Human transferrin receptor-conjugated antibody-peptide conjugate

Abstract

[Problem] To provide an art for crossing the blood-brain barrier. [Solution] A conjugate comprising the following: (1) a transferrin receptor-binding peptide, wherein (i) the peptide contains the amino acid sequence from the 1st to the 15th (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys) of the amino acid sequence given by SEQ ID NO: 1; (ii) the peptide has an amino acid sequence exhibiting a substitution, deletion, addition, and / or insertion of from 1 to 11 amino acid residues in the amino acid sequence from the 1st to the 15th of the amino acid sequence given by SEQ ID NO: 1; (iii) the peptide contains the amino acid sequence from the 1st to the 12th (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence given by SEQ ID NO: 14; or (iv) the peptide has an amino acid sequence exhibiting a substitution, deletion, addition, and / or insertion of from 1 to 8 amino acid residues in the amino acid sequence from the 1st to the 10th of the amino acid sequence given by SEQ ID NO: 14, and (2) a compound containing an antibody or an antigen-binding fragment thereof.

Description

[Technical Field]

[0001] This invention relates to a conjugate (hereinafter also referred to as a complex) comprising a peptide capable of binding to human transferrin receptors (hTfRs) and an antibody or its antigen-binding fragment. Furthermore, this invention relates to a conjugate of a cell-penetrating peptide and an antibody or its antigen-binding fragment. Moreover, this invention relates to a pharmaceutical product comprising such a conjugate. [Background technology]

[0002] Around the world, there is a high need for the development of treatments for brain-related diseases such as Alzheimer's disease and brain tumors, and research and development are progressing. In addition to conventional small-molecule drugs, the importance of large-molecule antibody drugs has recently increased. However, research and development of these drugs is fraught with difficulties. One of the reasons for this is the existence of the blood-brain barrier. Unlike capillaries in muscles and other tissues, the capillaries that supply blood to most of the brain tissue, with the exception of a few regions including the periventricular organs (pineal gland, pituitary gland, area 11, etc.), have endothelial cells that form their endothelium that are tightly bound together by strong intercellular junctions. As a result, passive transport of substances from the blood to the brain is hindered, and with some exceptions, substances other than highly lipid-soluble substances or substances with small molecular weights (200-500 Daltons or less) and electrically neutral at or near physiological pH are unlikely to cross from the capillaries into the brain. This mechanism that restricts the exchange of substances between the blood and the brain's tissue fluid via the capillary endothelium in the brain is called the blood-brain barrier (BBB). Furthermore, the blood-brain barrier restricts the exchange of substances not only in the brain but also between the tissue fluid of the central nervous system, including the brain and spinal cord, and the blood. The blood-brain barrier allows most cells in the central nervous system to maintain biochemical homeostasis, unaffected by fluctuations in the concentration of hormones, lymphokines, and other substances in the blood.

[0003] Various methods have been reported for enabling macromolecular substances to reach the brain via the blood-brain barrier, including modifying the macromolecular substance to have affinity for transferrin receptors, which are membrane proteins present on endothelial cells of brain capillaries (Patent Documents 1-3). For example, Patent Document 1 describes a blood-brain barrier shuttle that has affinity for transferrin receptors and can bind to them.

[0004] However, further methods are needed to allow macromolecules, especially antibodies, to cross the blood-brain barrier. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Special Publication No. 2015-528452 [Patent Document 2] Japanese Patent Publication No. H06-228199 [Patent Document 3] WO2016 / 208695 [Patent Document 4] WO2019 / 151539 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a conjugate of a peptide that binds to a transferrin receptor and an antibody. This conjugate has both the ability to bind to the transferrin receptor and the ability to bind to the antigen to which the antibody constituting the conjugate binds. Furthermore, the present invention aims to provide a technique for crossing the blood-brain barrier by conjugating an antibody or its antigen-binding fragment with a peptide that binds to the transferrin receptor and has the ability to cross the blood-brain barrier. More specifically, the present invention aims to provide a conjugate of a peptide that binds to the transferrin receptor and has the ability to cross the blood-brain barrier with an antibody. Furthermore, the present invention aims to provide a pharmaceutical product containing the above-mentioned conjugate. [Means for solving the problem]

[0007] The present invention was completed by discovering that by directly or via a linker, a conjugate having antigen-binding and transferrin receptor-binding ability can be created by linking a peptide having a specific structure with a compound containing an antibody, and furthermore, by using this conjugate, a desired antibody can be passed through the blood-brain barrier and introduced into cells.

[0008] One invention described in this specification relates to a conjugate comprising a peptide that binds to a transferrin receptor and an antibody or an antigen-binding fragment thereof.

[0009] The above peptide is a peptide that binds to the transferrin receptor, (i) A peptide containing the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys); (ii) A peptide having an amino acid sequence in which the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 has one to eleven amino acid residue substitutions, deletions, additions and / or insertions; (iii) A peptide having the amino acid sequence from the 1st to the 12th amino acid sequence of the amino acid sequence described in SEQ ID NO: 14 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or (iv) A peptide having an amino acid sequence in which the amino acid sequence described in Sequence ID No. 14 has one to eight amino acid residues substituted, deleted, added and / or inserted in the amino acid sequence from the 1st to the 10th amino acid sequence, That is the case. Further, the compound comprising an antibody or an antigen-binding fragment thereof may be IgG or an antigen-binding fragment derived from IgG, or may be an antibody selected from the group consisting of IgG1, IgG2 and IgG4 or an antigen-binding fragment thereof. Another example of the above peptide is: (I) substitution of the 1st alanine residue of SEQ ID NO: 1 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) substitution of the 2nd position of SEQ ID NO: 1 with any amino acid residue or any N-methyl amino acid; (III) substitution of the 3rd position of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring in a side chain thereof; (IV) substitution of the 5th position of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring in a side chain thereof; (V) substitution of the 6th asparagine residue of SEQ ID NO: 1 with a hydrophilic amino acid or alanine; (VI) substitution of the 8th tyrosine residue of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring in a side chain thereof; (VII) substitution of the 10th isoleucine residue of SEQ ID NO: 1 with any amino acid; (VIII) substitution of the 11th arginine residue of SEQ ID NO: 1 with any amino acid; (IX) substitution of the 12th arginine residue of SEQ ID NO: 1 with any amino acid; and (X) substitution of the 13th tyrosine residue of SEQ ID NO: 1 with any amino acid; (XI) substitution of the 14th N-methyltyrosine residue of SEQ ID NO: 1 with any amino acid; a peptide comprising an amino acid sequence having one or more substitutions selected from the above, or or; (I) substitution of the 1st alanine residue of SEQ ID NO: 14 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) substitution of the 2nd position of SEQ ID NO: 14 with any amino acid residue or any N-methyl amino acid; (III) Substitution of the third aromatic amino acid residue of Sequence ID No. 14 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (IV) Substitution of the fifth aromatic amino acid residue of Sequence ID No. 14 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (V) Substitution of the sixth asparagine residue of SEQ ID NO: 14 with a hydrophilic amino acid or alanine; (VI) Substitution of the 8th tyrosine residue of Sequence ID No. 14 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring in its side chain; (VII) Substitution of any amino acid at the 10th isoleucine residue of SEQ ID NO: 14; (X) Substitution of the 11th serine residue in sequence number 14 with a hydrophilic amino acid residue. It is a peptide comprising an amino acid sequence having one or more substitutions selected from the following. More specifically, the peptides are cyclic peptides consisting of one of the amino acid sequences from the 1st to 15th amino acids of SEQ ID NOs: 1-13, 15, 18-86, and 90-110, or from the 1st to 12th amino acid sequences of SEQ ID NOs: 14, 16, 17, and 87-89. In the conjugate of the present invention, it is preferable that the peptide is bound to the antibody via a linker. Furthermore, it is preferable that the linker is a peptide linker, a chemical linker, or a combination thereof. The specific linker is a linker having the sequence described in any of sequence numbers 111 to 161. The conjugate is preferably one in which the peptide and antibody are linked via a maleimide, hydrazide, or NHS site attached to the end of the linker. The conjugate is preferably one in which a linker-added peptide described in any of SEQ ID NOs. 1 to 110 is linked to an antibody or its antigen-binding fragment. This specification also provides a composition comprising any of the above-described conjugates, for delivering the conjugate into a cell or for crossing the blood-brain barrier. The composition may also contain various elements described in this specification in addition to the conjugate. This specification also provides pharmaceutical compositions comprising a conjugate or a salt thereof as described in any one of the above paragraphs as an active ingredient. The composition may also contain various elements described in this specification in addition to the conjugate or a salt thereof. The salt means a pharmaceutically acceptable salt of the conjugate. This specification also discloses a method for processing antibodies or their antigen-binding fragments so that they can be delivered into cells or cross the blood-brain barrier (a method for producing conjugates). This method includes the step of conjugating a peptide that binds to the transferrin receptor described above with the antibody or its antigen-binding fragment. Furthermore, this specification also provides methods for treating various diseases, which include the step of delivering the above-mentioned conjugate or composition into an intracellular space or passing it across the blood-brain barrier. [Effects of the Invention]

[0010] As demonstrated by the examples, the invention described in this specification provides a conjugate of a peptide, such as a peptide that binds to the human transferrin receptor (hTfR) or a cell-penetrating peptide, and a conjugate containing an antibody or its antigen-binding fragment. [Brief explanation of the drawing]

[0011] [Figure 1-1] Figure 1-1 is a photograph, instead of a diagram, showing the fluorescence intensity measurement results in cells treated with a trastuzumab-peptide conjugate. [Figure 1-2] Figure 1-2 is a photograph, replacing the diagram, showing the fluorescence intensity measurement results in cells treated with a trastuzumab-peptide conjugate. [Figure 1-3] Figures 1-3 are photographs, instead of diagrams, showing the fluorescence intensity measurements in cells treated with trastuzumab-peptide conjugate. [Figure 2-1] Figure 2-1 shows the plasma concentrations of the trastuzumab-peptide conjugate. [Figure 2-2]Figure 2-2 shows the concentrations of trastuzumab-peptide conjugate in various tissues. [Figure 2-3] Figure 2-3 shows the plasma concentrations of nivolumab-peptide conjugate. [Figure 2-4] Figure 2-4 shows the concentrations of nivolumab-peptide conjugate in various tissues. [Figure 3-1] Figure 3-1 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (cerebellum, 6 hours of treatment) using trastuzumab-hTfR_000894_PEG11_K(Maleimide) and trastuzumab-hTfR_000894_PEG36_K(Maleimide). [Figure 3-2] Figure 3-2 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (cerebellum, 6 hours of treatment) treated with nivolumab-894_3m_G4S2_K(Mal). [Figure 3-3] Figure 3-3 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (cerebellum, 6 hours of treatment) treated with nivolumab-894_3m_GGRGRS_K(Mal). [Figure 3-4] Figure 3-4 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (cerebellum, 24-hour treatment) treated with nivolumab-894_3m_GGRGRS_K(Mal). [Figure 3-5] Figure 3-5 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (hippocampus, 6 hours of treatment) using nivolumab-894_3m_GGRGRS_K(Mal). [Figure 3-6] Figure 3-6 is a photograph, replacing the diagram, showing the results of a mouse brain localization test (hippocampus, 24-hour treatment) treated with nivolumab-894_3m_GGRGRS_K(Mal).

[0012] (Detailed description of the invention) The following describes embodiments for carrying out the present invention. The present invention is not limited to the embodiments described below, but also includes modifications made to the embodiments described below to the extent that is obvious to those skilled in the art. In this specification, the entire text of the international patent application PCT / JP2021 / 006709 (International Publication WO2021-167107; not published at the time of the filing of the present application), which is identical in all respects to the applicant of this application, is incorporated by reference.

[0013] Conjugate (complex) The conjugate of the present invention is (1) A peptide that binds to a transferrin receptor, (i) A peptide having the amino acid sequence described in the amino acid sequence of the 1st to 15th amino acids of the amino acid sequence described in Sequence ID No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys); (ii) A peptide having an amino acid sequence in which the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 has one to eleven amino acid residue substitutions, deletions, additions and / or insertions; (iii) A peptide having the amino acid sequence described in the amino acid sequence of the 1st to 12th amino acids (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence described in SEQ ID NO: 14; or (iv) A peptide having an amino acid sequence in which the amino acid sequence described in SEQ ID NO: 14 has substitutions, deletions, additions and / or insertions of 1 to 8 amino acid residues in the amino acid sequence from the 1st to the 10th amino acid sequence, and (2) A compound containing an antibody or an antigen-binding fragment thereof. Here, the peptide and the compound containing the antibody or its antigen-binding fragment may be bound directly or via a linker, but preferably via a linker.

[0014] Transferrin receptor A transferrin receptor is a receptor that binds to transferrin, a protein found in plasma that binds to iron ions, and has the function of taking transferrin into cells. Transferrin receptors are expressed on various cells, including reticulocytes, placental trophoblast cells, and lymphocytes, and their expression has been suggested to be particularly high in tumor cells. Furthermore, because transferrin receptors have the property of triggering cellular endocytosis upon stimulation of iron ion binding in plasma, research is underway to use antibodies that bind to transferrin receptors as a drug delivery system to allow desired substances to cross the blood-brain barrier (BBB). In addition, type I and type II transferrin receptors are known, but in this invention, type I (Gene ID: 7037) is preferred as the transferrin receptor. In this specification, unless otherwise specified, human-type transferrin receptors are referred to as human TfR, hTfR, or simply TfR.

[0015] Peptides that bind to transferrin receptors To bind to a transferrin receptor (also called having binding activity or affinity) means to bind specifically to the transferrin receptor. Affinity is expressed by the equilibrium constant (KD) for the dissociation of the transferrin receptor and the binding peptide, which is a measure of the binding strength between the transferrin receptor and the binding peptide: as the value of KD decreases, the binding strength between the transferrin receptor and the binding peptide increases (alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD). As will be apparent to those skilled in the art (for example, based on further disclosure herein), affinity can be determined in a manner known in itself, depending on the type and properties of the substance being bound. Binding activity is also a measure of the strength of the binding between the transferrin receptor and the binding peptide. Binding activity relates to both the affinity between the transferrin receptor and its binding site on the binding peptide and the number of relevant binding sites present on the binding molecule.

[0016] The specific binding of the transferrin receptor to the binding peptide can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, as described herein, and different variants thereof. Preferably, the affinity between the peptide and the transferrin receptor in the present invention is less than 100 nM, preferably less than 50 nM KD, but is not limited to about 10 -5 M ~ about 10 -9 M, or in another embodiment, about 10 -7 M or less, for example, 10 -7 M~10 -13 M, for example 10 -9 M~10 -13 It can be M. A peptide that binds to a transferrin receptor is a peptide that specifically binds to a transferrin receptor as described above. (i) A peptide having the amino acid sequence described in the amino acid sequence of the 1st to 15th amino acids of the amino acid sequence described in Sequence ID No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys); (ii) A peptide having an amino acid sequence in which the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 has one to eleven amino acid residue substitutions, deletions, additions and / or insertions; (iii) A peptide having the amino acid sequence described in the amino acid sequence of the 1st to 12th amino acids (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence described in SEQ ID NO: 14; or (iv) The peptide is not particularly limited as long as it has an amino acid sequence in which the amino acid sequence of the first to tenth amino acid sequences of the amino acid sequence described in SEQ ID NO: 14 has one to eight amino acid residues substituted, deleted, added and / or inserted.

[0017] Capable of crossing the blood-brain barrier (BBB) Being able to cross the blood-brain barrier (BBB) ​​means, for example, that a substance can pass through the BBB into the brain, and that the substance or its metabolites can be detected in any part of the brain at a certain time after administration, or that findings can be obtained that suggest the substance had an effect in the brain.

[0018] Brain-related diseases Brain-related diseases are illnesses caused by some kind of abnormality in the brain, such as central nervous system (CNS) diseases. Examples of brain-related diseases, though not limited to these, include Alzheimer's disease, Parkinson's disease, prion diseases, Huntington's disease, lysosomal storage diseases, central nervous system disorders, tumors of the central nervous system including brain tumors, cerebral ischemia, diseases involving brain damage, traumatic central nervous system injuries, viral and bacterial central nervous system diseases, and mental illnesses such as schizophrenia and depression.

[0019] Compounds containing antibodies or their antigen-binding fragments Antibodies are glycoproteins produced by B cells, a type of lymphocyte, that play a crucial role in immune responses. They are also known as immunoglobulins, gamma-globulins, or Ig. Antibodies have a structure composed of polypeptides called light and heavy chains, which can be divided into an Fc region and a Fab region. The Fab region is known to have the ability to bind to antigens. The compounds containing the antigen-binding fragment refer to the Fab region of the antibody described above, which is the region that has the ability to bind to the antigen, and compounds containing that Fab region. Antibodies can be further divided into several isotypes; for example, in mammals, five types are known: IgG, IgA, IgM, IgD, and IgE. Among these, IgG (immunoglobulin G) is the most commonly found antibody in humans, particularly in the blood and tissues, and is mainly involved in secondary immune responses. Currently, almost all antibody drugs on the market are IgG. Furthermore, IgG is divided into four subclasses: IgG1, IgG2, IgG3, IgG4, and IgG5. In the present invention, the preferred antibody is IgG, and the more preferred antibody is IgG1 or IgG4. Furthermore, the preferred antigen-binding fragment-containing compound is a compound containing the Fab region of IgG, and more preferably a compound containing the Fab region of IgG1 or IgG4.

[0020] Cell-penetrating peptides Cell-permeable peptides are known, for example, as described in Japanese Patent Publication No. 6478632 and Japanese Patent Publication No. 6708770 (Peptides with Cell Permeability). As shown in the examples, the peptides in the present invention bind to transferrin receptors and are taken up into cells. Therefore, by using the complex of the present invention, it becomes possible to deliver the target active ingredient into cells, for example, to deliver nucleic acid drugs into cells.

[0021] peptide This term refers to a structure consisting of multiple amino acids in a sequence, and encompasses polypeptides and proteins. In this application, the term "amino acid" includes not only naturally occurring amino acids (natural amino acids) that are incorporated into peptide chains through mRNA translation within cells, but also amino acids that do not exist naturally (unnatural amino acids) that can form part of a peptide chain through peptide bonds. Amino acids may be artificially synthesized or naturally occurring. Furthermore, in this application, peptides include peptides in which a cyclic portion is formed by post-synthesis cyclization (also called cyclic peptides), and peptides obtained by further chemical modification of such peptides. In this specification, a cyclic peptide refers to a peptide whose amino acid sequence is formed by the bonding of two amino acids separated by one or more amino acid residues, resulting in a ring structure in whole or in part. There are no particular limitations on the type of bonding between the two amino acids, but cyclic peptides include those formed by amide bonds between the carboxyl group of one amino acid and the amino group of the other, thioether bonds between the carboxyl group of one amino acid and the thiol group of the other, thiol bonds between the thiol group of one amino acid and the thiol group of the other, lactam ring formation, macrocyclization reactions, and those having a lasso-peptide-like structure. However, in the case where the two amino acids are bonded by an amide bond, the amide bond is not limited to those formed by the bonding of the carboxyl group of one amino acid and the amino group of the other; it is sufficient if the bond is formed by an amide bond as a result of a synthetic reaction. The same applies to other types of bonding. In other words, in this application, a cyclic peptide is one in which a part of it forms a cyclic structure, and may have a linear portion.

[0022] In this specification, some amino acids may be modified for the purpose of cyclizing peptides. Such partially modified amino acids are also included in the amino acids of this application. For example, a chloroacetyl group may be added to the N-terminal amino acid and bound to a cysteine ​​residue in the peptide to form a cyclamen. Various (natural / unnatural) amino acids to which a chloroacetyl group has been added are also included in the amino acids of this application.

[0023] Non-natural amino acids refer to compounds other than natural amino acids that possess the characteristics of amino acids. Examples include, but are not limited to, β-amino acids, γ-amino acids, L-amino acids, D-amino acids (also called D-type amino acids), chemically modified amino acids such as amino acid mutants and amino acid derivatives, as well as amino acids that do not become building blocks of proteins in living organisms, such as norleucine, β-alanine, and ornithine. Other examples include N-methyl amino acids, N-ethyl amino acids, D-amino acids, histidine-like amino acids, amino acids with extra methylene groups or aromatic rings in their side chains, and amino acid derivatives in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group. Examples of non-natural amino acids and their abbreviations used herein are shown below. The CAS reference number or supplier name is shown in parentheses, and for newly synthesized amino acids, the synthesis example number is shown. Note that special amino acids are not limited to these; for example, those in which one or more hydrogen atoms in these molecules are substituted with alkyl groups are also considered special amino acids. When a hydrogen atom is substituted with an alkyl group, the alkyl group is preferably a methyl group or an ethyl group, and more preferably a methyl group. In this specification, amino acids preceded by Me or N-Me indicate N-methyl amino acids unless otherwise specified. For example, the N-methylated amino acid of alanine (Ala or A) is shown as MeAla, N-MeAla, MeA, or N-MeA. Furthermore, amino acids with a single-letter abbreviation preceded by d indicate D-amino acids. For example, the D-amino acid of alanine (Ala or A) is shown as da. Those without a CAS number or supplier information can be purchased as general reagents. Regarding the following amino acids, they can be used in peptide synthesis by protecting the alpha-amino group with Fmoc using known methods.

