaa2)H(serial number 4),

[0087] As a particularly preferred example, there can be mentioned 2) GPDCAYH(Xaa1)GELVWCTFH (SEQ ID NO: 2) (wherein Xaa1 is a lysine residue, Xaa2 is homocysteine, and preferably, disulfide bonds are formed between cysteines and / or between homocysteine ​​residues).

[0088] As described above, the IgG-binding peptide of the above formula (I) according to the present invention is characterized in that it has at least two cysteine ​​(C) residues separated in each amino acid sequence, and the cysteine ​​residues are arranged in a manner that a disulfide bond can be formed between these cysteine ​​residues. In the case of a preferred peptide, two cysteine ​​residues are bonded by a disulfide bond to form a cyclic peptide, and 1 to 3 arbitrary amino acid residues other than cysteine ​​may be present on the N-terminal side and the C-terminal side of each cysteine ​​residue. When there are 1 to 3 amino acid residues on the N-terminal side and the C-terminal side of each cysteine ​​residue, the amino acid residues at positions 1 to 2 and 16 to 17 from the N-terminal in the case of 17 amino acid residues are the amino acid residues exemplified above.

[0089] As described above, in the IgG binding peptide used in the present invention, Xaa1 is a protein-constituting amino acid such as a lysine residue, a cysteine ​​residue, an aspartic acid residue, and a glutamic acid residue, as well as a non-protein-constituting amino acid such as diaminopropionic acid and 2-aminosuberic acid, preferably a lysine residue. Xaa1 is preferably modified using a cross-linking agent described later. In this specification, "non-protein-constituting amino acid" refers to an amino acid that cannot be used to constitute a protein in an organism. In order to improve the site specificity when the IgG binding peptide used in the present invention is modified using a cross-linking agent, the IgG binding peptide used in the present invention preferably has no residues identical to Xaa1 in its sequence, or has almost no residues (for example, only 1 or 2). For example, when Xaa1 is a lysine residue, the IgG binding peptide used in the present invention preferably has no residues at all or has almost no lysine residues at positions other than Xaa1 in its sequence.

[0090] For the IgG binding peptide used in the present invention, the binding affinity to human IgG is about 10 times higher, preferably about 50 times higher, and more preferably about 200 times higher than that of other human immunoglobulins (IgA, IgE, IgM). The dissociation constant (Kd) associated with the binding of the IgG binding peptide used in the present invention to human IgG can be determined by surface plasmon resonance spectroscopy analysis (using, for example, the BIACORE system), for example, less than 1×10 -1 M~1×10 -3 M, preferably less than 1×10 -4 M, more preferably less than 1×10 -5 M.

[0091] The IgG binding peptide used in the present invention is bound to the Fc domain of the IgG antibody. The IgG binding peptide used in the present invention is as shown in the embodiments of WO2017 / 217347, in the above-mentioned Xaa1, and the specific region of IgG Fc, i.e., Lys248 residue based on Eu numbering in human IgG Fc (hereinafter, abbreviated as "Lys248" in this specification, equivalent to the 18th residue of human IgG CH2 (SEQ ID NO. 5)) or Lys246 residue (hereinafter, abbreviated as "Lys246" in this specification, equivalent to the 16th residue of human IgG CH2 (SEQ ID NO. 5)), preferably Lys248 is close.

[0092] The IgG binding peptide used in the present invention can be obtained by the method described in, for example, WO2017 / 217347. Specifically, peptide synthesis methods such as conventional liquid phase synthesis, solid phase synthesis, and peptide synthesis based on automatic peptide synthesizers can be used (Kelley et al., Genetics Engineering Principles and Methods, Setlow, JKeds., Plenum Press NY. (1990) Vol. 12, p. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989) WH Freeman Co.; Houghten, Proc. Natl. Acad. Sci. USA (1985) 82: p. 5132; "New Chemistry Experiment Lecture 1 Protein IV" (1992) Japan Biochemical Society, Tokyo Chemical Co-workers). Alternatively, a genetic recombination method using a nucleic acid encoding an IgG binding peptide used in the present invention, a phage display method, etc. can be used to manufacture a peptide. The target peptide can be produced by incorporating DNA encoding the amino acid sequence of the IgG binding peptide used in the present invention into an expression vector, introducing it into host cells and culturing them. The produced peptide can be recovered or purified by conventional methods, such as gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, chromatography such as HPLC, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption.

[0093] In peptide synthesis, for example, amino acids are prepared in which the functional groups of each amino acid (natural or non-natural) other than the α-amino group and α-carboxyl group to be bonded are protected, and a peptide bond forming reaction is performed between the α-amino group and the α-carboxyl group of each amino acid. Usually, the carboxyl group of the amino acid residue at the C-terminus of the peptide is previously bound to a solid phase via a suitable spacer group or linker. The protecting group at the amino terminus of the dipeptide obtained in this way is selectively removed, and a peptide bond is formed with the α-carboxyl group of the next amino acid. Such operations are continuously performed to produce peptides with protected side chain groups, and finally, all the protecting groups are removed and separated from the solid phase. The types of protecting groups, the protection methods, and the details of the peptide binding methods are described in detail in the above-mentioned literature.

