Carrier for immune cell migration and use thereof

Abstract

The present invention pertains to a carrier for transfer into immune cells. The present invention pertains to a reagent for transfer into immune cells. The present invention pertains to a method for transporting a carrier, which is for transfer into immune cells, into immune cells. The present invention pertains to a medicinal composition comprising a medicine and a carrier for transfer into immune cells.

Description

[Technical field]

[0001] The present invention relates to a carrier for immune cell migration.The present invention relates to a reagent for immune cell migration.The present invention relates to a method for transferring a carrier for immune cell migration to an immune cell.The present invention relates to a pharmaceutical composition comprising a medicine and a carrier for immune cell migration. [Background technology]

[0002] In recent years, immunotherapy has been attracting attention as a new cancer treatment method. Cancer immunotherapy can be broadly divided into passive immunotherapy and active immunotherapy. Passive immunotherapy is a treatment in which immune cells such as T cells taken from a patient are activated in vitro and then returned to the patient's body, and the activated immune cells attack cancer cells. Known examples of this include CAR-T cell therapy. Active immunotherapy is a treatment in which a substance that activates immune cells is administered to the patient, and the activated immune cells attack cancer cells. Known examples of this include cytokine therapy.

[0003] There is no system yet in place that can efficiently deliver substances that activate immune cells to immune cells in vivo without using ligands or antibodies that specifically bind to cell surface markers such as CD3. Various immune cells, including T cells, mainly perform immune reactions in lymph nodes to eliminate foreign substances in tissue fluid and lymph. Patent Document 1 describes that a dendrimer having a carboxyl group as a terminal group can migrate to lymph nodes, and that when the dendrimer was administered intradermally to mice, it accumulated in the sentinel lymph node and the secondary lymph node beyond it. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2015 / 170573 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors have found that the dendrimer of Patent Document 1 accumulates in lymph nodes of the body, but is not taken up by immune cells in the lymph nodes. Establishing a means capable of selectively transferring a desired physiologically active substance to immune cells of the body is expected to contribute to the development of cancer immunotherapy. An object of the present invention is to provide a carrier molecule capable of transferring to immune cells. [Means for solving the problem]

[0006] The present invention provides a carrier for immune cell migration comprising a complex molecule, the complex molecule comprising a branched polymer and a phenylalanine residue and having an anionic terminal structure, the phenylalanine residue being bound to an end group of the branched polymer.The present invention provides a reagent for immune cell migration comprising a carrier for immune cell migration.The present invention provides a method for transferring a carrier for immune cell migration to an immune cell, the method comprising contacting the carrier for immune cell migration with an immune cell in vivo (excluding humans), in vitro or ex vivo.The present invention provides a pharmaceutical composition comprising a medicine and a carrier for immune cell migration. Effect of the Invention

[0007] According to the present invention, a carrier capable of being transferred to immune cells is provided. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram showing the chemical structure of one of 64 terminal groups of dendrimers (G4-Suc and G4-CHex) having no phenylalanine residue at the end and having a linker attached to the end, and dendrimers (G4-Suc-Phe and G4-CHex-Phe) having a phenylalanine residue attached to the end via a linker. [Figure 2A] This is a distribution diagram obtained by flow cytometry (FCM) analysis of cells obtained from lymph nodes of mice administered a fluorescently labeled carrier for immune cell migration. [Figure 2B]The cells obtained from the lymph nodes of a mouse administered a fluorescently labeled carrier for immune cell migration were immunostained with a PE-labeled antibody and analyzed by FCM, resulting in a scattergram. [Diagram 3] 1 is a graph showing the percentage of cells containing carriers for immune cell migration among various types of immune cells detected with a PE-labeled antibody. [Figure 4] This is an image obtained by in vivo fluorescent imaging of a mouse administered with a fluorescently labeled carrier for immune cell migration. [Diagram 5] This is a graph obtained by measuring radiation emitted from organs etc. taken from mice administered with a carrier for immune cell migration labeled with a radioactive substance. [Figure 6] 1 is a graph showing the percentage of cells containing carriers for immune cell migration among various types of immune cells detected with a PE-labeled antibody. [Figure 7] 1 is a graph showing the percentage of cells containing carriers for immune cell migration among T cells and B cells under neutral or acidic conditions, detected with a PE-labeled antibody. [Figure 8] 1 is a graph showing the change in transmittance versus temperature for solutions (pH 4 to 7) of dendrimers (G4-Suc-Phe, G4-Ph-Phe, and G4-CHex-Phe) having phenylalanine residues bound to their ends via linkers. [Figure 9] 1 is a graph showing the change in transmittance versus temperature for a solution (pH 5 or pH 6.5) of a dendrimer (G4-Phe-SO3H) having a phenylalanine residue at its terminal and a sulfone terminal group. [Figure 10A] FIG. 1 is a graph showing the change in transmittance versus temperature for a solution (pH 4-7) of an immune cell migration carrier (G3.5-Phe30) in which a phenylalanine residue is directly bonded to the terminal group of a dendrimer and the anionic terminal group is a carboxyl group. [Figure 10B] FIG. 1 is a graph showing the change in transmittance versus temperature for a solution (pH 4-7) of a carrier for immune cell migration (G3.5-Phe41) in which a phenylalanine residue is directly bonded to the terminal group of a dendrimer and the anionic terminal group is a carboxyl group. [Figure 10C]FIG. 1 is a graph showing the change in transmittance versus temperature for a solution (pH 4-7) of a carrier for immune cell migration (G2.5-Phe18) in which a phenylalanine residue is directly bonded to the terminal group of a dendrimer and the anionic terminal group is a carboxyl group. [Figure 10D] FIG. 1 is a graph showing the change in transmittance versus temperature for a solution (pH 4-7) of a carrier for immune cell migration (G2.5-Phe14) in which a phenylalanine residue is directly bonded to the terminal group of a dendrimer and the anionic terminal group is a carboxyl group. [Figure 11] FIG. 1 is a schematic diagram showing the chemical structure of one of the 64 terminal groups of dendrimers having phenylalanine residues bound to their termini via linkers (G4-Suc-Phe and G4-CHex-Phe) and dendrimers having leucine residues bound to their termini via linkers (G4-Suc-Leu and G4-CHex-Leu). [Figure 12] 1 is a graph showing the average fluorescence intensity of cells obtained by FCM analysis of Jurkat cells that had been contacted with a fluorescently labeled carrier for immune cell migration. [Figure 13] FIG. 1 is a schematic diagram showing the chemical structure of one of the 64 terminal groups of dendrimers (G4-Phe-Suc and G4-Phe-CHex) in which a phenylalanine residue is directly bonded to the terminal and the anionic terminal group is a carboxyl group. [Figure 14] 1 is a graph showing the average fluorescence intensity of cells obtained by FCM analysis of Jurkat cells that had been contacted with a fluorescently labeled carrier for immune cell migration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The carrier for immune cell migration of this embodiment (hereinafter, also simply referred to as "carrier") includes a complex molecule having an anionic terminal structure formed by directly or indirectly binding a phenylalanine residue to the terminal group of a branched polymer. The branched polymer is not particularly limited as long as it is a polymer having a branched or dendritic structure. The terminal group of the branched polymer refers to the outermost group of the branched or dendritic structure of the branched polymer. The anionic terminal structure in the carrier of this embodiment refers to a portion containing a phenylalanine residue bound directly or indirectly to the terminal group of the branched polymer, and the group at the end of the portion is an anionic group. Examples of anionic groups include a carboxyl group, a sulfone group, a sulfate group, or a salt thereof. The present inventors found that the complex molecule can carry a substance of interest, and therefore the complex molecule can be used as a carrier for immune cell migration, and completed the present invention. Here, the "substance of interest" refers to a specific substance that is desired to be transferred to an immune cell. "Carrying a substance of interest" includes linking the substance of interest to an end group of a branched polymer in a complex molecule, incorporating the substance of interest in the internal space of the branched polymer, and linking the substance of interest to a phenylalanine residue.

[0010] In this embodiment, the branched polymer preferably has, as an end group, one or more functional groups that can be bonded to a phenylalanine residue or a linker described below. Examples of the functional group include an amino group, a carboxyl group, a hydroxyl group, and a sulfone group. Examples of the branched polymer include dendrimers, "hyperbranched polymers" with low branching regularity in the dendritic structure, "dendrigrafts" (also called random dendritic polymers), and star polymers.

[0011] Dendrimers are known to be compounds with a highly branched tree-like structure in three dimensions, and have a nearly spherical shape. In this specification, the term "dendrimer" also includes dendrons, which are partial structures that constitute dendrimers and have a structure in which at least one functional group in the core portion is not branched. Dendrimers generally consist of a core, several generations of branched portions, and terminal groups. The core of a dendrimer is derived from a compound having one or more functional groups. The functional groups include primary amino groups, secondary amino groups, hydroxy groups, carboxylic acid groups, thiol groups, ester groups, amide groups, ketone groups, aldehyde groups, and the like. Primary amino groups and secondary amino groups are preferred. Examples of compounds constituting the core include ammonia, ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,10-diaminodecane, and 1,12-diaminododecane. Alkyldiamines containing S-S bonds (cystamine) may also be used.

[0012] The branching portion of a dendrimer is composed of repeated branching structural units containing an atom with a valence of 3 or more. Examples of atoms with a valence of 3 or more include carbon, nitrogen, silicon, and phosphorus. For example, the following structure is known as the branching portion of a dendrimer.

[0013] [ka] (In the formula, n is an integer of 1 or more, and preferably an integer of 60 to 300.)

[0014] The dendrimers having the branched structures (A) to (C) above are described in the following documents: The branched portion of the dendrimer may contain two or more kinds of repeating units as described above. (A) Tomalia DA et al., Polym. J. 17, 117 (1985) and Tomalia DA et al., Angew. Chem. Int. Ed. Engl. 29, 138 (1990) (B) de Brabander-van den Berg EMM et al., Angew. Chem. Int. Ed. Engl. 32, 1308 (1993); de Brabander-van den Berg EMM et al., Macromol. Symp. 77, 51 (1994); Hummelen JC et al., Chem. Eng. J. 3, 1489 (1997) and Waner C. et al., Angew. Chem. Int. Ed. Engl. 32, 1300 (1993) (C) Ihre HR et al., Bioconjugate Chem. 13, 443-452 (2002) and Goodwin AP et al., J. Am. Chem. Soc., 129, 6994 (2007)

[0015] The structure of the terminal group of the dendrimer can be appropriately selected. For example, the terminal group may have the structure of the last repeating unit of the branched portion, or may have a structure different from that of the branched portion. The terminal group of the dendrimer preferably has one or more functional groups capable of binding to a phenylalanine residue or a linker described below.

