WO2021095781A1 - Carrier for transfer into immune cells and use thereof - Google Patents

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

Immune cell transfer carrier and its use

The present invention relates to a carrier for immune cell transfer. The present invention relates to reagents for immune cell transfer. The present invention relates to a method of transferring a carrier for immune cell transfer to immune cells. The present invention relates to a pharmaceutical composition comprising a drug and a carrier for transfer of immune cells.

In recent years, immunotherapy has been attracting attention as a new cancer treatment method. Immunotherapy for cancer can be broadly divided into passive immunotherapy and active immunotherapy. Passive immunotherapy is a treatment method in which immune cells such as T cells collected from a patient are activated in vitro and returned to the patient's body to attack cancer cells with the activated immune cells. For example, CAR- T cell therapy is known. Active immunotherapy is a treatment method in which a substance that activates immune cells is administered to a patient to attack cancer cells with the activated immune cells. For example, cytokine therapy is known.

There is still no system that efficiently delivers 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, carry out an immune response that eliminates foreign substances in tissue fluid and lymph fluid mainly in lymph nodes. Patent Document 1 states that a dendrimer having a carboxyl group as a terminal group can be transferred to a lymph node, and when the dendrimer is intradermally administered to a mouse, it accumulates in the sentinel lymph node and the secondary lymph node beyond the dendrimer. Are listed.

International Publication No. 2015/170573

The present inventors have found that the dendrimer of Patent Document 1 accumulates in the lymph nodes of a living body, but is not taken up by immune cells in the lymph nodes. It is expected that establishing a means capable of selectively transferring desired physiologically active substances and the like to immune cells of a living body will contribute to the development of cancer immunotherapy. An object of the present invention is to provide a carrier molecule that can be transferred to immune cells.

The present invention is a carrier for immune cell migration containing a complex molecule, wherein the complex molecule contains a branched polymer and a phenylalanine residue and has an anionic terminal structure, and the terminal group of the branched polymer. Provided is a carrier for immune cell transfer to which the phenylalanine residue is bound. The present invention provides a reagent for immune cell transfer, which comprises a carrier for immune cell transfer. The present invention provides a method of transferring an immune cell transfer carrier to an immune cell, comprising contacting the immune cell transfer carrier in vivo (but excluding humans), in vitro or ex vivo. The present invention provides a pharmaceutical composition comprising a drug and a carrier for immune cell transfer.

According to the present invention, a carrier that can be transferred to immune cells is provided.

Dendrimers (G4-Suc and G4-CHex) that do not have a phenylalanine residue at the end and have a linker attached to the end, and dendrimers (G4-Suc-Phe and G4-) that have a phenylalanine residue attached to the end via a linker. It is a schematic diagram which showed the chemical structure of one of 64 terminal groups which each has CHex-Phe). It is a distribution map obtained by analyzing cells obtained from the lymph nodes of mice to which a fluorescently labeled immune cell transfer carrier was administered by flow cytometry (FCM). It is a scattergram obtained by immunostaining cells obtained from the lymph nodes of mice to which a fluorescently labeled immune cell transfer carrier was administered with a PE-labeled antibody and analyzing with FCM. It is a graph which shows the ratio of the cell containing the carrier for immune cell transfer among various immune cells detected by PE-labeled antibody. 6 is an image obtained by in vivo fluorescence imaging of a mouse to which a fluorescently labeled immune cell transfer carrier was administered. It is a graph obtained by measuring the radiation emitted from the organ or the like collected from the mouse to which the carrier for transfer of immune cells labeled with a radioactive substance was administered. It is a graph which shows the ratio of the cell containing the carrier for immune cell transfer among various immune cells detected by PE-labeled antibody. It is a graph which shows the ratio of the cell containing the carrier for immune cell transfer in the T cell and B cell under the neutral or acidic conditions detected by the PE-labeled antibody. A graph showing the change in transmittance of a solution (pH 4 to 7) of a dendrimer (G4-Suc-Phe, G4-Ph-Phe and G4-CHex-Phe) in which a phenylalanine residue is bound to a terminal via a linker with respect to temperature. Is. It is a graph which shows the change of the transmittance with respect to the temperature of the solution (pH 5 or pH 6.5) of the _{dendrimer (G4-Phe-SO 3} H) which has a phenylalanine residue at the terminal and the terminal group is a sulfone group. Changes in permeability of a solution (pH _{4-7) of an immune cell transfer carrier (G3.5-Phe 30} ) in which a phenylalanine residue is directly attached to the terminal group of the dendrimer and the anionic terminal group is a carboxyl group. It is a graph which shows. Changes in permeability of a solution (pH _{4-7) of an immune cell transfer carrier (G3.5-Phe 41} ) in which a phenylalanine residue is directly attached to the terminal group of the dendrimer and the anionic terminal group is a carboxyl group. It is a graph which shows. Changes in permeability of a solution (pH _{4-7) of an immune cell transfer carrier (G2.5-Phe 18} ) in which a phenylalanine residue is directly attached to the terminal group of the dendrimer and the anionic terminal group is a carboxyl group. It is a graph which shows. Changes in permeability of a solution (pH _{4-7) of an immune cell transfer carrier (G2.5-Phe 14} ) in which a phenylalanine residue is directly attached to the terminal group of the dendrimer and the anionic terminal group is a carboxyl group. It is a graph which shows. Dendrimers with phenylalanine residues attached to the ends via a linker (G4-Suc-Phe and G4-CHex-Phe) and dendrimers with leucine residues attached to the ends via a linker (G4-Suc-Leu and G4-) It is a schematic diagram which showed the chemical structure of one of 64 terminal groups which each has CHex-Leu). It is a graph which shows the average value of the fluorescence intensity of the Jurkat cell which was in contact with the fluorescently labeled immune cell transfer carrier, and obtained by the analysis by FCM. A schematic 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 attached to the terminal and the anionic terminal group is a carboxyl group. It is a figure. It is a graph which shows the average value of the fluorescence intensity of the Jurkat cell which was in contact with the fluorescently labeled immune cell transfer carrier, and obtained by the analysis by FCM.

The carrier for immune cell transfer of the present embodiment (hereinafter, also simply referred to as “carrier”) is a complex having an anionic terminal structure in which a phenylalanine residue is directly or indirectly bonded to the terminal group of the branched polymer. Contains molecules. The branched polymer is not particularly limited as long as it is a polymer having a branched structure or a dendritic structure. The terminal group of the branched polymer means the outermost group of the branched structure or the dendritic structure of the branched polymer. The anionic terminal structure in the carrier of the present embodiment is a portion containing a phenylalanine residue that is directly or indirectly bonded to the terminal group of the branched polymer, and the group at the end of the portion is an anionic group. Refers to the part. Examples of the anionic group include a carboxyl group, a sulfone group, a sulfate group or a salt thereof. The present inventors have completed the present invention by finding that the complex molecule can be used as a carrier for immune cell transfer because the complex molecule can carry a substance of interest. Here, the "substance of interest" refers to a predetermined substance that is desired to be transferred to immune cells. “Carrying the substance of interest” means connecting the substance of interest to the terminal group of the branched polymer in the complex molecule, encapsulating the substance of interest in the internal space of the branched polymer, and phenylalanine residue. Includes linking the substance of interest to the group.

In the present embodiment, the branched polymer preferably has at least one functional group capable of binding to a phenylalanine residue or a linker described later as a terminal group. Examples of the functional group include an amino group, a carboxyl group, a hydroxy group, a sulfone group and the like. Examples of the branched polymer include dendrimers, "hyperbranched polymers" having low branching regularity in dendritic structures, "dendrigrafts" (also referred to as random dendritic polymers), and star polymers.

Dendrimer is a compound with a three-dimensionally highly branched dendritic structure, and is known to have an almost spherical morphology. As used herein, the term "dendrimer" also includes a dendron having a partial structure constituting the dendrimer, in which at least one functional group of the core portion is not branched. Dendrimers generally consist of a core, several generations of bifurcations, and end groups. The dendrimer core is derived from a compound having one or more functional groups. Examples of the functional group include a primary amino group, a secondary amino group, a hydroxy group, a carboxylic acid group, a thiol group, an ester group, an amide group, a ketone group, an aldehyde group and the like. Preferably, it is a primary amino group and a secondary amino group. Examples of the compounds constituting the core include ammonia, ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentano, 1,6-diaminohexane, 1,7-diaminoheptane, 1, Examples include 8-diaminooctane, 1,10-diaminodecane, and 1,12-diaminododecane. It may be an alkyldiamine (cystamine) containing an SS bond.

The branched portion of the dendrimer consists of repeating branched structural units containing atoms having a valence of 3 or more. Examples of the atom having a valence of 3 or more include carbon, nitrogen, silicon, phosphorus and the like. For example, the following structure is known as a branching portion of a dendrimer.