[0094] The manufacture using gene recombination method can be carried out, for example, by a method comprising the following steps: inserting the DNA encoding the peptide of the present invention into a suitable expression vector, introducing the vector into a suitable host cell, culturing the cell, and recovering the target peptide from the intracellular or extracellular fluid. The vector is not limited, and there are vectors such as plasmids, phages, clays, phagemids, and viruses. As plasmid vectors, there are no limitations, and plasmids derived from Escherichia coli (e.g., pET22b (+), pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, pBluescript, etc.), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, etc.), and plasmids derived from yeast (e.g., YEp13, YCp50, etc.), etc. can be cited. As phage vectors, there are no limitations, and examples include T7 phage display vectors (T7Select10-3b, T7Select1-1b, T7Select1-2a, T7Select1-2b, T7Select1-2c, etc. (Novagen)), and λ phage vectors (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, λZAPII, etc.). As viral vectors, there are no limitations, and examples include animal viruses such as retrovirus, adenovirus, adeno-associated virus, vaccinia virus, and Sendai virus, and insect viruses such as baculovirus. As cosmid vectors, there are no limitations, and examples include Lorist 6, Charomid9-20, and Charomid9-42. As phagemid vectors, there are no limitations, and examples include pSKAN, pBluescript, pBK, and pComb3H. The vector may contain a regulatory sequence, a screening marker for screening the vector containing the target DNA, a multiple cloning site for inserting the target DNA, etc. in a manner that can express the target DNA. Such regulatory sequences include promoters, enhancers, terminators, SD sequences or ribosome binding sites, replication origins, and poly A sites, etc. In addition, screening markers may use, for example, ampicillin resistance genes, neomycin resistance genes, kanamycin resistance genes, and dihydrofolate reductase genes, etc. The host cells used to introduce the vector are bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, insect cells, animal cells (for example, mammalian cells), and plant cells, etc., and the transformation or transfection to these cells includes, for example, calcium phosphate method, electroporation method, liposome transfection method, particle bombardment (particle gun) method, and PEG method, etc. The culture of the transformed cells is carried out according to the conventional method for culturing the host organism. For example, the culture solution of microorganisms such as Escherichia coli and yeast cells contains carbon sources, nitrogen sources, and inorganic salts that the host microorganism can assimilate. In order to facilitate the recovery of the IgG-binding peptide used in the present invention, the peptide produced by expression is preferably secreted outside the cell.This can be done by binding a DNA encoding a peptide sequence that enables secretion of the peptide from the cell to the 5' end of the DNA encoding the target peptide. The fusion peptide transferred to the cell membrane is cleaved by a signal peptidase, and the target peptide is secreted and released into the culture medium. Alternatively, the target peptide that has accumulated in the cell can be recovered. In this case, the cell is physically or chemically destroyed, and the target peptide is recovered using protein purification technology.

[0095] Therefore, the present invention also relates to nucleic acids encoding the peptides used in the present invention. Here, nucleic acids include DNA or RNA (eg, mRNA).

[0096] When the IgG binding peptide used in the present invention is fused with other proteins, the IgG binding peptide and other proteins can be prepared separately, and then the IgG binding peptide and the protein can be fused using a linker as needed, or it can be produced as a fusion protein by adding a suitable linker as needed using a gene recombination method. In this case, it is preferred to produce the fusion protein in a manner that does not impair the binding property of the IgG binding peptide of the present invention to IgG.

[0097] The above-mentioned IgG binding peptide used in the present invention is preferably modified with a cross-linking agent. As described above, the IgG binding peptide used in the present invention is as shown in the examples described later, in the above-mentioned Xaa1, close to Lys248 or Lys246 based on Eu numbering in a specific region of IgG Fc, i.e., human IgG Fc. Preferably, Lys248. Therefore, by modifying the Xaa1 of the IgG binding peptide used in the present invention with a cross-linking agent, it is cross-linked with IgG, so that a cross-linking structure can be formed site-specifically between the Xaa1 of the IgG binding peptide and Lys248 or Lys246, preferably Lys248 of IgGFc. As described above, the Xaa1 of the IgG binding peptide used in the present invention is modified with a cross-linking agent and various compounds, so that it is cross-linked with IgG, and various compounds can be specifically and simply introduced into IgG. In addition, according to the present invention, the compound of the above-mentioned formula (I) and other compounds can be introduced via the IgG binding peptide, and therefore, compounds of various structures can be introduced into IgG. In addition, the present invention has the advantage that the yield of the obtained product is high and there is no change in the antibody itself, so there is a low possibility of reducing the function of the antibody.

[0098] The IgG binding peptide used in the present invention can also be used for IgG of animals other than humans, preferably mammals. In this case, the site in IgG to which the IgG binding peptide used in the present invention binds can be easily determined by a person skilled in the art who has read this specification, for example, by comparing the sequence of human IgG with the sequence of IgG of other animals.

[0099] In the present invention, "crosslinking agent" is a chemical substance used to connect the IgG binding peptide used in the present invention to IgG Fc via a covalent bond. The crosslinking agent of the present invention can be appropriately selected by a person skilled in the art, and can be a compound having at least two sites capable of binding to a desired amino acid (e.g., a lysine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, 2-aminosuberic acid, or diaminopropionic acid, and arginine, etc.). Examples thereof include, but are not limited to, crosslinking agents preferably containing two or more succinimidyl groups, such as DSG (disuccinimidyl glutarate) and DSS (disuccinimidyl suberate); crosslinking agents preferably containing two or more imidic acid moieties, such as DMA (dimethyl adipimidate dihydrochloride), DMP (dimethyl pimelimidate dihydrochloride), and DMS (dimethylsuberimidate dihydrochloride); and crosslinking agents preferably containing two or more imidic acid moieties, such as DTBP (dimethyl 3,3'-dithiobispropionimidate dihydrochloride), DSP (succinimidyl propionate), succinimidyl propionate, ... propionate)) and other cross-linking agents having SS bonds.

[0100] The IgG-binding peptide modified with a cross-linking agent of the present invention can be produced by the method described in WO2017 / 217437.

[0101] In addition, the IgG-binding peptide used in the present invention may be modified by amidation of the C-terminus for the purpose of improving its stability.

[0102] The IgG binding peptide used in the present invention has a modified linker introduced into the N-terminus. Examples of the modified linker include a modified linker represented by the above formula (III).

[0103] The number of polyethylene glycol (PEG) molecules in the polyethylene glycol (PEG) linker of L1 is not particularly limited, and may be, for example, 1 to 50 molecules, 1 to 20 molecules, 2 to 10 molecules, or 2 to 6 molecules.

[0104] L2 is an amino acid sequence consisting of 0 to 5 amino acid residues, and preferably contains at least cysteine.

[0105] L3 only needs to be a group containing at least a DBCO (dibenzocyclooctyne) group, and can be selected from various commercially available DBCO reagents (for example, DBCO-C6-acid, dibenzylcyclooctyne amine, dibenzylcyclooctyne-maleimide, DBCO-PEG acid, DBCO-PEG-NHS ester, DBCO-PEG-alcohol, DBCO-PEG-amine, DBCO-PEG-NH-Boc, carboxyrhodamine-PEG-DBCO, sulfonylrhodamine-PEG-DBCO, TAMRA-PEG-DBCO, DBCO-PEG-biotin, DBCO-PEG-DBCO, DBCO-PEG-maleimide, TCO-PEG-DBCO, DBCO-mPEG, etc.) bonded to L1 or L2 to form a group. As an example, a group formed by the reaction of DBCO-maleimide represented by the above formula (IV) with the cysteine ​​residue of L2 can be cited.