[0016] A hyperbranched polymer is generally a polymer in which branches are developed by polymerizing AB2 type monomers. Here, A and B each represent a combination of functional groups capable of polymerization, such as a combination of a hydroxyl group and a carboxyl group, or an amino group and a carboxyl group. Alternatively, a substance that functions as a branch core may be used in combination. A hyperbranched polymer may also be produced by ring-opening polymerization of glycidol, ethyleneimine, or the like. Dendrigrafts are polymers synthesized by reacting, in stages, oligomers in which multiple monomers are bonded together, rather than monomers. These are collectively called random dendritic polymers. Random dendritic polymers have branched portions like dendrimers, but a core is not essential. In addition, the branched portions of random dendritic polymers may have irregular or discontinuous portions due to partial defects.

[0017] The branching unit of the random dendritic polymer may be a polylysine skeleton, a polyglycerin skeleton, or a skeleton of various sugars. An example of a random dendritic polymer in which the branching unit is lysine (-NH-CH(C4H2NH-)CO-) is PolyLysine-Dendri-graft (PLD: COLCOM). An example of a random dendritic polymer in which the branching unit is polyglycerin represented by the following formula is PGL X and PGL 10 (Daicel Chemicals).

[0018] [ka] (In the formula, n and m are independently an integer of 10 or more, preferably an integer of 20 or more.)

[0019] A star polymer is known as a polymer in which three or more polymer chains are branched radially from the center. Star polymers are roughly classified into regular type, in which the polymer chains are the same type, and asymmetric type, in which the polymer chains are different in type and molecular weight. Regular type star polymers can be obtained by living anionic polymerization of a living anionic polymer of styrene or 1,3-dienes with a multifunctional silyl chloride. Currently, polybutadiene star polymers having 3 to 128 polymer chains and star polystyrene having 33 polymer chains have been synthesized. In addition, as asymmetric type star polymers, four-component star polymers composed of polystyrene, polyisoprene, polybutadiene, and poly(4-methylstyrene) are known.

[0020] The star polymer may be of the multi-arm PEG type. Such star polymers are commercially available, for example, 4arm-PEG-NH2(C(CH2O(CH2CHO) n CH2CH2NH2HCl)4), 4arm-PEG-COOH (C(CH2O(CH2CH2O) nand 8arm-PEG-NH2 (R(O(CH2CH2O)4) (Merck), a multi-arm PEG type with eight branches. n CH2CH2NH2HCl)8, R is tripentaerythritol) and 8arm-PEG-COOH (R(O(CH2CH2O) n CH2COOH)8, R is tripentaerythritol) (Merck). In addition, among the SUNBRIGHT (registered trademark) series, the PTE series, HGEO series, and DX series represented by the following formulas (Yuka Sangyo Co., Ltd.) are also included.

[0021] [ka] (In the formula, X is -CO-CH2CH2-COO-N-hydroxysuccinimide (NHS), -CO-CH2CH2CH2-COO-NHS-(CH2)3-NHCO-CH2CH2-maleimide, -CH2CH2CH2CH2CH2-COO-NHS, -COO-p-nitrophenyl-NO2, -CH2CH2CH2NH2 or -(CH2)3-NHCO-CH2CH2-maleimide, and n is an integer of 10 or more.)

[0022] [ka] (In the formula, X is -CO-CH2CH2-COO-NHS, -CO-CH2CH2CH2-COO-NHS, -COO-p-nitrophenyl, -CH2CH2NH2·HCI or -CH2CH2SH, and n is an integer of 10 or more.)

[0023] [ka] (In the formula, X is -CH2CH2CH2-NH2, -CH2CH2-CHO or -CO-CH2CH2CH2-COO-NHS, and n is an integer of 10 or more.)

[0024] In this embodiment, it is preferable to use a dendrimer as the branched polymer. The generation number of the dendrimer can be appropriately selected depending on the structure of the core and branched portion of the dendrimer used. In this technical field, polyamidoamine (PAMAM) dendrimers of generations 0 to 10 are generally available. In addition, the method for producing a dendrimer is publicly known and is described in, for example, the above-mentioned documents. In this embodiment, it is preferable to use a dendrimer of generation 4 (G4), generation 5 (G5), or generation 6 (G6).

[0025] In the carrier of this embodiment, the end group of the branched polymer and the phenylalanine residue may be directly or indirectly bonded. The number of phenylalanine residues bonded to one end group of the branched polymer may be one or more. The phenylalanine residue may be either L- or D-isomer. The phenylalanine residue may be bonded to all end groups of the branched polymer, or to some end groups of the branched polymer. In this embodiment, the phenylalanine residue is bonded to 40% or more of the end groups of the branched polymer, preferably 45% or more, 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. For example, when the branched polymer is a G4 PAMAM dendrimer, of the 64 terminal groups of the dendrimer, 26 or more, preferably 29 or more, 32 or more, 36 or more, 39 or more, 42 or more, 45 or more, 48 or more, 52 or more, 55 or more, 58 or more, or 61 or more of the terminal groups have phenylalanine residues bonded to them.

[0026] When the end group of the branched polymer and the phenylalanine residue are directly bonded, the end group of the branched polymer may be reacted with the amino or carboxyl end of phenylalanine. Alternatively, the end group of the branched polymer may be reacted with a derivative of phenylalanine. In this embodiment, the type of bond between the end group of phenylalanine or its derivative and the end group of the branched polymer is not particularly limited. A preferred bond is a covalent bond, and examples of the bond include an amide bond (-NH-CO-), an ester bond (-CO-O-), a urethane bond (-NH-CO-O-), a urea bond (-NH-CO-NH-), a thiourea bond (-NH-CS-NH-), a sulfonamide bond (-SO2-NH-), and a sulfonate ester bond (-SO2-O-). In addition, the structure may be formed by the reaction of maleimide with a thiol. Examples of the derivative of phenylalanine include a compound in which the amino or carboxyl end of phenylalanine is derivatized with a group reactive to the end group of the branched polymer.

[0027] In one embodiment, the carrier for immune cell migration may be obtained by reacting the amino terminal of phenylalanine with the terminal group of the branched polymer. In this case, the anionic terminal structure in the carrier is composed of a phenylalanine residue directly bonded to the terminal group of the branched polymer, and the carboxyl group of the phenylalanine residue is the anionic group of the terminal structure. For example, when the terminal group of the branched polymer is a carboxyl group, this terminal group may be reacted with the amino terminal of phenylalanine to form an amide bond. When the terminal group of the branched polymer is an amino group, the amino terminal of phenylalanine may be derivatized with a group capable of forming an amide bond or a urethane bond with the amino group (e.g., a 4-nitrophenyl carbonate group or a carboxyl group). In addition, when the terminal of the branched polymer is a hydroxy group, the amino terminal of phenylalanine may be derivatized with a group capable of forming an ester bond with the hydroxy group (e.g., a carboxyl group). When the amino terminal of phenylalanine is reacted with the terminal group of the branched polymer, the carboxyl terminal of the phenylalanine may be protected in advance with a known protecting group.

[0028] In a further embodiment of the carrier for immune cell migration, when the end group of the branched polymer and the phenylalanine residue are directly bonded, a compound capable of imparting an anionic end group may be further bonded to the phenylalanine residue. For example, when the end group of the branched polymer is reacted with the carboxyl end of phenylalanine, in order to make the end structure of the resulting complex molecule anionic, it is preferable to react the amino end of the phenylalanine in the complex molecule with a compound capable of imparting an anionic end group. Examples of such compounds include compounds having a functional group capable of reacting with an amino group at one end of the compound and an anionic group (e.g., a carboxyl group, a sulfonic acid group, a sulfate group, etc.) at the other end, and intramolecular condensates thereof. For example, anhydrides of dicarboxylic acids such as succinic anhydride, 1,2-cyclohexanedicarboxylic anhydride, and phthalic anhydride are preferred. Alternatively, the compound capable of imparting an anionic end group may be a cyclic compound that reacts with an amino group to open the ring and form an anionic end group. Examples of such compounds include cyclic sulfonic acid esters such as 1,3-propane sultone.

[0029] In the carrier of this embodiment, the end group of the branched polymer and the phenylalanine residue may be bonded via a linker. The linker can be appropriately selected from hydrophobic compounds having a functional group at one end capable of reacting with the end group of the branched polymer and a functional group at the other end capable of reacting with phenylalanine or a derivative thereof. For example, a branched polymer having an amino or hydroxyl end group and an amino group of phenylalanine are bonded via the above-mentioned dicarboxylic anhydride as a linker. Examples of such linkers include succinic anhydride, 1,2-cyclohexanedicarboxylic anhydride, and phthalic anhydride. In this case, the synthesis scheme is briefly described as follows: first, the amino or hydroxyl group of the branched polymer is reacted with the dicarboxylic anhydride to obtain a branched polymer having a carboxyl end group. Then, the carboxyl group of the obtained branched polymer is reacted with the amino terminal of phenylalanine whose carboxyl terminal is protected with a protecting group to obtain a branched polymer having a phenylalanine residue bonded to the terminal. Finally, the protecting group of the phenylalanine residue is removed to obtain the carrier of this embodiment. Such a synthesis scheme itself is known and is described, for example, in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151. A crosslinking agent capable of binding the end group of the branched polymer to the amino end of phenylalanine can also be used. For example, when the end group of the branched polymer is an amino group, a compound having two isocyanate groups or isothiocyanate groups may be used. When the end group of the branched polymer is a carboxyl group or a 4-nitrophenyl carbonate group, a compound having an amino group and a carboxyl group or a succinimide group (which may be an amino acid) may be used. When the end group of the branched polymer is a mercapto group, a compound having a maleimide group and a carboxyl group or a succinimide group may be used. When the end group of the branched polymer is a maleimide group, a compound having a mercapto group and a carboxyl group or a succinimide group may be used.

[0030] In a preferred embodiment, the anionic terminal structure of the carrier for immune cell migration comprises a phenylalanine residue bound to the terminal group of a branched polymer and is represented by the following formula (I):

[0031] [ka] (In the formula, X1 is -NH-(C=O)- or -O-(C=O)-, and Y1 is -NH-(C=O)- or -NH-, R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, heterocycloalkylene group, or phenylene group which may have a substituent and have 3 to 8 carbon atoms; Z1 is a carboxyl group, a sulfonic group, a sulfate group, or a salt thereof.