(In the formula, n is an integer of 1 or more, preferably an integer of 60 to 300)

Each dendrimer having the above-mentioned branched structures (A) to (C) is described in the following documents. The branched portion of the dendrimer may include two or more types of repeating units as described above.
(A) Tomalia DA et al., Polym. J. 17, 117 (1985) and Tomalia DA et al., Angelw. Chem. Int. Ed. Engl. 29, 138 (1990)
(B) de Brabander-van den Berg EMM et al., Angelw. 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., Angelw. 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)

The structure of the terminal group of the dendrimer can be selected as appropriate. For example, the end group may have a 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 later.

Hyperbranched polymer is generally a polymer in which branching is developed by polymerizing an AB2 type monomer. Here, A and B each indicate a combination of functional groups capable of polymerization reaction, and examples thereof include a combination of a hydroxyl group and a carboxyl group, an amino group and a carboxyl group, and the like. Alternatively, a substance that functions as a branching core may be used in combination. Further, a hyperbranched polymer may be prepared by ring-opening polymerization of glycidol or ethyleneimine. A dendrigraft is not a monomer, but a polymer synthesized by stepwise reacting an oligomer in which a plurality of monomers are bonded. These are collectively called random dendritic polymers. Random dendrimers, like dendrimers, have bifurcations, but cores are not required. In addition, the branched portion of the random dendritic polymer may be partially defective and have irregular or discontinuous portions.

The branching unit of the random dendritic polymer may be a polylysine skeleton, a polyglycerin skeleton, or a skeleton of various sugars. Examples of the random dendritic polymer in which the branching unit is lysine (-NH-CH (C ₄ H ₂ NH-) CO-) include PolyLysine-Dendri-graft (PLD: COLCOM). Examples of the random dendritic polymer in which the branching unit is polyglycerin represented by the following formula include PGL X and PGL 10 (Daicel Chemistry).

(In the formula, n and m are independently integers of 10 or more, preferably 20 or more).

Star polymer is known as a polymer in which three or more polymer chains are radially branched from the center. Star polymers are roughly classified into a regular type in which the polymer chains are the same type and an asymmetric type in which the types and molecular weights of the polymer chains are different. Regular star polymers can be obtained by living anionic polymerization of styrene or 1,3-diene living anion polymers with polyfunctional silyl chloride. Currently, polybutadiene star polymers having 3 to 128 polymer chains, star polystyrene having 33 polymer chains, and the like are being synthesized. Further, as the asymmetrical star polymer, a four-component star polymer composed of polystyrene, polyisoprene, polybutadiene and poly (4-1 methylstyrene) is known.

The star polymer may be of the multi-arm PEG type. Such star polymers are commercially available, for example, 4arm-PEG-NH ₂ (C (CH ₂ O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ NH), which is a multi-arm PEG type with four branched chains. ₂ HCl) ₄ ), 4arm-PEG-COOH (C (CH ₂ O (CH ₂ CH ₂ O) _n CH ₂ COOH) ₄ ) (Merck), etc. Pentaerythritol core 8arm-PEG-NH ₂ (R (O (CH ₂ CH ₂ O) _n CH ₂ CH ₂ NH ₂ HCl) ₈ , R is tripentaerythritol) and 8arm-PEG-COOH (R (O (CH 2CH)) ₂ CH ₂ O) _n CH ₂ COOH) ₈ and R are tripentaerythritol) (Merck). In addition, among the SUNBRIGHT (registered trademark) series, the PTE series, HGEO series and DX series (Yuka Sangyo Co., Ltd.) represented by the following formulas can also be mentioned.

(In the formula, X is -CO-CH ₂ CH ₂ -COO-N hydroxysuccinimide (NHS) _{, -CO-CH 2} CH ₂ CH ₂ -COO- _{NHS- (CH 2} ) ₃ -NHCO-CH ₂ CH ₂ -Maleimide, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -COO-NHS, -COO-p Nitrophenyl-NO ₂ , -CH ₂ CH ₂ CH ₂ NH ₂ or-(CH ₂ ) ₃ -NHCO-CH ₂ CH ₂ -maleimide, where n is an integer greater than or equal to 10)

(In the formula, X is -CO-CH ₂ CH ₂ -COO-NHS, -CO-CH ₂ CH ₂ CH ₂ -COO-NHS, -COO-p nitrophenyl, -CH ₂ CH ₂ NH ₂ · HCI or -CH ₂ CH ₂ SH, where n is an integer greater than or equal to 10)

(In the equation, X is -CH ₂ CH ₂ CH ₂ -NH ₂ , -CH ₂ CH ₂ -CHO or -CO-CH ₂ CH ₂ CH ₂ -COO-NHS, and n is an integer greater than or equal to 10. )

In this embodiment, it is preferable to use a dendrimer as the branched polymer. The number of generations of the dendrimer can be appropriately selected according to the structure of the core and the branch portion of the dendrimer to be used. 0th to 10th generation polyamide amine (PAMAM) dendrimers are generally available in the art. Further, the method for producing a dendrimer itself is known, and is described in, for example, the above-mentioned literature. In this embodiment, it is preferable to use a 4th generation (G4), 5th generation (G5) or 6th generation (G6) dendrimer.

In the carrier of the present embodiment, the terminal group of the branched polymer and the phenylalanine residue may be directly bonded or indirectly bonded. The number of phenylalanine residues attached to one terminal group of the branched polymer may be one or two or more. The phenylalanine residue may be either L-form or D-form. The phenylalanine residue may be attached to all the end groups of the branched polymer, or may be attached to some end groups of the branched polymer. In this embodiment, the phenylalanine residue is 40% or more, preferably 45% or more, preferably 45% or more, 50% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more of the terminal group contained in the branched polymer. , 85% or more, 90% or more, or 95% or more. For example, when the branched polymer is a G4 PAMAM 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 among the 64 terminal groups of the dendrimer. A phenylalanine residue binds to 55 or more, 58 or more, or 61 or more terminal groups.

When the terminal group of the branched polymer and the phenylalanine residue are directly bonded, the terminal group of the branched polymer may be reacted with the amino terminal or the carboxyl terminal of phenylalanine. Alternatively, the terminal group of the branched polymer may be reacted with a derivative of phenylalanine. In the present embodiment, the type of bond between the terminal group of phenylalanine or a derivative thereof and the terminal group of the branched polymer is not particularly limited. Preferred bonds are covalent bonds, such as amide bonds (-NH-CO-), ester bonds (-CO-O-), urethane bonds (-NH-CO-O-), urea bonds (-NH-CO-NH). -), Thiourea bond (-NH-CS-NH-), sulfonamide bond (-SO ₂ -NH-), sulfonic acid ester bond (-SO ₂ -O-) and the like. Further, the structure may be formed by the reaction of maleimide and thiol. Examples of the derivative of phenylalanine include a compound in which the amino-terminal or carboxyl-terminal of phenylalanine is derivatized with a group having reactivity with the terminal group of the branched polymer.

In one embodiment, a carrier for immune cell transfer 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 consists of a phenylalanine residue directly bonded to the terminal group of the branched polymer, and the carboxyl group of the phenylalanine residue becomes 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 (for example, 4-nitrophenyl carbonate group or carboxyl group). 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 (for example, a carboxyl group). When the amino terminus of phenylalanine is reacted with the terminus of the branched polymer, the carboxyl terminus of the phenylalanine may be protected with a known protecting group in advance.

In the carrier for immune cell transfer of the further embodiment, when the terminal group of the branched polymer and the phenylalanine residue are directly bonded, even if a compound capable of imparting an anionic terminal group is further bonded to the phenylalanine residue. Good. For example, when the terminal group of the branched polymer is reacted with the carboxyl terminal of phenylalanine, the amino terminal of phenylalanine in the complex molecule is anionic in order to make the terminal structure of the obtained complex molecule anionic. It is preferable to react with a compound capable of imparting a terminal group. As such a compound, one end of the compound has a functional group capable of reacting with an amino group, and the other end has an anionic group (for example, a carboxyl group, a sulfone group, etc. Examples thereof include compounds having (sulfuric acid groups, etc.) and intramolecular condensates thereof. For example, succinic anhydride, 1,2-cyclohexanedicarboxylic acid anhydride, phthalic anhydride and other dicarboxylic acid anhydrides are preferred. Alternatively, the compound capable of imparting an anionic terminal group may be a cyclic compound that opens by reacting with an amino group to form an anionic terminal group. Such compounds include, for example, cyclic sulfonic acid esters such as 1,3-propane sultone.

In the carrier of the present embodiment, the terminal group of the branched polymer and the phenylalanine residue may be bonded via a linker. The linker can be appropriately selected from a hydrophobic compound having a functional group capable of reacting with the terminal group of the branched polymer at one end and a functional group capable of reacting with phenylalanine or a derivative thereof at the other end. For example, a branched polymer having an amino group or a hydroxyl group as a terminal group and an amino group of phenylalanine are bonded as a linker via the above-mentioned anhydride of dicarboxylic acid. Examples of such a linker include succinic anhydride, 1,2-cyclohexanedicarboxylic acid anhydride, phthalic anhydride and the like. To briefly describe the synthesis scheme in this case, first, the amino group or hydroxyl group of the branched polymer is reacted with the anhydride of the dicarboxylic acid to obtain a branched polymer having a carboxyl group as the terminal group. Then, the carboxyl group of the obtained branched polymer is reacted with the amino terminal of phenylalanine whose carboxyl terminal is protected by a protecting group to obtain a branched polymer having a phenylalanine residue bonded to the terminal. Finally, the protecting group for the phenylalanine residue can be removed to obtain the carrier of this embodiment. Such a synthetic scheme itself is known and is described, for example, in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151. Further, a cross-linking agent capable of binding the terminal group of the branched polymer and the amino terminal of phenylalanine can also be used. For example, when the terminal group of the branched polymer is an amino group, a compound having two isocyanate groups or isothiocyanate groups may be used. When the terminal 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 terminal 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 terminal 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.