[0106] The modified linker of formula (III) has DBCO that becomes a substrate for click reaction, and therefore, can be combined with a ligand having an azide group through a click reaction. The click reaction can use known conditions, but it only needs to be maintained at a temperature of 25 to 50° C. in a reaction solvent for 20 to 40 minutes. As a reaction solvent, in addition to buffers such as phosphate buffer, Tris buffer, acetate buffer, and citrate buffer, a mixed solvent in which an organic solvent such as dimethyl sulfoxide is mixed with a small amount of buffer can also be used.

[0107] <IgG antibody>

[0108] In one embodiment, the present invention relates to an IgG antibody combined with a modified IgG binding peptide of the present invention. The IgG antibody can be formed by mixing the modified IgG binding peptide with the IgG antibody and causing a cross-linking reaction between the modified IgG binding peptide and the IgG. Preferably, the amino acid residue of Xaa1 of the IgG binding peptide represented by the above formula (I) can cross-link with Lys248 or Lys246 of IgG Fc, preferably Lys248 in a site-specific manner. The cross-linking reaction can be implemented using the method described in WO2017 / 217437.

[0109] The IgG antibody applicable to the present invention can be any one that contains at least one heavy chain, and is not particularly limited. The molecular weight of the heavy chain is about 50 kDa, and it contains at least an antigen binding region and an Fc region. The heavy chain can be bound to a light chain with a molecular weight of about 25 kDa via a disulfide bond. The molecular weight of the IgG antibody of the present invention can be any one that satisfies the above-mentioned composition, and is not particularly limited, but is preferably about 100 to 170 kDa, and more preferably about 120 to 170 kDa.

[0110] In the present invention, "about" means a numerical range of ±10%.

[0111] The IgG antibody of the present invention is formed by performing a site-specific cross-linking reaction in the Fc region, and therefore, the cross-linking reaction is less likely to have a negative impact on the activity of IgG. In addition, by connecting a modified IgG binding peptide to IgG, new functionality can be added to IgG. Various antibody medicines can also be used in the form of IgG.

[0112] <Ligand>

[0113] In another aspect of the present invention, there is provided a peptide ligand represented by the above formula (II).

[0114] The compound of formula (II) can be obtained by reacting the NR 1 R 2 The group represented by the invention is combined with target molecule elements such as IgG binding peptides, modified IgG binding peptides, IgG antibodies, antibody fragments, and other ligands that can be combined with target molecules, and therefore, it can be used for radioactive metal labeling of various target molecule elements. In addition, the compound of the above-mentioned formula (II) has a peptide chain with phenylalanine residues and lysine residues as the main chain, and the main chain is metabolized in the liver, so the aggregation of radioactive metals to the liver is suppressed, and the excretion in the urine is promoted, and the in vivo dynamics is excellent. Thus, it can be applied to the radiotherapy drug not only using short half-life radioactive metal nuclides, but also using radioactive metal nuclides that release cell-damaging α rays and β rays, and in addition, since the radioactivity that becomes the background of the abdomen in the radioactive image diagnosis is less, therefore, the detection and diagnosis of the lesion site of the abdomen becomes easy.

[0115] In the above formula (II), R 1 and R 2 When the maleimide group is formed together with the nitrogen atom, it can be bonded to the SH group of cysteine. 1 and R 2 In the case where one of the two is a hydrogen atom and the other is a group represented by CO(CH2)4N3, it can be combined with an alkyne such as DBCO by a click reaction. Therefore, by using the modified linker represented by the above formula (III) and using the compound represented by the following formula (II-2) as a ligand, the above-mentioned modified IgG binding peptide, or the IgG antibody bound to the modified IgG binding peptide, can be combined with the ligand represented by the following formula (II-2).

[0116] [Chemical formula 4]

[0117]

[0118] (In formula (II-2), n is 0 or 1).

[0119] The ligand of the present invention may form a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt.

[0120] The acid addition salt may be any of an inorganic acid salt and an organic acid salt.

[0121] Examples of the inorganic acid salt include hydrochlorides, hydrobromides, sulfates, hydroiodides, nitrates, and phosphates.

[0122] Examples of the organic acid salt include citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0123] The base addition salt may be any of an inorganic base salt and an organic base salt.

[0124] Examples of the inorganic base salt include sodium salt, potassium salt, calcium salt, magnesium salt, and ammonium salt.

[0125] Examples of the organic base salt include triethylammonium salt, triethanolammonium salt, pyridinium salt, and diisopropylammonium salt.

[0126] <Modified Antibodies>

[0127] In another embodiment of the present invention, a modified antibody is provided, which comprises an IgG antibody and an IgG-binding peptide that binds to the IgG antibody, wherein the IgG-binding peptide comprises an amino acid sequence represented by the above-mentioned formula (I) and consisting of 13 to 17 amino acid residues, and the N-terminus of the IgG-binding peptide is connected to the compound represented by the above-mentioned formula (II-1) at the position of its lysine residue via a modified linker.

[0128] In the modified antibody of the present invention, it is preferred that the binding site between the lysine residue of the compound represented by the above formula (II-1) and the modified linker is formed by a click reaction between a DBCO group and an azide group. More preferably, a compound obtained by introducing an alkyl azide group having 1 to 10 carbon atoms into the lysine residue of the compound represented by the above formula (II-1) can be used, and it is further preferred to use a compound represented by the above formula (II-2). In addition, at this time, the modified linker is preferably a modified linker represented by the above formula (III). The click reaction can use known conditions, but as long as the temperature in the reaction solvent is maintained at 25 to 50°C for 20 to 40 minutes. As a reaction solvent, in addition to buffers such as phosphate buffer, Tris buffer, acetate buffer, and citrate buffer, a mixed solvent formed by mixing an organic solvent such as dimethyl sulfoxide with a small amount of buffer can also be used.