[0032] The terminal structure represented by formula (I) corresponds to a structure in which the terminal group of the branched polymer is directly bonded to a phenylalanine residue, and a compound capable of imparting an anionic terminal group to the phenylalanine residue is further bonded to the phenylalanine residue. In formula (I), X1 represents a bonding site between the terminal group of the branched polymer and the phenylalanine residue, and the left bond of X1 is bonded to the branched polymer. In formula (I), Y1, R, and Z1 correspond to a compound capable of imparting an anionic terminal group bonded to the phenylalanine residue. Y1 represents a bonding site between the terminal group of the phenylalanine residue and a compound capable of imparting an anionic terminal group. Z1 is a terminal group of the anionic terminal structure. The type of salt in Z1 is not particularly limited, but is preferably an alkali metal salt.

[0033] When R is an alkylene group having 1 to 10 carbon atoms, examples of such alkylene groups include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene, 2-ethylhexylene, nonylene, and decylene. Among these, alkylene groups having 1 to 4 carbon atoms are preferred. When R is an alkylene group having a substituent, the number of carbon atoms of the substituent is not included in the above number of carbon atoms.

[0034] When R is a cycloalkylene group or a heterocycloalkylene group, such a group may be a non-aromatic ring having 3 to 8 carbon atoms which may contain one or more heteroatoms selected from N, S, O, and P. Examples of such groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, pyrrolidinylene, piperidinylene, and morpholinylene. When R is a cycloalkylene group, heterocycloalkylene group, or phenylene group having a substituent, the number of carbon atoms of the substituent is not included in the number of carbon atoms.

[0035] Examples of the substituent in R include linear or branched saturated aliphatic hydrocarbon groups having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms.

[0036] In a further embodiment, the anionic terminal structure of the carrier for immune cell translocation comprises a phenylalanine residue bound to the terminal group of the branched polymer and is represented by the following formula (II):

[0037] [ka] (In the formula, X2 is -NH-(C=O)-, -(C=O)-NH-, -O-(C=O)-, -(C=O)-O-, -O-(C=O)-NH-, -NH-(C=O)-O-, -NH-(C=O)-NH-, -NH-(C=S)-NH-, or a structure obtained by a reaction of maleimide with a thiol; R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, heterocycloalkylene group, or phenylene group which may have a substituent and have 3 to 8 carbon atoms; Y2 is -(C=O)-NH-, -O-(C=O)-NH-, -NH-(C=O)-NH-, -NH-(C=S)-NH-, Z2 is a carboxyl group or a salt thereof.

[0038] The terminal structure represented by formula (II) corresponds to a structure in which the terminal group of the branched polymer and the phenylalanine residue are bonded via a linker, and the carboxyl group or a salt thereof of the phenylalanine residue forms an anionic terminal group. X2 represents a bonding site between the terminal group of the branched polymer and the linker, and the left bond of X2 is bonded to the branched polymer. In formula (II), R represents a linker moiety that intervenes between the terminal group of the branched polymer and the phenylalanine residue. The substituents in R and R are as described above. In formula (II), Y2 represents a bonding site between the linker and the amino group of the phenylalanine residue. Z2 is the terminal group of the anionic terminal structure.

[0039] In a further embodiment, the anionic terminal structure of the carrier for immune cell migration comprises a phenylalanine residue bound to the terminal group of the branched polymer and is represented by the following formula (III):

[0040] [ka] (wherein Z3 is a carboxyl group or a salt thereof).

[0041] The terminal structure represented by formula (III) corresponds to a structure in which the amino group of a phenylalanine residue is directly bonded to the terminal group of a branched polymer, and the carboxyl group or a salt thereof of the phenylalanine residue forms an anionic terminal group. In formula (III), the leftmost bond is bonded to the branched polymer. Z3 is the terminal group of the anionic terminal structure.

[0042] In this embodiment, the terminal structure represented by the above formula (I) is particularly preferably the structures represented by the following formulas (1) to (4). Furthermore, the terminal structure represented by the above formula (II) is particularly preferably the structures represented by the following formulas (5) to (7). The terminal structure represented by the above formula (III) is particularly preferably the structure represented by the following formula (8). In formulas (1) to (8), the leftmost bond is bonded to the branched polymer. In formulas (1) to (8), X +is a hydrogen ion or an alkali metal ion. Examples of the alkali metal ion include a sodium ion, a potassium ion, and a lithium ion. Among these, the sodium ion is preferred.

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] The particle size of the carrier in this embodiment is not particularly limited, but is usually 5 nm to 20 nm, preferably 5 nm to 15 nm. The particle size of the carrier is the average particle size of the smallest distribution in the volume-based particle size distribution obtained by analyzing the value measured based on the dynamic light scattering (DLS) method by the Marquardt method. The measuring device is ELSZ-DN2 (manufactured by Otsuka Electronics Co., Ltd.) or an equivalent product.

[0047] In a further embodiment, the carrier for immune cell migration may further include a substance of interest. That is, the carrier of this embodiment includes a complex molecule carrying a substance of interest. In this case, the substance of interest may be linked to the end group of the branched polymer in the complex molecule, may be encapsulated in the internal space of the branched polymer, or may be linked to a phenylalanine residue. When the substance of interest is linked to the end group or phenylalanine residue of the branched polymer in the complex molecule, the mode of linkage is not particularly limited. The linkage may be, for example, a covalent bond or a non-covalent bond. For example, the substance of interest may be directly or indirectly bound to the end group or phenylalanine residue of the branched polymer. When the branched polymer is a dendrimer, the method of encapsulating the substance of interest in the internal space of the dendrimer is known per se, and is described, for example, in Kojima C. et al., Bioconjuge Chem 11: 910-7 (2000), etc. In addition, the carrier of this embodiment may be used by adding it to another carrier carrying a substance of interest (for example, a liposome, a micelle, a polymeric micelle, etc.). For example, the carrier of this embodiment may be mixed with another carrier carrying a substance of interest, or the surface of the other carrier may be modified with the carrier of this embodiment via chemical or physical bonding.

[0048] The substance of interest may be, for example, a label, a nucleic acid, a protein, a peptide, etc. The label may be, for example, 11 C. 13 N, 15 O. 18 PET compounds containing radioisotopes such as F, 111Examples of the nucleic acid include a vector incorporating a gene encoding a desired protein, an antisense oligonucleotide, an siRNA, an shRNA, a ribozyme, an aptamer, and a decoy nucleic acid. Examples of the protein and peptide include a peptide and a protein containing an autoantigen and a non-autoantigen. Specifically, examples of the protein and peptide include a cancer preventive vaccine targeting a cancer-specific antigen (common cancer antigen, neoantigen) and an optimal cancer immunotherapy for each patient, and an immune tolerance targeting an allergy antigen that causes an autoimmune disease.

[0049] As described above, the carrier of this embodiment is characterized by migrating into immune cells, particularly immune cells in lymph nodes. The type of immune cells is not particularly limited, and examples thereof include T cells, B cells, dendritic cells, and macrophages. The carrier of this embodiment can reach lymph nodes and migrate into immune cells in lymph nodes by administration to a mammal. In addition, the carrier of this embodiment can migrate into immune cells by contacting the immune cells ex vivo or in vitro. As shown in the examples described later, the carrier of this embodiment is considered to migrate into immune cells by cellular endocytosis, not by membrane permeation.

[0050] As shown in the Examples below, the carrier of this embodiment has good transferability to T cells. In the art, there are few carriers capable of transferring to T cells, and the carrier of this embodiment can be suitably used to transfer a substance of interest to T cells.

[0051] As shown in the examples below, the carrier of this embodiment has improved migration to immune cells, particularly T cells, under acidic conditions. Examples of acidic conditions include a pH of 5 or more and 7 or less in the environment surrounding the immune cells. For example, in the case of in vitro or ex vivo, the pH of the liquid in contact with the cells, such as a culture medium, may be acidic. In the case of in vivo, it is known that the pH is acidic at sites where inflammation occurs (e.g., cancer tissue, etc.). Here, as described in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151, a complex molecule formed by binding a phenylalanine residue to the terminal group of a fourth-generation dendrimer via an ethylene group, a cyclohexylene group, or a phenylene group is known to undergo phase transition under acidic pH conditions and become more hydrophobic. It is considered that the property of phase transition in response to this pH may be involved in the improvement of migration to immune cells under acidic conditions.

[0052] When the carrier of this embodiment is administered to a subject (e.g., a mammal) in vivo, parenteral administration is preferred, and administration by injection is particularly preferred. Administration by injection may be any of intradermal, subcutaneous, intravenous, arterial, intramuscular, and intraperitoneal injections, and is preferably intradermal or subcutaneous, more preferably intradermal. When the carrier of this embodiment is administered to a subject (e.g., a cell) in vitro or ex vivo, it is preferred to contact the subject with the carrier by adding an appropriate amount of the carrier of this embodiment to a culture solution.

[0053] One embodiment of the present invention is a pharmaceutical composition comprising a medicine and a carrier for immune cell migration. The medicine refers to a substance used as an active ingredient or a candidate thereof. In a preferred embodiment, the medicine is a substance that can be carried by the carrier of this embodiment. That is, in the pharmaceutical composition of this embodiment, the medicine is preferably carried by the carrier for immune cell migration. Examples of the medicine include pharmaceutical compounds, proteins, peptides, and nucleic acids. The pharmaceutical compounds can be selected from known compounds used as active ingredients or candidates thereof. The proteins, peptides, and nucleic acids can be selected from known antibody drugs, peptide drugs, and nucleic acid drugs, respectively. The form of the pharmaceutical composition is preferably an injection containing a medicine and the carrier of this embodiment. The injection can be prepared by adding the carrier of this embodiment to purified water for injection or physiological saline. The content of the carrier for immune cell migration is, for example, 0.001 to 80 parts by weight, preferably 0.01 to 50 parts by weight, relative to 100 parts by weight of the injection. The injection may further contain a pharmaceutical acceptable additive. Such additives are themselves known, and examples thereof include preservatives, stabilizers, pH adjusters, isotonicity agents, solvents, solubilizing agents, and soothing agents.

[0054] One embodiment of the present invention is an immune cell migration reagent comprising the above-mentioned carrier for immune cell migration. The reagent of this embodiment is useful, for example, when migrating a substance of interest to an immune cell. The substance of interest can be transferred to an immune cell by supporting the substance of interest on the carrier for immune cell migration in the reagent of this embodiment and contacting it with an immune cell. The reagent of this embodiment may include a carrier for immune cell migration in a powder state (e.g., a freeze-dried state), or may include a carrier for immune cell migration in a state dispersed in an appropriate solvent. In the reagent of this embodiment, the carrier for immune cell migration may be contained in a container. Also, in the reagent of this embodiment, the container containing the carrier for immune cell migration may be packed in a box. The box may include a package insert. The package insert may describe the composition of the reagent, the structure of the carrier for immune cell migration, the method of use, and the like.