In a preferred embodiment, the anionic terminal structure of the carrier for immune cell transfer comprises a phenylalanine residue attached to the terminal group of the branched polymer and is represented by the following formula (I).

(In the equation, 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, a heterocycloalkylene group or a phenylene group having 3 to 8 carbon atoms which may have a substituent. Yes,
Z ₁ is a carboxyl group, a sulfone group, a sulfate group or a salt thereof)

The terminal structure represented by the formula (I) corresponds to a compound in which the terminal group of the branched polymer and the phenylalanine residue are directly bonded and a compound capable of imparting an anionic terminal group to the phenylalanine residue is further bonded. In formula (I), X ₁ indicates the binding site between the terminal group of the branched polymer and the phenylalanine residue, and _{the left binding site of X 1} is bound to the branched polymer. In formula (I), Y ₁ , R and Z ₁ correspond to compounds capable of imparting anionic end groups attached to phenylalanine residues. Y ₁ indicates the binding site between the terminal group of the phenylalanine residue and the compound capable of imparting an anionic terminal group. Z ₁ is the end group of the anionic end structure. In Z ₁ , the type of salt is not particularly limited, but is preferably an alkali metal salt.

When R is an alkylene group having 1 to 10 carbon atoms, such alkylene groups include, for example, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, neopentylene, hexylene, heptylene, octylene and 2-ethyl. Groups such as hexylene, nonylene and decylene can be mentioned. Among them, an alkylene group having 1 to 4 carbon atoms is preferable. When R is an alkylene group having a substituent, the above carbon number does not include the carbon number of the substituent.

When R is a cycloalkylene group or a heterocycloalkylene group, such groups may contain one or more heteroatoms selected from N, S, O and P and have 3 to 8 carbon atoms. It may be a non-aromatic ring. Examples thereof include groups such as cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptyrene, cyclooctylene, pyrrolidinylene, piperidinylene and morpholinylene. When R is a cycloalkylene group having a substituent, a heterocycloalkylene group or a phenylene group, the above carbon number does not include the carbon number of the substituent.

Examples of the substituent in R include a linear or branched saturated aliphatic hydrocarbon group having 1 or more and 6 or less carbon atoms, preferably 1 or more and 3 or less carbon atoms.

In a further embodiment, the anionic end structure of the immune cell translocation carrier comprises a phenylalanine residue attached to the end group of the branched polymer and is represented by the following formula (II).

(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 maleimide and thiol It is a structure obtained by the reaction,
R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, a heterocycloalkylene group or a phenylene group having 3 to 8 carbon atoms which may have a substituent. Yes,
Y ₂ is-(C = O) -NH-, -O- (C = O) -NH-, -NH- (C = O) -NH-, -NH- (C = S) -NH- Yes,
Z ₂ is a carboxyl group or a salt thereof)

The terminal structure represented by the formula (II) is such that the terminal group of the branched polymer and the phenylalanine residue are bonded via a linker, and the carboxyl group of the phenylalanine residue or a salt thereof forms an anionic terminal group. Equivalent to. X ₂ indicates the binding site between the end group of the branched polymer and the linker, and _{the left binding site of X 2} is bound to the branched polymer. In formula (II), R corresponds to the linker moiety intervening 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), Y ₂ indicates the binding site between the linker and the amino group of the phenylalanine residue. Z ₂ is the end group of the anionic end structure.

In a further embodiment, the anionic terminal structure of the immune cell transfer carrier contains a phenylalanine residue attached to the terminal group of the branched polymer and is represented by the following formula (III).

(In the formula, Z ₃ is a carboxyl group or a salt thereof)

The terminal structure represented by the formula (III) corresponds to one in which the amino group of the phenylalanine residue is directly bonded to the terminal group of the branched polymer, and the carboxyl group of the phenylalanine residue or a salt thereof forms an anionic terminal group. To do. In formula (III), the leftmost bond is attached to the branched polymer. Z ₃ is the end group of the anionic end structure.

In the present embodiment, as the terminal structure represented by the above formula (I), the structures represented by the following formulas (1) to (4) are particularly preferable. Further, as the terminal structure represented by the above formula (II), the structures represented by the following formulas (5) to (7) are particularly preferable. As the terminal structure represented by the above formula (III), the structure represented by the following formula (8) is particularly preferable. 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 sodium ion, potassium ion, lithium ion and the like. Among them, sodium ion is preferable.

The particle size of the carrier of the present embodiment is not particularly limited, but is usually 5 nm or more and 20 nm or less, preferably 5 nm or more and 15 nm or less. 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 by the dynamic light scattering (DLS) method by the Marquardt method. The measuring device is ELSZ-DN2 (manufactured by Otsuka Electronics Co., Ltd.) or its equivalent.

In a further embodiment, the immune cell transfer carrier may further comprise a substance of interest. That is, the carrier of the present embodiment contains a complex molecule carrying a substance of interest. In this case, the substance of interest may be linked to the terminal 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 terminal group or phenylalanine residue of the branched polymer in the complex molecule, the mode of linking is not particularly limited. The concatenation may be, for example, a covalent bond or a non-covalent bond. For example, the substance of interest may be attached directly or indirectly to the terminal group or phenylalanine residue of the branched polymer. When the branched polymer is a dendrimer, the method itself for encapsulating the substance of interest in the internal space of the dendrimer is known, and is described in, for example, Kojima C. et al., Bioconjuge Chem 11: 910-7 (2000). Further, it may be utilized by imparting the carrier of the present embodiment to another carrier (for example, liposome, micelle, polymer micelle, etc.) carrying the substance of interest. For example, it may be a mixture of another carrier carrying the substance of interest and the carrier of the present embodiment, or the carrier of the present embodiment may be modified on the surface of the other carrier via a chemical bond or a physical bond. May be good.

Examples of the substance of interest include labeling substances, nucleic acids, proteins, peptides and the like. Examples of labeling substances include PET compounds containing radioisotopes such as ¹¹ C, ¹³ N, ¹⁵ O, and ^{18 F, SPECT compounds containing radioisotopes such as 111} In, indocyanine green, and Patent Blue V. Examples thereof include low molecular weight organic dyes, fluorescent dyes such as indocyanine green, fluorescein, rhodamine, Alexa Fluor (trademark) and HiLyte (trademark) Fluor, and contrast agents for nuclear magnetic resonance imaging such as gadolinium chelating agents. Examples of the nucleic acid include a vector incorporating a gene encoding a desired protein, an antisense oligonucleotide, siRNA, shRNA, a ribozyme, an aptamer, a decoy nucleic acid and the like. Examples of proteins and peptides include peptides and proteins containing self-antigens and non-self-antigens. Specifically, cancer preventive vaccines targeting cancer-specific antigens (common cancer antigens, neoantigens), application to optimal cancer immunotherapy for individual patients, and targeting allergic antigens that cause autoimmune diseases The application to immune tolerance can be mentioned.

As described above, the carrier of the present embodiment is characterized in that it migrates into immune cells, particularly immune cells in lymph nodes. The type of immune cell is not particularly limited, and examples thereof include T cells, B cells, dendritic cells, and macrophages. When the carrier of the present embodiment is administered to a mammal, it can reach the lymph node and migrate into the immune cells in the lymph node. In addition, the carrier of the present embodiment can be transferred into the immune cells by contacting the immune cells in Exvivo or in vitro. As shown in Examples described later, the carrier of this embodiment is considered to migrate into immune cells by cell endocytosis rather than by membrane permeation.

As shown in Examples described later, 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 the present embodiment can be suitably used for transferring a substance of interest to T cells.

As shown in Examples described later, the carrier of this embodiment has improved transferability to immune cells, particularly T cells, under acidic conditions. As the acidic condition, the pH of the environment surrounding the immune cell is 5 or more and 7 or less. For example, in the case of in vitro or exvivo, the pH of the liquid in contact with cells such as a medium may be set to an acidic condition. In vivo, it is known that pH is an acidic condition at an inflamed site (eg, cancerous tissue). Here, as described in Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151, the terminal group of the 4th generation dendrimer is via an ethylene group, a cyclohexylene group or a phenylene group. It is known that the complex molecule formed by binding phenylalanine residues undergoes a phase transition under acidic pH conditions to become more hydrophobic. It is possible that the property of phase transition in response to this pH is involved in the improvement of the transferability to immune cells under acidic conditions.