[0129] <Radioactive metal-labeled antibodies>

[0130] In another aspect of the present invention, there is provided a radioactive metal-labeled antibody in which a radioactive metal is coordinated to the above-mentioned modified antibody.

[0131] Examples of radioactive metal nuclides include 111 In(Indium), 89 Zr (zirconium), 67 / 68 Ga (gallium), 64 Cu (copper), 90 Y(yttrium), 213 Bi (bismuth), 225 Ac(Actinium), 177 Lu (lutetium). The radioactive metal nuclide bound to the IgG binding peptide can be selected according to the use of the radioactive metal labeled antibody of the present invention. For example, the detection and diagnosis of cancer can be used 111 In, 89 Zr, 64 Cu and 67 / 68 Ga, such as PET (Positron Emission Tomography) can be used 89 Zr and 64 Cu, SPECT (Single Photon Emission Computed Tomography) can be used 111 In. For therapeutic use, it can be used 225 Ac and 177 Lu.

[0132] To coordinate the radioactive metal to the modified antibody of the present invention, for example, both can be maintained at 25 to 120° C. for 30 to 180 minutes in a reaction solvent. As the reaction solvent, phosphate buffer, Tris buffer, acetate buffer, citrate buffer, HEPES buffer, etc. can be used.

[0133] <Radioactive Medicine>

[0134] In another aspect of the present invention, a radiopharmaceutical comprising the above-mentioned radioactive metal-labeled antibody is provided.

[0135] The radiopharmaceutical of the present invention can be used as, for example, a nuclear medicine imaging diagnostic agent or a cancer diagnostic agent. In this case, the IgG antibody can be appropriately selected according to the type of cancer, for example, trastuzumab can be used for breast cancer, and cetuximab can be used for colorectal cancer. The nuclear medicine imaging diagnostic agent or the cancer diagnostic agent can be formulated according to conventional methods (see, for example, Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA).

[0136] Example

[0137] Hereinafter, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these contents.

[0138] Example 1: Preparation of IgG Antibody Containing DBCO

[0139] IgG antibodies to which the DBCO-modified IgG-binding peptides shown below were bound were prepared.

[0140] [Chemical formula 5]

[0141]

[0142] 1) Preparation of DBCO-modified IgG-binding peptide

[0143] The amino-PEG5-modified IgG binding peptide GPDCAYHKGELVWCTFH (sequence number 2, two Cys in the molecule were cross-linked via dichloroacetone, and the C-terminus was amidated) in which the amino group at the N-terminus was modified with an acetyl group was synthesized by the Fmoc solid phase synthesis method according to the conventional method. After deprotection, 9.1 mg of the purified IgG binding peptide was dissolved in 100 μL (33.3 mM) of DMSO containing 0.5% pyridine, and 50 μL of DMSO containing 100 mM DBCO-maleimide and 0.5% pyridine (1.5 times the amount based on the molar ratio of the peptide) was added thereto, and it was reacted at room temperature for 30 minutes. Then, 200 μL of DSG (500 mM) dissolved in acetonitrile was added to the peptide solution (final concentration was 286 mM, 30 times the molar equivalent relative to the peptide), and it was reacted at 50°C for 3 hours. The total amount (350 μL) was divided into two portions, diluted in 5 mL of 10% acetonitrile containing 0.1% TFA, and the supernatant after centrifugation was injected into an Inert Sustain (registered trademark) C18 column (7.6 mm 1×250 mm, GLScience), and eluted with a gradient from 10% acetonitrile containing 0.1% TFA to 60% acetonitrile containing 0.1% TFA. The eluate was subjected to mass spectrometry analysis, and after recovering the DBCO-modified IgG binding peptide as the target substance, the organic solvent was removed and then freeze-dried.

[0144] 2) Preparation of IgG antibody containing DBCO

[0145] The modified IgG binding peptide prepared in 1) above was dissolved in DMSO at a concentration of 4 mM, and 25.2 μL of the resulting solution was mixed with 16.8 mL of 20 μM anti-HER2 human antibody (trastuzumab) (Chugai Pharmaceutical) dissolved in 10 mM acetate buffer (pH 5.5), and reacted at 37°C for 3 hours (peptide to antibody molar ratio = 3:1). The DBCO-containing antibody prepared in this way was purified using a cation exchange chromatography column CIM multus SO3-8 (8 mL, BIA separation) in 10 mM acetate buffer (pH 5.5) with a gradient elution from 0 to 0.35 M NaCl. It was confirmed that in addition to unreacted antibodies, a monovalent antibody bound to one peptide and a divalent antibody bound to two peptides were generated. The peak of the monovalent antibody was separated here, and then centrifuged at 3000 g on Vivaspin (molecular weight cutoff of 10000 Da, GE Healthcare), thereby implementing desalting and concentration.

[0146] Example 2: Synthesis of DO3ABn-Phe-Lys((CH2)4-N3)-OH

[0147] DO3ABn-Phe-Lys((CH2)4-N3)-OH (a compound in which n is 0 in formula (II-2)) was synthesized by the following route (wherein the product of step (a) is compound (4b), and the product of step (b) is compound (5b)). 1 A JEOLECS-400 spectrometer (JEOL Ltd.) was used for H-NMR analysis, and a HPLC1200 series 6130 quadrupole LC / MS mass spectrometer (Agilent Technologies Japan, Ltd.) was used for ESI-MS analysis.

[0148] [Chemical formula 6]

[0149]

[0150] Synthesis of Ethyl 5-azidovalerate (Compound (1))

[0151] Ethyl 5-bromovalerate (1 g, 4.78 mmol) was dissolved in N,N-dimethylformamide (DMF, 3 mL), and NaN3 (0.5 g, 7.7 mmol) was added, and stirred at 85°C overnight. The suspension was diluted with water (40 mL) and extracted with diethyl ether (40 mL × 3). The solution was concentrated under reduced pressure and the resulting solution was used directly in the next reaction.

[0152] Synthesis of 5-Azidovaleric acid (Compound (2))

[0153] After adding 1N NaOH aqueous solution (8.2 mL) to the solution of compound (1), methanol was added until the mixture became homogeneous. After stirring at room temperature for 4 hours, the reaction solution was concentrated under reduced pressure, and the obtained aqueous solution was washed with diethyl ether (5 mL). Then, 5% citric acid aqueous solution was added to make the solution acidic, and then extracted with diethyl ether (5 mL×3). After adding Na2SO4 to the organic layer and drying it, the solvent was distilled off under reduced pressure to obtain compound (2) (360 mg, 52.6%).