[0055] One embodiment of the present invention is a method for transferring the carrier for immune cell transfer to immune cells, comprising contacting the carrier for immune cell transfer with immune cells in vivo (excluding humans), in vitro, or ex vivo. The details of the immune cells are as described above. The method for contacting the carrier for immune cell transfer with immune cells is not particularly limited. For example, in the case of in vivo, the carrier for immune cell transfer can be administered to an animal excluding humans to bring the carrier for immune cell transfer into contact with immune cells. The details of administration are as described above. In the case of in vitro, the carrier for immune cell transfer can be added to immune cells in a medium to bring the carrier for immune cell transfer into contact with immune cells. In the case of ex vivo, an organ containing immune cells (e.g., spleen, thymus, lymph nodes, etc.) can be collected from an animal, and the obtained organ can be perfused with, for example, a liquid containing the carrier for immune cell transfer, to bring the carrier for immune cell transfer into contact with immune cells. When a carrier for immune cell transfer carries a substance of interest, the transfer method of this embodiment makes it possible to transfer the substance of interest to immune cells in vitro or ex vivo via the carrier for immune cell transfer.

[0056] One embodiment of the present invention is a use of a complex molecule for the manufacture of a carrier for immune cell migration, the complex molecule comprising a branched polymer and a phenylalanine residue, and having an anionic terminal structure, the phenylalanine residue being bound to an end group of the branched polymer. Details of the carrier for immune cell migration and the complex molecule are as described above.

[0057] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0058] Manufacturing Example 1: Preparation of carrier for immune cell migration (1) Preparation of carboxy-terminated dendrimers Carboxy-terminated dendrimers were prepared by the method described in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151. The specific procedure was as follows. A methanol solution of amino-terminated G4 PAMAM dendrimer (Sigma-Aldrich) was evaporated under reduced pressure and lyophilized. The dendrimer (56 mg, 3.9 μmol) was dissolved in 125 mM NaHCO3 buffer (5.5 mL) and an excess of succinic anhydride or cis-1,2-cyclohexanedicarboxylic anhydride (100 equivalents each) was added. Each solution was stirred at room temperature overnight and the pH was adjusted to 8-10 using a 4N NaOH aqueous solution. The solution containing the carboxy-terminated dendrimer was purified by dialysis in distilled water using a dialysis membrane (molecular weight cut-off (MWCO): 1,000). The purified solution was lyophilized to obtain a white powder of each carboxy-terminated dendrimer. The yield of the dendrimer with succinic acid added to the end (G4-Suc) was 95 mg, a yield of 111%. The yield of the dendrimer with cyclohexanedicarboxylic acid added to the end (G4-CHex) was 62 mg, a yield of 64%. Each solution of G4-Suc and G4-CHex was analyzed at room temperature using a 400 MHz JNM-LA 400 JNM-ECX (JEOL). 1 The results of H-NMR spectrum measurement are shown below.

[0059] G4-Suc (NaOD in DO): 2.24-2.28 (br, NCH2C of the inner and terminal chains of PAMAM and methylene of succinic acid H2 CONHCH2CH2N), 2.46 (br, NCH2CH2CONHCH2C in the inner chain of PAMAM H2 N), 2.65 (br, NC of inner and terminal chains of PAMAM H2 CH2CONHCH2CH2N) and 3.13 (br, NCH2CH2CONHC in the inner and terminal chains of PAMAM H2 CH2N, and NCH2CH2CONHCH2C at the end of the PAMAM chain H2 N).

[0060] G4-CHex (NaOD in DO): 1.21-1.83 (m, methylene of cyclohexanedicarboxylic acid), 2.26 (br, NCH2C of PAMAM) H2 CONHCH2CH2N), 2.41-2.55 (br, NCH2CH2CONHCH2C of the inner chain of PAMAM and the methine of cyclohexanedicarboxylic acid H2 N), 2.66 (br, NC of the inner and terminal chains of PAMAM H2 CH2CONHCH2CH2N) and 3.14 (br, NCH2CH2CONHC in the inner and end chains of PAMAM H2 CH2N, and NCH2CH2CONHCH2C at the end of the PAMAM chain H2 N).

[0061] (2) Preparation of dendrimers with phenylalanine residues attached to the ends (2.1) Addition of a phenylalanine residue to G4-Suc The carboxy terminus of G4-Suc was reacted with the amino terminus of the carboxy-terminally protected phenylalanine residue. The specific procedure was as follows. G4-Suc (83 mg, 3.8 μmol) was dispersed in DMSO (5 mL) and stirred overnight. Then, 3-phenyl-L-alanine benzyl ester 4-toluenesulfonate (Phe-OBzl·Tos) (0.12 g, 0.30 mmol), 1-[bis(dimethylamino)methylene]-1H-benzotriazole-3-oxide hexafluorophosphate (HBTU) (0.11 g, 0.28 mmol) and triethylamine (TEA) (41 μL, 0.29 mmol) were added to the solution of G4-Suc and stirred at room temperature. After stirring for 4 days, distilled water (1 mL) was added and the resulting solution was purified by dialysis in methanol using a dialysis membrane (molecular weight cut-off (MWCO): 1,000). Methanol was removed from the purified solution under reduced pressure, and the solution was freeze-dried to obtain a powder of G4-Suc with Phe-OBzl added to the end (G4-Suc-Phe-OBzl). The yield was 88 mg, and the yield was 66%. 1 The results of H-NMR spectrum measurement are shown below.

[0062] G4-Suc-Phe-OBzl (MeOD containing DCl): 2.33 (s, CH3 of tosyl), 2.41-2.49 (methylene of succinic acid), 2.70-3.04 (m, NCH2C of inner and terminal chains of PAMAM) H2 CONHCH2CH2N, and NCH2CH2CONHCH2C in the inner chain of PAMAM H2 N, and Hβ of Phe), 3.25-3.62 (br, NC of inner and terminal chains of PAMAM H2 CH2CONHCH2CH2N, and NCH2CH2CONHC in the inner and end chains of PAMAM. H2 CH2N, and NCH2CH2CONHCH2C at the end of the PAMAM chain H2 N), 4.62 (br, Hα of Phe), 5.02 (br, benzyl of Phe), 7.11-7.26 (m, phenyl of Phe and tosyl), and 7.69 (m, phenyl of tosyl).

[0063] (2.2) Addition of a phenylalanine residue to G4-CHex In the same manner as described in (2.1) above, the carboxy terminus of G4-CHex was reacted with the amino terminus of the carboxy-terminal-protected phenylalanine residue. Specifically, G4-CHex (53 mg, 2.11 μmol) was reacted with Phe-OBzl·Tos (0.12 g, 0.30 mmol), HBTU (0.09 g, 0.24 mmol), and TEA (41 μL, 0.29 mmol) in DMSO (5 mL) to obtain G4-CHex with Phe-OBzl attached to the terminus (G4-CHex-Phe-OBzl). Distilled water (1 mL) was added thereto, and the resulting solution was purified by dialysis in methanol using a dialysis membrane (molecular weight cutoff (MWCO): 1,000). Methanol was removed from the purified solution under reduced pressure, and the solution was lyophilized to obtain a powder of G4-CHex-Phe-OBzl. The yield was 84 mg, which was 109%. A solution of G4-CHex-Phe-OBzl 1 The results of H-NMR spectrum measurement are shown below.

[0064] G4-CHex-Phe-OBzl (MeOD containing DCl): 1.27-2.03 (m, methylene of CHex), 2.34 (s, CH3 of tosyl), 2.50-3.12 (m, NCH2C of inner and terminal chains of PAMAM) H2 CONHCH2CH2N, and NCH2CH2CONHCH2C in the inner chain of PAMAM H2 N, and Hβ of Phe), 3.44-3.58 (br, NC of inner and terminal chains of PAMAM H2 CH2CONHCH2CH2N, and NCH2CH2CONHC in the inner and end chains of PAMAM. H2 CH2N, and NCH2CH2CONHCH2C at the end of the PAMAM chain H2 N, and CHex methine), 4.56-4.65 (br, Hα of Phe), 5.00 (br, benzyl of Phe), 7.15-7.28 (m, phenyl of Phe and tosyl), and 7.67-7.69 (m, phenyl of tosyl).

[0065] (2.3) Deprotection of phenylalanine residues at the termini of the dendrimer G4-Suc-Phe-OBzl (101 mg, 2.9 μmol) and G4-CHex-Phe-OBzl (84 mg, 2.2 μmol) were dissolved in methanol (4 mL), and 4M NaOH methanol solution (500 μL) was added. After stirring at 4° C. for 2 hours, the dispersion of each dendrimer was purified by dialysis in distilled water using a dialysis membrane (molecular weight cut-off (MWCO): 1,000). The purified solution was freeze-dried to obtain white solids of dendrimers (G4-Suc-Phe and G4-CHex-Phe) with phenylalanine residues added to the ends. The yield of G4-Suc-Phe was 57 mg, and the yield was 64%. The yield of G4-CHex-Phe was 55 mg, and the yield was 77%.

[0066] Here, G4 PAMAM is a dendrimer having 64 terminal groups. 1As a result of H-NMR spectrum measurement, the number of acid anhydrides (Suc or CHex) bonded to the terminal groups and the number of phenylalanine residues bonded to the acid anhydrides were as shown in Table 1. Moreover, a schematic diagram showing one of the chemical structures of the 64 terminal groups possessed by each dendrimer of G4-Suc, G4-CHex, G4-Suc-Phe, and G4-CHex-Phe is shown in Figure 1. In Figure 1, "den" represents G4 PAMAM.

[0067] [Table 1]

[0068] Table 1 shows that dendrimers G4-Suc-Phe and G4-CHex-Phe with phenylalanine residues added to the termini were obtained as carriers for immune cell migration. It was also shown that in G4-Suc-Phe and G4-CHex-Phe, the termini of the dendrimer and the phenylalanine residue were bound via ethylene and cyclohexylene groups, respectively, as linkers.

[0069] Example 1: Imaging of fluorescent dye-linked carriers for immune cell migration (1) Preparation of fluorescent dye-linked carriers for immune cell migration (1.1) Preparation of G5-CHex-Phe and G5-Suc A dendrimer having succinic acid attached to the end (G5-Suc) and a dendrimer having phenylalanine residues attached to the end using a cyclohexylene group as a linker (G5-CHex-Phe) were prepared in the same manner as in the above production example, except that G5 PAMAM dendrimer (Sigma-Aldrich) was used instead of G4 PAMAM dendrimer.