[0024] Yph (S)-2-amino-3-(4-phenoxyphenyl)propanoic acid (CAS number: 150351-64-7) W7OMe (S)-2-amino-3-(7-methoxy-1H-indole-3-yl)propanoic acid (CAS number: 25198-03-2) W7N (S)-2-amino-3-(1H-pyrrolo[2,3-β]pyridin-3-yl)propanoic acid (CAS number: 49758-35-2) W7F (S)-2-amino-3-(7-fluoro-1H-indole-3-yl)propanoic acid (CAS number: 138514-97-3) W6N (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (Kishida Chemical Co., Ltd.) (CAS number: 149704-63-2) W6F (S)-2-amino-3-(6-fluoro-1H-indole-3-yl)propanoic acid (CAS number: 19310-00-0) W5OMe 5-Methoxy-L-tryptophan (CAS number: 25197-96-0) W5F (S)-2-amino-3-(5-fluoro-1H-indole-3-yl)propanoic acid (CAS number: 16626-02-1) W4OMe 4-Methoxy-L-tryptophan (CAS number: 406938-53-2) W4N (S)-2-amino-3-(1H-pyrrolo[3,2-β]pyridin-3-yl)propanoic acid (CAS number: 149818-23-5) W4F (S)-2-amino-3-(4-fluoro-1H-indole-3-yl)propanoic acid (CAS number: 106034-22-4) W4C (S)-2-amino-3-(4-chloro-1H-indole-3-yl)propanoic acid (CAS number: 52448-14-3) W2N (S)-2-amino-3-(1H-indole-3-yl)propanoic acid (CAS number: 53538-54-8) W1iPr 1-Isopropyl-L-tryptophan (CAS number: 1219485-46-7) W1Et7Cl (S)-2-amino-3-(7-chloro-1-ethyl-1H-indole-3-yl)propanoic acid W1Et 1-Ethyl-L-tryptophan (CAS number: 168471-23-6) Tbg (S)-2-amino-3,3-dimethylbutanoic acid (CAS number: 158059-28-0) pHPeG N-(4-hydroxyphenethyl)glycine (CAS number: 169836-45-7) PeG N-(2-phenylethyl)-glycine (CAS number: 7738-38-7) NVA L-norvaline (CAS number: 6600-40-4) Nle L-norleucine (CAS number: 327-57-1) Nal2 β-(2-naphthyl)L-alanine (CAS number: 58438-03-2) Nal1 β-(1-naphthyl)L-alanine (CAS number: 55516-54-6) MeoBph N-α-methyl-2-phenyl-L-phenylalanine MeNal2 N-α-methyl-β-(2-naphthyl)-L-alanine (CAS number: 179385-30-9) MeNal1 N-α-methyl-β-(1-naphthyl)-L-alanine (CAS number: 2137057-01-1) MemBph N-α-methyl-3-phenyl-L-phenylalanine Hph L-homophenylalanine (CAS number: 943-73-7) Hly (S)-2,7-diaminoheptanoic acid (CAS number: 498-56-6) F4OMe (S)-2-amino-3-(4-methoxyphenyl)propanoic acid (CAS number: 7635-29-2) F4G (4-Guazinyl)-L-Phenylalanine (CAS No.: 59574-11-7) F4F 4-Fluoro-L-phenylalanine (CAS number: 1132-68-9) F4C 4-Chloro-L-phenylalanine (CAS number: 14173-39-8) F3OMe (S)-2-amino-3-(3-methoxyphenyl)propanoic acid (CAS number: 98813-19-5) F3F 3-Fluoro-L-phenylalanine (CAS number: 19883-77-3) F3C 3-Chloro-L-phenylalanine (CAS number: 80126-51-8) F2OMe (S)-2-amino-3-(2-methoxyphenyl)propanoic acid (CAS number: 193546-31-5) F2C (S)-2-amino-3-(2-chlorophenyl)propanoic acid (CAS number: 103616-89-3) MeF4OMe (S)-3-(4-methoxyphenyl)-2-(methylamino)propanoic acid (CAS number: 52939-33-0) MeF4F N-α-methyl-4-fluoro-L-phenylalanine (CAS number: 347851-71-2) MeF: N-methylphenylalanine MeF3F N-α-methyl-3-fluoro-L-phenylalanine (CAS number: 347851-71-2) MeF3C N-α-methyl-3-chloro-L-phenylalanine (CAS number: 2255324-91-3) MeBph N-α-methyl-4-phenyl-L-phenylalanine Me4Py N-α-methyl-4-pyridyl-L-alanine Me3Py N-α-methyl-3-pyridyl-L-alanine Dr. D-Arginine dp D-proline dc D-cysteine dk D-lysine Dap L-α,β-diaminopropionic acid (CAS number: 515-94-6) Dab (S)-2,4-diaminobutanoic acid (CAS number: 1758-80-1) Cit 2-amino-5-ureidopentanoic acid (CAS number: 627-77-0) Cha β-cyclohexyl-L-alanine (CAS number: 4441-50-3) CeG N-(2-carboxyethyl)-glycine (CAS number: 505-72-6) Cbg (S)-2-amino-2-cyclobutylacetic acid (CAS number: 49607-08-1) Cba Cyclobutylalanine (CAS number: 1201593-65-8) aMeY α-methyl-L-tyrosine (CAS number: 658-48-0) aMeW α-methyltryptophan (CAS number: 153-91-3) aMeK α-methyl-lysine (CAS number: 111717-28-3) aMeC α-methyl-L-cysteine ​​(CAS number: 441317-73-3) Aib α-methylalanine (CAS number: 62-57-7) Ahp / Alahp (S)-2-aminoheptanoic acid (CAS number: 1115-90-8) Abu L-α-aminobutanoic acid (CAS number: 1492-24-6) A4paa (S)-2-amino-3-(1-(carboxymethyl)piperazine-4-yl)propanoic acid (Kishida Chemical Co., Ltd.) 5Ind (S)-2-amino-3-(1H-indole-5-yl)propanoic acid (CAS number: 460096-38-2) 4Py2NH2 (S)-2-amino-3-(2-aminopyridine-4-yl)propanoic acid (Kishida Chemical Co., Ltd.) 4Py 4-Pyridyl-L-Alanine (CAS number: 1956-21-4) 3Py6NH2 (S)-2-amino-3-(6-aminopyridine-3-yl)propanoic acid 3Py 3-Pyridyl-L-Alanine (CAS number: 17470-24-5) W1aa 1-(carboxymethyl)-L-tryptophan (CAS number: 773823-50-0) KCOpipzMe N6-(4-methylpiperazine-1-carbonyl)-L-lysine (Kishida Chemical Co., Ltd.) W1mCON 1-(2-amino-2-oxoethyl)-L-tryptophan W1EtOH 1-(2-hydroxyethyl)-L-tryptophan 3Py6OMe (S)-2-amino-3-(6-methoxypyridine-3-yl)propanoic acid (CAS number: 1270317-99-1) Epyrl2RCOO 2-amino-(5-((R)-2-((allyloxy)carbonyl)pyrrolidine-1-yl)-5-oxopentanoic acid (Glu(d-Pro-O-allyl)-OH) Dpyrl2RCOO 2-Amino-(4-((R)-2-((allyloxy)carbonyl)pyrrolidine-1-yl)-4-oxobutanoic acid (Asp(d-Pro-O-allyl)-OH) MeF3COO 3-carboxy-N-methylphenylalanine (CAS number: 1499826-56-0) 3Imp 2-amino-3-(imidazo[1,2-a]pyridine-3-yl)propanoic acid (CAS number: 2276942-95-9) KaAc N6-Glycyl-L-Lysine (Lys(Gly-O-allyl)-OH) A1Me4pip 4-amino-1-methylpiperazine-4-carboxylic acid (CAS number: 15580-66-2) Har N6-Carbamimidoyl-L-lysine (CAS number: 156-86-5) Acpr (S)-2-amino-3-cyclopropylpropanoic acid (CAS number: 1492156-90-7) Atb (S)-2-amino-4,4-dimethylpentanoic acid (CAS number: 1934633-35-8) MeF35dC (S)-3-(3,5-dichlorophenyl)-2-(methylamino)propanoic acid (CAS number: 1542508-65-5) Adod 12-aminododecanoic acid (CAS number: 693-57-2) Hly L-homolisine (CAS number: 37689-89-7) W5C 5-chloro-L-tryptophan (CAS number: 52448-15-4) F3COO L-3-carboxyphenylalanine (CAS number: 13861-02-4) F3CON L-3-Carbamoylphenylalanine (CAS number: 1217651-22-3) Hgl L-2-aminoadipic acid (CAS number: 1118-90-7) Ndm N,N-dimethyl-L-asparagine (CAS number: 62937-43-3) KN3 or LysN3 6-azido-L-norleucine (CAS number: 159610-92-1) KAc N6-acetyl-L-lysine (CAS number: 692-04-6) dorn D-ornithine (CAS number: 348-66-3) F3H 3-Hydroxy-L-phenylalanine (CAS number: 587-33-7) Yae O-(2-aminomethyl)-L-tyrosine (CAS number: 1909283-20-0) F4aao O-(2-carboxymethyl)-L-tyrosine (CAS number: 24558-63-2) F4OEt O-ethyl-L-tyrosine (CAS number: 32795-52-1) F34dOMe 3,4-dimethoxy-L-phenylalanine (CAS number: 142995-28-6) alT L-alrosreonine (CAS number: 28954-12-3) alI L-alloisoleucine (CAS number: 1509-34-8) MeK N-methyl-L-lysine (CAS number: 7431-89-2) Tbg (S)-2-amino-3,3-dimethylbutyrate (CAS number: 20859-02-3) NVA L-norvaline (CAS number: 6600-40-4) Abu (S)-(+)-2-aminobutyric acid (CAS number: 1492-24-6) da D-alanine Bph 4-Phenyl-L-phenylalanine (CAS number: 155760-02-4) ds D-serine de D-glutamic acid MeA N-methyl-L-alanine (CAS number: 3913-67-5) MeR N-methyl-L-arginine (CAS number: 2480-28-6) MeW N-methyl-L-tryptophan (CAS number: 526-31-8) MeY N-methyl-L-tyrosine MeG N-methylglycine K(Maleimide) N6-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)methyl)cyclohexane-1-carbonyl)-L-lysine dk(Maleimide) N6-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)methyl)cyclohexane-1-carbonyl)-D-lysine KTrzMal (S)-2-amino-6-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)methyl)-1H-1,2,3-triazole-1-yl)hexanoic acid gAbu 4-aminobutanoic acid Furthermore, these newly synthesized amino acids are useful because they have the potential to add new functions to various peptides when producing various peptide derivatives.

[0025] The peptide in this invention is (i) A peptide containing the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys); (ii) A peptide having an amino acid sequence in which the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 has one to eleven amino acid residue substitutions, deletions, additions and / or insertions; (iii) A peptide containing the 1st to 12th amino acid sequence (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence described in SEQ ID NO: 14; or (iv) A peptide having an amino acid sequence in which the amino acid sequence described in Sequence ID No. 14 has one to eight amino acid residues substituted, deleted, added and / or inserted in the amino acid sequence from the 1st to the 10th amino acid residue. The peptide is a peptide that binds to a transferrin receptor. Note that each of the above options (i) to (iv) can be selected in any combination. Preferred examples of this peptide are peptides that can cross the blood-brain barrier or peptides that have cell permeability.

[0026] About peptide sequences The number of amino acid substitutions, deletions, additions, and / or insertions in the amino acid sequence described in Sequence ID No. 1 may be between 1 and 10 in the amino acid sequence from the 1st to the 15th amino acid sequence, with a lower limit of 1. The upper limits are 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, and 2, with a minimum of 1. In the amino acid sequence described in Sequence ID No. 14, the amino acid sequence from the 1st to the 12th amino acid may contain between 1 and 8 amino acids, with a lower limit of 1. The upper limit is 8, 7, 6, 5, 4, 3, or 2 amino acids, with a minimum of 1. Such amino acid substitutions are preferably conservative amino acid substitutions.

[0027] Conservative amino acid substitutions A "conservative amino acid substitution" refers to a substitution with a functionally equivalent or similar amino acid. Generally, substitutions within a given group can be considered structurally and functionally conservative. However, as is obvious to those skilled in the art, the role played by a particular amino acid residue can be determined by its significance in the three-dimensional structure of the molecule containing that amino acid. For example, a cysteine ​​residue can take the less polar oxidized (disulfide) form compared to its reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute a structurally and functionally important feature. Also, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic or cation-pi interactions. In such cases, substitution of amino acids with these side chains with amino acids belonging to the acidic or nonpolar group may still be structurally and functionally conservative. Residues such as proline, glycine, and cysteine ​​(disulfide form) can have a direct effect on the three-dimensional structure of the main chain and often cannot be substituted without structural distortion.

[0028] Conservative amino acid substitutions include specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0029] Preferred examples of this peptide are groups 1 and 2 below: Group 1: (I) Substitution of the first alanine residue of SEQ ID NO: 1 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) Substitution of any second amino acid residue or any N-methylamino acid in SEQ ID NO: 1; (III) Substitution of the third aromatic amino acid residue of Sequence ID No. 1 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (IV) Substitution of the fifth aromatic amino acid residue of Sequence ID No. 1 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (V) Substitution of the sixth asparagine residue of SEQ ID NO: 1 with a hydrophilic amino acid or alanine; (VI) Substitution of the 8th tyrosine residue of Sequence ID No. 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring as a side chain; (VII) Substitution of the 10th isoleucine residue of Sequence ID No. 1 with any amino acid; (VIII) Substitution of the 11th arginine residue of SEQ ID NO: 1 with any amino acid; (IX) Substitution of the 12th arginine residue of Sequence ID No. 1 with any amino acid; and (X) Substitution of the 13th tyrosine residue of SEQ ID NO: 1 with any amino acid; (XI) Substitution of the 14th N-methyltyrosine residue of SEQ ID NO: 1 with any amino acid; An amino acid sequence having one or more substitutions selected from; and Group 2: (I) Substitution of the first alanine residue of SEQ ID NO: 14 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) Substitution of any second amino acid residue or any N-methylamino acid in SEQ ID NO: 14; (III) Substitution of the third aromatic amino acid residue of Sequence ID No. 14 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (IV) Substitution of the fifth aromatic amino acid residue of Sequence ID No. 14 with a methylated aromatic amino acid residue or an amino acid residue having an aromatic ring in its side chain; (V) Substitution of the sixth asparagine residue of SEQ ID NO: 14 with a hydrophilic amino acid or alanine; (VI) Substitution of the 8th tyrosine residue of Sequence ID No. 14 with an aromatic amino acid residue, a methylated aromatic amino acid residue, or an amino acid residue having an aromatic ring in its side chain; (VII) Substitution of any amino acid at the 10th isoleucine residue of SEQ ID NO: 14; (X) Substitution of the 11th serine residue in sequence number 14 with a hydrophilic amino acid residue. Includes an amino acid sequence having one or more substitutions selected from.

[0030] "Methylation" refers to N-methylation, that is, the addition of a methyl group to the amino group of the amino acid in question. For example, methylated alanine refers to N-methylalanine (MeA). "Having an aromatic ring in its side chain" means an amino acid that has an aromatic ring in its side chain, which may be a fused ring or a heterocycle. The aromatic ring may also have substituents. For example, F4C is a type of amino acid that has an aromatic ring in its side chain because its side chain contains a benzyl group in which the carbon at position 4 is substituted with a carbon bonded to chlorine.

[0031] Naturally occurring amino acids can be divided into the following groups based on the common properties of their side chains. (1) Hydrophobic (also called nonpolar) amino acids: Amino acids that exhibit hydrophobicity (nonpolarity), including alanine ("Ala" or simply "A"), glycine ("Gly" or simply "G"), valine ("Val" or simply "V"), leucine ("Leu" or simply "L"), isoleucine ("Ile" or simply "I"), proline ("Pro" or simply "P"), phenylalanine ("Phe" or simply "F"), tryptophan ("Trp" or simply "W"), tyrosine ("Tyr" or simply "Y"), and methionine ("Met" or simply "M"). Furthermore, hydrophobic amino acids can be further divided into the following groups. Aliphatic amino acids: Amino acids having fatty acids or hydrogen in their side chains, including Ala, Gly, Val, Ile, and Leu. Aliphatic branched-chain amino acids: Amino acids having branched fatty acids in their side chains, including Val, Ile, and Leu. Aromatic amino acids: Amino acids having an aromatic ring in their side chain, including Trp, Tyr, and Phe.

[0032] (2) Hydrophilic (also called polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine ("Ser" or simply "S"), threonine ("Thr" or simply "T"), cysteine ​​("Cys" or simply "C"), asparagine ("Asn" or simply "N"), glutamine ("Gln" or simply "Q"), aspartic acid ("Asp" or simply "D"), glutamic acid ("Glu" or simply "E"), lysine (also written as lysine; "Lys" or simply "K"), arginine ("Arg" or simply "R"), and histidine ("His" or simply "H"). Furthermore, hydrophilic amino acids can be further divided into the following groups. Acidic amino acids: Amino acids whose side chains exhibit acidity, including Asp and Glu. Basic amino acids: Amino acids whose side chains exhibit basicity, including Lys, Arg, and His. Neutral amino acids: Amino acids whose side chains are neutral, including Ser, Thr, Asn, Gln, and Cys. Furthermore, Gly and Pro can be classified as "amino acids that affect the orientation of the main chain," and Cys and Met, amino acids containing sulfur molecules in their side chains, can be classified as "sulfur-containing amino acids." Furthermore, groups with aromatic side chains include Trp, Tyr, and Phe.

[0033] Examples of preferred peptides in this specification are peptides that can bind to human transferrin receptors (hTfRs), similar to the peptides described above. Other preferred examples are peptides that can cross the blood-brain barrier or peptides that have cell permeability.

[0034] Examples of preferred peptides in this specification include peptides comprising the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys), and also peptides having an amino acid sequence in which any of the amino acid residues 1, 2, 3, 5, 6, 8, 10 to 14th in SEQ ID NO: 1 are substituted. "An amino acid residue has been substituted" means that a specific amino acid residue has been replaced with an amino acid residue that may be modified in other ways.

[0035] In a peptide containing the amino acid sequence from the 1st to the 15th amino acid sequence (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys) of the amino acid sequence described in Sequence ID No. 1, The first alanine residue of SEQ ID NO: 1 is either a modified alanine (Ala) or a modified glutamic acid (Glu). The second valine residue in SEQ ID NO: 1 is either a modified valine (Val) or a modified glutamic acid (Glu). The third phenylalanine residue in Sequence ID No. 1 is a modified phenylalanine (Phe), The fifth tryptophan residue in sequence number 1 is a modified tryptophan (Trp), The sixth asparagine residue in sequence number 1 is alanine. The eighth tyrosine residue in SEQ ID NO: 1 is a modified phenylalanine. The peptide in Sequence ID No. 1 has a modified isoleucine (Ile), a modified alanine (Ala), or a modified valine (Val). The 11th arginine residue in sequence number 1 is any amino acid residue. A peptide in which the 12th isoleucine residue of SEQ ID NO: 1 is a modified isoleucine (Ile) or a modified valine (Val), That's fine.

[0036] "May be modified" means that known amino acid modifications or alterations may be made. Examples of modifications include N-methylation (also called methylation), amino acid modifications having abbreviations disclosed herein, modification (conversion) to the D type, and conversion to known derivatives of the amino acid in question.