[0154] 1 H NMR (CDCl3): δ1.62-1.77 (4H, m, CH2), 2.39-2.43 (2H, t, CH2), 3.29-3.33 (2H, t, CH2)

[0155] 2,3,5,6-tetrafluorophenyl 5-azidopentanoate Synthesis of azidovalerate) (compound (3))

[0156] Compound (2) (17.9 mg, 0.125 μmol), 2,3,5,6-tetrafluorophenol (44.0 mg, 0.250 μmol), and N,N-diisopropylethylamine (DIEA, 53.2 μL, 0.312 μmol) were dissolved in CH2Cl2 (2.0 mL), and then 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 48.0 mg, 0.250 μmol) was added under ice cooling. The solution was returned to room temperature and stirred for 1.5 hours, then ethyl acetate (10 mL) was added, and the mixture was washed with a saturated ammonium chloride solution (3 mL×3). After adding Na2SO4 to the organic layer and drying, the solvent was distilled off under reduced pressure to obtain Compound (3) (39.9 mg). The obtained crude product contained 2,3,5,6-tetrafluorophenol, but was used directly in the next reaction.

[0157] Synthesis of DO3ABn-Phe-Lys-OH (Compound (4b))

[0158] Phe-Lys was conjugated to Cl-Trt(2-Cl)-Reisn (Watanabe Chemical Industry, product number: A00187) by peptide extension reaction. t Bu)3DO3ABn-COOH (16-20 mg), 1.3 equivalents of a resin bonded with Phe-Lys, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 2.5 equivalents), 1-hydroxy-7-azabenzotriazole (HOAt, 2.5 equivalents), DIEA (5.0 equivalents), and stirred overnight at room temperature in DMF (200 μL). The mixture was washed 8 times with DMF, then washed 4 times with CH2Cl2, and then the resin was dried under reduced pressure. A mixture of trifluoroacetic acid (TFA) / triisopropylsilane / water = 95 / 2.5 / 2.5 was added to the dried resin, and stirred at room temperature for 3 hours. After the solvent was removed by distillation under reduced pressure, diethyl ether (5 mL) was added to crystallize it. The crystals were collected by filtration, washed with diethyl ether, and then dried under reduced pressure to obtain 7.7 mg (25.7%) of a TFA salt of compound (4b) as white crystals.

[0159] Synthesis of DO3ABn-Phe-Lys((CH2)4-N3)-OH (Compound (5b))

[0160] Compound (4b) was dissolved in DMF (0.5 mL), and compound (3) (7.5 equivalents) and DIEA (4.0 equivalents) were added, followed by stirring at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and the mixture was dissolved again in water (5 mL). The obtained aqueous solution was washed with CHCl3 (3 mL×3), concentrated under reduced pressure, and purified by HPLC by separation and extraction, thereby obtaining 1.7 mg (20.0%) of the TFA salt of compound (5b) as white crystals.

[0161] ESI-MS (M+H) + :m / z 881, found:881

[0162] It should be noted that in the purification by HPLC for separation and extraction, Imtakt Cadenza C18 (20 mm×150 mm) was used as a chromatographic column, 0.1% TFA / water was used for phase A, 0.1% TFA / methanol was used for phase B, and a gradient system was used in which the phase B increased from 10% to 50% in 0 to 45 minutes and from 50% to 100% in 45 to 55 minutes, and the peak with a retention time of 38.0 minutes was separated at a flow rate of 5 mL / min.

[0163] In addition, mass spectrometry analysis was carried out in the following manner: Imtakt US-C18 (4.6 mm×150 mm) was used as a chromatographic column, 0.1% TFA / water was used for phase A, 0.1% TFA / acetonitrile was used for phase B, and a gradient system was used in which the concentration of phase B increased from 0 to 45% within 0 to 30 minutes and from 45 to 100% within 30 to 40 minutes. The peak with a retention time of 25.0 minutes was analyzed at a flow rate of 1 mL / min.

[0164] Example 3: Synthesis of DO3ABn-Phe-Gly-Lys((CH2)4-N3)-OH

[0165] DO3ABn-Phe-Lys((CH2)4-N3)-OH (a compound wherein n is 1 in formula (II-2)) was synthesized by the route shown in Example 2 (wherein the product of step (a) is compound (4c), and the product of step (b) is compound (5c)).

[0166] Synthesis of DO3ABn-Phe-Gly-Lys-OH (Compound (4c))

[0167] The same procedure as for compound (4b) in Example 2 was carried out except that a resin to which Phe-Gly-Lys was bonded was used instead of Phe-Lys, to obtain 30.0 mg (76.8%) of a TFA salt of compound (4c) as white crystals.

[0168] Synthesis of DO3ABn-Phe-Gly-Lys((CH2)4-N3)-OH (Compound (5c))

[0169] The same procedures as for Compound (5b) in Example 2 were carried out except that Compound (4c) was used instead of Compound (4b) to obtain 4.6 mg (14.0%) of a TFA salt of Compound (5c) as white crystals.

[0170] ESI-MS(M+H)+:m / z 938, found:938

[0171] It should be noted that, in the purification by HPLC, unlike Example 2, Imtakt Cadenza C18 (20 mm×150 mm) was used as the chromatographic column, 0.1% TFA / water was used for phase A, 0.1% TFA / acetonitrile was used for phase B, and a gradient system was used in which the phase B increased from 10% to 50% in 0 to 45 minutes and from 50% to 100% in 45 to 55 minutes, and the peak with a retention time of 33.9 minutes was extracted at a flow rate of 5 mL / min.

[0172] In addition, the conditions for mass spectrometry analysis were the same as those in Example 2, but the retention time was 24.7 minutes.

[0173] Comparative Example 1: Synthesis of DO3ABn-Gly-Lys((CH2)4-N3)-OH

[0174] DO3ABn-Gly-Lys((CH2)4-N3)-OH (wherein n is 1 and does not have a phenyl residue in the formula (II-2)) was synthesized by the route shown in Example 2 (wherein the product of step (a) is compound (4a) and the product of step (b) is compound (5a)).