When the carrier of the present embodiment is administered to a subject (for example, a mammal) in vivo, parenteral administration is preferable, and administration by injection is particularly preferable. The administration by injection may be any of intradermal injection, subcutaneous injection, intravenous injection, arterial injection, intramuscular injection, and intraperitoneal injection, but is preferably intradermal injection, subcutaneous injection, and more preferably intradermal injection. .. When the carrier of the present embodiment is administered to a subject (for example, cells) in vitro or exvivo, it is preferable to bring the subject and the carrier into contact with each other by adding an appropriate amount of the carrier of the present embodiment to the culture solution.

One embodiment of the present invention is a pharmaceutical composition containing a drug and a carrier for immune cell transfer. A drug is a substance used as an active ingredient or a candidate thereof. In a preferred embodiment, the drug is a substance that can be supported by the carrier of this embodiment. That is, in the pharmaceutical composition of the present embodiment, it is preferable that the pharmaceutical is supported on a carrier for immune cell transfer. Examples of the medicine include pharmaceutical compounds, proteins, peptides, nucleic acids and the like. The pharmaceutical compound can be selected from, for example, an active ingredient or a known compound used as a candidate thereof. The protein, peptide and nucleic acid can be selected from known antibody drugs, peptide drugs and nucleic acid drugs, respectively. As the form of the pharmaceutical composition, an injection containing the drug and the carrier of the present embodiment is preferable. The injection can be prepared by adding the carrier of the present embodiment to purified water for injection or physiological saline. The content of the carrier for immune cell transfer is, for example, 0.001 to 80 parts by weight, preferably 0.01 to 50 parts by weight, based on 100 parts by weight of the injection. The injection may further contain pharmaceutically acceptable additives. Such additives themselves are known and include, for example, preservatives, stabilizers, pH regulators, tonicity agents, solvents, solubilizers, soothing agents and the like.

One embodiment of the present invention is an immune cell transfer reagent containing the above-mentioned immune cell transfer carrier. The reagents of this embodiment are useful, for example, when transferring a substance of interest to immune cells. By supporting the substance of interest on the carrier for transfer of immune cells in the reagent of the present embodiment and contacting the substance with the immune cells, the substance of interest can be transferred to the immune cells. The reagent of the present embodiment may contain an immune cell transfer carrier in a powder state (for example, a lyophilized state), or may include an immune cell transfer carrier in a state of being dispersed in a suitable solvent. In the reagent of the present embodiment, the carrier for immune cell transfer may be contained in a container. Further, in the reagent of the present embodiment, the container containing the carrier for immune cell transfer 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 transfer, the method of use, and the like.

One embodiment of the present invention transfers an immune cell transfer carrier to an immune cell, comprising contacting the immune cell transfer carrier in vivo (excluding humans), in vitro or ex vivo with the immune cell transfer carrier described above. How to do it. Details of immune cells are as described above. The method of contact between the immune cell transfer carrier and the immune cell is not particularly limited. For example, in the case of in vivo, the immune cell transfer carrier and the immune cell can be obtained by administering the immune cell transfer carrier to an animal other than human. Can be contacted. The details of administration are as described above. In the case of in vitro, the immune cell transfer carrier and the immune cell can be brought into contact with each other by adding the immune cell transfer carrier to the immune cells in the medium. In the case of Exvivo, immune cells are collected by collecting organs containing immune cells (for example, spleen, thymus, lymph nodes, etc.) from animals and perfusing the obtained organs with a liquid containing, for example, a carrier for immune cell transfer. The transfer carrier can be contacted with immune cells. When the carrier for transfer of immune cells carries a substance of interest, the transfer method of the present embodiment allows the substance of interest to be transferred to immune cells in vitro or exvivo via the carrier for transfer of immune cells.

One embodiment of the present invention is the use of a complex molecule for the production of a carrier for immune cell translocation, wherein the complex molecule comprises a branched polymer and a phenylalanine residue and has an anionic terminal structure. The use, wherein the phenylalanine residue is attached to the terminal group of the branched polymer. Details of the immune cell transfer carrier and complex molecule are as described above.

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

Production Example 1: Preparation of carrier for immune cell transfer
(1) Preparation of carboxy-terminal dendrimer The carboxy-terminal dendrimer was 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 an amino-terminal G4 PAMAM dendrimer (Sigma-Aldrich) was distilled off under reduced pressure and freeze-dried. Dendrimer (56 mg, 3.9 μmol) _{was dissolved in 125 mM LVDS 3} buffer (5.5 mL) and an excess amount of succinic anhydride or cis-1,2-cyclohexanedicarboxylic acid anhydride (100 equivalents each) was added. .. Each solution was stirred at room temperature overnight and the pH was adjusted to 8-10 with an aqueous 4N NaOH solution. A solution containing a carboxy-terminated dendrimer was purified by dialysis in distilled water using a dialysis membrane (molecular weight cutoff (MWCO): 1,000). The purified solution was lyophilized to give a white powder of each carboxy-terminated dendrimer. The yield of dendrimer (G4-Suc) with succinic acid added to the end was 95 mg, and the yield was 111%. The yield of dendrimer (G4-CHex) with cyclohexanedicarboxylic acid added to the end was 62 mg, and the yield was 64%. ^{The results of 1} H-NMR spectrum measurement of each solution of G4-Suc and G4-CHex at room temperature using 400 MHz JNM-LA 400 JNM-ECX (manufactured by JEOL) are shown below.

G4-Suc (NaOD-containing D2O): 2.24-2.28 (br, NCH2C H2 CONHCH2CH2N in the inner and terminal chains of PAMAM and methylene succinate), 2.46 (br, NCH2CH2CONHCH2C H2 N in the inner chain of PAMAM), 2.65 ( br, NC H2 CH2 CH2CONHCH2CH2N in the inner and terminal strands of PAMAM) and 3.13 (br, NCH2CH2CONHC H2 CH2N in the inner and terminal strands of PAMAM, and NCH2CH2CONHCH2C H2 N in the terminal strand of PAMAM).

G4-CHex (NaOD-containing D2O): 1.21-1.83 (m, methylene of cyclohexanedicarboxylic acid), 2.26 (br, NCH2C H2 CONHCH2CH2N of PAMAM), 2.41-2.55 (br, inner chain of PAMAM and methine of cyclohexanedicarboxylic acid) NCH2CH2CONHCH2C H2 N), 2.66 (br, NC H2 CH2CONHCH2CH2N of the inner and terminal chains of PAMAM) and 3.14 (br, NCH2CH2CONHC H2 CH2N of the inner and terminal chains of PAMAM, and NCH2CH2CONHCH2C H2 N of the terminal chains of PAMAM) ..

(2) Preparation of dendrimer with phenylalanine residue added to the end
(2.1) Addition of phenylalanine residue to G4-Suc The carboxy terminus of G4-Suc was reacted with the amino terminus of the phenylalanine residue that protected the carboxy terminus. The specific procedure was as follows. G4-Suc (83 mg, 3.8 μmol) was dispersed in DMSO (5 mL) and stirred overnight. Then, in a solution of G4-Suc, 3-phenyl-L-alanine benzyl ester 4-toluene sulfonate (Phe-OBzl · Tos) (0.12 g, 0.30 mmol), 1- [bis (dimethylamino) methylene] -1H-benzo Triazole-3-oxide hexafluorophosphate (HBTU) (0.11 g, 0.28 mmol) and triethylamine (TEA) (41 μL, 0.29 mmol) were added 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 cutoff (MWCO): 1,000). Methanol was removed from the purified solution under reduced pressure and lyophilized to obtain a powder of G4-Suc (G4-Suc-Phe-OBzl) having Phe-OBzl added to the end. The yield was 88 mg and the yield was 66%. ^{The results of 1} H-NMR spectrum measurement of the solution of G4-Suc-Phe-OBzl are shown below.

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

(2.2) Addition of 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 phenylalanine residue that protected the carboxy terminus. .. Specifically, in DMSO (5 mL), G4-CHex (53 mg, 2.11 μmol), Phe-OBzl · Tos (0.12 g, 0.30 mmol), HBTU (0.09 g, 0.24 mmol) and TEA (41 μL, By reacting with 0.29 mmol), G4-CHex (G4-CHex-Phe-OBzl) to which Phe-OBzl was added to the end was obtained. Distilled water (1 mL) was added thereto, and the obtained 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 lyophilized to obtain a powder of G4-CHex-Phe-OBzl. The yield was 84 mg and the yield was 109%. ^{The results of 1} H-NMR spectrum measurement of the solution of G4-CHex-Phe-OBzl are shown below.

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 H2 CONHCH2CH2N of inner and terminal chains of PAMAM and NCH2CH2CONHCH2C H2 N of the inner chain PAMAM and Hβ of Phe), 3.44-3.58 (br, NC H2 CH2CONHCH2CH2N strands of the inner and end of PAMAM, and the inner and end of the chain of PAMAM NCH2CH2CONHC H2 CH2N, and NCH2CH2CONHCH2C H2 N in the terminal chain of PAMAM, and methine in CHex), 4.56-4.65 (br, Phe Hα), 5.00 (br, Phe benzyl), 7.15-7.28 (m, Phe and Tosyl phenyl), and 7.67-7.69 (m, Tosyl phenyl).