[0070] (1.2) Addition of amino groups to G5-CHex-Phe and G5-Suc G5-CHex-Phe (25.34 mg, 0.376 μmol), N-(tert-butoxycarbonyl)-1,2-diaminoethane (Boc-ethylenediamine) (Tokyo Chemical Industry Co., Ltd., 0.3 μL, 1.88 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) (Sigma-Aldrich, 1.23 mg, 3.37 μmol) were dissolved in pure water (2.5 mL) and reacted overnight at room temperature with stirring. The reaction solution was purified by ultrafiltration (MWCO: 3000, 10 °C, 7500 g, 125 mM NaHCO3 aqueous solution). The resulting solution was lyophilized to obtain a white powder (G5-CHex-Phe-Boc). The yield was 23.42 mg, which was 90%. G5-CHex-Phe-Boc (21.03 mg, 0.305 μmol) was dissolved in trifluoroacetic acid (TFA) (1.2 mL) and stirred at 4°C overnight to remove the Boc group. The solvent was then removed under reduced pressure, and pure water was added and removed under reduced pressure several times to wash the product. The product was vacuum-dried overnight and then freeze-dried to obtain white crystals of G5-CHex-Phe with an amino group at the end. The yield was 34.48 mg, a 166% yield. G5-Suc with an amino group at the end was obtained in the same manner as above.

[0071] (1.3) Fluorescent dye conjugation to G5-CHex-Phe and G5-Suc G5-CHex-Phe (20 mg, 0.29 μmol) with an amino group at the end was dissolved in 125 mM NaHCO3 aqueous solution (1 mL, pH 8.3). A DMSO solution (1 mL) of green fluorescent dye HiLyte™ Fluor 488 acid SE (Anaspec, 0.41 mg, 0.587 μmol) was added to the dendrimer in an amount of 4 equivalents, and the mixture was stirred for 24 hours at room temperature in the dark. The mixture was then purified by ultrafiltration (MWCO: 3000, 4°C, 7500 g, 125 mM NaHCO3 aqueous solution). The resulting solution was freeze-dried to obtain crystals of G5-CHex-Phe with a fluorescent dye linked to the end. The yield was 9.24 mg, and the yield was 45%. G5-Suc with an amino group at the end was also obtained in the same manner as above, to obtain G5-Suc with a fluorescent dye linked to the end.

[0072] (1.4) NMR and UV-visible absorption measurements Each dendrimer (approximately 4 mg) linked to a fluorescent dye was dissolved in DO (30 μL) and NaOD (20 μL), and the compounds were analyzed at room temperature using a 400 MHz JNM-LA 400 JNM-ECX (JEOL). 1 H NMR and 31P NMR measurements were performed. The number of fluorescent dyes bound to one dendrimer molecule was identified using a V-630 UV-Vis spectrophotometer (JASCO). An aqueous solution (1 mL) of each dendrimer linked to a fluorescent dye was placed in a measurement cell, and the absorption spectrum (350-650 nm, 25 °C) was measured. Here, G5 PAMAM is a dendrimer having 128 terminal groups. From the results of NMR measurements, in G5-CHex-Phe, CHex was bound to 126 of the 128 terminal groups, and Phe was further bound to 111 of the CHex-bound terminal groups. In addition, in G5-Suc, Suc was bound to 128 of the 128 terminal groups. From the results of UV-vis measurements, two molecules of fluorescent dye were bound to one molecule of G5-CHex-Phe, and one or three molecules of fluorescent dye were bound to one molecule of G5-Suc. Hereinafter, G5-CHex-Phe with two molecules of fluorescent dye linked to its terminus will be referred to as “G5-CHex-Phe(dye2)”, and G5-Suc with one and three molecules of fluorescent dye linked to its terminus will be referred to as “G5-Suc(dye1)” and “G5-Suc(dye3)”, respectively.

[0073] (2) Imaging of fluorescent dye-linked carriers for immune cell migration (2.1) Animals All animal experiments were performed using BALB / cCrSlc mice (8–10 weeks old, female, 18–21 g, Japan SLC Co., Ltd.) in compliance with the university's regulations regarding animal experiments.

[0074] (2.2) Migration to immune cells in lymph nodes G5-CHex-Phe (dye2), G5-Suc (dye1) or G5-Suc (dye3) was added to physiological saline to give a dye concentration of 10 nmol / 100 μL to prepare a dispersion of each dendrimer. The dispersion of each dendrimer (20 μL on the hands, 30 μL on the feet) was administered intradermally to the backs of both hands and feet of mice. After administration, the administration site was immediately massaged for 30 seconds. After 3 and 24 hours, the mice were euthanized and the axillary and popliteal lymph nodes were collected. As a control, lymph nodes were collected in the same manner from mice that had not been administered the dendrimer. The collected lymph nodes were immersed in a 24-well plate filled with 1 mL of serum-free RPMI under ice cooling. The RPMI in the wells was removed, the lymph nodes were cut with scissors, and 1 mg / mL collagenase solution (1 mL) was added and left to stand at 37 °C for 30 minutes. The lymph node tissue was then ground on a cell strainer to obtain cells. The liquid containing the cells was centrifuged (4000 rpm, 5 min) to remove the supernatant, and serum-free RPMI (5 mL) was added to the cells to disperse them. The liquid containing the cells was centrifuged (4000 rpm, 5 min) to remove the supernatant, and stain buffer (1% FBS, 0.09% NaN3, 2 mM EDTA, and PBS) (0.5 mL) was added to obtain a cell suspension. The cell suspension was prepared so that the cell count was 2 × 10 7Stain buffer was added so that the cell concentration was 1.5 mL / mL, and 50 μL was dispensed into 1.5 mL tubes. The cell suspension was centrifuged (9200 rpm, 5 min) to remove the supernatant. A solution (50 μL each) of anti-CD3 antibody, anti-CD45R antibody, anti-CD11c antibody, and anti-F4 / 80 antibody labeled with yellow fluorescent dye phycoerythrin (PE) was added thereto, and the mixture was left in the dark for 30 minutes under ice cooling. Here, CD3, CD45R, CD11c, and F4 / 80 are molecular markers for detecting T cells, B cells, dendritic cells, and macrophages, respectively. For comparison, cells to which no labeled antibodies were added were also prepared. Then, stain buffer (100 μL) was added, and the mixture was centrifuged (9200 rpm, 5 min, 4° C.) to remove the supernatant, and the cells were washed. Stain buffer (1 mL) was added to the cells reacted with each labeled antibody to obtain a sample for flow cytometry (FCM) analysis. Each specimen was analyzed using a BD FACS™ Calibur (BD Biosciences). Based on the analysis results of 10,000 cells from each specimen, a distribution diagram was created with the number of cells on the vertical axis and the intensity of green fluorescence (530 nm) on the horizontal axis (see FIG. 2A), and a scattergram was created with the intensity of yellow fluorescence (585 nm) on the vertical axis and the intensity of green fluorescence (530 nm) on the horizontal axis (see FIG. 2B). In the analysis, in order to remove dead cells and impurities, a gate was determined to measure only live cells in the lymph node from a distribution diagram of the size and shape of cells measured by forward scattering and side scattering.

[0075] As shown in Figure 2A, G5-Suc (dye 1) or G5-Suc (dye 3) showed the same distribution as the control (Control) where no dendrimer was administered, suggesting that they hardly migrated to cells in the lymph nodes. In contrast, G5-CHex-Phe (dye 2) detected more cells with high green fluorescence intensity than the control. Therefore, it is suggested that G5-CHex-Phe (dye 2) migrated to cells in the lymph nodes.

[0076] As shown in the control scattergram in Figure 2B, most of the immune cells in the collected lymph nodes were T cells and B cells, with relatively few dendritic cells and macrophages. As shown in the scattergram of G5-Suc (dye 3) without the addition of PE-labeled antibodies (No staining), few cells showed high green fluorescence intensity, indicating that G5-Suc (dye 3) hardly migrates to cells in the lymph nodes. This is consistent with Figure 2A. In addition, as shown in the scattergram of G5-Suc (dye 3) with the addition of each PE-labeled antibody, few immune cells showed high values ​​for both green and yellow fluorescence intensity. This suggests that G5-Suc (dye 3) is not taken up much by any of T cells, B cells, dendritic cells, or macrophages. On the other hand, in the scattergrams in which each PE-labeled antibody was added, more immune cells were detected with G5-CHex-Phe (dye2) that showed high values ​​for both green and yellow fluorescence intensity compared to G5-Suc (dye3). Based on FIG. 2B, a graph (FIG. 3) was created showing the ratio of immune cells (cells that had incorporated a carrier for immune cell migration) that showed high green fluorescence intensity among immune cells (immune cells detected with PE-labeled antibodies) that showed high yellow fluorescence intensity. In FIG. 3, "T" represents T cells, "B" represents B cells, "DC" represents dendritic cells, and "MΦ" represents macrophages. As shown in FIG. 3, G5-CHex-Phe (dye2) migrated well into all of T cells, B cells, dendritic cells, and macrophages.

[0077] (2.3) In vivo fluorescence imaging G5-CHex-Phe (dye2) or G5-Suc (dye3) was added to physiological saline to give a dye concentration of 10 nmol / 100 μL to prepare a dendrimer dispersion. Each dendrimer dispersion (30 μL) was administered to the paws of mice. Immediately after administration, the administration site was massaged for 30 seconds. Four and 24 hours after administration, the mice were euthanized and the epidermis was peeled off. The hands and feet were fixed, and in vivo fluorescence imaging was performed using a fluorescence microscope FluoVivo (INDEC Biosystems). The results are shown in Figure 4.

[0078] In Figure 4, the areas surrounded by ellipses indicate the administration sites of each dendrimer, the areas indicated by arrows indicate lymph nodes, and the areas indicated by arrowheads indicate lymphatic vessels. Lymph nodes could be detected in mice 4 hours after administration of G5-CHex-Phe (dye2) and G5-Suc (dye3). In addition, lymphatic vessels connecting the primary and secondary lymph nodes could be confirmed in mice 4 hours after administration of G5-CHex-Phe (dye2). However, 24 hours after administration, lymph nodes could not be detected in mice administered G5-Suc (dye3). On the other hand, lymph nodes could be detected in mice administered G5-CHex-Phe (dye2). Here, although all the images shown in Figure 4 were taken with the same exposure time, it can be seen that G5-CHex-Phe (dye2) emits clearly stronger fluorescence than G5-Suc (dye3). This suggests that the accumulation of G5-CHex-Phe (dye 2) in lymph nodes is greater than that of G5-Suc (dye 3).