[0037] Furthermore, in a peptide containing the amino acid sequence from the 1st to the 15th amino acid sequence of the amino acid sequence described in Sequence ID No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys), The first amino acid residue of Sequence ID No. 1 is Ala, Aib, Abu, Glu, Gly, Ser, Phe, Pro, or MeA, and is particularly preferably Ala or Abu. The second amino acid residue of Sequence ID No. 1 is Val, Glu, Ala, Arg, Lys, Asp, Phe, Dap, Har, Abu, Nva, AcPr, Atb, Ahp, or Hgl, and is particularly preferably Val, Glu, or Hgl. The third amino acid residue of Sequence ID No. 1 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO, or MeF3C, and is particularly preferably Phe, MeF, or MeF3C. The fifth amino acid residue of SEQ ID NO: 1 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1iPr, Yph, W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON, or W6F, and is particularly preferably Trp or MeTrp. The sixth amino acid residue of Sequence ID No. 1 is Asn, Ala, or Asp, and is particularly preferably Ala or Asn. The eighth amino acid residue of SEQ ID NO: 1 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nal1, and is particularly preferably Tyr, Ahp, or F4OMe. The 10th amino acid residue of SEQ ID NO: 1 is Ala, Abu, Acpr, Ahp, Aib, alI, alT, Atb, Dab, Dap, dorn, Gln, Hly, Ile, Lys, KCOpipzMe, Leu, Nle, Nva, Pro, Arg, Ser, Thr, Tbg, Val, or Tyr, and is particularly preferably Ile or Ala. The 11th amino acid residue of SEQ ID NO: 1 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva, or Orn, and is particularly preferably Ala, Glu, Lys, Arg, or Hly. The 12th amino acid residue of Sequence ID No. 1 is Lys, Glu, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap, or Dab, and is particularly preferably Glu, Arg, or dr. The 13th amino acid residue of SEQ ID NO: 1 is Ala, Phe, Asn, Tyr, or pHPeG, and is particularly preferably Phe or Tyr. The peptide in Sequence ID No. 1 is one of the following: the 14th amino acid residue is MeY, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW, or pHPeG, particularly preferably Tyr or MeTyr. That's fine.

[0038] Another preferred example of this peptide is a peptide having an amino acid sequence in which the amino acid sequence of the first to twelfth amino acids of the amino acid sequence described in SEQ ID NO: 14 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) has 1 to 8 amino acid residue substitutions, deletions, and / or insertions.

[0039] A peptide containing the amino acid sequence from the 1st to the 12th amino acid sequence of the amino acid sequence described in SEQ ID NO: 14 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) may also be a peptide having an amino acid sequence in which any of the amino acid residues at positions 1, 2, 3, 5, 6, 8, 10, or 11 of SEQ ID NO: 14 is substituted.

[0040] In a peptide containing the amino acid sequence from the 1st to the 12th amino acid sequence (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence described in SEQ ID NO: 14, The first alanine residue of SEQ ID NO: 14 is either a modified alanine (Ala) or a modified glutamic acid (Glu). The second valine residue in SEQ ID NO: 14 is either a modified valine (Val) or a modified glutamic acid (Glu). The third phenylalanine residue in SEQ ID NO: 14 is a modified phenylalanine (Phe), The fifth tryptophan residue in sequence number 14 is a modified tryptophan (Trp), The sixth asparagine residue in sequence number 14 is alanine. The 8th tyrosine residue of SEQ ID NO: 14 is a modified phenylalanine. The 10th isoleucine residue of Sequence ID No. 14 may be modified isoleucine (Ile), may be modified alanine (Ala), or may be modified valine (Val). The 11th serine residue in sequence number 14 is histidine (His) or asparagine (Asn). Peptides are also acceptable.

[0041] These peptides preferably have a peptide length of 12 to 15, more preferably 12 to 14, more preferably 12 to 13, and most preferably 12. In this case, peptide length refers to the number of amino acids in the cyclic structure and does not include the number of amino acid linkers.

[0042] Furthermore, in a peptide containing the amino acid sequence from the 1st to the 12th amino acid sequence (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) of the amino acid sequence described in Sequence ID No. 14, The first amino acid residue of SEQ ID NO: 14 is Ala, Aib, Abu, Glu, Gly, Ser, Phe, Pro, or MeA, and is particularly preferably Ala. The second amino acid residue of Sequence ID No. 14 is Val, Glu, Ala, Arg, Lys, Asp, Phe, Dap, Har, Abu, Nva, AcPr, Atb, Ahp, or Hgl, and is particularly preferably Val. The third amino acid residue of Sequence ID No. 1 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO, or MeF3C, and is particularly preferably Phe. The fifth amino acid residue of SEQ ID NO: 1 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1iPr, Yph, W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON, or W6F, and is particularly preferably Trp. The sixth amino acid residue of Sequence ID No. 1 is Asn, Ala, or Asp, and is particularly preferably Asn. The eighth amino acid residue of SEQ ID NO: 1 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nal1, and is particularly preferably Tyr. The 10th amino acid residue of SEQ ID NO: 1 is Ala, Abu, Acpr, Ahp, Aib, alI, alT, Atb, Dab, Dap, dorn, Gln, Hly, Ile, Lys, KCOpipzMe, Leu, Nle, Nva, Pro, Arg, Ser, Thr, Tbg, Val, or Tyr, and is particularly preferably Ile. The 11th amino acid residue of SEQ ID NO: 1 is Ser, His, or Asn, and is particularly preferably Ser. Peptides are also acceptable.

[0043] Preferred examples of this peptide are peptides consisting of the amino acid sequences from the 1st to the 15th amino acids of SEQ ID NOs: 1-13, 15, 18-86, 90-110, or the amino acid sequences from the 1st to the 12th amino acids of SEQ ID NOs: 14, 16, 17, 87-89.

[0044] A preferred example of this peptide is any of the peptides described above, which is a cyclic peptide. Preferably, the peptide is one in which the first amino acid in the 1st to 15th amino acid sequences of SEQ ID NOs: 1-13, 15, 18-86, 90-110, or the 1st to 12th amino acid sequences of SEQ ID NOs: 14, 16, 17, 87-89, is chloroacetylated, and the acetyl group is cyclically formed with the cysteine ​​at the end of the amino acid sequence.

[0045] A preferred example of this peptide is an amino acid sequence or a complex of an amino acid sequence and a linker (a linker-added peptide as described below) described in any of SEQ ID NOs: 1 to 110, which is either (1) a peptide containing the 1st to 15th amino acid sequence portion of SEQ ID NOs: 1 to 13, 15, 18 to 86, 90 to 110, wherein the portion has a cyclic structure, or (2) a peptide containing the 1st to 12th amino acid sequence portion of SEQ ID NOs: 14, 16, 17, 87 to 89, wherein the portion has a cyclic structure.

[0046] About cyclic peptides A cyclic peptide is defined as a peptide in which two amino acids are linked together, and all or part of the peptide is cyclic. In this application, however, peptides in which amino acids form a cross-linking structure, peptides in which a cyclic structure is formed by lactam ring formation or macrocyclization reactions, and peptides having a lasso-peptide-like structure are also included. In other words, in this application, a cyclic peptide only needs to have a part of it that forms a cyclic structure, and it may also have a linear portion. Furthermore, it may have a complex cyclic structure, such as a bicyclic structure in which two amino acids contained in a single cyclic peptide are further linked. Peptides generally have poor metabolic stability in vivo and their large size makes them difficult to permeate cell membranes. To address these challenges, methods such as cyclization of peptides have been employed. Cyclization of peptides has been shown to improve protease resistance and metabolic stability, and to restrict conformational changes, thereby increasing rigidity and improving membrane permeability and affinity to target proteins.

[0047] cyclization method Peptide cyclization can be carried out according to known methods. While not limited to these methods, for example, by designing a peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed after translation via disulfide bonds. Alternatively, cyclization can be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus using genetic code reprogramming techniques, as described by Goto et al. (Y. Goto, et al. Acss Chem. Biol. 3 120-129 (2008)), and then placing cysteine ​​residues within the peptide. This allows for spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization via thioether bonds. Cyclization can also be achieved by placing other amino acid combinations that form a ring within the peptide using genetic code reprogramming techniques. Furthermore, cyclization can be achieved by synthesizing a peptide with a cycloamide at the N-terminus and placing Hgl residues within the peptide. Thus, any known cyclization method is not particularly limited.

[0048] The peptide has a cyclic structure in which the N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which the N-terminal amino acid (the first amino acid residue) is bonded to the 15th or 12th cysteine ​​residue contained in the peptide. In another embodiment, the peptide has a cyclic structure in which the chloroacetylated N-terminal amino acid (the first amino acid residue) is bonded to the 15th or 12th cysteine ​​residue contained in the peptide. "Chloroacetylation" may also be "halogen acetylation" with other halogens. Also, "acetylation" may also be "acylation" with acyl groups other than acetyl groups.

[0049] In this specification, some amino acids may be modified for the cyclization of peptides. Such partially modified amino acids are also included in the amino acids of this application. For example, as described above, a chloroacetyl group may be added to the N-terminal amino acid and bound to a cysteine ​​residue in the peptide to form a cyclamen. Such various (natural / unnatural) amino acids to which a chloroacetyl group has been added are also included in the amino acids of this application.

[0050] A preferred example of the peptide of the present invention is any of the above-mentioned peptides, which consist of 15 amino acid residues or 12 amino acid residues.

[0051] Peptide length The number of amide bonds (number and length of amino acids) in the peptide and peptide moiety is not particularly limited, but it is preferable that the total number of amino acid residues (excluding amino acids if the substance bound to the peptide or the linker connecting the substance to the peptide contains amino acids) is 20 or less. Preferably, there are 6 or more amino acids, 7 or more, 8 or more, 9 or more, 10 or more, and 11 or more, and preferably 19 or less, 18 or less, 17 or less, 16 or less, and 15 or less amino acids.

[0052] Linker The conjugate (complex) of the present invention contains a peptide and an antibody or its antigen-binding fragment, and it is preferable that the peptide and the antibody or its antigen-binding fragment are linked via a linker. The linker between the peptide and the antibody or its antigen-binding fragment may be formed by a chemical bond between the linker of a linker-containing peptide (hereinafter also referred to as a linker-added peptide) and the antibody or its antigen-binding fragment, using any suitable reactive group including a reactive functional group described later.

[0053] The following describes linker-added peptides. Examples of linkers in linker-added peptides include those with an amino acid length of 1 to 15, and that contain one or more selected from glycine (Gly) and serine (Ser). Preferred examples of this linker are cysteine ​​(Cys) or lysine (Lys) whose N-terminus may be modified.

[0054] Another example of a linker in a linker-added peptide is one with an amino acid length of 1 to 5, and containing one or more selected from D-glutamic acid (de) and methylated glycine (MeG). Preferred examples of this linker are cysteine ​​(Cys) or lysine (Lys) whose N-terminus may be modified.

[0055] Another example of a linker is a PEG linker containing polyethylene glycol (PEG) or a derivative of polyethylene glycol. Derivatives of polyethylene glycol include all known PEG linkers, and some may have substituted or added functional groups. Examples include, but are not limited to, methyl groups, amino groups, and azide groups. Preferably, the PEG linker is PEG4c, PEG12c, PEG8c, PEG36, PEG4c-PEG4c-PEG4c, or PEG4c-PEG4c. Furthermore, it is preferable that the PEG linker further contains one or more selected from glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), gAbu, KTrzMal, and lysine (Lys). Furthermore, the linker may also contain reactive functional groups for bonding the desired substance. Examples of reactive functional groups include maleimides, hydrazides, NHS, and functional groups used for click reactions.

[0056] Another example of a linker in a linker-added peptide is a linker having a sequence shown as Linker SEQ in Table 1, or a sequence shown by any of SEQ ID NOs. 111-161 in Table 5.

[0057] A preferred example of a linker in a linker-added peptide is: Polyethylene glycol (PEG), The linker has an amino acid sequence represented by one of the following: a G linker, which is a peptide linker consisting of Gly or MeG; a GS linker, which is a peptide linker consisting of Gly or MeG and Ser; a sequence shown as Linker SEQ in Table 1; or a sequence shown by any of the sequence numbers 111 to 161 shown in Table 5.

[0058] A linker (also called a crosslinker) in this specification represents an intermolecular link between a peptide bound to a transferrin receptor and an antibody or its antigen-binding fragment, and may be any known or described linker. In certain embodiments, the linker may be, for example, a chemical linker, a fatty acid linker, or a peptide linker (polypeptide linker). It may also be a complex of, for example, a chemical linker and a peptide linker. For example, it may be a linker structure having both PEG and an amino acid residue or a peptide moiety, as shown in the sequence shown as Linker SEQ in Table 1, or any of SEQ ID NOs. 111-161 shown in Table 5. The linker may be something that separates or decouples depending on the environment or conditions, or it may maintain a stable structure.

[0059] Chemical linkers: In some embodiments, the linker may be a chemical linker. Chemical linkers include, but are not limited to, substituted or unsubstituted alkylenes, substituted or unsubstituted heteroalkylenes, substituted or unsubstituted cycloalkylenes, substituted or unsubstituted heterocycloalkylenes, substituted or unsubstituted arylenes and / or substituted or unsubstituted heteroarylenes. The peptide and linker may also be conjugated via sulfhydryl groups, amino groups (amines), and / or carbohydrates or any suitable reactive group. Homobifunctional and heterobifunctional crosslinkers (conjugation agents) are available from many commercial sources. The crosslinker may contain a flexible arm, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Examples of crosslinkers include BSS3 (bis(sulfosuccinimidyl)sverate), NHSS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide), sulfo-EMCSS ([Ne-maleimidocaproic acid]hydrazide), hydrazides, and SSATA (N-succinimidyl-SS-acetylthioacetic acid).

[0060] A preferred example of a chemical linker is a PEG (Polyethyleneglycol) linker. For example, a PEG linker may consist of 1 to 24 ethylene glycol units.

[0061] Fatty acid linker: The linker may be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. For example, the fatty acid linker may be a linker having 12-aminododecanoic acid.

[0062] Peptide linkers: Peptide linkers contain at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is one amino acid (e.g., any natural amino acid such as Cys). In other embodiments, glycine-rich peptides are used, such as peptides containing the sequence [Gly-Gly-Gly-Gly-Ser]n (wherein n is 1, 2, 3, 4, 5, or 6), as described in U.S. Patent No. 7,271,149. In other embodiments, serine-rich peptide linkers are used, such as those described in U.S. Patent No. 5,525,491. Examples of serine-rich peptide linkers include those of the formula [XXXX-Gly]y (wherein up to two of X are Thr, the remaining X are Ser, and y is 1 to 5) (e.g., Ser-Ser-Ser-Ser-Gly (wherein y is 2 or more)). In some cases, the linker is a single amino acid (for example, any amino acid such as Gly or Ala).

[0063] Alternatively, other known linkers include those described in WO2021 / 054370, WO2020 / 209285, WO2020 / 028832, WO2017 / 221883, WO2015 / 194520, WO2012 / 150960, WO2012 / 029986, etc.

[0064] Manufacturing method The peptides in this invention can be produced by known peptide production methods, such as chemical synthesis methods including liquid-phase methods, solid-phase methods, and hybrid methods combining liquid-phase and solid-phase methods; and genetic engineering methods.

[0065] The solid-phase method involves, for example, esterifying the hydroxyl group of a resin containing a hydroxyl group with the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group. Known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used as esterification catalysts. Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid, in which all functional groups except the carboxyl group of the main chain are protected, is added. The carboxyl group is then activated to bond the first and second amino acids. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid, in which all functional groups except the carboxyl group of the main chain are protected, is added. The carboxyl group is then activated to bond the second and third amino acids. This process is repeated until a peptide of the desired length is synthesized, at which point all functional groups are deprotected.

[0066] Examples of solid-phase synthesized resins include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.). Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid or hydrogenation, the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine. For α-carboxyl group protection, methyl esters, ethyl esters, benzyl esters, tert-butyl esters, cyclohexyl esters, etc., can be used. Other functional groups of amino acids include the hydroxyl groups of serine and threonine, which can be protected with benzyl or tert-butyl groups, and the hydroxyl group of tyrosine, which can be protected with 2-bromobenzyloxycarbonyl or tert-butyl groups. The amino groups of the lysine side chains, and the carboxyl groups of glutamic acid and aspartic acid, can be protected in the same way as α-amino and α-carboxyl groups.

[0067] The carboxyl group can be activated using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

[0068] Peptide chains can be cleaved from resin by treatment with acids such as TFA and hydrogen fluoride (HF).

[0069] The production of peptides by genetic engineering (translation synthesis system) can be carried out using the nucleic acid encoding the peptide in this invention. The nucleic acid encoding the peptide in this invention may be DNA or RNA. The nucleic acids encoding the peptides in the present invention can be prepared by known methods or similar methods. For example, they can be synthesized by an automated synthesis apparatus. Restriction enzyme recognition sites may be added to the obtained DNA for insertion into a vector, or a base sequence encoding an amino acid sequence for cleaving the resulting peptide chain with an enzyme may be incorporated. As described above, when the peptide in the present invention is fused with a membrane-permeable peptide, etc., the nucleic acid also includes the nucleic acid encoding the membrane-permeable peptide. To suppress degradation by host-derived proteases, a chimeric protein expression method can be used, in which the target peptide is expressed as a chimeric peptide with another peptide. In this case, the nucleic acids used are those encoding the target peptide and the peptide that binds to it.

[0070] Next, an expression vector is prepared using the nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector, either as is, digested with restriction enzymes, or by adding a linker. Examples of vectors include E. coli plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), cosmids, etc.

[0071] The promoter can be appropriately selected depending on the type of host. If the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or CMV (cytomegalovirus) can be used. If the host is E. coli, the trp promoter, T7 promoter, lac promoter, etc., can be used. Expression vectors can also incorporate nucleic acids that encode DNA replication origins (ori), selection markers (antibiotic resistance, nutritional requirements, etc.), enhancers, splicing signals, polyalpha alpha addition signals, and tags (FLAG, HA, GST, GFP, etc.).

[0072] Next, suitable host cells are transformed with the expression vector described above. The host can be appropriately selected in relation to the vector, and examples include Escherichia coli, Bacillus subtilis, Bacillus species, yeast, insects or insect cells, and animal cells. Examples of animal cells that can be used include HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be carried out according to known methods such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun, depending on the type of host. By culturing the transformed cells according to conventional methods, the target peptide is expressed.

[0073] Peptide purification from transformant cultures involves harvesting the cultured cells, suspending them in a suitable buffer, disrupting the cells by methods such as sonication or freeze-thaw cycles, and obtaining a crude extract by centrifugation or filtration. If peptides are secreted into the culture medium, the supernatant is collected. Purification from the crude extract or culture supernatant can also be carried out by known methods or similar methods (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reverse-phase high-performance liquid chromatography, etc.). The obtained peptide may be converted from a free form to a salt, or from a salt to a free form, by known or equivalent methods.

[0074] The translation synthesis system may be a cell-free translation system. A cell-free translation system may include, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), termination factor (RF), and ribosomal regeneration factor (RRF), as well as other factors necessary for translation. E. coli extract or wheat germ extract may be added to increase expression efficiency. Rabbit red blood cell extract or insect cell extract may also be added. By continuously supplying energy to a system containing these components using dialysis, proteins can be produced in quantities ranging from several hundred μg to several mg / mL. A system including RNA polymerase may also be used to facilitate transcription from gene DNA. Commercially available cell-free translation systems include Roche Diagnostics' RTS-100 (registered trademark), Gene Frontier's PURESYSTEM, and New England Biolabs' PURExpress In Vitro Protein Synthesis Kit, all derived from E. coli, and systems from Zoygene and CellFree Science, among others, which utilize wheat germ extract. Cell-free translation systems allow for the acquisition of highly purified expression products without the need for purification.

[0075] In cell-free translation systems, artificial aminoacyl-tRNAs may be used instead of aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases, by cylating (acylating) a desired amino acid or hydroxy acid to the tRNA. Such aminoacyl-tRNAs can be synthesized using artificial ribozymes. Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894 “A Examples include "flexizyme that selectively charges amino acids activated by a water-friendly leaving group" (and WO2007 / 066627, etc.). Flexizyme is also known by the original flexizyme (Fx), and modified versions thereof such as dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), and aminoflexizyme (aFx).

[0076] By using tRNA linked to a desired amino acid or hydroxy acid, generated by Flexizyme, the desired codon can be translated in association with the desired amino acid or hydroxy acid. Special amino acids may be used as the desired amino acid. For example, unnatural amino acids necessary for the cyclization described above can also be introduced into the bound peptide by this method.

[0077] The chemical synthesis of cyclic peptides and their analogues in the present invention can be performed using a variety of commonly used methods in the art, including stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformationally supported re-ligation, and chemical ligation. The synthesis of peptides and their analogues described herein is chemical synthesis using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on a combination of an Fmoc group that temporarily protects the α-amino group and allows for selective removal by a base, and a protecting group that temporarily protects the side-chain functional group and is stable under de-Fmoc conditions. Such common peptide side chain selections are known from the aforementioned Peptide Synthesis and Applications, 2nd edition, GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214, etc. Preferred peptide side chain protecting groups include Boc and Mtt groups for amino groups such as lysine, tert-butyl groups for carboxyl groups of glutamic acid and aspartic acid, and Trt and Mmt groups for thiol groups of cysteine.