[0175] Synthesis of DO3ABn-Gly-Lys-OH (Compound (4a))

[0176] The same procedure as for compound (4b) in Example 2 was carried out except that a resin to which Gly-Lys was bonded was used instead of Phe-Lys, to obtain 8.3 mg (25.3%) of a TFA salt of compound (4a) as white crystals.

[0177] Synthesis of DO3ABn-Gly-Lys((CH2)4-N3)-OH (Compound (5a))

[0178] The same procedures as for Compound (5b) in Example 2 were carried out except that Compound (4a) was used instead of Compound (4b) to obtain 1.5 mg (16.3%) of a TFA salt of Compound (5a) as white crystals.

[0179] ESI-MS(M+H)+:m / z 791, found:791

[0180] The retention time in HPLC purification was 22.6 minutes, and the retention time in mass spectrometry analysis was 19.3 minutes.

[0181] Preparation Example 1: Preparation of DBCO-NGA

[0182] Using the same method as Journal of Medicinal Chemistry 1997, 40, 2643-2652, a 10 mg / mL NGA solution obtained by introducing a thiol group using 2-iminothiolane was prepared. 2 μL of a DMSO solution in which 50 equivalents of DBCO-maleimide (purchased from Aldrich) were dissolved relative to galactose-binding albumin (NGA) was added to 100 μL of the solution, and the mixture was reacted at 37°C for 1.5 hours. After the reaction, the solution was purified using a spin column using sephadex G-50 equilibrated with D-PBS (-) as a carrier, thereby preparing DBCO-NGA to which DBCO was bound at an average binding number of 2.1.

[0183] Production Example 2: 111 Preparation of In-DO3ABn-Phe-Lys-NGA

[0184] DO3ABn-Phe-Lys((CH2)4-N3)-OH 111 In labeling and click reaction with DBCO-NGA were synthesized according to the following route (wherein the product of step (a) is compound (6b), and the product of step (b) is compound (7b)).

[0185] [Chemical formula 7]

[0186]

[0187] 111 Preparation of In-DO3ABn-Phe-Lys((CH2)4-N3)-OH (Compound (6b))

[0188] Towards 111 InCl3 solution (1.48 MBq / 60 μL) was added with 1 M acetate buffer (pH 5.5, 20 μL), and the mixture was allowed to stand at room temperature for 5 minutes. -4Compound (5b) was dissolved in 0.1M acetate buffer (pH 5.5, 20μL) at a concentration of 1.5M. The solution was reacted at 95°C for 5 minutes, then returned to room temperature, 10μL of 0.2M DTPA solution was added, and the mixture was allowed to stand at room temperature for 5 minutes. The reaction solution was purified by analytical HPLC. The fraction containing the target substance was solvent-replaced with methanol using Sep-pak, 30μL of D-PBS(-) was added to the resulting solution, and the solvent was concentrated under reduced pressure. 0.05M acetate buffer (pH5.5) was used for phase A, and methanol was used for phase B. A gradient system was used in which phase B increased from 20% to 60% in 0-30 minutes and from 60% to 100% in 30-35 minutes. The peak with a retention time of 20.2 minutes was fractionated at a flow rate of 1mL / min, and purification was performed to obtain compound (6b) with a radiochemical yield of >99%.

[0189] 111 Preparation of In-DO3ABn-Phe-Lys-NGA (7b)

[0190] 30 μL of D-PBS(-) solution of compound (6b) was added to 10 μL of DBCO-NGA solution and reacted at room temperature for 14 hours. The solution after the reaction was purified using a spin column with sephadex G-50 equilibrated with D-PBS(-) as a carrier, thereby obtaining the target compound (6b) with a radiochemical yield of 43.0%. 111 In-labeled NGA (compound (7b)).

[0191] Production Example 3: 111 Preparation of In-DO3ABn-Phe-Gly-Lys-NGA

[0192] DO3ABn-Phe-Gly-Lys((CH2)4-N3)-OH 111 In labeling and click reaction with DBCO-NGA were synthesized according to the route shown in Preparation Example 2 (wherein the product of step (a) is compound (6c), and the product of step (b) is compound (7c)).

[0193] 111 Preparation of In-DO3ABn-Phe-Gly-Lys((CH2)4-N3)-OH (Compound (6c))

[0194] The same procedures as for Compound (6b) in Preparation Example 2 were carried out except that Compound (5c) was used instead of Compound (5b), to give Compound (6c) in a radiochemical yield of 98.7%.

[0195] The retention time in HPLC purification was 18.7 minutes.

[0196] 111 Preparation of In-DO3ABn-Phe-Gly-Lys-NGA(7c)

[0197] The same procedures as for Compound (6b) in Preparation Example 2 were carried out except that Compound (6c) was used instead of Compound (6b), to give Compound (7c) in a radiochemical yield of 50.0%.

[0198] Production Example 4: 111 Preparation of In-DO3ABn-Gly-Lys-NGA

[0199] DO3ABn-Gly-Lys((CH2)4-N3)-OH 111 In labeling and click reaction with DBCO-NGA were synthesized according to the route shown in Preparation Example 2 (wherein the product of step (a) is compound (6a), and the product of step (b) is compound (7a)).

[0200] 111 In-DO3ABn-Gly-Lys((CH 2 ) 4 -N 3 Preparation of )-OH (Compound (6a))

[0201] The same procedures as for Compound (6b) in Production Example 2 were carried out except that Compound (5a) was used instead of Compound (5b), to give Compound (6a) in a radiochemical yield of 93.2%.

[0202] In the purification by analytical HPLC, Imtakt US-C18 (4.6 mm × 150 mm) was used as a chromatographic column, 0.05 M acetic acid buffer (pH 5.5) was used as phase A, methanol was used as phase B, and a gradient system was used in which the B phase increased from 0 to 40% in 0 to 30 minutes and from 40% to 100% in 30 to 35 minutes. The peak with a retention time of 19.1 minutes was fractionated at a flow rate of 1 mL / min to perform purification.

[0203] The same procedures as for Compound (6b) in Preparation Example 2 were carried out except that Compound (6a) was used instead of Compound (6b) to give Compound (7a) in a radiochemical yield of 38.0%.