[0079] Example 2: Pharmacokinetics of radioactive substance-linked carriers for immune cell migration (1) Preparation of radioactive material-linked carriers for immune cell migration (1.1) Preparation of chelating agent-linked carriers for immune cell migration In Example 2, G5-Suc and G5-CHex-Phe with amino groups attached to the ends, prepared in Example 1, were used. G5-CHex-Phe with amino groups attached to the ends (12.16 mg, 0.212 μmol) was dissolved in 125 mM NaHCO3 aqueous solution (900 μL, pH 8.3). A 125 mM NaHCO3 aqueous solution (900 μL) containing 7 equivalents of the chelating agent p-SCN-Bn-DTPA (0.967 mg, 1.49 μmol) relative to the dendrimer was added thereto, and the mixture was stirred at 37° C. for 24 hours. The mixture was then purified by ultrafiltration (MWCO: 3000, 4° C., 7500 g, 125 mM NaHCO3 aqueous solution). The resulting solution was freeze-dried to obtain crystals of G5-CHex-Phe with fluorescent dyes attached to the ends. The yield was 5.27 mg, which was 35%. G5-Suc with an amino group at its end was also obtained in the same manner as above, with a chelating agent linked to its end. As in Example 1, each dendrimer linked to a radioactive substance was subjected to UV-vis measurement, and the number of chelating agents bound to one dendrimer molecule was identified using a V-630 UV-visible spectrophotometer (JASCO). The results of the UV-vis measurement showed that two molecules of the chelating agent were bound to one molecule of both G5-CHex-Phe and G5-Suc.

[0080] (1.2) Association of chelating agents with radioactive substances G5-CHex-Phe (20 nmol) with an amino group at the end was dissolved in 0.3 M ammonium acetate aqueous solution (200 μL). 111 InCl3 (200 μL, ~0.18 mCi) was added and stirred at room temperature for 1 hour. Then, it was purified with a PD-10 column (PBS) and ultrafiltered (MWCO: 3000). The obtained solution was diluted with saline so that the concentration of the radioactive substance was 10 μCi / 100 μL, and the radioactive substance ( 111 A solution of G5-CHex-Phe with terminal cation (In) was obtained. G5-Suc with terminal amino group was also prepared in the same manner as above to obtain G5-Suc with terminal cation (radioactive substance).

[0081] (2) Pharmacokinetic analysis of immune cell transfer carriers Animal experiments were performed using BALB / cCrSlc mice (8-10 weeks old, female, 18-21 g, Japan SLC Co., Ltd.) in compliance with the university's regulations regarding animal experiments. A dispersion (30 μL) of each dendrimer linked to a radioactive substance was intradermally administered to the mouse's foot. After administration, the administration site was immediately massaged for 30 seconds. The mice were euthanized 3 hours and 24 hours after administration, and the administration site, popliteal lymph nodes, blood, heart, lungs, liver, spleen, and kidneys were collected and weighed. The limbs were fixed, and the radioactivity of the organs collected from the mice was measured using an Autowell Gamma Counter (PerkinElmer). The results are shown in Figure 5. As can be seen from Figure 5, mice administered with G5-CHex-Phe had significantly higher radioactivity in the lymph nodes than mice administered with G5-Suc. In other words, it was suggested that G5-CHex-Phe accumulates in the lymph nodes more significantly than G5-Suc. In addition, radiation was detected in the liver and kidneys of mice administered G5-CHex-Phe, suggesting that some of the administered G5-CHex-Phe reached the blood vessels from the lymph nodes and circulated throughout the body.

[0082] Example 3: Imaging of fluorescent dye-linked carriers for immune cell migration (2) (1) Preparation of fluorescent dye-linked carriers for immune cell migration In Example 3, G4-Suc-Phe and G4-CHex-Phe prepared in Production Example 1 were used as carriers for immune cell migration. For comparison, G4-Suc and G4-CHex prepared in Production Example 1 were also used. In the same manner as in Example 1, G4-Suc-Phe, G4-CHex-Phe, G4-Suc, and G4-CHex were each reacted with 4 equivalents of Boc-ethylenediamine and 6 equivalents of DMT-MM, and the Boc group was removed with an appropriate amount of TFA to give an amino group to the end of each dendrimer. As a result of measuring these by NMR, 4 to 5 molecules of ethylenediamine were bound to the end of each molecule of each dendrimer. Each dendrimer with an amino group at the end was dissolved in DMSO (1 mL). Four equivalents of green fluorescent dye FITC (Tokyo Chemical Industry Co., Ltd.) and 6 equivalents of TEA were added to the dendrimer, and the mixture was stirred overnight at room temperature in a light-shielded state. The solution was then purified by ultrafiltration (MWCO: 2000, DMSO, methanol). The resulting solution was freeze-dried to obtain crystals of G4-Suc-Phe, G4-CHex-Phe, G4-Suc, and G4-CHex, each of which had a fluorescent dye linked to its end. UV-vis measurement of these crystals revealed that one or two molecules of FITC were bound to the end of each dendrimer molecule.

[0083] (2) In vitro migration of immune cells into the spleen (2.1) Collection of spleen cells and contact with carriers for immune cell transfer Animal experiments were performed using BALB / c Slc-nu / nu nude mice (female, with MDA-MB-231 cancer tumor, Japan SLC Co., Ltd.) in compliance with the university's regulations regarding animal experiments. Mice were euthanized and spleens were collected. The collected spleens were immersed in serum-free RPMI under ice cooling and cut into pieces with scissors. 1 mg / mL collagenase solution (1 mL) was added to the cut spleen tissue and left to stand at 37°C for 30 minutes. The spleen tissue was then crushed on a cell strainer to obtain cells. The liquid containing the cells was centrifuged (4000 rpm, 5 minutes) to remove the supernatant, and serum-free RPMI (5 mL) was added to the cells to disperse them. The liquid containing the cells was centrifuged (4000 rpm, 5 minutes) to remove the supernatant, and serum-free RPMI (10 mL) was added to obtain a cell suspension. The cell suspension was placed in a 96-well plate at 1 × 10 4 Cells / well were seeded. Each dendrimer linked to FITC was added to serum-free RPMI to give a dye concentration of 5 μM to prepare a dispersion of each dendrimer. 100 μL of each dendrimer dispersion was added per well and incubated at 4°C for 4 hours, or at 37°C for 1 hour or 4 hours.

[0084] (2.2) Fluorescence imaging of cells The 96-well plate was centrifuged (4000 rpm, 20 seconds) to remove the supernatant, and the cells were washed with PBS. The cells were observed with an inverted microscope IX71 (Olympus Corporation, objective lens magnification 10x) and analyzed with imaging software CellScens Standard (Olympus Corporation). The results are shown in Table 2. In the table, "x" indicates that cells emitting green fluorescence were not detected, and "o" indicates that cells emitting green fluorescence were detected.

[0085] [Table 2]

[0086] In Example 3, the spleen of a nude mouse was used as a specimen. Since nude mice do not have a thymus and B cells are abundant in the spleen, this experiment mainly observed the transfer of the carrier for immune cell transfer to B cells. As shown in Table 2, when incubated at 37°C, only the dendrimer having a phenylalanine residue bound to the end was able to transfer to cells. On the other hand, at 4°C, none of the dendrimers were transferred to cells. This suggests that the dendrimer having a phenylalanine residue bound to the end was not transferred to cells by penetrating the cell membrane, but was transferred to cells via endocytosis of the cells.

[0087] Example 4: Imaging of fluorescent dye-linked carriers for immune cell migration (3) (1) Fluorescent dye-linked carrier for immune cell migration In Example 4, G4-Suc-Phe and G4-CHex-Phe linked to the termini of FITC prepared in Example 3 were used as carriers for immune cell migration. For comparison, G4-Suc and G4-CHex linked to the termini of FITC were also used.

[0088] (2) In vitro migration of immune cells into the spleen (2.1) Collection of spleen cells, contact with carrier for immune cell transfer, and immunostaining The animal experiment was performed using BALB / c mice (6 weeks old, female, Japan SLC Co., Ltd.) in compliance with the university's regulations regarding animal experiments. The mice were euthanized and the spleens were collected. A cell suspension was obtained from the collected spleens in the same manner as in Example 3. The number of cells was 1.6 × 10 6The cell suspension was separated into 1.5 mL tubes so that the cell suspension was 10 cells / tube. Each dendrimer linked to FITC was added to serum-free RPMI so that the dye concentration was 5 μM to prepare a dispersion of each dendrimer. Each dendrimer dispersion was added to a 1.5 mL tube containing cells and incubated at 37°C for 3 hours. In addition, the dispersion of each dendrimer was added to a 1.5 mL tube containing cells in the same manner as above, except that the pH of serum-free RPMI was adjusted to 6.02, and incubated at 37°C for 3 hours. The pH of serum-free RPMI without pH adjustment was 7.95. The liquid containing the cells was centrifuged (3000 rpm, 5 minutes, 4°C) to remove the supernatant, and stain buffer (1% FBS, 2 mM EDTA and PBS) (0.5 mL) was added to obtain a cell suspension. The number of cells was 2 × 10 5 The cell suspension was separated into 1.5 mL tubes so as to obtain cells / tube. A solution of PE-labeled anti-CD3 antibody, anti-CD45R antibody, anti-CD11c antibody, and anti-F4 / 80 antibody (antibody: serum-free RPMI = 1:49, total volume 50 μL) was added, and the mixture was left in the dark for 30 minutes under ice cooling. Then, stain buffer (100 μL) was added, and the mixture was centrifuged (3000 rpm, 5 minutes, 4 ° C.) to remove the supernatant, and the cells were washed. Stain buffer (0.5 mL) was added to the cells reacted with each labeled antibody to obtain a sample for FCM analysis. Each sample was analyzed using BD FACS Calibur (BD Biosciences). In the same manner as in Example 1, a graph (FIG. 6) was created showing the proportion of cells containing carriers for immune cell migration in immune cells detected with PE-labeled antibodies from the analysis results. In addition, a graph (FIG. 7) was prepared showing the ratio of cells containing carriers for immune cell migration among T cells and B cells detected with PE-labeled antibodies when the pH was set to 6.02.

[0089] In FIG. 6, "T" represents T cells, "B" represents B cells, "DC" represents dendritic cells, and "MΦ" represents macrophages. As shown in FIG. 6, G4-Suc-Phe migrated to all of the spleen-derived T cells, B cells, dendritic cells, and macrophages. G4-CHex-Phe also migrated to all of the immune cells (not shown). On the other hand, G4-Suc and G4-CHex hardly migrated to any of the cells (not shown). In FIG. 7, "T" represents T cells, and "B" represents B cells. As shown in FIG. 7, the proportion of G4-Suc-Phe that migrated to T cells increased by adjusting the pH of the medium to 6.02. G4-CHex-Phe also showed a similar tendency (not shown). On the other hand, there was no change in the proportion of G4-Suc-Phe that migrated to B cells. This suggests that G4-Suc-Phe and G4-CHex-Phe have improved migration to T cells under acidic conditions.