(2.3) Deprotection of phenylalanine residue at the terminal of 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), respectively. , 4M NaOH methanol solution (500 μL) was added. After stirring at 4 ° C. for 2 hours, the dispersion of each dendrimer was purified by dialyzing in distilled water using a dialysis membrane (molecular weight cutoff (MWCO): 1,000). The purified solution was lyophilized to give 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%.

Here, G4 PAMAM is a dendrimer having 64 end groups. ^{As a result of 1} H-NMR spectrum measurement of each solution of G4-Suc-Phe and G4-CHex-Phe, the number of bonds of acid anhydride (Suc or CHex) to the terminal group and the phenylalanine residue to the acid anhydride The number of bonds was as shown in Table 1. Further, FIG. 1 shows a schematic diagram showing the chemical structure of one of the 64 terminal groups of each of the G4-Suc and G4-CHex, G4-Suc-Phe and G4-CHex-Phe dendrimers. In FIG. 1, "den" indicates G4 PAMAM.

From Table 1, it was shown that G4-Suc-Phe and G4-CHex-Phe, which are dendrimers having a phenylalanine residue added to the end, were obtained as carriers for immune cell transfer. In addition, in G4-Suc-Phe and G4-CHex-Phe, it was shown that the terminal of the dendrimer and the phenylalanine residue were bound via an ethylene group and a cyclohexylene group as linkers, respectively.

Example 1: Imaging of a carrier for immune cell transfer to which a fluorescent dye is linked
(1) Preparation of carrier for immune cell transfer to which a fluorescent dye is linked
(1.1) Preparation of G5-CHex-Phe and G5-Suc In the same manner as in the above production example, succinic acid is added to the end except that G5 PAMAM dendrimer (Sigma-Aldrich) is used instead of G4 PAMAM dendrimer. An added dendrimer (G5-Suc) and a dendrimer (G5-CHex-Phe) having a phenylalanine residue added to the end using a cyclohexylene group as a linker were prepared.

(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 Kasei Kogyo Co., Ltd., 0.3 μL, 1.88 μmol) and 4- (4,6-dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (DMT-MM) ( Sigma-Aldrich, 1.23 mg, 3.37 μmol) was dissolved in pure water (2.5 mL) and reacted by stirring overnight at room temperature. The reaction mixture was purified by ultrafiltration (MWCO: 3000, 10 ° C., 7500 g, 125 mM LVDS ₃ aqueous solution). The obtained solution was lyophilized to give a white powder (G5-CHex-Phe-Boc). The yield was 23.42 mg and the yield 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. Then, the solvent was distilled off under reduced pressure, pure water was added, and the distillation under reduced pressure was carried out a plurality of times to wash the product. The product was vacuum dried overnight and then lyophilized to give white crystals of G5-CHex-Phe end-added with an amino group. The yield was 34.48 mg and the yield was 166%. As for G5-Suc, G5-Suc having an amino group added to the terminal was obtained in the same manner as above.

(1.3) Binding of Fluorescent Dye to G5-CHex-Phe and G5-Suc G5-CHex-Phe (20 mg, 0.29 μmol) with an amino group added to the end was added to 125 mM acrylamide ₃ aqueous solution (1 mL, pH 8.3). Dissolved. To this, add 4 equivalents of DMSO solution (1 mL) of green fluorescent dye HiLyte ™ Fluor 488 acid SE (Anaspec, 0.41 mg, 0.587 μmol) to the dendrimer, and stir at room temperature for 24 hours in a light-shielded state. did. Then, it was purified by ultrafiltration (MWCO: 3000, 4 ° C., 7500 g, 125 mM LVDS _{3 aqueous solution).} The obtained solution was freeze-dried to obtain crystals of G5-CHex-Phe having a fluorescent dye linked to the end. The yield was 9.24 mg and the yield was 45%. As for G5-Suc having an amino group added to the terminal, G5-Suc having a fluorescent dye linked to the terminal was obtained in the same manner as described above.

(1.4) NMR measurement and ultraviolet-visible absorption (UV-vis) measurement Each dendrimer (about 4 mg) linked with a fluorescent dye _{was dissolved in D 2} O (30 μL) and NaOD (20 μL), and 400 MHz JNM-LA 400 JNM- ¹ H NMR and ³¹ P NMR measurements of the compound were performed at room temperature using ECX (manufactured by JEOL Ltd.). The number of fluorescent dyes bound to one molecule of dendrimer was identified using a V-630 ultraviolet-visible spectrophotometer (manufactured by JASCO). An aqueous solution (1 mL) of each dendrimer concatenated with a fluorescent dye was taken in a measurement cell, and the absorption spectrum (350-650 nm, 25 ° C.) was measured. Here, G5 PAMAM is a dendrimer having 128 end groups. From the results of NMR measurement, in G5-CHex-Phe, CHex was bound to 126 end groups out of 128 end groups, and Phe was further bound to 111 of these end groups. In G5-Suc, Suc was bound to 128 of the 128 end groups. From the results of UV-vis measurement, two molecules of fluorescent dye are bound to one molecule of G5-CHex-Phe, and one or three molecules of fluorescent dye are bound to one molecule of G5-Suc. It was. Hereinafter, G5-CHex-Phe in which two molecules of fluorescent dye are linked to the end is referred to as "G5-CHex-Phe (dye2)", and G5-Suc in which one molecule and three molecules of fluorescent dye are linked to the terminal is referred to as "G5-Suc", respectively. They are called "G5-Suc (dye1)" and "G5-Suc (dye3)".

(2) Imaging of a carrier for immune cell transfer to which a fluorescent dye is linked
(2.1) Animals All animal experiments were conducted using BALB / cCrSlc mice (8-10 weeks old, female, 18-21 g, Nippon SLC Co., Ltd.) in compliance with the university regulations regarding animal experiments. ..

(2.2) Translocation to immune cells in lymph nodes G5-CHex-Phe (dye2), G5-Suc (dye1) or G5-Suc (dye3) in saline so that the pigment concentration is 10 nmol / 100 μL. To prepare a dispersion of each dendrimer. A dispersion of each dendrimer (20 μL in the hand and 30 μL in the foot) was intradermally administered to the insteps of both hands and feet of the mice. Immediately after administration, the administration site was massaged for 30 seconds. Mice were euthanized after 3 and 24 hours and axillary lymph nodes and below-knee lymph nodes were collected. As a control, lymph nodes were collected from mice not receiving dendrimers in the same manner. The collected lymph nodes were immersed in a 24-well plate filled with 1 mL serum-free RPMI under ice-cooling. The RPMI in the well was removed, the lymph nodes were separated with scissors, 1 mg / mL collagenase solution (1 mL) was added, and the mixture was allowed to stand at 37 ° C. for 30 minutes. Then, the lymph node tissue was ground on a cell strainer to obtain cells. The cell-containing solution was centrifuged (4000 rpm, 5 minutes) to remove the supernatant, and the cells were dispersed by adding serum-free RPMI (5 mL). Centrifuge the cell-containing solution (4000 rpm, 5 minutes) to remove the supernatant _{, add stain buffer (1% FBS, 0.09% NaN 3} , 2 mM EDTA and PBS) (0.5 mL) to make a cell suspension. Obtained. Stain buffer was added to the cell suspension so that the number of cells was 2 × 10 ⁷ cells / mL, and 50 μL was dispensed into a 1.5 mL tube. The cell suspension was centrifuged (9200 rpm, 5 minutes) to remove the supernatant. A solution of anti-CD3 antibody, anti-CD45R antibody, anti-CD11c antibody and anti-F4 / 80 antibody (50 μL each) labeled with the yellow fluorescent dye phycoerythrin (PE) was added thereto, and the mixture was cooled on ice for 30 minutes. It was left in the dark. 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 none of the labeled antibodies were added were also prepared. Then, stain buffer (100 μL) was added, and the cells were washed by centrifugation (9200 rpm, 5 minutes, 4 ° C.) to remove the supernatant. Stain buffer (1 mL) was added to the cells reacted with each labeled antibody to obtain a sample for flow cytometry (FCM) analysis. Each sample was analyzed using BD FACS ™ Calibur (manufactured by BD Biosciences). Based on the analysis results of 10,000 cells in each sample, the vertical axis shows the number of cells, the horizontal axis shows the intensity of green fluorescence (530 nm) (see FIG. 2A), and the vertical axis shows yellow fluorescence (see FIG. 2A). A scattergram (see FIG. 2B) was prepared with an intensity of 585 nm) and a green fluorescence (530 nm) intensity on the horizontal axis. In the analysis, in order to remove dead cells and impurities, a gate for measuring only live lymph node cells was determined from the distribution map of cell size and shape measured by forward scattering and side scattering.

From FIG. 2A, G5-Suc (dye1) or G5-Suc (dye3) shows the same distribution as the control without dendrimer administration, and it can hardly be transferred to the cells in the lymph node. It was suggested. On the other hand, in G5-CHex-Phe (dye2), many cells showing a high green fluorescence intensity were detected as compared with the control. Therefore, it was suggested that G5-CHex-Phe (dye2) was transferred to the cells in the lymph nodes.