[0078] The peptides and their analogues described in the present invention can be synthesized stepwise on the solid-phase resin described above. The α-amino protecting group must be selectively removed from the C-terminal amino acid and all amino acids and peptides used in the synthesis process. Preferably, the synthesis is initiated by using the solid-phase resin described above, activating the C-terminal carboxyl group of a peptide whose N-terminus is appropriately protected with Fmoc or the like, or the C-terminal carboxyl group of an amino acid protected with Fmoc, using an appropriate reagent, and then adding it to an amino group on the solid-phase resin. Subsequent peptide chain extension can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and then the condensation of the protected amino acid derivative, according to the amino acid sequence of the target peptide. In addition, the target peptide can be released in the final stage. For example, conditions for liberation include using a TFA solution containing water / silyl hydride / thiol as a scavenger in TFA, as described in Teixeira, WE Benckhuijsen, PE de Koning, ARPM Valentijn, JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0079] The synthesis of the peptide analogs described herein can be carried out using a single or multi-channel peptide synthesizer, such as CEM's Liberty Blue synthesizer or Biotage's Syro I synthesizer.

[0080] The carboxyl group can be activated using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

[0081] Conjugate (complex) The conjugate of the present invention comprises the aforementioned peptide and an antibody or its antigen-binding fragment, and it is preferable that the peptide and the antibody or its antigen-binding fragment are linked via a linker. The linker between the peptide and the antibody or its antigen-binding fragment may be formed by a chemical bond between the linker of a linker-containing peptide (hereinafter also referred to as a linker-added peptide) and the antibody or its antigen-binding fragment, using any suitable reactive group containing a reactive functional group.

[0082] Since the conjugate of the present invention contains a peptide that has binding affinity to hTfR and can pass through the blood-brain barrier, the conjugate of the present invention has binding affinity to hTfR and can deliver compounds containing antibodies or antigen-binding fragments thereof into the blood-brain barrier, as shown in the examples. Furthermore, since the conjugate of the present invention contains a cell-penetrating peptide, as shown in the examples, the conjugate of the present invention has cell-penetrating properties and can deliver compounds containing antibodies or their antigen-binding fragments into cells. Therefore, the conjugate of the present invention may be particularly useful for the prevention or treatment of brain-related diseases.

[0083] Pharmaceutical composition

[0084] Another embodiment disclosed in this specification relates to a pharmaceutical composition. This pharmaceutical comprises the TfR-binding peptide-antibody conjugate described above and a pharmaceutically acceptable salt or solvate thereof (for simplicity, these will also be referred to below simply as a compound containing the antibody or its antigen-binding fragment). The pharmaceutical composition preferably contains an effective amount of the conjugate described above as an active ingredient.

[0085] In this specification, the form of administration of the pharmaceutical composition is not particularly limited and may be administered orally or parenterally. Examples of parenteral administration include injection (such as intramuscular injection, intravenous injection, or subcutaneous injection), transdermal administration, and transmucosal administration (through the nose, mouth, eye, lung, vagina, or rectum).

[0086] The above pharmaceutical composition can be modified in various ways, taking into account the easily metabolized and excreted nature of polypeptides. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood retention time and reduce its antigenicity. Alternatively, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., may be used as sustained-release bases, and polypeptides may be encapsulated within them. When administered transdermally, a weak electric current can be applied to the skin surface to allow penetration through the stratum corneum (iontophoresis).

[0087] The above-mentioned pharmaceutical composition may use the active ingredient as is, or it may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, poultices, etc. Formulation can be carried out by conventional methods, for example, by using excipients, binders, disintegrants, lubricants, solvents, solubilizers, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., as appropriate. Examples of ingredients used in formulation include, but are not limited to, purified water, saline solution, phosphate buffer, dextrose, glycerol, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropylcellulose, starch, corn starch, anhydrous silicic acid, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc. In view of the fact that peptides are poorly absorbed through the mucous membrane, the above pharmaceutical composition may contain an absorption enhancer to improve the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0088] Pills or tablets may also be coated with sugar, gastric-soluble, or enteric-soluble substances. The injectable preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, humectants, emulsifiers, dispersants, stabilizers, solvents, solubilizers, preservatives, etc. may be added.

[0089] The dosage of the pharmaceutical composition of the present invention when administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited. For example, 30 μg to 100 g, 100 μg to 500 mg, or 100 μg to 100 mg can be administered in one dose or in several divided doses. In the case of injection administration, depending on the patient's weight, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg may be administered in one dose or in several divided doses.

[0090] This specification provides the use of a conjugate for manufacturing a pharmaceutical product for the prevention or treatment of brain-related diseases. In this case, the conjugate may be any of the above-described types.

[0091] Another embodiment disclosed in this specification is a method for conjugating the peptide of the present invention with a compound comprising an antibody or an antigen-binding fragment thereof, (i) A step of reducing the disulfide bond in a compound containing an antibody or an antigen-binding fragment thereof; (ii) A step of preparing the peptide to which a linker having maleimide at its terminus is attached; (iii) A step of contacting a compound containing an antibody or antigen-binding fragment thereof, whose disulfide bond has been reduced in (i), with the peptide prepared in (ii), This is a method that includes Regarding step (i), since each chain of the antibody is linked by disulfide bonds, the thiol groups can be exposed by reducing these bonds. Disulfide bond reduction can be performed using known reducing agents, and is not limited to those mentioned above, but DTT (dithiothreitol), 2-MEA (ethylamine), BME (β-mercaptoethanol), TCEP (tris(2-carboxyethyl)phosphine), etc., can be preferably used. With respect to step (ii), the linker may be the linker described above. Maleimide can be attached to the ends of these linkers in a known manner. Regarding step (iii), the maleimide group reacts with the thiol group in a solution of pH 6.5 to 7.5 to form a stable thioether group, thereby enabling the compound containing the antibody or its antigen-binding fragment to be bound to the peptide. Since antibodies typically have multiple disulfide bonds, multiple peptides can be bound to the target antibody or compound containing its antigen-binding fragment by the above method.

[0092] Another embodiment disclosed in this specification is a method for conjugating the peptide of the present invention with a compound comprising an antibody or an antigen-binding fragment thereof, (i) A step of oxidizing the sugar chain of a compound containing an antibody or its antigen-binding fragment; (ii) A step of preparing the peptide to which a linker having a hydrazide at its terminus is attached; (iii) A step of contacting a compound containing an antibody whose sugar chain has been oxidized in (i) or an antigen-binding fragment thereof with the peptide prepared in (ii), This is a method that includes Regarding step (i), antibodies are known to have sugar chains, which can be oxidized to generate carbonyl groups. While known oxidizing agents can be used and are not limited to those mentioned above, sodium periodate is preferably used. With respect to step (ii), the linker may be the linker described above. The hydrazide can be bonded to the ends of these linkers in a known manner. Regarding step (iii), the hydrazide group reacts with the carbonyl group in a pH 5-7 solution to form a stable hydrazine bond, thereby enabling the compound containing the antibody or its antigen-binding fragment to be bound to the peptide.

[0093] Furthermore, another embodiment disclosed in this specification is a method for conjugating the peptide of the present invention with a compound comprising an antibody or an antigen-binding fragment thereof, (i) A step of preparing the peptide to which a linker having an N-hydroxysuccinimide (NHS) at its terminus is attached; (ii) A step of contacting a compound containing an antibody or an antigen-binding fragment thereof with the peptide prepared in (ii), This is a method that includes With respect to step (i), the linker may be one of the linkers described above. The NHS can be coupled to the ends of these linkers in a known manner. Regarding step (ii), since NHS and amines react efficiently at a pH of neutral or higher to form an amide bond, the peptide can be attached to the target antibody or a compound containing its antigen-binding fragment by binding the NHS of the peptide-linker to the amine present in the antibody. Furthermore, this specification also provides a method for producing peptide-antibody conjugates using the above-described conjugation method.

[0094] Abbreviation Fmoc as 9-fluorenylmethyloxycarbonyl; HOAt as 1-hydroxybenzotriazole; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate as HATU; MeCN as acetonitrile; Acetyl as Ac; Bovine serum albumin (BSA) As chloroacetyl, it is called ClAc; CO2 as carbon dioxide; 1,8-Diazabicyclo"5.4.0"-7-Undecene as DBU; N,N-diisopropylethylamine is referred to as DIPEA or DIEA; DODT as 3,6-dioxa-1,8-octane-dithiol; Dimethyl sulfoxide (DMSO) DMF as N,N-dimethylformamide; EDC as ethylene dichloride; Ethylenediaminetetraacetic acid (EDTA); Fetal bovine serum (FBS) is used as a substitute for fetal bovine serum. HEPES as hydroxyethylpiperazine ethanesulfonic acid; IC50 as the 50% inhibitory concentration; Mtt as monomethyltrityl; Mmt as monomethoxytrityl; o-Ns as 2-nitrobenzenesulfonyl; Trifluoroacetic acid (TFA); TIS as triisopropylsilane; Tritil as Trt Tris(2-carboxyethyl)phosphine is TCEP; mL (unit): milliliters; M as the unit of Mohler; volume / volume as v / v; DIPCI as N,N'-diisopropylcarbodiimide Oxyma pure as ethyl cyano(hydroxyimino)ethyl acetate; PBS as phosphate-buffered saline; Phosphate-buffered saline - Tween20 as PBST; AA as an amino acid; HOSu; as N-hydroxysuccinimide Dichloromethane (DCM); As N-(chloroacetoxy)succinimide, it is called ClaAcOSu; Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) as SMCC; The unit is millimolar (mM); The unit is micrometer (μm); mm (unit): millimeters; nanometer (unit: nm); Angstrom (unit): Å; Minutes (unit): min; Mass spectrometry (MS); Concentration (conc;) mmol (unit): millimoles; mg (unit): milligrams; Rotations per minute (in rpm); Time (unit): h; Gravitational acceleration (unit: G); HPLC as a high-performance liquid chromatography method; Liquid chromatography-mass spectrometry, such as LC-MS or LC / MS; DMAP as 4-dimethylaminopyridine; EDCI·HCl is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. hydrazide NHS ester or NHS N-hydroxysuccinimide PEG4c, PEG3, or PEG4 1-amino-3,6,9,12-tetraoxapentadecane-15-euic acid PEG8c 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-euic acid PEG12c or PEG11 or PEG12 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-euic acid PEG36 1-amino-3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108-Hexatriacontaoxaundecahectane-111-Eucic Acid [Examples]

[0095] The present invention will be further described in detail by the following reference examples and embodiments, but these are not intended to limit the present invention and may be modified without departing from the scope of the present invention. In this specification, we refer to International Application Number PCT / JP2021 / 006709 (International Publication WO2021-167107; not published at the time of the application of the present application), which has the exact same applicant as the present application.

[0096] chemical synthesis In the following examples, all raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis were either commercially available or synthesized by those skilled in the art using organic chemical methods. Unless otherwise specified, amino acids containing protecting groups were commercially available.

[0097] The following describes general methods for peptide synthesis, cyclization, purification, and analysis, but the conditions can be adjusted as appropriate depending on the sequence, etc. If a method is described in the examples, that method was used. Peptide chain elongation in solid-phase resins was carried out using the resins described in each example as starting materials, under commonly used peptide coupling and Fmoc removal reaction conditions. The reactions were performed using the CEM Liberty Blue automated peptide synthesizer, following the manufacturer's manual. Common amino acids used are listed below, with side-chain protecting groups indicated in parentheses.

[0098] Fmoc-Trp(Boc)-OH;Fmoc-Thr(tBu)-OH;Fmoc-N-Me-Gly-OH;Fmoc-Asp(OtBu)-OH;Fmoc-N-Me-Phe-OH;Fmoc-Ala-OH;Fmoc-N-Me-Ala-OH;Fmoc-His(Trt )-OH;Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH;Fmoc-HydPro(tBu)-OH;Fmoc-Cys(Trt)-OH;Fmoc-Lys(Mtt)-OH;Fmoc-Ser(tBu)-OH;Fmoc-N-Me-Ser(tBu)-OH.

[0099] The introduction of the chloroacetyl group was performed by removing the Fmoc group from the α-amino group in the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, and then adding chloroacetic acid (3 equivalents) to 3 equivalents of a DMF solution of N,N'-diiropropylcarbodiimide (0.5M) and 3 equivalents of a DMF solution of HOAt (0.5M), and shaking at room temperature for 40 minutes.

[0100] For side chain deprotection and cleavage from the solid phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times each with DMF and methylene chloride and dried under reduced pressure. Next, the reaction vessel containing the solid phase resin was mixed with reagent cocktail-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) and shaken at room temperature for 150 minutes. The reaction solution was filtered and recovered through the frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered through the frit and mixed with the aforementioned filtrate. When this filtrate was added to excess diethyl ether cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0°C, and the obtained solid was used in the next cyclization reaction.

[0101] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the number of moles of the solid phase resin, then adding 6 or 7 equivalents of triethylamine and stirring at room temperature for approximately 16 hours. The resulting reaction solution was acidified with acetic acid and concentrated under reduced pressure using Biotage® V-10 (Biotage Japan Co., Ltd.).

[0102] For the purification of the obtained crude peptide, reverse-phase preparative HPLC was performed using a Waters AutoPurification System-SQD2 single quadruple mass spectrometer, and elution was carried out while monitoring the m / z ions derived from the target product. It was confirmed that the mass spectrum obtained in ESI-positive scan mode and the mass spectrum including polyvalent ions calculated from the molecular formula of the target product were in agreement within the error range of the mass spectrometer used. The purification conditions, including the column used, are shown in each example.

[0103] The structure of chemically synthesized peptides was determined by calculating the molecular weight, considering the amino acids used according to the target sequence and the building blocks used as needed, and confirming this by ESI-MS(+) in mass spectrometry. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. The detected mass was reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as divalent or trivalent ions. The analytical methods are described in each example. [Example 1]

[0104] In this example, a peptide that binds to hTfR was synthesized. The synthesized peptide is shown in Table 1. Table 5 shows only the linker sequence extracted from Table 1. The synthesis was carried out in the same manner as described above and in Example 2. Note that for SEQ ID NO: 23, 894_Bicycle_002_GGRGRS_K(Mal), in addition to cyclization by the 1st Ala residue and the 15th Cys residue, it has a bicycle structure in which the 2nd Hgl residue and the 11th Hly residue are bound. The synthesized peptide was analyzed under the analytical conditions described in each example, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observations and the value of X when expressed as the number of proton additions (M+XH)X+ are shown in Table 1.

[0105] Furthermore, naked peptides (peptides without a linker) consisting of the amino acid sequences of positions 1 to 15 of the peptides listed in Table 1 (i.e., peptides having the amino acid sequences of SEQ ID NOs. 1 to 110) were synthesized using the method described in Example 7 of International Application No. PCT / JP2021 / 006709 (International Publication WO2021-167107), and their binding ability to hTfR was confirmed by SPR. The KD values ​​obtained from these SPR measurements are denoted as A if the KD value is less than 1 nM, B if it is between 1 nM and 100 nM, C if it is between 100 nM and 1 mM, and D if it is 1 mM or greater. These results are listed in the column for the peptide containing the corresponding naked peptide in Table 1. Here, ND indicates No Data (meaning that data was not obtained). For SEQ ID NOs. 104 to 107, SPR measurements were performed on those with a linker bound, and binding ability to hTfR was confirmed in all cases. As a result, it was confirmed that all of the naked peptides listed in Table 1 possess hTfR binding ability. Furthermore, when the hTfR binding ability of peptide 894_3m_PEG12_dk(Maleimide) (a peptide of sequence number 36 with the linker sequence of sequence number 114 attached) listed in Table 1 was confirmed by SPR using the same method as above, it fell into category B of the above classification. This indicates that even when a linker is attached to an hTfR-binding peptide (naked), the ability to bind to hTfR can be maintained.

[0106] [Table 1] [Table 1-1-1] [Table 1-1-2] [Table 1-1-3] [Table 1-2-1] [Table 1-2-2] [Table 1-2-3] [Table 1-3-1] [Table 1-3-2] [Table 1-3-3] [Table 1-4-1] [Table 1-4-2] [Table 1-4-3]

[0107] [Table 5] [Table 5-1] [Table 5-2]

[0108] [Example 2-1] Synthesis of 894_3m_G4S2_K(Mal)(SEQ ID NO: 37) This example describes the synthesis of 894_3m_G4S2_K(Mal) (a peptide of sequence number 37 with the corresponding linker listed in Table 1 attached). [ka]

[0109] Using sieber amide resin (Watanabe Chemical, 0.52 mmol / g, 2.4 g x 3), the target peptide was synthesized following a general procedure, starting with the removal of Fmoc. A CEM Liberty Blue solid-phase synthesizer was used, and the procedure followed the manufacturer's manual. For the introduction of each residue, the basic condition was to react one equivalent of resin with Fmoc-AA / DIPCI / Oxyma pure (5.3 equivalents / 10 equivalents / 5 equivalents) in DMF at 75°C for 10 minutes. However, the 15th residue was reacted at 50°C for 20 minutes. The 3rd, 4th, 8th, 10th, 13th, and 14th residues were reacted twice at 75°C for 10 minutes each. The 11th and 12th residues were reacted twice at 50°C for 20 minutes each. The 2nd residue was reacted three times at 75°C for 60 minutes each. The reaction for the first residue was carried out twice at 75°C for 20 minutes each. The basic condition for Fmoc removal was to react with a 20% piperidine DMF solution at 75°C for 3 minutes. However, for the 15th, 16th, and 17th residues, the reaction was carried out at room temperature for 5 minutes, followed by a reaction at 75°C for 3 minutes. For the 2nd and 13th residues, the reaction was carried out at 25°C for 5 minutes, followed by a reaction for 10 minutes. For the introduction of the chloroacetyl group, the Fmoc group of the α-amino group was removed from the solid resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, chloroacetic acid (5 equivalents), DIPCI (5 equivalents), and HOSu (5 equivalents) were stirred in DCM, and the same amount of DMF as the DCM was added to prepare a DCM / DMF solution of ClAcOSu (0.015M). This solution was added to the solid resin and shaken at room temperature for 180 minutes. For deprotection of the side chains and cleavage from the solid phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Then, reaction cocktail-A (200 mL, a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered through the frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered through the frit, and mixed with the aforementioned filtrate.When this filtrate was added to excess diisopropyl ether cooled to 0°C, a cloudy white precipitate formed. The mixture was collected by filtration, washed with diethyl ether cooled to 0°C, and the obtained solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO (containing 5% water) such that the final concentration of the peptide was 4.9 mM based on the number of moles of the solid-phase resin, then 7 equivalents of triethylamine was added, and the mixture was shaken at room temperature for about 1 hour. 1.05 equivalents of SMCC was added to the obtained reaction solution, and the mixture was shaken at room temperature for 1.5 hours. The obtained reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0110] The obtained crude product was purified under the following conditions (column: Waters Xbridge® C18 5μm® 50×250 mm; mobile phase: A = 0.1% TFA in H₂O, B = 0.1% TFA in MeCN; temperature: 60°C; gradient (%B concentration): 0-0% over 5.1 minutes, then 0-5% over 1.9 minutes, then 5-29% over 5 minutes, then 29-34% over 13.5 minutes, then 34-60% over 1.5 minutes; flow rate: 1-1 mL / min over 5.1 minutes, then 1-119 mL / min over 1.9 minutes, then 119 mL / min).

[0111] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions, and was 94.1%. Analytical conditions: retention time = 11.33 min; column: Kinetex EVO C18 2.6 μm 2.1×150 mm, 100Å; mobile phase: A = 0.025% TFA in H₂O, B = 0.025% TFA in MeCN; temperature: 40°C; gradient (%B concentration): 20-60% over 20 minutes, then 60-95% over 1 minute, then 95% over 5 minutes; flow rate: 0.25 mL / min ESI-MS (+) observed m / z = 1423.95 (M+2H) 2+

[0112] [Example 2-2] Synthesis of 894_3m_GGRGRS_K(Mal)(SEQ ID NO: 82) This example describes an example of the synthesis of 894_3m_GGRGRS_K(Mal) (the peptide of SEQ ID NO: 82 bound to the corresponding linker described in Table 1).