[0090] Manufacturing Example 2: Preparation of carrier for immune cell migration (2) (1) Preparation of G4-Ph-Phe A carrier for immune cell migration (G4-Ph-Phe) having a phenylalanine residue at the terminal and a phthalic acid linker was prepared by the method described in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151. The synthesis scheme is shown below.

[0091] [ka]

[0092] PAMAM dendrimer G4 (56.0 mg, 3.94 μmol) was dissolved in DMSO-DMF mixed solvent (2.3 mL), and an excess of phthalic anhydride (0.3170 g, 1.90 mmol) was added, followed by TEA (200 μL, 1.44 mmol), and the mixture was stirred overnight at room temperature. After dialysis against DMSO (MWCO: 1000), the mixture was dialyzed against distilled water. Finally, the mixture was lyophilized to obtain G4-Ph (yield: 67.3 mg, yield: 69%). Next, G4-Ph (34.5 mg, 1.46 μmol) was weighed out, dispersed in DMSO (5 mL), and stirred overnight. After stirring, Phe-OMe·HCl (0.1200 g, 280 μmol), HBTU (0.1100 g, 290 μmol), and TEA (40.7 μL, 294 μmol) were added and stirred for 4 days. Distilled water (1 mL) was then added, and the resulting solution was purified by dialysis in methanol using a dialysis membrane (molecular weight cut-off (MWCO): 1,000). Methanol was removed from the purified solution under reduced pressure, and the solution was lyophilized to obtain G4-Ph-(Phe-OMe) (yield: 30.5 mg, yield: 60%). G4-Ph-(Phe-OMe) was then dissolved in methanol (4 mL), and 4 M NaOH methanol solution (500 μL) was added. After stirring for 2 h at 4 °C, the dendrimer dispersion was purified by dialysis in distilled water using a dialysis membrane (molecular weight cut-off (MWCO): 1,000). The purified solution was lyophilized to obtain G4-Ph-Phe (yield: 63.2 mg, yield: 90.2%). 1 H-NMR spectroscopy revealed that the number of phthalic acid (Ph) bonds to the terminal group of G4 PAMAM was 64, and the number of phenylalanine residues (Phe) bonds was 58.

[0093] Example 5: Measurement of the change in transmittance of G4-Suc-Phe, G4-Ph-Phe, and G4-Chex-Phe with respect to temperature Solutions of G4-Suc-Phe, G4-Ph-Phe and G4-Chex-Phe with various pH values ​​were prepared, and the transmittance was measured by changing the temperature of these solutions. Measurement conditions: measurement wavelength: 500 nm, heating rate: 1°C / min, measurement interval: 0.1°C, bandwidth: 1.5 nm. After adjusting the pH with glycine hydrochloric acid buffer (pH 3.5 or less), acetate buffer (pH 5.5 or less) and phosphate buffer (pH 6 or more), the temperature change of the transmittance was measured using a UV / Vis Spectrophotometer (V-630, JASCO). A Peltier holder (ETC-717, JASCO) was used to adjust the temperature. The results are shown in Figure 8.

[0094] For all dendrimers, the transmittance decreased when the pH of the solution became acidic. The transmittance of G4-Chex-Phe, G4-Ph-Phe, and G4-Suc-Phe decreased near body temperature in aqueous solutions of pH 6.5, pH 6, and pH 5. In addition, under these pH conditions, the transmittance increased when heated, showing an upper critical solution temperature (UCST) type of behavior.

[0095] Manufacturing Example 3: Preparation of carrier for immune cell migration (3) (1) Preparation of G4-Phe-SO3H An immune cell migration carrier (G4-Phe-SO3H) having a phenylalanine residue at the terminal and a sulfone group as an anionic terminal group was synthesized according to the following scheme with reference to Chen HT. et al., J. Am. Chem. Soc., 2004, vol. 126(32), pp. 10044-10048.

[0096] [ka]

[0097] G4-Phe was obtained by reacting the amino terminal of G4 PAMAM dendrimer (Sigma-Aldrich) with the carboxyl terminal of an amino-protected phenylalanine residue. 1,3-propane sultone (142.3 mg, 1.17 mmol) was dissolved in acetonitrile (2.8 mL). G4-Phe (92.2 mg, 2.27 μmol) was dissolved in 125 mM NaHCO3 buffer (2.8 mL). These two solutions were mixed, nitrogen bubbling was performed, and the mixture was stirred at room temperature. The mixture was then left for two days, and the pH was measured on the third day. Since the pH was below 1, a 4M NaOH aqueous solution was added to make the pH basic, and nitrogen bubbling was performed and the mixture was stirred. The pH was measured on the fourth day. Since the pH was 7-8, a 4M NaOH aqueous solution was added to raise the pH to about 9. The pH was measured on the fifth day. Since the pH was about 8, an HCl aqueous solution was added to lower the pH, and the pH was then raised to about 8. After reducing the volume of the solution with an evaporator, distilled water (2.1 mL) was added and dialyzed against distilled water. After lyophilization, G4-Phe-SO3H was obtained (yield: 60.9 mg, yield: 95%). 1 H-NMR spectroscopy revealed that the number of bonds between phenylalanine residues (Phe) and the terminal group of G4 PAMAM was 49 and 56, respectively, of sulfonic acids bonded to the terminal group and phenylalanine residues of G4 PAMAM.

[0098] (2) Measurement of the change in transmittance of G4-Phe-SO3H with respect to temperature A 1 mg / mL (buffer concentration 20 mM) G4-Phe-SO3H solution was prepared, and the temperature of the solution was changed to measure the transmittance. Measurement conditions: measurement wavelength: 500 nm, temperature rise rate: 1°C / min, measurement interval: 0.1°C, bandwidth: 1.5 nm. After adjusting the pH with glycine hydrochloric acid buffer (pH 3.5 or less), acetate buffer (pH 5.5 or less), and phosphate buffer (pH 6 or more), the temperature change of the transmittance was measured using a UV / Vis Spectrophotometer (V-630, JASCO). A Peltier holder (ETC-717, JASCO) was used to adjust the temperature. The results are shown in Figure 9.

[0099] As shown in FIG. 9, G4-Phe-SO3H showed a lower critical solution temperature (LCST) type phase transition (LCST=52°C) at pH 5 and a UCST type phase transition (UCST=36°C) at pH 6.5. G4-Phe-SO3H was a dendrimer that sensitively caused LCST and UCST type phase transitions under acidic conditions. The transmittance of the G4-Phe-SO3H solution was 3% at room temperature and pH 6.5, but increased to 91% at about 40°C and pH 6.5. Also, referring to Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151, the transmittance of the G4-Phe-SO3H solution at about 37°C and pH 6.5 was comparable to that of the G4-CHex-Phe solution under the same conditions (about 30%).

[0100] Manufacturing Example 4: Preparation of carrier for immune cell migration (4) (1) Preparation of G3.5-Phe and G2.5-Phe Carriers for immune cell migration (G3.5-Phe and G2.5-Phe) in which phenylalanine residues are directly bonded to the terminal groups of the dendrimers and the anionic terminal groups are carboxyl groups were synthesized according to the following scheme. In this production example, PAMAM dendrimers (G3.5: 64 terminals, and G2.5: 32 terminals) in which the terminal groups are carboxyl groups were reacted with the amino groups of phenylalanine residues with protected carboxyl groups.

[0101] [ka]

[0102] (1.1) Preparation of G3.5-Phe PAMAM dendrimer G3.5 (38.7 mg, 2.99 μmol) was dissolved in 125 mM NaHCO3 aqueous solution (pH 9.1, 100 μL), and DMSO (4 mL) was added. DMT-MM (215.2 mg, 777 μmol) was added. Phe-OBzl·Tos (156.1 mg, 365 μmol) was added and stirred at room temperature for 3 days. Next, dialysis (MWCO: 2000) was performed with methanol. Methanol was distilled off under reduced pressure, and the mixture was vacuum dried for 3 hours, followed by lyophilization to obtain G3.5-(Phe-OBzl) (yield: 39.1 mg, yield: 47.5%). Next, the synthesized G3.5-(Phe-OBzl) (39.1 mg, 1.42 μmol) was dissolved in methanol (4 mL), and a methanol solution containing 4M NaOH (500 μL) was added and stirred at 4°C for 2 hours. After stirring, the mixture was dialyzed against distilled water (MWCO: 2000) and then freeze-dried to obtain G3.5-Phe (yield: 25.0 mg, yield: 101%). 1 As a result of H NMR (D2O containing DCl) spectrum measurement, the number of phenylalanine residues (Phe) bonded to the terminal group of PAMAM dendrimer G3.5 was 30. G3.5-Phe obtained by these reactions is hereinafter referred to as "G3.5-Phe". 30 " It is called.

[0103] G3.5-Phe was also prepared under different synthetic conditions, that is, the condensation reaction was carried out in the same manner using DMSO (1.5 mL) / water (1 mL) as the solvent to give G3.5-Phe (yield: 39.5 mg, yield: 71.0%). 1 As a result of H NMR (D2O containing DCl) spectrum measurement, the number of phenylalanine residues (Phe) bonded to the terminal group of PAMAM dendrimer G3.5 was 41. G3.5-Phe obtained by these reactions is hereinafter referred to as "G3.5-Phe". 41 " It is called.

[0104] (1.2) Preparation of G2.5-Phe PAMAM dendrimer G2.5 (38.0 mg, 6.06 μmol) was dissolved in distilled water (100 μL). Next, DMSO (4 mL) was added to disperse G2.5. Then, DMT-MM (198.9 mg, 718 μmol) and Phe-OBzl·Tos (137.2 mg, 321 μmol) were added, and the reaction and purification were performed in the same manner as above to obtain G2.5-(Phe-OBzl) (yield: 57.8 mg, 71.9%). Next, the synthesized G2.5-(Phe-OBzl) (55.8 mg, 4.21 μmol) was reacted and purified in the same manner as above (1.1) to obtain G2.5-Phe (yield: 25.2 mg, 92.2%). 1 As a result of H NMR (D2O containing DCl) spectrum measurement, the number of phenylalanine residues (Phe) bonded to the terminal group of PAMAM dendrimer G2.5 was 18. G2.5-Phe obtained by these reactions is hereinafter referred to as "G2.5-Phe". 18 " It is called.

[0105] G2.5-Phe was also prepared under different synthetic conditions. That is, 0.5 equivalents of Phe-OBzl·Tos was added to the terminal of PMAMA dendrimer (G2.5) and the reaction was carried out in the same manner to obtain G2.5-Phe (yield: 13.7 mg, yield: 53.3%). 1 As a result of H NMR (D2O containing DCl) spectrum measurement, the number of phenylalanine residues (Phe) bonded to the terminal group of PAMAM dendrimer G2.5 was 14. G2.5-Phe obtained by these reactions is hereinafter referred to as "G2.5-Phe". 14 " It is called.