As shown in the control scattergram of FIG. 2B, most of the immune cells in the collected lymph nodes were T cells and B cells, and there were relatively few dendritic cells and macrophages. G5-Suc (dye3) was used because few cells showed high green fluorescence intensity, as shown in the scattergram of G5-Suc (dye3) without PE-labeled antibody (No staining). It has been shown that it hardly migrates to cells in the lymph nodes. This is also consistent with FIG. 2A. In addition, for G5-Suc (dye3), few immune cells showed high values in both green fluorescence intensity and yellow fluorescence intensity, as shown in the scattergram to which each PE-labeled antibody was added. This suggests that G5-Suc (dye3) is not well taken up by T cells, B cells, dendritic cells and macrophages. On the other hand, for G5-CHex-Phe (dye2), in the scattergram to which each PE-labeled antibody was added, immune cells showing higher values in both green fluorescence intensity and yellow fluorescence intensity than G5-Suc (dye3). Many were detected. Based on FIG. 2B, among immune cells showing high yellow fluorescence intensity (immune cells detected with PE-labeled antibody), immune cells showing high green fluorescence intensity (cells incorporating a carrier for transfer to immune cells). A graph (Fig. 3) showing the ratio of In FIG. 3, "T" represents a T cell, "B" represents a B cell, "DC" represents a dendritic cell, and "MΦ" represents a macrophage. As shown in FIG. 3, G5-CHex-Phe (dye2) successfully translocated to all T cells, B cells, dendritic cells and macrophages.

(2.3) In vivo fluorescence imaging G5-CHex-Phe (dye2) or G5-Suc (dye3) was added to physiological saline so that the dye concentration was 10 nmol / 100 μL to prepare a dispersion of dendrimer. A dispersion of each dendrimer (30 μL) was administered to the paws of the mice. Immediately after administration, the administration site was massaged for 30 seconds. Mice were euthanized and epidermis stripped 4 and 24 hours after dosing. The limbs were fixed and in vivo fluorescence imaging was performed using a fluorescence microscope FluoVivo (manufactured by INDEC Biosystems). The results are shown in FIG.

In FIG. 4, the part surrounded by an ellipse indicates the administration site of each dendrimer, the part indicated by the arrow indicates the lymph node, and the part indicated by the arrowhead indicates the lymphatic vessel. Lymph nodes could be detected in mice 4 hours after administration of G5-CHex-Phe (dye2) and G5-Suc (dye3). In addition, in mice 4 hours after administration of G5-CHex-Phe (dye2), lymphatic vessels connecting the primary and secondary lymph nodes could be confirmed. However, 24 hours after administration, lymph nodes could not be detected in mice administered with G5-Suc (dye3). On the other hand, lymph nodes could be detected in mice treated with G5-CHex-Phe (dye2). Here, all the images shown in FIG. 4 were taken at the same exposure time, but G5-CHex-Phe (dye2) clearly emitted stronger fluorescence than G5-Suc (dye3). Understand. This suggests that the amount of lymph node accumulation in G5-CHex-Phe (dye2) is higher than that in G5-Suc (dye3).

Example 2: The pharmacokinetics of a carrier for immune cell transfer to which a radioactive substance is linked
(1) Preparation of carrier for immune cell transfer to which radioactive substances are linked
(1.1) Preparation of Carrier for Immune Cell Translocation Linked with Chelating Agent In Example 2, G5-Suc and G5-CHex-Phe prepared in Example 1 having an amino group added to the end were used. G5-CHex-Phe (12.16 mg, 0.212 μmol) end-added with an amino group _{was dissolved in a 125 mM acrylamide 3} aqueous solution (900 μL, pH 8.3). To this, 7 equivalents of 7 equivalents of the chelating agent p-SCN-Bn-DTPA (0.967 mg, 1.49 μmol) in 125 mM LVDS ₃ aqueous solution (900 μL) was added to the dendrimer, and the mixture was stirred at 37 ° C. for 24 hours. Then, it was purified by ultrafiltration (MWCO: 3000, 4 ° C., 7500 g, 125 mM LVDS _{3 aqueous solution).} The obtained solution was freeze-dried to obtain crystals of G5-CHex-Phe having a fluorescent dye linked to the end. The yield was 5.27 mg and the yield was 35%. As for G5-Suc having an amino group added to the terminal, G5-Suc having a chelating agent linked to the terminal was obtained in the same manner as described above. In the same manner as in Example 1, each dendrimer to which the radioactive substance was linked was measured by UV-vis, and the number of bonds of the chelating agent to one molecule of the dendrimer was measured using a V-630 ultraviolet-visible spectrophotometer (manufactured by JASCO Corporation). Identified. From the results of UV-vis measurement, two molecules of chelating agent were bound to both one molecule of G5-CHex-Phe and G5-Suc.

(1.2) Association of chelating agent and radioactive substance G5-CHex-Phe (20 nmol) imparted with an amino group at the end was dissolved in a 0.3 M aqueous ammonium acetate solution (200 μL). ¹¹¹ InCl ₃ (200 μL, ~ 0.18 mCi) was added thereto, and the mixture was stirred at room temperature for 1 hour. Then, it was purified by PD-10 column (PBS) and ultrafiltered (MWCO: 3000). The obtained solution was diluted with physiological saline so that the concentration of the radioactive substance was 10 μCi / 100 μL to obtain a solution of G5-CHex-Phe ^{in which the radioactive substance (111 In) was ligated to the end.} As for G5-Suc having an amino group added to the terminal, G5-Suc having a radioactive substance linked to the terminal was obtained in the same manner as above.

(2) Dynamics analysis of carrier for immune cell transfer Animal experiments are conducted using BALB / cCrSlc mice (8-10 weeks old, female, 18-21 g, Nippon SLC Co., Ltd.). I went in compliance with. A dispersion (30 μL) of each dendrimer linked with a radioactive substance was intradermally administered to the paws of mice. Immediately after administration, the administration site was massaged for 30 seconds. Mice were euthanized 3 and 24 hours after administration, and the site of administration, below-knee lymph nodes, blood, heart, lungs, liver, spleen and kidneys were collected and weighed. The limbs were fixed, and the radioactivity of organs collected from mice was measured using an autowell gamma counter (manufactured by PerkinElmer). The results are shown in FIG. As can be seen from FIG. 5, the mice administered with G5-CHex-Phe had significantly higher radioactivity in the lymph nodes than the mice administered with G5-Suc. That is, it was suggested that G5-CHex-Phe was remarkably accumulated in the lymph nodes as compared with G5-Suc. In addition, since radiation was detected in the liver and kidneys of mice to which G5-CHex-Phe was administered, part of the administered G5-CHex-Phe could reach blood vessels from lymph nodes and circulate throughout the body. It was suggested.

Example 3: Imaging of a carrier for immune cell transfer to which a fluorescent dye is linked (2)
(1) Preparation of carrier for immune cell transfer to which a fluorescent dye is linked In Example 3, G4-Suc-Phe and G4-CHex-Phe prepared in Production Example 1 were used as carriers for immune cell transfer. 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 are each reacted with 4 equivalents of Boc-ethylenediamine and 6 equivalents of DMT-MM. Boc groups were removed by an amount of TFA to add amino groups to the ends of each dendrimer. As a result of NMR measurement of these, 4 to 5 molecules of ethylenediamine were bonded to the end of each dendrimer of 1 molecule. Each dendrimer with an amino group attached to the end was dissolved in DMSO (1 mL). To this, 4 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. Then, it was purified by ultrafiltration (MWCO: 2000, DMSO, methanol). The obtained solution was freeze-dried to obtain crystals of G4-Suc-Phe, G4-CHex-Phe, G4-Suc and G4-CHex having a fluorescent dye linked to the end. As a result of UV-vis measurement of these, one or two molecules of FITC were bound to the end of each dendrimer of one molecule.

(2) Transfer to immune cells in the spleen in vitro
(2.1) Collection of spleen cells and contact with a carrier for immune cell transfer In animal experiments, BALB / c Slc-nu / nu nude mice (female, with MDA-MB-231 cancer tumor, Japan SLC Co., Ltd.) ) Was used to comply with the university's regulations regarding animal experiments. Mice were euthanized and the spleen was harvested. The collected spleen was immersed in serum-free RPMI under ice-cooling and separated with scissors. A 1 mg / mL collagenase solution (1 mL) was added to the divided spleen tissue, and the mixture was allowed to stand at 37 ° C. for 30 minutes. Then, the spleen tissue was ground on a cell strainer to obtain cells. The cell-containing solution was centrifuged (4000 rpm, 5 minutes) to remove the supernatant, and the cells were dispersed by adding serum-free RPMI (5 mL). The cell-containing solution 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 seeded on a 96-well plate at 1 × 10 ⁴ cells / well. Each dendrimer linked with FITC was added to serum-free RPMI to 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 37 ° C for 1 or 4 hours.

(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 10 times) and analyzed with imaging software CellScens Standard (Olympus Corporation). The results are shown in Table 2. In the table, "x" indicates that no cells emitting green fluorescence were detected, and "○" indicates that cells emitting green fluorescence were detected.