Chemical Formula

[0113] Using sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.53 g), the target peptide was synthesized following a general procedure, starting with the removal of Fmoc. A CEM Liberty Blue HT was used as the solid-phase synthesizer, and the procedure followed the manufacturer's manual. For the introduction of each residue, the basic condition was to use 1 equivalent of resin and Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) and react twice in DMF at 75°C for 10 minutes each. However, the second residue was reacted twice at 75°C for 30 minutes each. The 5th, 6th, 7th, 16th, 17th, 19th, 21st, and 22nd residues were reacted once at 75°C for 10 minutes each. The 11th residue was reacted twice at 50°C for 15 minutes each. The reaction at residues 12, 15, 18, and 20 was carried out once at 50°C for 15 minutes. Fmoc removal was primarily performed by reacting a 20% piperidine DMF solution with the resin at 75°C for 3 minutes. However, Fmoc removal at residues 2 and 13 was carried out by reacting the resin twice at room temperature for 5 minutes each. Chloroacetyl group introduction was performed by stirring chloroacetic acid (5 equivalents), DIPCI (5 equivalents), and HOSu (5 equivalents) in DCM, adding the same amount of DMF as the DCM to prepare a DCM / DMF solution of ClAcOSu (0.25M), adding it to the solid resin obtained in the previous step, and shaking at room temperature for 60 minutes. For side chain deprotection and cleavage from the solid resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail-A (10 mL, a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid phase resin, and the mixture was shaken at room temperature for 150 minutes. The reaction solution was filtered and recovered using frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered using frit, and mixed with the aforementioned filtrate. When this filtrate was added to a mixed solvent of excess diethyl ether and hexane cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (9500 rpm, 1 min), the supernatant was decanted, and washed with diethyl ether cooled to 0°C. The resulting solid was used in the next cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in DMSO (containing 5% water) such that the final concentration of the peptide was 5 mM based on the number of moles of solid phase resin, then adding 7 equivalents of triethylamine and shaking at room temperature for about 3 hours. 1.1 equivalents of SMCC was added to the obtained reaction solution, followed by shaking at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0114] The obtained crude product was purified under the following conditions (column: Waters Xbridge® C18 5 μm 50×150 mm; mobile phase: A=0.1% TFA in H₂O, B=0.1% TFA in MeCN; temperature: 40°C; gradient (%B concentration): 5-29% over 3 minutes, then 29-34% over 8 minutes, then 34-60% over 1 minute; flow rate: 120 mL / min).

[0115] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analysis conditions, and was 95.4%. Analysis conditions: retention time = 9.53 min; column: Kinetex EVO C18 2.6 μm 2.1×150 mm, 100 Å; mobile phase: A=0.025% TFA in H₂O, B=0.025% TFA in MeCN; temperature: 40°C; gradient (%B concentration): 20-60% over 20 minutes, then 60-95% over 1 minute, then hold at 95% for 5 minutes; flow rate: 0.25 mL / min ESI-MS(+) observed m / z = 1005.73 (M+3H) 3+

[0116] [Example 2-3] Synthesis of hTfR_000894_PEG11_(Hydrazine)(SEQ ID NO: 6) This example describes a synthesis example of hTfR_000894_and_PEG11_(Hydrazine) (the peptide of SEQ ID NO: 6 bound to the corresponding linker described in Table 1).

Chemical Formula

[0117] Using NH2-NH-Trt(2-Cl)-resin (0.77 mmol / g, 0.13 g) prepared according to a general method rather than commercially available Cl-Trt(2-Cl)-resin, the target peptide was synthesized starting with the removal of Fmoc, following the aforementioned general method. A CEM Liberty Blue HT was used as the solid-phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / HATU / DIEA (5.3 equivalents / 5 equivalents / 10 equivalents) was used per equivalent of resin, and the condensation reaction was carried out once at 25°C for 30 minutes. For residues 11, 12, 13, and 14, the reaction was carried out twice at 25°C for 30 minutes each. For residue 16, the reaction was carried out once at 25°C for 60 minutes. De-Fmocation was performed by reacting 20% ​​piperidine in DMF at 25°C for 5 minutes, followed by a 10-minute reaction. The introduction of chloroacetyl groups was carried out by removing the Fmoc groups from the α-amino groups in the solid resin containing the Fmoc-protected peptide obtained in the previous step, using the method described above. Then, 5 equivalents of 0.2 M chloroacetic acid in DMF, 5 equivalents of 0.5 M HATU in DMF, and 10 equivalents of 1 M DIEA in DMF were added to the solid resin and shaken at room temperature for 30 minutes. For side chain deprotection and cleavage from the solid resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with methylene chloride, and then dried under reduced pressure. Subsequently, a reaction cocktail (4 mL, a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid resin, and the mixture was shaken at room temperature for 90 minutes. The reaction mixture was filtered and recovered from the frit. The solid resin remaining in the reaction vessel was shaken again with the cutting cocktail, the solution components were recovered from the frit, and mixed with the aforementioned filtrate. When this filtrate was added to excess diethyl ether / hexane (1 / 1) cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The obtained solid was washed with a small amount of diethyl ether cooled again to 0°C, and the resulting solid was dried and used for the next cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO so that the final concentration was 5 mM based on the number of moles of solid resin, and then 6 equivalents of triethylamine were added and shaken at room temperature for about 15 hours.The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVac.

[0118] The obtained crude product was purified using the following conditions: (Column: Waters Xbridge® C18 5μm 19x150mm; Mobile phase: A=0.1% TFA in H2O, B=0.1% TFA in MeCN; Temperature: 40℃; Gradient (%B conc): 5-30% over 3 minutes, then 30-35% over 8 minutes, then 35-60% over 1 minute; Flow rate: 17 mL / min.

[0119] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions and was found to be 76.5%. Analytical conditions: Retention time = 10.28 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40°C; Gradient (%B conc) 20-60% over 20 minutes, then 60-95% over 1 minute, then 95-95% over 5 minutes; Flow rate: 0.25 mL / min. ESI-MS(+) Observed value m / z=899.92(M+3H) 3+

[0120] [Examples 2-4] Synthesis of hTfR_000894_PEG11_K(Maleimide)(SEQ ID NO: 1) This example describes the synthesis of hTfR_000894_PEG11_K(Maleimide) (the peptide of SEQ ID NO: 1, with the corresponding linker listed in Table 1 attached). [ka]

[0121] Using Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.38 mmol / g, 0.66 g), the target peptide was synthesized following a general procedure, starting with the removal of Fmoc. A CEM Liberty Blue HT was used as the solid-phase synthesizer, and the procedure followed the manufacturer's manual. For the introduction of each residue, the basic condition was to use 1 equivalent of resin with Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) and react once at 75°C for 10 minutes. However, for residues 11 and 12, the reaction was carried out twice at 25°C for 20 minutes. For residues 13 and 14, the reaction was carried out twice at 75°C for 10 minutes. For residue 15, the reaction was carried out once at 25°C for 30 minutes. For residue 16, the reaction was carried out once at 25°C for 60 minutes. Furthermore, the basic condition for Fmoc removal was to react the peptide with a 20% piperidine DMF solution at 75°C for 3 minutes once. However, for the 13th and 15th residues, Fmoc removal was performed by reacting at 5°C for 5 minutes, followed by a 10-minute reaction. For the introduction of chloroacetyl groups, the Fmoc groups of the α-amino groups were first removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equivalents of 0.2 M chloroacetic acid DMF solution, 5 equivalents of 0.5 M HATU DMF solution, and 10 equivalents of 1 M DIEA DMF solution were added to the solid-phase resin and shaken at room temperature for 30 minutes. For deprotection of the side chains and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail-A (10 mL, a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered using frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered using frit, and mixed with the aforementioned filtrate. When this filtrate was added to excess diethyl ether / hexane cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (10000 rpm, 1 min), and the solution was decanted. After washing with diethyl ether cooled to 0°C, the obtained solid was used in the next cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in DMSO to a final peptide concentration of 5 mM based on the moles of the solid-phase resin, adding 10 equivalents of triethylamine, and shaking at room temperature for about 15 hours. 1.2 equivalents of SMCC based on the moles of the solid-phase resin was added to the obtained reaction solution, and the mixture was shaken at room temperature for 3.5 hours. Acetic acid was added to the resulting reaction solution, and the solution was concentrated under reduced pressure using a Genevac EZ-2 Elite.

[0122] The obtained crude product was purified under the following conditions (column: Waters Xbridge® C18 5 µm 50×150 mm; mobile phase: A=0.1% TFA in H2O, B=0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 8-33% over 3 minutes, then 33-38% over 8 minutes, then 38-60% over 1 minute; flow rate: 120 mL / min).

[0123] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions, and was found to be 92.2%. Analytical conditions: retention time=12.03 min; column: Kinetex EVO C18 2.6 µm 2.1×150 mm, 100 Å; mobile phase: A=0.025% TFA in H2O, B=0.025% TFA in MeCN; temperature: 40°C; gradient (%B conc): 20-60% over 20 minutes, then 60-95% over 1 minute, then 95%-95% over 5 minutes; flow rate: 0.25 mL / min ESI-MS (+) observed m / z=1010.68 (M+3H) 3+

[0124] [Example 2-5] Synthesis of 894_PEG12_(NHS)(SEQ ID NO: 19) This example describes the synthesis of 894_PEG12_(NHS) (a peptide of SEQ ID NO: 19 bound to the corresponding linker described in Table 1). Chemical structure

[0125] Using Fmoc-Wang Resin (1.2 mmol / g, 0.21 g), the target peptide was synthesized following a general procedure, starting with the removal of Fmoc. A CEM Liberty Blue solid-phase synthesizer was used, and the procedure followed the manufacturer's manual. For the introduction of each residue, the basic condition was to use 1 equivalent of resin with Fmoc-AA / HATU / DIPEA (4.2 equivalents / 4 equivalents / 8 equivalents) and react once at 75°C for 10 minutes. However, residues 11 and 12 were reacted twice at 25°C for 20 minutes each. Residues 13 and 14 were reacted twice at 75°C for 10 minutes each. Residue 15 was reacted once at 25°C for 30 minutes. Residue 16 was reacted once with Fmoc-AA / DIPCI / DMAP (3 equivalents / 3 equivalents / 0.75 equivalents) at 25°C for 60 minutes. Furthermore, Fmoc removal was performed by reacting the peptide with a 20% piperadine DMF solution at 75°C for 3 minutes once. However, residues 13 and 15 were reacted at 25°C for 5 minutes, followed by a 10-minute reaction. For the introduction of the chloroacetyl group, the Fmoc group of the α-amino group was first removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equivalents of a 0.2 M chloroacetic acid DMF solution, 5 equivalents of a 0.5 M HATU DMF solution, and 10 equivalents of a 1 M DIEA DMF solution were added to the solid-phase resin and shaken at 25°C for 30 minutes. For the deprotection of the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail-A (10 mL, a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid phase resin, and the mixture was shaken at 25°C for 90 minutes. The reaction solution was filtered and recovered using frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered using frit, and mixed with the aforementioned filtrate. When this filtrate was added to excess diethyl ether / hexane = 1 / 1 cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (10000 rpm, 1 min), and the solution was decanted. After washing with diethyl ether cooled to 0°C, the obtained solid was used in the next cyclization reaction.The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the number of moles of the solid phase resin, then adding 6 equivalents of triethylamine and shaking at 25°C for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVac. The obtained crude product was purified using the following conditions (Column: Waters Xbridge® C18 5μm 50x150mm; Mobile phase: A=0.1%TFA in H2O, B=0.1%TFA in MeCN; Temperature: 40℃; Gradient (%B conc): 7-32% over 3 minutes, then 32-37% over 8 minutes, then 37-60% over 1 minute; Flow rate: 120mL / min). After lyophilization, the obtained cyclic peptide (51.9 mg, 19.3 μmol) was dissolved in DMSO / water (0.7 mL, 9 / 1), then N-hydroxysuccinimide (11 mg, 96.5 μmol) and EDCI (18.5 mg, 96.5 μmol) were added, and the mixture was stirred at 25°C for 3 hours, followed by quenching with acetic acid.

[0126] The resulting reaction mixture was purified under the following conditions (Column: Waters Xbridge® C18 5μm 30x150mm; Mobile phase: A=0.1%TFA in H2O, B=0.1%TFA in MeCN; Temperature: 40℃; Gradient (%B conc): 9-35% over 3 minutes, then 35-40% over 8 minutes, then 40-60% over 1 minute; Flow rate: 45mL / min).

[0127] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions and was found to be 88.0%. Analytical conditions: Retention time = 12.74 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40°C; Gradient (%B conc): 20-60% over 20 minutes, then 60-95% over 1 minute, then 95% over 5 minutes; Flow rate: 0.25 mL / min ESI-MS(+) Observed value m / z=1390.89(M+2H) 2+

[0128] [Example 3] The peptide obtained in Example 1 and the antibody conjugate were synthesized, purified, and analyzed by the following method.

[0129] General method

[0130] [RP-UPLC-MS chromatography] Reverse-phase chromatography (RP-UPLC) [Analysis conditions A] Antibodies and antibody-peptide conjugates were separated using an ACQUITY UPLC® Protein BEH C4 (Cat. No. 186004495, Waters) column with dimensions of 2.1 mm × 50 mm, a pore size of 300 Å, and an average particle size of 1.7 μm. Mobile phase: A = water / 0.1% trifluoroacetic acid, B = acetonitrile / 0.1% trifluoroacetic acid. Analysis was performed under the following conditions: column temperature 80°C, flow rate 0.4 mL / min, gradient (%B conc.) as described in Table 2-1, and absorption wavelength 220 nm.

[0131] [Table 2-1]

[0132] [Analysis conditions B] Antibodies and antibody-peptide conjugates were separated using an ACQUITY UPLC® Protein BEH C4 (Cat. No. 186004495, Waters) column with dimensions of 2.1 mm × 50 mm, a pore size of 300 Å, and an average particle size of 1.7 μm. Analysis was performed under the following conditions: mobile phase A = water / 0.1% trifluoroacetic acid, B = acetonitrile / 0.1% trifluoroacetic acid, column temperature 80°C, flow rate 0.4 mL / min, gradient (%B conc.) as described in Table 2-2, and absorption wavelength 280 nm. [Table 2-2]

[0133] [Analysis conditions C] Antibodies and antibody-peptide conjugates were separated using an ACQUITY UPLC® Protein BEH C4 (Cat. No. 186004495, Waters) column with dimensions of 2.1 mm × 50 mm, a pore size of 300 Å, and an average particle size of 1.7 μm. Mobile phase: A = water / 0.1% trifluoroacetic acid, B = acetonitrile / 0.1% trifluoroacetic acid. Analysis was performed under the following conditions: column temperature 80°C, flow rate 0.4 mL / min, gradient (%B conc.) as described in Table 2-3, and absorption wavelength 280 nm. [Table 2-3]

[0134] [Analysis conditions D] Antibodies and antibody-peptide conjugates were separated using an ACQUITY UPLC® Protein BEH C4 (Cat. No. 186004495, Waters) column with dimensions of 2.1 mm × 50 mm, a pore size of 300 Å, and an average particle size of 1.7 μm. Mobile phase: A = water / 0.1% trifluoroacetic acid, B = acetonitrile / 0.1% trifluoroacetic acid. Analysis was performed under the following conditions: column temperature 80°C, flow rate 0.4 mL / min, gradient (%B conc.) as described in Table 2-4, and absorption wavelength 220 nm. [Table 2-4]

[0135] [Analysis conditions E] Antibodies and antibody-peptide conjugates were separated using an ACQUITY UPLC® Protein BEH C4 (Cat. No. 186004495, Waters) column with dimensions of 2.1 mm × 50 mm, a pore size of 300 Å, and an average particle size of 1.7 μm. Mobile phase: A = water / 0.1% trifluoroacetic acid, B = acetonitrile / 0.1% trifluoroacetic acid. Analysis was performed under the following conditions: column temperature 80°C, flow rate 0.4 mL / min, gradient (%B conc.) as described in Table 2-4, and absorption wavelength 220 nm. [Table 2-5]

[0136] Mass spectrometry (MS) Mass spectrometry data for antibodies and antibody-peptide conjugates were obtained using Xevo® G2-XSQTof (Waters) in positive ESI mode in the range of 500–4000 m / z. Source temperature and desolvation gas temperature were 150°C and 500°C, respectively, and desolvation gas and nebulizer gas flow rates were 800 L / hour and 50 L / hour, respectively. Capillary voltage was set to 3.00 kV. MassLynx TM MaxEnt software TM The data was converted to a mass spectrum using a single deconvolution algorithm according to the manufacturer's specified method.

[0137] Concentration measurement of antibodies and antibody-peptide conjugates Antibody concentrations were measured using a UV meter (DeNovix DS-11) according to the manufacturer's specified method. A 280 nM molar extinction coefficient, which varied depending on the type of antibody, was used.

[0138] Purification of antibody-peptide conjugates [Purification method A] 70 μL of antibody-peptide conjugate and 4 mL of 100 mM HEPES buffer pH 7.8 were added to each column of an Amicon Ultra-4 (30,000 MWCO, Merck Millipore Ltd) vessel, and centrifugation (5000 G x 15 minutes) was performed using a centrifuge (Tommy Seikou). The antibody-peptide conjugate was purified by repeating the addition of the buffer and centrifugation a total of three times.

[0139] [Purification method B] 125 μL of antibody-peptide conjugate and 4 mL of 20% aqueous acetic acid solution (pH 2.0) were added to each column of an Amicon Ultra-4 (30,000 MWCO, Merck Millipore Ltd) vessel, and centrifugation (5000 G x 15 min) was performed using a centrifuge (Tommy Seikou). The addition of the same solution to the antibody-peptide conjugate and centrifugation were repeated a total of three times. Subsequently, 4 mL of 100 mM HEPES buffer (pH 8.5) was added to the column, and centrifugation (5000 G x 15 min) was performed once using a centrifuge (Tommy Seikou) to purify the antibody-peptide conjugate.

[0140] [Purification method C] 60 μL of antibody-peptide conjugate and 4 mL of 10% aqueous acetic acid solution (pH 2.0) were added to each column of an Amicon Ultra-4 (50,000 MWCO, Merck Millipore Ltd) vessel, and centrifugation (5000 G x 15 min) was performed using a centrifuge (Tommy Seikou). The addition of the same solution to the antibody-peptide conjugate and centrifugation were repeated a total of three times. Subsequently, 4 mL of 100 mM HEPES buffer (pH 8.5) was added to the column, and centrifugation (5000 G x 15 min) was performed once using a centrifuge (Tommy Seikou) to purify the antibody-peptide conjugate. [Purification method D] 150 μL of antibody-peptide conjugate and 4 mL of 50 mM HEPES buffer pH 7.8 were added to each column of an Amicon Ultra-4 (30,000 MWCO, Merck Millipore Ltd) vessel, and centrifugation (5,000 G x 15 minutes) was performed using a centrifuge (Tommy Seikou). The antibody-peptide conjugate was purified by repeating the addition of the buffer and centrifugation a total of three times.

[0141] [Example 3-1] Nivolumab buffer exchange and concentration For the antibody nivolumab, the following procedure was performed before conjugation with the peptide.

[0142] [Buffer exchange method A] Buffer exchange for nivolumab (Selleckchem, Cat. No. A2002) was performed using a Superdex 200 Increse 10 / 300GL (Cat. No. 28-9909-44, GE Healthcare) column with an average particle size of 8.6 μm and a column size of 10 mm × 300 mm. Using the above column and AKTA pure25 M2 (GE Healthcare), a 50 mM HEPES (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.) solution was prepared as the mobile phase. At a detection wavelength of 220 nm and a flow rate of 0.5 mL / min, the fraction eluted between 11.5 and 13.5 minutes was collected. The collected fraction was concentrated using Amicon Ultra (30, 000 MWCO, Merck Millipore Ltd) to obtain a final 13.0 g / L nivolumab solution.

[0143] [Buffer replacement method B] Buffer exchange for nivolumab (Selleckchem, Cat. No. A2002) was performed using Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). Nivolumab dissolved at 5 g / L in 100 μL of 1x PBS was added to a column packed with 1 mL of Bio-Gel P-30 swollen with 100 mM HEPES pH 7.8 (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.) per column, and 4.78 g / L of nivolumab was recovered after buffer exchange using a plate centrifuge (Plate Spin, Kubota) at 1000 G for 4 minutes. [Buffer exchange method C] Buffer exchange for nivolumab (Celec Biotech Co., Ltd., Cat. No. A2002) was performed using a Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL nivolumab was added to 60 mL of a 50 mM HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer prepared at pH 7.8, and centrifugation was performed using a centrifuge (Tommy Seikou) for 2,300 g x 15 minutes. After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final nivolumab solution of 11.1 g / L.

[0144] [Example 3-2] Buffer exchange and concentration of trastuzumab For the antibody trastuzumab, the following procedure was performed before conjugation with the peptide. [Buffer exchange method A] Buffer exchange for trastuzumab (Chugai Pharmaceutical Co., Ltd., Cat. No. 002224228) was performed using a Superdex 200 Increse 10 / 300GL column (Cat. No. 28-9909-44, GE Healthcare) with an average particle size of 8.6 μm and a column size of 10 mm × 300 mm. Using the above column and AKTA pure25 M2 (GE Healthcare), HEPES (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.) prepared at 50 mM, pH 7.8 was used as the mobile phase, and the fraction eluted at a flow rate of 0.5 mL / min was collected between 20 and 35 minutes. The collected fraction was concentrated using Amicon Ultra (30, 000 MWCO, Merck Millipore Ltd). Ultimately, we obtained two trastuzumab solutions: one at 17.0 g / L and the other at 19.9 g / L. [Buffer replacement method B] Buffer exchange for trastuzumab (Chugai Pharmaceutical Co., Ltd., Cat. No. 002224231) was performed using a Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck Company, Cat. No. UFC703008). 1 mL of 5 mg / mL trastuzumab was added to 60 mL of a 50 mM, pH 7.8 HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer, and centrifugation (2,300 g x 15 minutes) was performed using a centrifuge (Tommy Seikou). After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final 10.0 g / L trastuzumab solution.