[0106] (2) Measurement of the change in transmittance of G3.5-Phe and G2.5-Phe with respect to temperature In the same manner as in Production Example 2, solutions of G3.5-Phe and G2.5-Phe having various pH values ​​were prepared, and the transmittance of the solutions was measured while changing the temperature. The results are shown in Figures 10A to 10D. As shown in Figure 10A, the transmittance of G3.5-Phe 30The transmittance of the solution did not change at pH 7, but at pH 6, it increased gradually with heating from 5°C to 55°C. This was UCST-type behavior. On the other hand, at pH 5, the transmittance was high at low temperatures and decreased around 45°C. This was LCST-type behavior. At pH 4, LCST-type behavior was also observed, but a decrease in transmittance was already observed at 5°C. As shown in Figure 10B, the transmittance of G3.5-Phe 41 The transmittance of the solution did not change at pH 7 or pH 6, but at pH 5, it increased gradually with heating from 35°C to 85°C. This was UCST-type behavior. On the other hand, at pH 4, the transmittance was high at low temperatures and decreased from 5°C. This was LCST-type behavior.

[0107] As shown in FIG. 10C, G2.5-Phe 18 The transmittance of the solution did not change at pH 7, but increased at pH 6 in the range of 5°C to 25°C, with high transmittance over the entire temperature range. At pH 5, the transmittance increased gradually with heating from 5°C to 95°C. This was similar to the behavior of the UCST type. On the other hand, at pH 4, the transmittance was low at 63% from 5°C, and decreased with heating. This was the behavior of the LCST type. As shown in Figure 10D, the transmittance of G2.5-Phe 14 The transmittance of the solution did not change at pH 7, but increased gradually with heating from 5 to 65°C at pH 6 and from 45 to 95°C at pH 5. This was UCST-type behavior. On the other hand, at pH 4, the transmittance was high at low temperatures and decreased around 35°C. This was LCST-type behavior.

[0108] Example 6: Comparison of amino acid residues in carriers for immune cell migration (1) Fluorescent dye-linked carrier for immune cell migration In Example 6, G4-Suc-Phe and G4-CHex-Phe, which were prepared in Example 3 and had FITC linked to the end, were used as carriers for immune cell migration. For comparison, carriers (G4-Suc-Leu and G4-CHex-Leu) in which leucine residues were linked to dendrimers were prepared, and FITC was linked to the end. G4-Suc-Leu and G4-CHex-Leu were prepared in the same manner as G4-Suc-Phe and G4-CHex-Phe, except that leucine benzyl ester p-toluenephosphonate was used instead of 3-phenyl-L-alanine benzyl ester 4-toluenesulfonate in Production Example 1. The number of leucine residues linked in each of G4-Suc-Leu and G4-CHex-Leu was 64. Schematic diagrams of G4-Suc-Phe, G4-CHex-Phe, G4-Suc-Leu, and G4-CHex-Leu are shown in Figure 11. FITC was linked to G4-Suc-Leu and G4-CHex-Leu in the same manner as in Example 3. Each dendrimer linked with FITC was added to serum-free RPMI (pH 8.85 and pH 6) so that the dye concentration was 5 μM, to prepare a dispersion of each dendrimer.

[0109] (2) In vitro migration to immune cells Jurkat cells, a human T-cell leukemia-derived cell line, were used as immune cells. The cell suspension was placed in a microtube at 1 × 10 5 The cells were placed in a ratio of cells / tube, and the microtubes were centrifuged (3000 rpm, 3 min) to remove the supernatant. 25 μL of each dendrimer dispersion was added per tube, and incubated at 37° C. for 3 hours. The microtubes were centrifuged (3000 rpm, 3 min) to remove the supernatant, and the cells were washed with PBS. After removing the supernatant, 1 mL of serum-free RPMI was added to each microtube to obtain samples for FCM analysis. Each sample was analyzed using a BD FACS Calibur (BD Biosciences). A graph (FIG. 12) was created showing the average fluorescence intensity of the cells to which each dendrimer was added.

[0110] As can be seen from A and B in Figure 12, the fluorescence intensity of G4-Suc-Phe was higher than that of G4-Suc-Leu. Also, as can be seen from C and D in Figure 12, the fluorescence intensity of G4-CHex-Phe was higher than that of G4-CHex-Leu. Although both phenylalanine and leucine are amino acids with non-polar side chains, Figure 12 shows that dendrimers bound to phenylalanine residues have better migration to immune cells than dendrimers bound to leucine residues.

[0111] Example 7: Imaging of fluorescent dye-linked carriers for immune cell migration (4) (1) Preparation of fluorescent dye-linked carriers for immune cell migration In Example 7, G4-Phe-Suc and G4-Phe-CHex with FITC linked to the end were used as carriers for immune cell migration. G4-Phe-Suc and G4-Phe-CHex were prepared as follows. In the same manner as in Production Example 3, the amino end of G4 PAMAM dendrimer (Sigma-Aldrich) was reacted with the carboxyl end of a phenylalanine residue with a protected amino group to obtain G4-Phe. G4-Phe (125 mg, 5.57 μmol) was dissolved in 125 mM NaHCO3 buffer (3 mL), and an excess of succinic anhydride or cis-1,2-cyclohexanedicarboxylic anhydride (500 equivalents for each) was added. Each solution was stirred overnight at room temperature, and the pH was adjusted to 8 to 10 using a 4N NaOH aqueous solution. The solution containing the dendrimer was purified by dialysis in distilled water using a dialysis membrane (MWCO: 1,000). The purified solution was freeze-dried to obtain G4-Phe-Suc and G4-Phe-CHex. The yield of G4-Phe-Suc was 68 mg, which was a 56% yield. The yield of G4-Phe-CHex was 65 mg, which was a 72% yield. Each solution of G4-Phe-Suc and G4-Phe-CHex was freeze-dried. 1H-NMR spectrum was measured. In G4-Phe-Suc, the number of phenylalanine residues (Phe) bound to the terminal group of G4 PAMAM was 56, and the number of succinic acid bound to the terminal group and phenylalanine residue of G4 PAMAM was 57. In addition, in G4-Phe-CHex, the number of phenylalanine residues (Phe) bound to the terminal group of G4 PAMAM was 64, and the number of cyclohexanedicarboxylic acid bound to the terminal group and phenylalanine residue of G4 PAMAM was 64. Schematic diagrams of G4-Phe-Suc and G4-Phe-CHex are shown in FIG. 13. FITC was linked to G4-Phe-Suc and G4-Phe-CHex in the same manner as in Example 3.

[0112] (2) In vitro migration to immune cells The transferability of G4-Phe-Suc and G4-Phe-Chex into Jurkat cells was examined in the same manner as in Example 6. A graph showing the average fluorescence intensity of cells to which each dendrimer was added (FIG. 14) was prepared. As shown in FIG. 14, it was shown that G4-Phe-Suc and G4-Phe-Chex were transferred into Jurkat cells.

Claims

1. A carrier for immune cell migration comprising a complex molecule, the complex molecule comprises a branched polymer and a phenylalanine residue and has an anionic terminal structure, the phenylalanine residue being bound to an end group of the branched polymer; the anionic terminal structure is a carboxyl group of the phenylalanine residue or a salt thereof, or an anionic terminal group bonded via the phenylalanine residue; the anionic end group is a carboxyl group, a sulfonic acid group, a sulfate group, or a salt thereof; The branched polymer is a dendrimer. Carrier for immune cell migration.

2. The carrier according to claim 1, wherein the end group of the branched polymer is directly bonded to the phenylalanine residue.

3. The carrier according to claim 2, further comprising a compound capable of imparting said anionic terminal group to said phenylalanine residue.

4. 2. The carrier according to claim 1, wherein the anionic terminal group is a carboxyl group or a salt thereof.

5. The carrier according to claim 1, wherein the end group of the branched polymer and the phenylalanine residue are bonded via a linker.

6. The anionic terminal structure comprises the phenylalanine residue attached to an end group of the branched polymer and has the following formula (I): 【Chemistry 1】 (In the formula, X 1 is -NH-(C=O)- or -O-(C=O)-, and Y 1 is -NH-(C=O)- or -NH-, R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, heterocycloalkylene group, or phenylene group which may have a substituent and have 3 to 8 carbon atoms; Z 1 is a carboxyl group, a sulfonic group, a sulfate group, or a salt thereof) or the following formula (II): 【Chemistry 2】 (In the formula, X 2 is -NH-(C=O)-, -(C=O)-NH-, -O-(C=O)-, -(C=O)-O-, -O-(C=O)-NH-, -NH-(C=O)-O-, -NH-(C=O)-NH-, -NH-(C=S)-NH-, or a structure obtained by the reaction of maleimide with a thiol, R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, heterocycloalkylene group, or phenylene group which may have a substituent and have 3 to 8 carbon atoms; Y 2 is -(C=O)-NH-, -O-(C=O)-NH-, -NH-(C=O)-NH- or -NH-(C=S)-NH-, Z 2 is a carboxyl group or a salt thereof) or the following formula (III): 【Chemistry 3】 (In the formula, Z 3 is a carboxyl group or a salt thereof) The carrier according to any one of claims 1 to 5, wherein the carrier is represented by the formula:

7. The carrier according to any one of claims 1 to 6, wherein the immune cells are immune cells present in lymph nodes.

8. The carrier according to any one of claims 1 to 7, wherein the immune cells are at least one type selected from the group consisting of T cells, B cells, dendritic cells, and macrophages.

9. The carrier according to any one of claims 1 to 8, wherein the transferability to T cells changes in response to pH.

10. A reagent for immune cell migration, comprising the carrier for immune cell migration according to any one of claims 1 to 9.

11. A method for transferring a carrier for immune cell transfer to an immune cell, comprising contacting the carrier for immune cell transfer according to any one of claims 1 to 9 with an immune cell in vivo (excluding humans), in vitro or ex vivo.

12. The method of claim 11 , wherein the carrier for immune cell migration comprises a substance of interest.

13. The method of claim 12, wherein the substance of interest is at least one selected from a labeling substance, a nucleic acid, a protein, and a peptide.

14. The method according to claim 13, wherein the labeling substance is at least one selected from the group consisting of a radioisotope, a low molecular weight organic dye, a fluorescent dye, and a contrast agent for nuclear magnetic resonance imaging.

15. A pharmaceutical composition comprising a medicine and the carrier for immune cell migration according to any one of claims 1 to 9.

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