In Example 3, the spleen of a nude mouse was used as a sample. Since nude mice do not have thymus and B cells are abundant in the spleen, this experiment mainly observes the transfer of carriers for immune cell transfer to B cells. As shown in Table 2, when incubated at 37 ° C., only dendrimers with phenylalanine residues attached to the ends could be transferred to cells. On the other hand, at 4 ° C., none of the dendrimers had migrated to the cells. This suggests that the dendrimer with the phenylalanine residue attached to the terminal does not penetrate the cell membrane and translocate into the cell, but translocates into the cell via endocytosis of the cell. It was.

Example 4: Imaging of a carrier for immune cell transfer to which a fluorescent dye is linked (3)
(1) Carrier for Immune Cell Transfer to which Fluorescent Dye is Linked In Example 4, G4-Suc-Phe and G4-CHex-Phe to which FITC was ligated prepared in Example 3 prepared in Example 3 were used as the carrier for immune cell transfer. Using. For comparison, G4-Suc and G4-CHex with FITC ligated at the ends were also used.

(2) Transfer to immune cells in the spleen in vitro
(2.1) Collection of spleen cells, contact with a carrier for immune cell transfer, and immunostaining For animal experiments, BALB / c mice (6 weeks old, female, Nippon SLC Co., Ltd.) were used at the University of Animal Experiments. It was done in compliance with the regulations of. Mice were euthanized and the spleen was harvested. A cell suspension was obtained from the collected spleen in the same manner as in Example 3. The cell suspension was divided into 1.5 mL tubes so that the number of cells was 1.6 × 10 ^{6 cells / tube.} Each dendrimer linked with FITC was added to serum-free RPMI to a dye concentration of 5 μM to prepare a dispersion of each dendrimer. The dispersion of each dendrimer was added to a 1.5 mL tube containing the cells and incubated at 37 ° C. for 3 hours. The dispersion of each dendrimer was added to a 1.5 mL tube containing cells and incubated at 37 ° C. for 3 hours in the same manner as above except that the pH of serum-free RPMI was adjusted to 6.02. The pH of serum-free RPMI without adjusting the pH was 7.95. The cell-containing solution 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 cell suspension was divided into 1.5 mL tubes so that the number of cells was 2 × 10 ^{5 cells / tube.} Add 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) and place in the dark for 30 minutes under ice-cooling. It was left in place. Then, stain buffer (100 μL) was added, and the cells were washed by centrifugation (3000 rpm, 5 minutes, 4 ° C.) to remove the supernatant. 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 (manufactured by BD Biosciences). In the same manner as in Example 1, a graph (FIG. 6) showing the proportion of cells containing a carrier for immune cell transfer in the immune cells detected by the PE-labeled antibody was prepared from the analysis results. In addition, a graph (FIG. 7) showing the ratio of cells containing a carrier for immune cell transfer among T cells and B cells detected by a PE-labeled antibody when the pH was set to 6.02 was prepared.

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

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

PAMAM dendrimer G4 (56.0 mg, 3.94 μmol) was dissolved in DMSO-DMF mixed solvent (2.3 mL), excess phthalic anhydride (0.3170 g, 1.90 mmol) was added, TEA (200 μL, 1.44 mmol) was added. Stirred overnight at room temperature. After dialysis with DMSO (MWCO: 1000), dialysis was performed with distilled water. Finally, freeze-drying was performed to obtain G4-Ph (yield: 67.3 mg, yield: 69%). Next, G4-Ph (34.5 mg, 1.46 μmol) was weighed, 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 after stirring for 4 days, distilled water (1 mL) was added to obtain. The 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 lyophilized to obtain G4-Ph- (Phe-OMe) (yield: 30.5 mg, yield: 60%). Subsequently, G4-Ph- (Phe-OMe) was dissolved in methanol (4 mL), and a 4M NaOH methanol solution (500 μL) was added. After stirring at 4 ° C. for 2 hours, the dendrimer dispersion was purified by dialysis in distilled water using a dialysis membrane (molecular weight cutoff (MWCO): 1,000). The purified solution was lyophilized to give G4-Ph-Phe (yield: 63.2 mg, yield: 90.2%). ^{As a result of 1} H-NMR spectrum measurement of G4-Ph-Phe, the number of phthalic acid (Ph) bound to the terminal group of G4 PAMAM was 64, and the number of phenylalanine residue (Phe) bound was 58.

Example 5: Measurement of change in transmittance of G4-Suc-Phe, G4-Ph-Phe and G4-Chex-Phe with respect to temperature G4-Suc-Phe, G4-Ph-Phe and G4-Chex having various pHs -Each solution of Phe was 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 hydrochloride buffer (pH 3.5 or less), acetate buffer (pH 5.5 or less) and phosphate buffer (pH 6 or more), the transmittance is measured by UV / Vis Spectrophotometer (V-630, JASCO). The temperature change was measured. A Peltier holder (ETC-717, JASCO) was used to adjust the temperature. The results are shown in FIG.

All dendrimers showed a behavior that 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 this pH condition, the behavior of the upper critical solution temperature (UCST) type, in which the transmittance increases when heated, was observed.

Production Example 3: Preparation of carrier for immune cell transfer (3)
(1) Preparation of G4-Phe-SO _{3 H An immune cell transfer carrier (G4-Phe-SO 3} H) having a phenylalanine residue at the terminal and an anionic terminal group being a sulfone group was prepared by Chen HT. et al. , J.Am.Chem.Soc., 2004, vol.126 (32), pp.10044-10048, and synthesized by the following scheme.

The amino terminus of G4 PAMAM dendrimer (Sigma-Aldrich) was reacted with the carboxyl terminus of the amino group-protected phenylalanine residue to give G4-Phe. 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 acrylamide 3} buffer (2.8 mL). These two solutions were mixed, nitrogen bubbling and then stirring at room temperature. Then, it was left for 2 days, and the pH was measured on the 3rd day. Since the pH was 1 or less, a 4M NaOH aqueous solution was added to make the pH basic, nitrogen bubbling was performed, and the mixture was stirred. The pH was measured on the 4th day. Since the pH was 7 to 8, the pH was raised to about 9 by adding a 4M NaOH aqueous solution. The pH was measured on the 5th day. Since the pH was about 8, the pH was lowered to about 8 by adding an aqueous HCl solution. After reducing the amount of the solution with an evaporator, distilled water (2.1 mL) was added, and the mixture was dialyzed against distilled water. After lyophilization, G4-Phe-SO ₃ H was obtained (yield: 60.9 mg, yield: 95%). ^{As a result of 1} H-NMR spectrum measurement of the solution of G4-Phe-SO ₃ H, the number of phenylalanine residue (Phe) bound to the terminal group of G4 PAMAM was 49, and it was found to be the terminal group of G4 PAMAM and the phenylalanine residue. The number of sulfonic acid bonds was 56.

(2) Measurement of change in transmittance of G4-Phe-SO ₃ H with respect to temperature A 1 mg / mL (buffer concentration 20 mM) G4-Phe-SO ₃ H solution was prepared, and the temperature of the solution was changed. The transmittance was measured. 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 hydrochloride buffer (pH 3.5 or less), acetate buffer (pH 5.5 or less) and phosphate buffer (pH 6 or more), the transmittance is measured by UV / Vis Spectrophotometer (V-630, JASCO). The temperature change was measured. A Peltier holder (ETC-717, JASCO) was used to adjust the temperature. The results are shown in FIG.

As shown in FIG. 9, G4-Phe-SO ₃ H exhibits 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. ℃) was shown. G4-Phe-SO ₃ H was a dendrimer that sensitively undergoes LCST-type and UCST-type phase transitions under acidic conditions. The transmittance of the G4-Phe-SO ₃ H solution was 3% at pH 6.5 at room temperature, but improved to 91% at pH 6.5 at about 40 ° C. Also, referring to Tamaki M. et al., RSC Adv., 2018, vol.8, pp.28147-28151, _{the transmittance of the G4-Phe-SO 3} H solution at pH 6.5 at about 37 ° C is the same. It was about the same as the transmittance of the G4-CHex-Phe solution under the conditions (about 30%).

Production Example 4: Preparation of carrier for immune cell transfer (4)
(1) Preparation of G3.5-Phe and G2.5-Phe An immune cell transfer carrier (G3.5-Phe and) in which a phenylalanine residue is directly bound to the terminal group of the dendrimer and the anionic terminal group is a carboxyl group. G2.5-Phe) was synthesized by the following scheme. In this production example, a PAMAM dendrimer having a carboxyl group as a terminal group (G3.5: 64 terminals and G2.5: 32 terminals) was reacted with an amino group of a phenylalanine residue having a protected carboxyl group. ..