[0204] Preparation Example 5: Preparation of CHX-A"-DTPA-NGA

[0205] NGA was dissolved in 0.1 M boric acid buffer (pH 8.5) to prepare an NGA solution (250 μg / 50 μL). Separately, p-SCN-CHX-A″-DTPA ((R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid, (R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid, purchased from Aldrich) was dissolved in 0.1 M boric acid buffer (pH 8.5) (6 mg / mL), 0.1 N NaOH was added to adjust the pH to 8 to 9, and then the solution was diluted with 0.1 M boric acid buffer (pH 8.5) to prepare a p-SCN-CHX-A″-DTPA solution (5 mg / mL). 20 equivalents of p-SCN-CHX-A"-DTPA solution were added to the NGA solution and reacted at room temperature overnight.

[0206] After the reaction, the solution was purified using a spin column using sephadex G-50 as a support equilibrated with 0.25 M acetate buffer (pH 5.5) to obtain the target CHX-A″-DTPA-NGA solution.

[0207] Production Example 6: 111 Preparation of In-CHX-A”-DTPA-NGA

[0208] Will 111 InCl3 solution (1.48MBq / 20μL) was added to 1M acetate buffer (pH5.5, 5μL), and the mixture was allowed to stand at room temperature for 5 minutes. A CHX-A"-DTPA-NGA solution (30μg / 15μL) was added to the resulting solution. The solution was reacted at 37°C for 1.5 hours, then returned to room temperature, 0.2M DTPA solution (10μL) was added, and the mixture was allowed to stand at room temperature for 5 minutes. The reaction solution was purified using a centrifugal column using sephadex G-50 equilibrated with D-PBS(-) as a carrier, thereby obtaining the target 111 In-CHX-A”-DTPA-NGA.

[0209] Example 4: Radioactive metal labeling of IgG antibodies 111 In] The Making of H-FGK-DOTA

[0210] 1.4 mg (1.5 nmol) of the DBCO-containing IgG antibody obtained in Example 1 and 0.1 mg (75 nmol) of DO3ABn-Phe-Gly-Lys((CH2)4-N3)-OH obtained in Example 3 were subjected to a click reaction in 167.5 μL of 10 mM acetic acid buffer at room temperature. After the reaction was completed, it was purified by ultrafiltration to produce a modified antibody (hereinafter referred to as "H-FGK-DOTA") in which trastuzumab was bound to DOTA-FGK via a DBCO-modified IgG binding peptide. The obtained H-FGK-DOTA was 111 200 μL of InCl3 solution (19.7 MBq, pH 6.5) and 50 μL of 100 mM HEPES buffer were mixed. 111 In] was prepared in such a way that the molar number of H-FGK-DOTA was 20 to 100 times that of H-FGK-DOTA. The complex formation reaction was carried out by reacting it at 45°C for 2 hours. After the complex was formed, it was purified by ultrafiltration. The radioactivity was measured using a radioisotope activity meter. As a result, the obtained [ 111 The radioactivity of In]H-FGK-DOTA was 2.33 MBq, and the radiochemical yield calculated after correction for decay was 14.0%.

[0211] In addition, 111 In]H-FGK-DOTA was dropped onto filter paper and then developed with 100 mM sodium citrate buffer. After development, the radiochemical purity was measured using a TLC scanner (Gita Star, Raytest).

[0212] Example 5: Radioactive metal labeling of IgG antibodies 111 In]H-DOTA production

[0213] The DBCO-containing IgG antibody obtained in Example 1 and 0.04 mg (75 nmol) of 1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid)-10-(azidopropylethylacetamide) (Azide-mono-amide-DOTA, Macrocyclics) were subjected to a click reaction in 167.5 μL of acetic acid buffer at room temperature. After the reaction was completed, it was purified by ultrafiltration to produce an IgG antibody (hereinafter referred to as "H-DOTA") in which trastuzumab was bound to DOTA via a DBCO-modified IgG binding peptide. The obtained H-DOTA was added to 111 200 μL of InCl3 solution (21.3 MBq, pH 6.5) and 50 μL of 100 mM HEPES buffer were mixed to perform a complex formation reaction. After the complex was formed, it was purified by ultrafiltration to obtain [111 In] H-DOTA 2.99 MBq. The radiochemical yield and radiochemical purity were calculated in the same manner as in Example 3. As a result, the radiochemical yield was 19.5% and the radiochemical purity was 99.6%.

[0214] Evaluation 1: Animal testing (1)

[0215] Each of the products obtained in Preparation Examples 2 to 4 and 6 was 111 In-labeled NGA solution was diluted and adjusted with D-PBS (-) to a concentration of (11.1 kBq / 8 μg protein / 100 μL / mouse) and administered via the tail vein of ddY, 6-week-old male mice. Five mice in a group were killed 5 minutes or 1 hour after administration, blood and organs of interest were recovered, and the weight of the organs was measured, and the radioactivity was measured using an Auto-well Gamma Counter.

[0216] [Table 1]

[0217] Table 1

[0218] Gly-Lys Phe-Lys Phe-Gly-Lys SCN blood 0.1±0.2 0.1±0.0 0.1±0.1 0.1±0.0 liver 89.5±3.4 91.1±6.2 93.3±3.5 95.0±4.1 spleen 0.2±0.0 0.2±0.0 0.2±0.0 0.0±0.0 kidney 0.1±0.0 0.2±0.0 0.1±0.0 0.1±0.0 pancreas 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 heart 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 lung 0.1±0.1 0.1±0.0 0.1±0.0 0.1±0.0 Stomach 0.1±0.1 0.1±0.1 0.0±0.0 0.1±0.1 Small Intestine 0.1±0.1 0.1±0.1 0.1±0.1 0.2±0.1

[0219] [Table 2]

[0220] Table 2

[0221] Gly-Lys Phe-Lys Phe-Gly-Lys SCN blood 0.0±0.0 0.3±0.1 0.3±0.1 0.0±0.0 liver 89.1±1.2 49.5±5.6 47.8±4.0 79.9±2.0 spleen 0.2±0.0 0.2±0.1 0.2±0.0 0.0±0.0 kidney 0.2±0.0 0.9±0.3 1.1±0.3 0.1±0.0 pancreas 0.0±0.0 0.0±0.1 0.0±0.0 0.0±0.0 heart 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 lung 0.0±0.0 0.1±0.1 0.1±0.0 0.0±0.0 Stomach 0.1±0.1 1.1±0.7 0.3±0.2 0.3±0.3 Small Intestine 2.5±0.7 27.8±6.5 36.3±3.7 8.9±1.2

[0222] Table 1 shows the radioactivity (%ID) of each organ 5 minutes after administration. Table 2 shows the radioactivity (%ID) of each organ 1 hour after administration. Gly-Lys represents the administration of the 111 In-DO3ABn-Gly-Lys-NGA results, Phe-Lys indicates that the 111 In-DO3ABn-Phe-Lys-NGA results, Phe-Gly-Lys indicates that the 111 The results of In-DO3ABn-Phe-Gly-Lys-NGA, SCN represents the 111 Results for In-CHX-A”-DTPA-NGA.