[0145] [Example 3-3] Pertuzumab buffer exchange and concentration For the antibody pertuzumab, the following procedure was performed before conjugation with the peptide. Buffer exchange for pertuzumab (Celec Biotech Co., Ltd., Cat. No. A2008) was performed using a Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL pertuzumab was added to 60 mL of a 50 mM, pH 7.8 HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer, and centrifugation (2,300 g x 15 minutes) was performed using a centrifuge (Tommy Seikou). After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final pertuzumab solution of 8.2 g / L.

[0146] [Examples 3-4] Cetuximab buffer exchange and concentration For the antibody cetuximab, the following procedure was performed before conjugation with the peptide. Buffer exchange for cetuximab (Celec Biotech Co., Ltd., Cat. No. A2000) was performed using a Sentricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL cetuximab was added to 60 mL of a 50 mM, pH 7.8 HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer, and centrifugation (2,300 g x 15 minutes) was performed using a centrifuge (Tommy Seikou). After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final cetuximab solution of 6.7 g / L.

[0147] [Examples 3-5] Ipilimumab buffer exchange and concentration For the antibody ipilimumab, the following procedure was performed before conjugation with the peptide. Buffer exchange for ipilimumab (CEREC Biotech Co., Ltd., Cat. No. A2001) was performed using a Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL ipilimumab was added to 60 mL of a 50 mM HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer at pH 7.8, and centrifugation (2,300 g x 15 minutes) was performed using a centrifuge (Tommy Seikou). After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final ipilimumab solution of 6.4 g / L.

[0148] [Examples 3-6] Atezolizumab buffer exchange and concentration For the antibody atezolizumab, the following procedure was performed before conjugation with the peptide. Buffer exchange for atezolizumab (CEREC Biotech Co., Ltd., Cat. No. A2004) was performed using a Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL cetuximab was added to 60 mL of a 50 mM HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer prepared at pH 7.8, and centrifugation was performed using a centrifuge (Tommy Seikou) at 2,300 g for 15 minutes. After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final atezolizumab solution of 6.4 g / L.

[0149] [Examples 3-7] Buffer exchange and concentration of pembrolizumab For the antibody pembrolizumab, the following procedure was performed before conjugation with the peptide. Buffer exchange for pembrolizumab (CEREC Biotech Co., Ltd., Cat. No. A2005) was performed using Centricon Plus-70, Ultracel-PL membrane, 30kDa (Merck, Cat. No. UFC703008). 1 mL of 5 mg / mL cetuximab was added to 60 mL of a 50 mM HEPES (Nacalai Tesque Co., Ltd., Cat. No. 02443-05) buffer prepared at pH 7.8, and centrifugation was performed using a centrifuge (Tommy Seikou) at 2,300 g for 15 minutes. After centrifugation, 60 mL of the same solution was added and centrifuged again. This procedure was repeated a total of three times to obtain a final 9.5 g / L pembrolizumab solution.

[0150] [Examples 3-8] Method for creating nivolumab-894_3m_G4S2_K(Mal) conjugate (conjugate number 11) using maleimide. 13 g / L nivolumab, which had been buffer-exchanged using [Buffer Exchange Method A] described in Example 3-1, was diluted to 5.2 g / L with 100 mM HEPES pH 7.8 (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 24 equivalents per antibody molecule) was added to the diluted nivolumab, and the disulfide bonds in the antibody were reduced by incubation at 30°C for 60 hours. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). After repeating this procedure one more time, a solution of the compound obtained in Example 2-1 dissolved in 10 mM dimethyl sulfoxide (8.7 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between nivolumab and the peptide was confirmed using [Analytical Condition A] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below. The nivolumab-peptide conjugate obtained from the above reaction was purified using [Purification Method A] described in the section [Purification of Antibody-Peptide Conjugate] to obtain nivolumab-peptide conjugate at a concentration of 4.37 g / L and an antibody yield of 1.75 mg. Furthermore, the peptides with maleimide as the reactive functional group in the linker portion, as listed in Table 1, were reacted with nivolumab in the same manner as described above to create the nivolumab-peptide conjugates listed in Table 3-1.

[0151] [Examples 3-9] Method for creating nivolumab-894_3m_GGRGRS_K(Mal) conjugate (conjugate number 43) using maleimide. 13 g / L nivolumab, which had been buffer-exchanged using [Buffer Exchange Method A] described in Example 3-1, was diluted to 5.2 g / L with 100 mM HEPES pH 7.8 (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 24 equivalents per antibody molecule) was added to the diluted nivolumab, and the disulfide bonds in the antibody were reduced by incubation at 30°C for 60 hours. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). After repeating this procedure one more time, a solution of the compound obtained in Example 2-2 dissolved in 10 mM dimethyl sulfoxide (8.7 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between nivolumab and the peptide was confirmed using [Analytical Condition A] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below. The nivolumab-peptide conjugate obtained from the above reaction was purified using [Purification Method A] described in the section [Purification of Antibody-Peptide Conjugate] to obtain nivolumab-peptide conjugate at a concentration of 4.85 g / L and an antibody yield of 2.42 mg.

[0152] [Examples 3-10] Method for creating trastuzumab-hTfR_000894_PEG11_K(Maleimide) conjugate (conjugate number 1) using maleimide. 19.9 g / L trastuzumab, which had been buffer-exchanged using [Buffer Exchange Method A] described in Example 3-2, was diluted to 5.2 g / L with 100 mM HEPES pH 7.8 (Cat. No. 02443-05, Nacalai Tesque Co., Ltd.). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 3.6 equivalents per antibody molecule) was added to the diluted trastuzumab, and the disulfide bonds in the antibody were reduced by incubation at 25°C for 30 minutes. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Cat. 1504150). A solution of the compound obtained in Example 2-4 dissolved in 10 mM dimethyl sulfoxide (8.7 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between trastuzumab and the peptide was carried out under [Analytical Condition D] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below. The trastuzumab-peptide conjugate obtained from the above reaction was purified by the method described in [Purification of Antibody-Peptide Conjugate] to obtain a trastuzumab-peptide conjugate at a concentration of 5.87 g / L and an antibody yield of 0.88 mg. The peptides with maleimide as the reactive functional group in the linker, as listed in Table 1, were reacted with trastuzumab in the same manner as described above (analytical conditions are described in Table 3-1) to prepare the trastuzumab-peptide conjugates listed in Table 3-1.

[0153] [Example 3-11] Method for creating trastuzumab-hTfR_000894_PEG11_(Hydradine) conjugate (conjugate number 60) using hydrazide 19.9 g / L trastuzumab, which had been buffer-exchanged using [Buffer Exchange Method A] described in Example 3-2, was diluted to 10.4 g / L with 100 mM citrate buffer (pH 3.5). 200 uL of the diluted trastuzumab was mixed with the same amount of sodium periodate (Thermo Fisher Scientific), diluted to 20 mM with 100 mM citrate buffer, as the antibody diluent, and reacted at room temperature for 30 minutes to oxidize the sugar chains on the antibody. After the oxidation reaction, the reaction solution was desalted by adding 100 μL of the above reaction solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Cat. 1504150). The compound obtained in Example 2-3 (peptide of SEQ ID NO: 6 with the linker described in Table 1) was dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody), and 7.2 μL of 50% diluted aniline (Wako Pure Chemical Industries) diluted with DMSO was added per 100 μL of the desalted sample solution. The mixture was incubated at 25°C for 3-4 hours to react the purified carbonyl group with the antibody's sugar chain. The reaction between trastuzumab and the peptide was carried out using [Analytical Method C] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-2 below. The trastuzumab-peptide conjugate obtained from the above reaction was purified by the method described in [Purification of Antibody-Peptide Conjugate] to obtain a trastuzumab-peptide conjugate at a concentration of 4.6 g / L and an antibody yield of 0.23 mg. Furthermore, peptides with hydrazide reactive functional groups in the linker portion listed in Table 1 (peptides of SEQ ID NOs. 7 and 11 with the linkers listed in Table 1 attached) were reacted with trastuzumab in the same manner as described above to prepare the trastuzumab-peptide conjugates listed in Table 3-2.

[0154] [Example 3-12] Method for preparing nivolumab-894_PEG12(NHS) conjugate (conjugate number 64) using N-hydroxysuccinimide (NHS) To 4.9 g / L nivolumab, which had been buffer-exchanged using the [Buffer Exchange Method B] described in Example 3-1, a solution of the compound obtained in Example 2-5 (the peptide of SEQ ID NO: 19 with the linker described in Table 1) dissolved in 10 mM dimethyl sulfoxide (15.0 equivalents per molecule of antibody) was added, and the mixture was incubated at 25°C for 60 minutes to allow the amine group of the antibody to react with the linker of the compound. The reaction between nivolumab and the peptide was carried out under [Analytical Condition D] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-3 below. The nivolumab-peptide conjugate obtained from the above reaction was purified by the method described in [Purification Method B] of [Purification of Antibody-Peptide Conjugate] to obtain nivolumab-peptide conjugate at a concentration of 2.2 g / L and an antibody yield of 0.11 mg. Furthermore, the peptides in the linker portion whose reactive functional group is N-hydroxysuccinimide (the peptide of SEQ ID NO: 4 with the linker listed in Table 1) were reacted with nivolumab using the method described above (analytical conditions are described in Table 3-3) to prepare the nivolumab-peptide conjugates listed in Table 3-3.

[0155] [Example 3-13] Method for preparing trastuzumab-894_PEG12(NHS) conjugate (conjugate number 63) using N-hydroxysuccinimide (NHS) To 17 g / L trastuzumab, which had been buffer-exchanged using [Buffer Exchange Method A] in Example 3-2, a solution of the compound obtained in Example 2-5 (the peptide of SEQ ID NO: 19 with the linker described in Table 1) dissolved in 10 mM dimethyl sulfoxide (4.0 equivalents per molecule of antibody) was added, and the solution was incubated at 25°C for 60 minutes to allow the amine group of the antibody to react with the linker of the compound. The reaction between nivolumab and the peptide was performed by [RP-UPLC-MS chromatography], and the results are shown in Table 3-3 below. The trastuzumab-peptide conjugate obtained from the above reaction was purified by the method described in [Purification Method C] of [Purification of Antibody-Peptide Conjugate] to obtain a trastuzumab-peptide conjugate at a concentration of 1.2 g / L and an antibody yield of 0.12 mg.

[0156] [Examples 3-14] Method for creating a pertuzumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 66) using maleimide. 8.2 g / L pertuzumab, which had been buffered using the [pertuzumab buffer exchange and concentration] method described in Example 3-3, was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 12 equivalents per antibody molecule) was added to the diluted pertuzumab, and the mixture was incubated at room temperature for 1 hour to cleave the disulfide bonds in the antibody. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between pertuzumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0157] [Example 3-15] Method for creating a cetuximab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 67) using maleimide. 8.2 g / L cetuximab, which had been buffered using the [Buffer Exchange and Concentration of Cetuximab] method described in Example 3-4, was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 12 equivalents per antibody molecule) was added to the diluted cetuximab, and the mixture was incubated at room temperature for 1 hour to cleave the disulfide bonds in the antibody. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between cetuximab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0158] [Example 3-16] Method for creating the ipilimumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 68) using maleimide. 6.4 g / L ipilimumab, which had been buffered using the [buffer exchange and concentration of ipilimumab] method described in Example 3-5, was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 12 equivalents per antibody molecule) was added to the diluted ipilimumab, and the mixture was incubated at room temperature for 1 hour to cleave the disulfide bonds in the antibody. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between ipilimumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0159] [Example 3-17] Method for creating the atezolizumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 69) using maleimide. 6.4 g / L atezolizumab, which had been buffered using the method described in Example 3-6 [Buffer exchange and concentration of atezolizumab], was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 12 equivalents per antibody molecule) was added to the diluted atezolizumab, and the mixture was incubated at room temperature for 1 hour to cleave the disulfide bonds in the antibody. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between atezolizumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0160] [Example 3-18] Method for creating a pembrolizumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 70) using maleimide. 6.4 g / L pembrolizumab, which had been buffered using the method described in Example 3-7 [Buffer exchange and concentration of pembrolizumab], was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 125 mM tris(2-carboxyethyl)phosphine (TCEP, 120 equivalents per antibody molecule) was added to the diluted cetuximab, and the mixture was incubated at 37°C for 1 hour to cleave the disulfide bonds in the antibody. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). This desalting treatment was performed a total of two times. A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between pembrolizumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0161] [Example 3-19] Method for creating a trastuzumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 71) using maleimide. 10.0 g / L tratusuzumab, which had been buffer-exchanged using the [Buffer Exchange Method B] described in Example 3-2, was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 12.5 mM tris(2-carboxyethyl)phosphine (TCEP, 12 equivalents per antibody molecule) was added to the diluted cetuximab, and the disulfide bonds in the antibody were cleaved by incubation at 37°C for 1 hour. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and the mixture was incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between trastuzumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0162] [Examples 3-20] Method for creating a nivolumab-894_3m_PEG12_dk(Maleimide) conjugate (conjugate number 72) using maleimide. 11.1 g / L nivolumab, which had been buffer-exchanged using the [Buffer Exchange Method C] described in Example 3-1, was diluted to 5.2 g / L with 50 mM HEPES pH 7.8 (Nacalai Tesque Co., Ltd., Cat. No. 02443-05). 125 mM tris(2-carboxyethyl)phosphine (TCEP, 120 equivalents per antibody molecule) was added to the diluted cetuximab, and the disulfide bonds in the antibody were cleaved by incubation at 37°C for 1 hour. Desalting was performed by adding 70 μL of the above solution per column to a column packed with 1 mL of swollen Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150). This desalting treatment was performed a total of two times. A solution of the compound obtained in Example 1 dissolved in 10 mM dimethyl sulfoxide (10 equivalents per molecule of antibody) was added, and incubated at 30°C for 30 minutes to react the disulfide bonds in the antibody with the linker of the compound. The reaction between nivolumab and the peptide was confirmed using [Analytical Condition E] described in [RP-UPLC-MS Chromatography], and the results are shown in Table 3-1 below.

[0163] [Example 3-21] Mass spectrometry of antibody-peptide conjugates prepared using maleimide Antibody-peptide conjugates were prepared using the peptides listed in Table 3-1 according to the conjugation procedures described in Examples 3-8 to 3-10 and 3-14 to 3-20. The prepared antibody-peptide conjugates were analyzed using the method described in the [RP-UPLC-MS chromatography] section, and the analysis results are shown in Table 3-1. The conditions used for the analysis of each antibody-peptide conjugate are shown in the "Analysis Conditions" column of the table. In antibody-peptide conjugates prepared using the thiol-maleimide reaction, the disulfide bond where the peptide is bound is cleaved, and under the conditions used for the analysis of the antibody-peptide conjugates in the table, they are analyzed as "heavy chain-peptide fragment," "light chain-peptide fragment," or "light chain and heavy chain complex-peptide fragment." In the table, "L" represents the light chain of the antibody, "H" represents the heavy chain, "P" in "nP" represents the added peptide, and "n" (where n is an integer) represents the number of added peptides. In the table, "LP" represents a fragment with one peptide attached to the light chain, "H3P" represents a fragment with three peptides attached to the heavy chain, "H4P" represents a fragment with four peptides attached to the heavy chain, "LH2P" represents a fragment with two peptides attached to the light-heavy chain complex, and "LH3P" represents a fragment with three peptides attached to the light-heavy chain complex. "ND" in the table indicates that it was not detected. The molecular weight (calculated value) of each fragment of each antibody-peptide conjugate was calculated from the molecular weights of the heavy and light chains obtained by analyzing trastuzumab or nivolumab that had undergone the reduction treatment described in the conjugation procedure described in Examples 3-3, 4, and 5 using the analytical method described in the table, and the molecular weight of the peptide indicated by the SEQ ID No. in Table 3-1. In the table, each antibody-peptide conjugate fragment with a numerical value listed in the "Measured Value" column has an area ratio of 10% or more in the liquid chromatography chromatogram; fragments with a ratio lower than 10% are marked "ND" or not listed. In addition, the proportion of unreacted antibodies in all conjugates listed in the table is 5% or less in terms of area ratio in the chromatogram.

[0164] [Examples 3-22] Mass spectrometry of antibody-peptide conjugates prepared using hydrazides Antibody-peptide conjugates were prepared using the peptides listed in Table 3-2, following the conjugation procedure described in Example 3-11. The prepared antibody-peptide conjugates were analyzed using the method described in the [RP-UPLC-MS chromatography] section, and the analysis results are shown in Table 3-2. The conditions used for the analysis of each antibody-peptide conjugate are shown in the "Analysis Conditions" column of the table. In the table, "P" in "nP" represents the added peptide, and "n" (where n is an integer) represents the number of added peptides. In the table, the "Antibody" column shows the type of antibody used in the conjugation, and "1P" to "9P" indicate the number of peptides added to one antibody molecule. That is, "1P" represents a structure with one peptide molecule added to the antibody, "2P" represents a structure with two peptide molecules added to the antibody, and so on. "ND" in the table indicates that it was not detected. In the table, the values ​​for each conjugate are divided into upper and lower rows. The upper row shows the molecular weight (calculated value) of each antibody-peptide conjugate, which was calculated from the molecular weight obtained by analyzing trastuzumab or nivolumab using the analytical methods described in the table, and the molecular weight of the peptide indicated by the SEQ ID No. in Table 3-2. Although there are multiple types of glycans in trastuzumab, the molecular weight of G0F / G1F was used when determining the molecular weight (calculated value) of the trastuzumab-peptide conjugate. The lower section shows the "measured values." Antibody-peptide conjugates with numerical values ​​listed in the "measured values" column are those with an area ratio of 10% or more in the liquid chromatography chromatogram. The proportion of unreacted antibodies in all conjugates listed in the table is 5% or less in terms of area ratio in the chromatogram. In other words, in the case of conjugate number 60 (trastuzumab-hTfR_000894_PEG11_(Hydradine) conjugate), the upper panel 150914 of 1P, which represents a structure in which one peptide molecule is attached to trastuzumab, is a calculated value, while the lower panel measurement value 151031 was obtained, indicating that trastuzumab with one peptide molecule attached was indeed obtained.

[0165] [Examples 3-23] Mass spectrometry of antibody-peptide conjugates prepared using N-hydroxysuccinimide (NHS) Antibody-peptide conjugates were prepared using the peptides listed in Table 3-3, following the conjugation procedures described in Examples 3-12 and 3-13. The prepared antibody-peptide conjugates were analyzed using the method described in the [RP-UPLC-MS Chromatography] section, and the analysis results are shown in Table 3-3. The conditions used for the analysis of each antibody-peptide conjugate are shown in the "Analysis Conditions" column of the table. In the table, "P" in "nP" represents the added peptide, and "n" (where n is an integer) represents the number of added peptides. In the "Antibody" column of the table, the type of antibody used in the conjugation is shown, and "1P" to "9P" indicate the number of peptides added to one antibody molecule, respectively. That is, "1P" represents a component with one peptide molecule added to the antibody, "2P" represents a component with two peptide molecules added to the antibody, and so on. "ND" in the table indicates that it was not detected. In the table, the values ​​for each conjugate are divided into upper and lower rows. The upper row shows the molecular weight (calculated value) of each antibody-peptide conjugate, which was calculated from the molecular weight obtained by analyzing trastuzumab or nivolumab using the analytical methods described in the table and the molecular weight of the peptide indicated by the SEQ ID No. in Table 3-3. Although there are multiple types of glycans in trastuzumab, when determining the molecular weight (calculated value) of the trastuzumab-peptide conjugate, the molecular weight of G0F / G1F was used (as abbreviations for glycans, G0 is used for glycans without galactose, G1 for those with one, G2 for those with two, and if fucose is also present, it is expressed as G0F, G1F, or G2F. G0F / G1F refers to an antibody with one G0F and one G1F glycan attached). The lower section shows the "measured values." The antibody-peptide conjugates listed in the "measured values" column are those with an area ratio of 10% or more in the liquid chromatography chromatogram. The proportion of unreacted antibodies in all the conjugates listed in the table is 5% or less in terms of area ratio in the chromatogram. In other words, in the case of conjugate number 63 (trastuzumab-894_PEG12_(NHS) conjugate), the upper value of 150887 in 1P, which is a structure in which one peptide molecule is attached to trastuzumab, is a calculated value, while the lower value was not obtained. Therefore, trastuzumab with one peptide molecule attached was not obtained. However, in 2P, which is a structure in which two peptide molecules are attached, the calculated value was 153551, while the measured value obtained was 153506, indicating that trastuzumab with two peptide molecules attached was indeed obtained.