[0223] As shown in Table 1, radioactivity accumulation was observed in the liver at 5 minutes after administration, and since the amino acid sequences of Phe-Lys and Phe-Gly-Lys were included, excretion from the liver was observed.

[0224] Comment 2: Animal testing (2)

[0225] For the use of the [ 111 In]H-FGK-DOTA and [ 111 To investigate the effectiveness of the FGK sequence using In]H-DOTA, an in vivo experiment was performed.

[0226] The solvent was replaced with phosphate buffered saline (PBS) [ 111 In]H-FGK-DOTA and 111 In]H-DOTA was adjusted to 200 kBq per 100 μL, and 100 μL was administered to each of three 6-week-old male mice (ddY) through the tail vein under isoflurane anesthesia. After administration, the mice were killed by bleeding to death at 1 hour, 6 hours, and 24 hours. The organs (heart, lung, liver, spleen, kidney, stomach, small intestine, large intestine, lower limb bones, muscles, and bladder) were removed, and the weight was measured together with the blood, urine, feces, and the rest of the body, and the radioactivity was determined. The mean value and standard deviation of the radioactivity distribution (injection volume % (ID)) at each hour time point are shown in Figure 1. In addition, Figure 2 shows the in vivo dynamics (% ID) of the organ of interest. Statistical analysis was performed using the Student's t test. In addition, significant differences were determined as P < 0.05.

[0227] Figure 1A This is a graph showing the results 1 hour after administration. Figure 1B This is a graph showing the results 6 hours after administration. Figure 1C The graph shows the results 24 hours after administration.

[0228] Figure 2A is a graph showing the temporal changes in radioactivity distribution (%ID) in the liver. Figure 2B It is a graph showing the time-dependent change of radioactivity distribution (%ID) in kidney. Figure 2C In the table, “kidney and urinary system” indicates the temporal changes in the total radioactivity distribution (%ID) in the kidney, bladder, and urine, and “liver and biliary system” indicates the temporal changes in the total radioactivity distribution (%ID) in the liver, small intestine, large intestine, and feces.

[0229] According to the 6-hour time point after administration [ 111 In]H-DOTA and 111 In]H-FGK-DOTA liver uptake was different, and at the 24-hour time point after administration [ 111 The significant increase in the excretion of In]H-FGK-DOTA into urine indicates that the FGK sequence successfully promoted the excretion of radionuclides and reduced their accumulation in the liver.

[0230] This application claims priority based on Japanese patent application No. 2018-78487 filed on April 16, 2018, the entire disclosure of which is incorporated herein by reference.

Claims

1. A modified antibody comprising an IgG antibody and an IgG-binding peptide that binds to the IgG antibody, wherein: The IgG binding peptide consists of the following amino acid sequence: GPDCAYHKGELVWCTFH (serial number 2) In the amino acid sequence, optionally, two cysteine ​​residues are bonded to each other by a disulfide bond, and optionally, the C-terminus is amidated, The N-terminus of the IgG binding peptide is linked to a compound represented by the following formula (II-1) at the position of the lysine residue via a modified linker. In formula (II-1), n ​​is 0 or 1.

2. The modified antibody according to claim 1, wherein The binding site between the lysine residue of the compound represented by the formula (II-1) and the modified linker is formed by a click reaction between a DBCO (dibenzocyclooctyne) group and an azide group.

3. The modified antibody according to claim 2, wherein The modified linker is a modified linker represented by the following formula (III): L1-L2-L3(III) L1 is a polyethylene glycol linker bound to the N-terminus of the IgG binding peptide, L2 is an amino acid sequence consisting of 0 or more and 5 or less amino acid residues, L3 is a group having a DBCO (dibenzocyclooctyne) group at the terminal, By introducing an alkyl azide group having 1 to 10 carbon atoms into the lysine residue of the compound represented by the formula (II-1), a binding site between the lysine residue and the modified linker is formed through a click reaction.

4. The modified antibody according to claim 3, wherein In the formula (III), L2 has a cysteine ​​residue, and L3 is formed by reacting DBCO-maleimide represented by the following formula (IV) with the cysteine ​​residue of L2, 5. A radioactive metal-labeled antibody comprising the modified antibody according to any one of claims 1 to 3 coordinated with a radioactive metal.

6. A radioactive drug comprising the radioactive metal-labeled antibody according to claim 5.

7. A polypeptide-containing compound or a salt thereof, wherein a polypeptide capable of binding to a target molecule is bound to a compound represented by the following formula (II-1): In formula (II-1), n ​​is 0 or 1, in, The polypeptide is a modified IgG binding peptide, or an IgG antibody bound to the modified IgG binding peptide, wherein the modified IgG binding peptide is composed of an IgG binding peptide and a modified linker bound to the N-terminus of the IgG binding peptide, and the modified linker is represented by the following formula (III): The IgG binding peptide is composed of the following amino acid sequence: GPDCAYHKGELVWCTFH (SEQ ID NO: 2), wherein, in the amino acid sequence, optionally, two cysteine ​​residues are disulfide bonded to each other, and optionally, the C-terminus is amidated, L1-L2-L3(III) In formula (III), L1 is a polyethylene glycol linker bound to the N-terminus of the IgG binding peptide, L2 is an amino acid sequence consisting of 0 or more and 5 or less amino acid residues, and L3 is a group having a DBCO (dibenzocyclooctyne) group at the terminal.

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