[0166] [Table 3-1] [Table 3-1-1] [Table 3-1-2] [Table 3-1-3] [Table 3-1-4] [Table 3-2] [Table 3-2] [Table 3-3] [Table 3-3]

[0167] [Example 4-1] AlphaLISA study to evaluate the binding compatibility of PD-1 to nivolumab-peptide conjugate. The binding affinity of various trastuzumab-peptide conjugates to the anti-PD-1 antibody (nivolumab)-peptide conjugate against the PD-1 antigen was measured using AlphaLISA. The measurements were performed according to the manual included with the AlphaLISA Human PD-1 and PD-L1 binding kit (PerkinElimer, AL356F). Specifically, 2.5 μL of nivolumab-peptide conjugate, diluted to 5.31E-02 g / L, 1.77E-02 g / L, 5.90E-03 g / L, 1.97E-03 g / L, 6.56E-04 g / L, 2.19E-04 g / L, 7.29E-05 g / L, 2.43E-05 g / L, 8.10E-06 g / L, and 2.70E-06 g / L with the buffer included in the kit, is added to 2.5 μL of 20 nM 4× His Tagged PD-L1 and 2.5 μL of 20 nM 4× Biotinylated PD-1 included in the kit. Finally, 5 mg / mL Anti-6× His Acceptor beads, 5 mg / mL Streptavidin Donor beads, and 1× Immunoassay beads included in the kit are added. After adding 2.5 μL of a solution prepared by mixing buffer in a ratio of 1:2:122, the plate was incubated at room temperature in the dark for 90 minutes. After incubation, a plate reader (Enspir) was used. TM Fluorescence was measured using a Perkin Elmer fluoride analyzer. The IC50 values ​​for each nivolumab-peptide conjugate were calculated from the obtained measurements and are shown in Table 4.

[0168] [Table 4] [Table 4]

[0169] These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0170] [Example 4-2] Evaluation of intermolecular interactions between HER-2 and trastuzumab-peptide conjugate using surface plasmon resonance (SPR) For various trastuzumab-peptide conjugates, intermolecular interactions via surface plasmon resonance (SPR) of anti-HER-2 antibody (trastuzumab)-peptide conjugates against the HER-2 antigen were tested using the method described below. The specific test method is described below.

[0171] An NTA sensor chip (Global Life Science Technologies Japan Co., Ltd.) was inserted into a Biacore T200 (Global Life Science Technologies Japan Co., Ltd.), and priming was performed three times with running buffer: 10mM HEPES pH8.0 (Nacalai Tesque Co., Ltd.), 150mM NaCl (Nacalai Tesque Co., Ltd.), 0.05% Tween 20 (Nacalai Tesque Co., Ltd.), and 0.1% BSA (SIGMA-ALDRICH). Equilibration was performed at a flow rate of 30 μL / min. A 350mM EDTA solution was reacted at a flow rate of 10 μL / min for 60 seconds, followed by a 0.5mM NiCl2 solution (Kishida Chemical) at a flow rate of 10 μL / min for 60 seconds. Finally, the NTA sensor chip was washed with a 3mM EDTA solution (Nacalai Tesque Co., Ltd.) at a flow rate of 10 μL / min for 60 seconds. 50 μL each of 60 mM EDC solution (Global Life Science Technologies Japan Co., Ltd.) and 650 mM NHS solution (Global Life Science Technologies Japan Co., Ltd.) were mixed and reacted at a flow rate of 10 μL / min for 420 seconds. 150 μL of 0.1 μM HER-2 (R&D SYSTEMS) solution was prepared by diluting it with running buffer and reacted at a flow rate of 10 μL / min for 60 seconds to immobilize HER-2 on the NTA sensor chip. After immobilization, capping was performed by reacting with a 1.0 M ethanolamine aqueous solution (Global Life Science Technologies Japan Co., Ltd.) at a flow rate of 10 μL / min for 420 seconds. The trastuzumab-peptide conjugate (conjugate number 1; trastuzumab-hTfR_000894_PEG11_K(Maleimide)) solution prepared in Examples 3-5, which was prepared to 10 mM in DMSO solution, was diluted with running buffer to a final concentration of 10 μM, and then diluted solutions of 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM were prepared. Using the above samples, the peptide kinetics against HER-2 were obtained by SPR measurement.The kinetics evaluation model was Single Cycle Kinetics, and the KD value was determined by curve fitting using the least squares method with Biacore T200 Evaluation Software Version 3.0 (Global Life Science Technologies Japan Co., Ltd.). The result of evaluating the binding affinity of conjugate number 1, trastuzumab-peptide conjugate to HER-2, was 1.00E-10M (0.1nM) in Kd. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0172] [Example 5-1] ELISA-based study of intermolecular interactions between HER-2 and pertuzumab-peptide conjugate The binding affinity of the pertuzumab-peptide conjugate to the HER2 antigen was measured by ELISA. 50 μg of HER-2 (R&D SYSTEMS, Cat. No. 10126-ER) was dissolved in PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 1 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved HER-2, and the mixture was incubated overnight at 4°C to biotinylate the HER-2. Unreacted NHS-PEG4-Biotin was removed by adding 1 mL of Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150) to a column packed with a solution (PBST) made by adding polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) to PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 0.1%. 100 μL of the above solution was added to each column. Randomly biotinylated HER-2 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to immobilize the HER-2. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of pertuzumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8 - 0.1% Block Ace and pertuzumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0173] [Example 5-2] ELISA-based study of intermolecular interactions between EGFR and cetuximab-peptide conjugate The binding affinity of the cetuximab-peptide conjugate to the antigen EGFR was measured by ELISA. 50 μg of EGFR (R&D SYSTEMS, Cat.No.AVI10493-50) was dissolved in PBS to a final concentration of 0.1 g / L. EGFR was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of cetuximab-peptide conjugate and cetuximab, diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8-0.1% Block Ace, were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0174] [Example 5-3] ELISA-based study of intermolecular interactions between CTLA-4 and ipilimumab-peptide conjugate The binding affinity of the ipilimumab-peptide conjugate to the antigen CTLA-4 was measured by ELISA. 100 μg of CTLA-4 (Abcam, Cat. No. ab167727) was dissolved in PBS (Nacalai Tesque, Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 10 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved CTLA-4, and the CTLA-4 was biotinylated by incubation at 4°C overnight. Unreacted NHS-PEG4-Biotin was removed by adding 100 μL of the above solution per column to a column packed with 1 mL of Bio-Gel P-6 (Bio-Rad, Cat. No. 1504130) that had been swollen with a solution of PBS with polyoxyethylene sorbitan monolaurate (Nacalai Tesque, Cat. No. 28353-85) to a final concentration of 0.1% (PBST). Randomly biotinylated CTLA-4 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of ipilimumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8 - 0.1% Block Ace and ipilimumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0175] [Example 5-4] ELISA-based study of intermolecular interactions between PD-L1 and atezolizumab-peptide conjugate The binding affinity of the atezolizumab-peptide conjugate to the antigen PD-L1 was measured by ELISA. 100 μg of PD-L1 (R&D SYSTEMS, Cat. No. 9049-B7) was dissolved in PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 10 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved PD-L1, and the mixture was incubated overnight at 4°C to biotinylate the PD-L1. Unreacted NHS-PEG4-Biotin was removed by adding 1 mL of Bio-Gel P-6 (Bio-Rad, Cat. No. 1504130), which had been swollen with a solution (PBST) of PBS to which polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) had been added to a column to a final concentration of 0.1%. Randomly biotinylated PD-L1 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of atezolizumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8-0.1% Block Ace and atezolizumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0176] [Example 5-5] ELISA-based study of intermolecular interactions between PD-1 and pembrolizumab-peptide conjugate The binding affinity of pembrolizumab-peptide conjugate to the antigen PD-1 was measured by ELISA. 100 μg of PD-1 (R&D SYSTEMS, Cat. No. 8986-PD-100) was dissolved in PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 10 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved PD-1, and the mixture was incubated overnight at 4°C to biotinylate the PD-1. Unreacted NHS-PEG4-Biotin was removed by adding 1 mL of Bio-Gel P-6 (Bio-Rad, Cat. No. 1504130), which had been swollen with a solution (PBST) of PBS to which polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) had been added to a column to a final concentration of 0.1%. Randomly biotinylated PD-1 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of pembrolizumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8 - 0.1% Block Ace and pembrolizumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0177] [Examples 5-6] ELISA-based study of intermolecular interactions between HER-2 and trastuzumab-peptide conjugate The binding affinity of the trastuzumab-peptide conjugate to the HER2 antigen was measured by ELISA. 50 μg of HER-2 (R&D SYSTEMS, Cat. No. 10126-ER) was dissolved in PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 1 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved HER-2, and the mixture was incubated overnight at 4°C to biotinylate the HER-2. Unreacted NHS-PEG4-Biotin was removed by adding 1 mL of Bio-Gel P-30 (Bio-Rad, Cat. No. 1504150) to a column packed with a solution (PBST) made by adding polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) to PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 0.1%. 100 μL of the above solution was added to each column. Randomly biotinylated HER-2 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to immobilize the HER-2. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of trastuzumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8-0.1% Block Ace and trastuzumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0178] [Examples 5-7] ELISA-based study of intermolecular interactions between PD-1 and nivolumab-peptide conjugate The binding affinity of the nivolumab-peptide conjugate to the antigen PD-1 was measured by ELISA. 100 μg of PD-1 (R&D SYSTEMS, Cat. No. 8986-PD-100) was dissolved in PBS (Nacalai Tesque Co., Ltd., Cat. No. 27575-31) to a final concentration of 1 g / L. Five equivalents of 10 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) were added to the dissolved PD-1, and the mixture was incubated overnight at 4°C to biotinylate the PD-1. Unreacted NHS-PEG4-Biotin was removed by adding 1 mL of Bio-Gel P-6 (Bio-Rad, Cat. No. 1504130), which had been swollen with a solution (PBST) of PBS to which polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) had been added to a column to a final concentration of 0.1%. Randomly biotinylated PD-1 was added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of nivolumab-peptide conjugate diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8 - 0.1% Block Ace and nivolumab were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the antibody's ability to bind to the antigen is not lost even when conjugated with a peptide.

[0179] [Examples 5-8] ELISA-based intermolecular interaction testing of TfR and various antibody-peptide conjugates The binding affinity of various antibody-peptide conjugates to TfR was measured by ELISA. Biotinization was performed by adding 10 equivalents of 1 mM NHS-PEG4-Biotin (Thermo Scientific, Cat. No. A39259) to 100 μg of human TfR and incubating overnight at 4°C. For the human TfR, recombinant hTfR described in Example 2 of WO2018 / 124121 was used. Unreacted NHS-PEG4-Biotin was removed by adding 100 μL of the above solution per column to a column packed with 1 mL of Bio-Gel P-6 (Bio-Rad, Cat. No. 1504130) swollen with a solution of PBS with polyoxyethylene sorbitan monolaurate (Nacalai Tesque Co., Ltd., Cat. No. 28353-85) to a final concentration of 0.1% (PBST). Randomly biotinylated TfRs were added at a rate of 1 pmol per well to IMMOBILIZER STREPTAVIDIN F96 clear (NUNC, Cat. No. 436014) and incubated overnight at 4°C to solidify the phase. The plates were washed with 900 μL of PBST using a plate washer (TECAN, HydroSpeed). Blocking treatment was performed by adding 300 μL of 1% Block Ace (KAC Corporation, Cat. No. UK-B80) per well and incubating at room temperature for 90 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of various antibody-peptide conjugates and unmodified antibodies, diluted to a final concentration of 100 nM with 50 mM HEPES pH 7.8-0.1% Block Ace, were added and incubated at room temperature for 60 minutes. The plates were then washed with 900 μL of PBST using a plate washer. 50 μL of a solution of Goat Anti-Human IgG H&L (HRP) (Abcam, Cat. No. ab97165), diluted 50,000 times in PBST-0.1% Block Ace solution, was added and incubated at room temperature for 45 minutes. The plate was washed with 900 μL of PBST using a plate washer. 50 μL of TMB Solution (Sara care, Cat. No. 5150-0077), returned to room temperature, was added and allowed to develop color in the dark at room temperature for 10 minutes. Then, 50 μL of TMB Stop Solution (Sara care, Cat. No. 5150-0021) was added to stop the color development. Plate reader (Enspir) TM The absorbance at 450 nm was measured using a Perkin Elmer instrument. This measurement was repeated twice, and the average absorbance is shown in Table 6. These results demonstrate that the binding ability of the TfR-binding peptide to TfR is not lost even when conjugated with an antibody.

[0180] [Table 6] [Table 6-1]

[0181] [Table 6-2]

[0182] [Example 6] Cell permeability test The cell permeability of the antibody-peptide conjugates obtained in Examples 1 to 3 was confirmed.

[0183] [Cell culture] Human breast cancer cells BT-549 were cultured in RPMI-1640 medium containing 10% FBS and 2 mmol / L L-Glutamine. Culturing was performed at 37°C under 5% CO2 conditions.

[0184] [Cell seeding] Collagen Type I (Corning) was diluted with 20 mmol / L acetic acid to a concentration of 50 μg / mL. One sterile coverslip was placed in each well of a 24-well plate, and the diluted Collagen Type I solution was added. The plates were then incubated at 37°C for 1 hour. After removing the Collagen Type I solution, the plates were washed three times with PBS. 1 × 10⁶ per well. 5 Human breast cancer cells BT-549 were seeded and cultured overnight at 37°C and 5% CO2.

[0185] [Preparation of antibody-peptide conjugate solution and its addition to cells] Dilution medium (RPMI 1640 medium containing 0.5% bovine serum albumin and 20 μg / mL human transferrin holotype) was used to dilute the antibody-peptide conjugate. The antibody-peptide conjugate was diluted with dilution medium to concentrations of 2.5, 5, and 10 μg / mL, or 1, 3, and 9 μg / mL. After confirming that BT-549 cells cultured overnight in a 24-well plate were adhered to the coverslip, they were washed twice with RPMI 1640 medium. 500 μL / well of RPMI 1640 medium containing 0.5% bovine serum albumin was added and incubated on ice for 15 minutes, then removed. 500 μL / well of diluted antibody-peptide conjugate solution was added and incubated at 37°C under 5% CO2 conditions for 3 hours. As a negative control, only the antibody (nivolumab or trastuzumab) was added instead of the antibody-peptide conjugate.

[0186] [Cell fixation and permeabilization] The antibody-peptide conjugate solution was removed from the 24-well plate, and the BT-549 cells were washed three times with PBS. 500 μL / well of 4% paraformaldehyde-phosphate buffer (Fujifilm Wako Pure Chemical Industries) was added, and after standing at room temperature for 15 minutes, the cells were washed three times with PBS. 500 μL / well of 0.1% Triton X-100 solution was added, and after standing at room temperature for 10 minutes, the samples were washed three times with PBS.

[0187] [Immunostaining] 250 μL / well of PBS containing 10% donkey serum and 1% bovine serum albumin was added, and after standing at room temperature for 1 hour, the samples were washed three times with PBS. Goat anti-human IgG h+l (Bethil Laboratories) was diluted to 5 μg / mL in PBS-T containing 0.5% bovine serum albumin to prepare the primary antibody solution. 200 μL of the primary antibody solution was added to each well, and after standing at room temperature for 1 hour, the samples were washed three times with PBS. The secondary antibody solution was prepared by diluting Donkey anti-goat IgG h+l and DyLight488 conjugated (Bethil Laboratories) 300-fold in PBS-T containing 0.5% bovine serum albumin. 200 μL of the secondary antibody solution was added to each well, and after standing at room temperature and protected from light for 1 hour, the samples were washed three times with PBS.

[0188] [Nuclear staining and mounting] 500 μL / well of Hoechst 33342 (Thermo Fisher Scientific), diluted to 2 μg / mL in PBS, was added and allowed to stand for 10 minutes at room temperature in the dark, then washed three times with PBS. Cover slips were removed from the 24-well plate and mounted on glass slides using Fluorescent Mounting Medium (Agilent), and allowed to stand overnight at room temperature in the dark. Observation was performed using an inverted fluorescence microscope DMI6000B (Leica Microsystems) at wavelengths for DyLight488 and DAPI detection. The results are shown in Figure 1.

[0189] Figure 1-1 shows the results of adding the amount of trastuzumab-894_PEG12_(NHS) conjugate (conjugate number 63) indicated in the figure. Figure 1-1 demonstrates that trastuzumab-894_PEG12_(NHS) conjugate has the ability to penetrate cells.

[0190] Figure 1-2 shows the results of adding trastuzumab-hTfR_000894_PEG11_(Hydradine) (conjugate number 60) in the amounts indicated in the figure. Figure 1-2 demonstrates that the conjugate of hTfR_000894_PEG11_(Hydradine) and trastuzumab has the ability to cross into cells.

[0191] Figures 1-3 show the results of adding trastuzumab-hTfR_000894_PEG11_K(Maleimide) (conjugate number 1);a and trastuzumab-hTfR_000894_PEG36_K(Maleimide) (conjugate number 2);b at a concentration of 5 μg / mL. Figures 1-3 demonstrate that the conjugate of hTfR_000894_PEG(11 / 36)_(Hydradine) and trastuzumab has the ability to penetrate cells.

[0192] [Example 7]

[0193] Brain transferability assessment test The brain penetration of the antibody-peptide conjugates obtained in Examples 1 to 3 was confirmed in knock-in mice (KI mice).

[0194] [Example 7-1] Brain penetration evaluation study of trastuzumab-peptide conjugate using hTfR-KI mice

[0195] [Preparation of the administration solution] Trastuzumab administration solution: Trastuzumab was diluted with physiological saline to a concentration of 1 mg / mL. Trastuzumab-hTfR_000894_PEG11_K(Maleimide) administration solution: Trastuzumab-hTfR_000894_PEG11_K(Maleimide) (conjugate number 1) synthesized in Example 3-5 was diluted with physiological saline to a concentration of 1 mg / mL. Trastuzumab-hTfR_000894_PEG36_K(Maleimide) administration solution: Trastuzumab-hTfR_000894_PEG36_K(Maleimide) (conjugate number 2), synthesized in the same manner as in Examples 3-5, was diluted with physiological saline to a concentration of 1 mg / mL.

[0196] [Administration to hTfR-KI mice, tissue sampling] Next, a brain penetration evaluation test was conducted using hTfR-KI mice. Human TfR-expressing hTfR-KI mice (male, 13-15 weeks old) were rapidly administered trastuzumab solution, trastuzumab-hTfR_000894_PEG11_K(Maleimide) solution, or trastuzumab-hTfR_000894_PEG36_K(Maleimide) solution at a dose of 5 mg / kg via the tail vein (6 mice / group). Blood was collected from the tail vein 1 and 3 hours after administration (2 mice / time point). 6 and 24 hours after administration, the mice were anesthetized with isoflurane, blood was collected from the right ventricle, and then the blood was drained by perfusing the left ventricle with physiological saline for 4-5 minutes. Blood collection was performed using EDTA-2K treated needles and syringes, and the collected blood was stored on ice until it was separated into plasma. After blood removal, the brain was collected and split into left and right halves. The left brain was embedded in a cryogenic embedding medium (OCT compound, Sakura FineTech) for immunohistochemical staining, and a frozen block was prepared. The right brain, heart, lungs, liver, spleen, kidneys, and quadriceps femoris muscle were collected for tissue concentration measurement of test substances. After weighing, they were rapidly frozen in liquid nitrogen and stored at -70°C or below.

[0197] [Preparation of tissue extract] Lysis buffer was prepared by adding Protease Inhibitor Cocktail (P8340, Sigma-Aldrich Co., LLC.) to RIPA Buffer (Fujifilm Wako Pure Chemical Corporation) to a final concentration of 1%. Metal beads, tissue, and 20 times the weight of the tissue in Lysis Buffer were added to a tube, and the tissue was crushed using a bead crusher (Tytec Co., Ltd.) to obtain tissue lysate. The tissue lysate was centrifuged (11,000 × g, 4°C, 5 min), and the supernatant was collected as tissue extract.

[0198] [Preparation of the reagent] Capture antibody solution: Anti-Human Kappa Light Chain Goat IgG Biotin (100 μg / mL, Immunobiological Laboratories Co., Ltd.) was diluted to 1 μg / mL using Blocker Casein in PBS. SULFO-labeled antibody solution: SULFO-TAG Anti-Human Antibody (Goat) (500 μg / mL, Meso Scale Diagnostics, LLC) was diluted to 1 μg / mL using Blocker Casein in PBS. 2× Read Buffer T: An appropriate amount of MSD Read Buffer T (4×) (Meso Scale Diagnostics, LLC) was diluted with an equal volume of sterile water for injection.

[0199] [Preparation of calibration curve samples] Trastuzumab, trastuzumab-hTfR_000894_PEG11_K(Maleimide), or trastuzumab-hTfR_000894_PEG36_K(Maleimide) were diluted with blank plasma or tissue extract (plasma or tissue extract from untreated individuals) to prepare calibration curve samples with concentrations ranging from 0.195 to 200 ng / mL.

[0200] [Preparation of the measurement sample] Each tissue extract was appropriately diluted with a blank tissue extract to prepare the measurement samples.

[0201] [Measurement of test substance concentrations in plasma and tissue] 150 μL / well of Blocker Casein in PBS (Thermo Fisher Scientific, Inc.) was added to a Strept Avidin plate (Meso Scale Diagnostics, LLC), and the plate was shaken for 60 minutes (26°C, 500 rpm) using a plate shaker (Biosan) to block the contents. The Blocker Casein in PBS was removed from the Strept Avidin plate, and 200 μL / well of PBST was added to each plate for washing. 25 μL / well of Capture antibody solution was added to the Strept Avidin plate, and the plate was shaken for 60 minutes (26°C, 500 rpm). The Capture antibody solution was removed, and 200 μL / well of PBST was added to each plate for washing. This washing procedure was repeated three times. 25 μL / well of calibration curve sample and measurement sample were added to the Strept Avidin plate, and the plate was shaken for 60 minutes (26°C, 500 rpm). After removing the calibration curve sample and the measurement sample, PBST was added to each well and washed. This washing procedure was repeated three times. SULFO-labeled antibody solution was added to each well of the Strept Avidin plate and shaken in a plate shaker for 60 minutes (26°C, 500 rpm). After removing the SULFO-labeled antibody solution, PBST was added to each well and washed. This washing procedure was repeated three times. 2× Read Buffer T was added to each well of the Strept Avidin plate and measured using a plate reader (Sector S600, Meso Scale Diagnostics, LLC) to calculate the concentration of each test substance in the sample.

[0202] [Results of plasma test substance concentration measurement] Figure 2-1 shows the results of plasma concentration measurements of trastuzumab peptide conjugates. Trastuzumab, trastuzumab-hTfR_000894_PEG11_K(Maleimide), and trastuzumab-hTfR_000894_PEG36_K(Maleimide) were detected in plasma at all time points evaluated and showed a biphasic change in blood concentration, with relatively rapid decay up to 6 hours after administration and gradual decay thereafter. Trastuzumab-peptide conjugates, trastuzumab-hTfR_000894_PEG11_K(Maleimide) and trastuzumab-hTfR_000894_PEG36_K(Maleimide), showed lower values ​​than trastuzumab at all time points.

[0203] Figure 2-1 is a graph that replaces the diagram showing the plasma concentrations of trastuzumab-hTfR_000894_PEG11_K(Maleimide) (conjugate number 1) and trastuzumab-hTfR_000894_PEG36_K(Maleimide) conjugate (conjugate number 2).

[0204] [Results of measurement of test substance concentration in tissue] Figure 2-2 shows the results of measuring the concentration of transtuzumab peptide conjugate in the tissue. Trastuzumab, trastuzumab-hTfR_000894_PEG11_K(Maleimide), and trastuzumab-hTfR_000894_PEG36_K(Maleimide) were detected in all tissues. The concentrations of trastuzumab-hTfR_000894_PEG11_K(Maleimide) and trastuzumab-hTfR_000894_PEG36_K(Maleimide) were higher than the concentration of trastuzumab in brain tissue. This indicates that the trastuzumab-peptide conjugate has brain penetration.

[0205] Figure 2-2 is a graph that replaces the diagram showing the tissue concentrations of trastuzumab-hTfR_000894_PEG11_K(Maleimide) (conjugate number 1) and trastuzumab-hTfR_000894_PEG36_K(Maleimide) conjugate (conjugate number 2).

[0206] [Example 7-2] Brain penetration evaluation study of nivolumab-peptide conjugate using hTfR-KI mice

[0207] [Preparation of the administration solution] Nivolumab administration solution: Nivolumab was diluted with physiological saline to a concentration of 1 mg / mL. Nivolumab-894_3m_G4S2_K(Mal) administration solution: Nivolumab-894_3m_G4S2_K(Mal) (conjugate number 11) synthesized in Example 3-3 was diluted with physiological saline to a concentration of 1 mg / mL.

[0208] [Administration to hTfR-KI mice, tissue sampling] Next, a brain penetration evaluation test was conducted using hTfR-KI mice. Human TfR-expressing hTfR-KI mice (male, 15 weeks old) were rapidly administered nivolumab solution or nivolumab-894_3m_G4S2_K(Mal) solution at a dose of 5 mg / kg via the tail vein (6 mice / group). Blood was collected from the tail vein 1 and 3 hours after administration (2 mice / time point). 6 and 24 hours after administration, the mice were anesthetized with isoflurane, blood was collected from the right ventricle, and then blood was drained by perfusing the left ventricle with physiological saline for 4-5 minutes. Blood collection was performed using EDTA-2K treated needles and syringes, and the collected blood was stored on ice until it was separated into plasma. After blood drainage, the brain was collected and divided into left and right hemispheres. The left brain was embedded in a cryogenic embedding medium (OCT compound, Sakura FineTech) for immunohistochemical staining, and a cryogenic block was prepared. For measuring the concentration of the test substance in tissue, samples were taken from the right brain, heart, lungs, liver, spleen, kidneys, and quadriceps femoris muscle. After weighing, the samples were rapidly frozen with liquid nitrogen and stored at -70°C or below.

[0209] [Preparation of tissue extract] Lysis buffer was prepared by adding Protease Inhibitor Cocktail (P8340, Sigma-Aldrich Co., LLC.) to RIPA Buffer (Fujifilm Wako Pure Chemical Corporation) to a final concentration of 1%. Metal beads, tissue, and 20 times the weight of the tissue in Lysis Buffer were added to a tube, and the tissue was crushed using a bead crusher (Tytec Co., Ltd.) to obtain tissue lysate. The tissue lysate was centrifuged (11,000 × g, 4°C, 5 min), and the supernatant was collected as tissue extract.

[0210] [Preparation of the reagent] Capture antibody solution: Anti-Human Kappa Light Chain Goat IgG Biotin (100 μg / mL, Immunobiological Laboratories Co., Ltd.) was diluted to 1 μg / mL using Blocker Casein in PBS. SULFO-labeled antibody solution: SULFO-TAG Anti-Human Antibody (Goat) (500 μg / mL, Meso Scale Diagnostics, LLC) was diluted to 1 μg / mL using Blocker Casein in PBS. 2× Read Buffer T: An appropriate amount of MSD Read Buffer T (4×) (Meso Scale Diagnostics, LLC) was diluted with an equal volume of sterile water for injection.

[0211] [Preparation of calibration curve samples] As a control, nivolumab or nivolumab-894_3m_G4S2_K(Mal) (conjugate number 11) was diluted with blank plasma or tissue extract (plasma or tissue extract from untreated individuals) to prepare calibration curve samples with concentrations ranging from 0.195 to 200 ng / mL.

[0212] [Preparation of the measurement sample] Each tissue extract was appropriately diluted with a blank tissue extract to prepare the measurement samples.

[0213] [Measurement of test substance concentrations in plasma and tissue] 150 μL / well of Blocker Casein in PBS (Thermo Fisher Scientific, Inc.) was added to a Strept Avidin plate (Meso Scale Diagnostics, LLC), and the plate was shaken for 60 minutes (26°C, 500 rpm) using a plate shaker (Biosan) to block the contents. The Blocker Casein in PBS was removed from the Strept Avidin plate, and 200 μL / well of PBST was added to each plate for washing. 25 μL / well of Capture antibody solution was added to the Strept Avidin plate, and the plate was shaken for 60 minutes (26°C, 500 rpm). The Capture antibody solution was removed, and 200 μL / well of PBST was added to each plate for washing. This washing procedure was repeated three times. 25 μL / well of calibration curve sample and measurement sample were added to the Strept Avidin plate, and the plate was shaken for 60 minutes (26°C, 500 rpm). After removing the calibration curve sample and the measurement sample, PBST was added to each well and washed. This washing procedure was repeated three times. SULFO-labeled antibody solution was added to each well of the Strept Avidin plate and shaken in a plate shaker for 60 minutes (26°C, 500 rpm). After removing the SULFO-labeled antibody solution, PBST was added to each well and washed. This washing procedure was repeated three times. 2× Read Buffer T was added to each well of the Strept Avidin plate and measured using a plate reader (Sector S600, Meso Scale Diagnostics, LLC) to calculate the concentration of each test substance in the sample.

[0214] [Results of plasma test substance concentration measurement] Figure 2-3 shows the results of plasma nivolumab-peptide conjugate concentration measurements. Nivolumab and nivolumab-894_3m_G4S2_K(Mal) were detected in plasma at all time points evaluated, showing a biphasic change in blood concentration with relatively rapid decay up to 6 hours after administration and gradual decay thereafter. Nivolumab-894_3m_G4S2_K(Mal), the nivolumab-peptide conjugate, showed lower values ​​than nivolumab at all time points.

[0215] Figure 2-3 is a graph that replaces the diagram showing the plasma concentration of nivolumab-894_3m_G4S2_K(Mal) conjugate (conjugate number 11).

[0216] [Results of measurement of test substance concentration in tissue] Figure 2-4 shows the results of measuring nivolumab-peptide conjugate concentrations in tissues. Nivolumab and nivolumab-894_3m_G4S2_K(Mal) conjugate were detected in all tissues. The concentration of nivolumab-894_3m_G4S2_K(Mal) conjugate was higher than that of nivolumab in brain tissue. This indicates that nivolumab-peptide conjugate has brain penetration capabilities.

[0217] Figure 2-4 is a graph that replaces the diagram showing the tissue concentrations of nivolumab-894_3m_G4S2_K(Mal) conjugate (conjugate number 11).

[0218] [Example 7-3]

[0219] [Immunostaining of trastuzumab in tissue] Using a cryostat (Leica Microsystems Inc.), 7 μm thick sections were prepared from the frozen brain blocks of each individual prepared in Example 7-1 and mounted on peel-resistant glass slides (Matsunami Glass Industry Co., Ltd.). After air drying, the tissue sections were fixed by immersion in 4% Paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) for 5 minutes. After washing with PBST (3 minutes, 3 times), the sections were immersed in 0.3% H2O2 / MeOH for 20 minutes to block endogenous peroxidase. After washing the sections with PBST (3 minutes, 3 times), SuperBlock Blocking Buffer in PBS was added to the sections and blocked at room temperature for 100 minutes. After washing the sections with PBST (3 minutes, 3 times), HRP-labeled anti-human IgG antibody (A80-219P, Bethyl Laboratories, Inc.) moderately diluted with CanGet Signal Immunostain solution A (Toyobo Co., Ltd.) was added dropwise, and the sections were treated at room temperature for 120 minutes. After washing the sections with PBST (3 minutes, 3 times), sensitization treatment with the TSA-Plus Fluorescein System (PerkinElmer, Inc.) was performed according to the package insert. After washing the sections with PBST (3 minutes, 3 times), Anti-Fluorescein-HRP (Dako) was added dropwise, and the sections were treated at room temperature for 15 minutes. After washing the sections with PBST (3 minutes, 3 times), DAB substrate solution (Dako) prepared according to the package insert was added dropwise, and the sections were treated until appropriate color development occurred. The sections were washed with running water and counterstained with Mayer's Hematoxylin (Fujifilm Wako Pure Chemical Industries, Ltd.). The sections were washed with running water, dehydrated and cleared using an ethanol elevation series and xylene, and Eukitt (ORSAtec GmbH aka Kindler GmbH) was added to the sections as a mounting medium. Coverslips were then placed over the sections to prepare the tissue specimens.

[0220] [Immunostaining of nivolumab in tissue] Using a cryostat (Leica Microsystems Inc.), 7 μm thick sections were prepared from the frozen brain blocks of each individual prepared in Example 7-2 and mounted on peel-resistant glass slides (Matsunami Glass Industry Co., Ltd.). After air drying, the tissue sections were fixed by immersion in 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd.) for 5 minutes. After washing with PBST (3 minutes, 3 times), the sections were immersed in 0.3% H2O2 / MeOH for 20 minutes to block endogenous peroxidase. After washing the sections with PBST (3 minutes, 3 times), SuperBlock Blocking Buffer in PBS was added to the sections and blocked at room temperature for 100 minutes. After washing the sections with PBST (3 minutes, 3 times), an anti-human IgG antibody appropriately diluted with CanGet Signal Immunostain solution A (Toyobo Co., Ltd.) was added for 894_3m_G4S2_K(Mal)-nivolumab conjugate (conjugate number 11), and an anti-nivolumab antibody (A01931-40, Genscript Biotech Corporation) appropriately diluted with CanGet Signal Immunostain solution A (Toyobo Co., Ltd.) was added for 894_3m_GGRGRS_K(Mal)-nivolumab (conjugate number 43), and the sections were left to stand overnight at 4°C. After washing the sections with PBST (3 minutes, 3 times), CSAII Rabbit Link (Dako) diluted 20-fold with CanGet Signal Immunostain solution A was added to the sections and treated at room temperature for 30 minutes. After washing the sections with PBST (3 minutes, 3 times), sensitization treatment with the TSA-Plus Fluorescein System (PerkinElmer, Inc.) was performed according to the package insert. After washing the sections with PBST (3 minutes, 3 times), Anti-Fluorescein-HRP (Dako) was added to the sections and treated at room temperature for 15 minutes. After washing the sections with PBST (3 minutes, 3 times), DAB substrate solution (Dako) prepared according to the package insert was added to the sections and treated until appropriate color development occurred. The sections were washed with running water and counterstained with Mayer's Hematoxylin stain (Fujifilm Wako Pure Chemical Industries, Ltd.).The sections were washed with running water, dehydrated and cleared using an ethanol elevation series and xylene, Eukitt (ORSAtec GmbH aka Kindler GmbH) was added to the sections as a mounting medium, and a coverslip was placed over them to prepare the tissue specimens.

[0221] [Results of immunohistochemical staining] Result 1: Figure 3-1 shows the results of immunohistochemical staining in the cerebellum 6 hours after treatment with trastuzumab-hTfR_000894_PEG11_K(Maleimide) (conjugate number 1) and trastuzumab-hTfR_000894_PEG36_K(Maleimide) (conjugate number 2). The lower figure is an enlarged view of the square area in the upper figure. These results demonstrate that the hTfR_000894_PEG11 / 36_K(Maleimide)-trastuzumab conjugate has brain penetration.

[0222] Figure 3-1 is a photograph replacing the diagram showing trastuzumab-hTfR_000894_PEG11 / 36_K(Maleimide) (conjugate number 1 / 2) in the cerebellum at 6 hours. Of the parts in Figure 3-1, a-1 is trastuzumab only. b-1 is trastuzumab-hTfR_000894_PEG11_K(Maleimide), c-1 represents trastuzumab-hTfR_000894_PEG36_K(Maleimide). The figure below is a magnified view of a portion of the figure above.

[0223] Result 2: Figure 3-2 shows the results of immunohistochemical staining in the cerebellum 6 hours after treatment with nivolumab-894_3m_G4S2_K(Mal) (conjugate number 11). The lower figure is an enlarged view of the rectangular area in the upper figure. These results indicate that the nivolumab-894_3m_G4S2_K(Mal) conjugate has brain penetration. Figure 3-2 is a photograph replacing the diagram showing the cerebellar response to nivolumab-894_3m_G4S2_K(Mal) (conjugate number 11) at 6 hours. Of the images in Figure 3-2, a-2 is nivolumab only. b-2 is nivolumab-894_3m_G4S2_K(Mal). The figure below is a magnified view of a portion of the figure above.

[0224] Result 3: Figure 3-3 shows the results of immunohistochemical staining in the cerebellum 6 hours after treatment with nivolumab-894_3m_GGRGRS_K(Mal) (conjugate number 43), and Figure 3-4 shows the results after 24 hours. The lower figure is an enlarged view of the rectangular area in the upper figure. These results indicate that the nivolumab-894_3m_GGRGRS_K(Mal) conjugate has brain penetration. Figure 3-3 is a photograph replacing the diagram showing nivolumab-894_3m_GGRGRS_K(Mal) (conjugate number 43) cerebellar 6 hours. Of Figure 3-3, a-3 is nivolumab only. b-3 is nivolumab-894_3m_GGRGRS_K(Mal). The figure below is a magnified view of a portion of the figure above.

[0225] Figure 3-4 is a photograph replacing the diagram showing the cerebellar response to nivolumab-894_3m_GGRGRS_K(Mal) (conjugate number 43) over 24 hours. Of the images in Figure 3-4, a-4 is nivolumab only. b-4 is nivolumab-894_3m_GGRGRS_K(Mal). The figure below is a magnified view of a portion of the figure above.

[0226] Results 4: Figure 3-5 shows the results of immunohistochemical staining in the hippocampus 6 hours after treatment with nivolumab-894_3m_GGRGRS_K(Mal) (conjugate number 43), and Figure 3-6 shows the results after 24 hours. The lower figure is an enlarged view of the rectangular area in the upper figure. These results indicate that the nivolumab-894_3m_GGRGRS_K(Mal) conjugate has the ability to penetrate the hippocampus. Figure 3-5 is a photograph replacing the diagram showing the hippocampal response to nivolumab-894_3m_GGRGRS_K(Mal) (conjugate number 43) at 6 hours. Of the images in Figure 3-5, a-5 is nivolumab only. b-5 is nivolumab-894_3m_GGRGRS_K(Mal). The figure below is a magnified view of a portion of the figure above.

[0227] Figure 3-6 is a photograph replacing the diagram showing the hippocampal response to 894_3m_GGRGRS_K(Mal)-nivolumab (conjugate number 43) over 24 hours. Of the figures in Figure 3-6, a-6 is nivolumab only. b-6 is 894_3m_GGRGRS_K(Mal)-nivolumab.

Claims

1. Conjugates including the following: (1) A peptide that binds to the transferrin receptor, A cyclic peptide consisting of any of the amino acid sequences of the first to 15th amino acids in SEQ ID NOs: 1-13, 15, 18-86, 90-110, and the first to 12th amino acid sequences in SEQ ID NOs: 14, 16, 17, 87-89, and (2) A compound containing an antibody or an antigen-binding fragment thereof.

2. The conjugate according to claim 1, wherein the peptide is bound to the antibody via a linker.

3. The conjugate according to claim 2, wherein the linker is a peptide linker, a chemical linker, or a combination thereof.

4. The conjugate according to claim 2, wherein the linker is a linker having the sequence described in any of sequence numbers 111 to 161.

5. The conjugate according to claim 2, wherein the peptide and the antibody are linked via a maleimide, hydrazide, or NHS site bound to the end of the linker.

6. A conjugate comprising the following: A conjugate comprising a peptide that binds to a transferrin receptor, wherein a linker-added peptide described in any of SEQ ID NOs: 1 to 110 is bound to an antibody or its antigen-binding fragment.

7. A composition comprising the conjugate described in any one of claims 1 to 6, for delivering the conjugate into an intracellular space or for passing it across the blood-brain barrier.

8. A pharmaceutical composition comprising the conjugate or salt thereof according to any one of claims 1 to 6 as an active ingredient.

9. A peptide that binds to the transferrin receptor, A method for processing an antibody or its antigen-binding fragment so that it can be delivered into a cell or pass through the blood-brain barrier, comprising the step of conjugating a cyclic peptide consisting of any of the amino acid sequences of the first to 15th amino acids of SEQ ID NOs: 1-13, 15, 18-86, 90-110, and the amino acid sequences of the first to 12th amino acids of SEQ ID NOs: 14, 16, 17, 87-89, to an antibody or its antigen-binding fragment.

10. A method for conjugating a cyclic peptide, which binds to a transferrin receptor and comprises any of the amino acid sequences of the first to 15th amino acids of SEQ ID NOs: 1-13, 15, 18-86, 90-110, and the first to 12th amino acid sequences of SEQ ID NOs: 14, 16, 17, 87-89, to an antibody or a compound containing an antigen-binding fragment thereof, (i) A step of reducing the disulfide bond in the antibody or a compound containing the antigen-binding fragment thereof; (ii) the step of preparing the peptide to which a linker having maleimide at its terminus is attached; and (iii) A step of contacting a compound containing an antibody or antigen-binding fragment thereof, in which the disulfide bond has been reduced in (i), with the peptide prepared in (ii), A method that includes this.

Images