(1.1) Preparation of G3.5-Phe PAMAM dendrimer G3.5 (38.7 mg, 2.99 _{μmol) was dissolved in 125 mM LVDS 3} 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 the mixture was stirred at room temperature for 3 days. Next, dialysis (MWCO: 2000) was performed with methanol. Methanol was distilled off under reduced pressure, vacuum dried for 3 hours, and then freeze-dried 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), a methanol solution containing 4M NaOH (500 μL) was added, and the mixture was stirred at 4 ° C. for 2 hours. did. After stirring, the mixture was dialyzed against distilled water (MWCO: 2000). Then, it was freeze-dried to obtain G3.5-Phe (yield: 25.0 mg, yield: 101%). ^{As a result of 1} H NMR (DCl-containing D ₂ O) spectrum measurement, the number of phenylalanine residue (Phe) bound to the terminal group of PAMAM dendrimer G3.5 was 30. The G3.5-Phe obtained by these reactions is hereinafter referred to as "G3.5-Phe ₃₀ ".

G3.5-Phe was also prepared under different synthetic conditions. That is, in the condensation reaction, DMSO (1.5 mL) / water (1 mL) was used as a solvent in the same manner, and G3.5-Phe was obtained (yield: 39.5 mg, yield: 71.0%). ^{As a result of 1} H NMR (DCl-containing D ₂ O) spectrum measurement, the number of phenylalanine residue (Phe) bound to the terminal group of PAMAM dendrimer G3.5 was 41. The G3.5-Phe obtained by these reactions is hereinafter referred to as "G3.5-Phe ₄₁ ".

(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 carried out in the same manner as above to obtain G2.5- (Phe-OBzl) (yield: 57.8 mg, yield: 71.9%). Next, the synthesized G2.5- (Phe-OBzl) (55.8 mg, 4.21 μmol) was reacted and purified in the same manner as in (1.1) above to obtain G2.5-Phe (yield: 25.2 mg). , Yield: 92.2%). ^{As a result of 1} H NMR (DCl-containing D ₂ O) spectrum measurement, the number of phenylalanine residues (Phe) bound to the terminal group of PAMAM dendrimer G2.5 was 18. The G2.5-Phe obtained by these reactions is hereinafter referred to as "G2.5-Phe ₁₈ ".

G2.5-Phe was also prepared under different synthetic conditions. That is, 0.5 equivalent of Phe-OBzl · Tos was added to the end 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). %). ^{As a result of 1} H NMR (DCl-containing D ₂ O) spectrum measurement, the number of phenylalanine residues (Phe) bound to the terminal group of PAMAM dendrimer G2.5 was 14. The G2.5-Phe obtained by these reactions is hereinafter referred to as "G2.5-Phe ₁₄ ".

(2) Measurement of 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 pHs were prepared. It was prepared and the transmittance was measured by changing the temperature of the solution. The results are shown in FIGS. 10A to 10D. As shown in FIG. 10A, _{the transmittance of the solution of G3.5-Phe 30} did not change at pH 7, and at pH 6, it gradually increased from 5 ° C to 55 ° C by heating. This was a UCST type behavior. On the other hand, at pH 5, the transmittance was high at low temperatures, and the transmittance decreased from around 45 ° C. This was an LCST type behavior. The LCST type behavior was also observed at pH 4, but the transmittance had already decreased at 5 ° C. As shown in FIG. 10B, _{the transmittance of the solution of G3.5-Phe 41} did not change at pH 7 and pH 6, and at pH 5, it gradually increased from 35 ° C. to 85 ° C. by heating. This was a UCST type behavior. On the other hand, at pH 4, the transmittance was high at low temperatures, and the transmittance decreased from 5 ° C. This was an LCST type behavior.

As shown in FIG. 10C, _{the permeability of the solution of G2.5-Phe 18} did not change at pH 7, increased in the range of 5 ° C to 25 ° C at pH 6, but was high in the entire temperature range. It became. At pH 5, it gradually increased from 5 ° C to 95 ° C with heating. This was similar to the UCST type behavior. On the other hand, at pH 4, the transmittance was as low as 63% from 5 ° C, and the transmittance decreased with heating. This was an LCST type behavior. As shown in FIG. 10D, _{the permeability of the solution of G2.5-Phe 14} remains unchanged at pH 7, from 5 ° C to 65 ° C at pH 6 and from 45 ° C to 95 ° C at pH 5 by heating. It rose moderately. This was a UCST type behavior. On the other hand, at pH 4, the transmittance was high at low temperatures, and the transmittance decreased from around 35 ° C. This was an LCST type behavior.

Example 6: Comparison of Amino Acid Residues in Immune Cell Translocation Carriers
(1) Carrier for Immune Cell Transfer to which Fluorescent Dye is Linked In Example 6, G4-Suc-Phe and G4-CHex-Phe to which FITC was ligated prepared in Example 3 prepared in Example 3 were used as the carrier for immune cell transfer. Using. For comparison, carriers (G4-Suc-Leu and G4-CHex-Leu) in which leucine residues were bound to dendrimers were prepared, and FITC was ligated to the ends. G4-Suc-Leu and G4-CHex-Leu are G4- except that leucine benzyl ester p-toluene phosphonate was used instead of 3-phenyl-L-alanine benzyl ester 4-toluene sulfonate in Production Example 1. It was prepared in the same manner as Suc-Phe and G4-CHex-Phe. The number of leucine residues bound to each of G4-Suc-Leu and G4-CHex-Leu was 64. A schematic diagram of G4-Suc-Phe, G4-CHex-Phe, G4-Suc-Leu and G4-CHex-Leu is shown in FIG. The 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) to a dye concentration of 5 μM to prepare a dispersion of each dendrimer.

(2) Transition to immune cells in vitro 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 cells / tube, and the microtube was centrifuged (3000 rpm, 3 minutes) 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 minutes) to remove the supernatant and the cells were washed with PBS. After removing the supernatant, 1 mL of serum-free RPMI was added to the microtubes to obtain a sample for FCM analysis. Each sample was analyzed using BD FACS Calibur (manufactured by BD Biosciences). A graph (FIG. 12) showing the average value of the fluorescence intensity of the cells to which each dendrimer was added was prepared.

As can be seen from A and B in FIG. 12, the fluorescence intensity of G4-Suc-Phe was higher than that of G4-Suc-Leu. Further, as can be seen from C and D in FIG. 12, the fluorescence intensity of G4-CHex-Phe was higher than that of G4-CHex-Leu. Both phenylalanine and leucine are amino acids having non-polar side chains, but from FIG. 12, the dendrimer to which a phenylalanine residue is bound has better transferability to immune cells than the dendrimer to which a leucine residue is bound. It has been shown.

Example 7: Imaging of a carrier for immune cell transfer to which a fluorescent dye is linked (4)
(1) Preparation of Carrier for Immune Cell Transfer to which Fluorescent Dye is Linked In Example 7, G4-Phe-Suc and G4-Phe-CHex to which FITC was ligated at the end were used as carriers for immune cell transfer. G4-Phe-Suc and G4-Phe-CHex were prepared as follows. In the same manner as in Production Example 3, the amino terminus of G4 PAMAM dendrimer (Sigma-Aldrich) was reacted with the carboxyl terminus of the amino group-protected phenylalanine residue to obtain G4-Phe. Dissolve G4-Phe (125 mg, 5.57 μmol) in 125 mM LVDS ₃ buffer (3 mL) and add an excess of succinic anhydride or cis-1,2-cyclohexanedicarboxylic acid anhydride (both 500 eq). Added. Each solution was stirred at room temperature overnight and the pH was adjusted to 8-10 with an aqueous 4N NaOH solution. The solution containing the dendrimer was purified by dialysis in distilled water using a dialysis membrane (MWCO: 1,000). The purified solution was lyophilized to give G4-Phe-Suc and G4-Phe-CHex. The yield of G4-Phe-Suc was 68 mg, and the yield was 56%. The yield of G4-Phe-CHex was 65 mg, and the yield was 72%. Each solution of G4-Phe-Suc and G4-Phe-CHex ^{was measured by 1} H-NMR spectrum. In G4-Phe-Suc, the number of phenylalanine residue (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 G4-Phe-CHex, the number of bonds of phenylalanine residue (Phe) to the terminal group of G4 PAMAM is 64, and the number of bonds of cyclohexanedicarboxylic acid to the terminal group of G4 PAMAM and phenylalanine residue is 64. there were. A schematic diagram of G4-Phe-Suc and G4-Phe-CHex is shown in FIG. The FITC was linked to G4-Phe-Suc and G4-Phe-CHex in the same manner as in Example 3.

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

Claims (16)

1. A carrier for immune cell migration containing a complex molecule.
   The complex molecule contains a branched polymer and a phenylalanine residue, has an anionic terminal structure, and the phenylalanine residue is attached to the terminal group of the branched polymer.
   Carrier for immune cell transfer.
2. The carrier according to claim 1, wherein the terminal group of the branched polymer and the phenylalanine residue are directly bonded.
3. The carrier according to claim 2, wherein a compound capable of imparting an anionic end group is further bound to the phenylalanine residue.
4. The carrier according to claim 3, wherein the anionic end group is a carboxyl group, a sulfone group, a sulfate group, or a salt thereof.
5. The carrier according to claim 1, wherein the terminal group of the branched polymer and the phenylalanine residue are bonded via a linker.
6. The anionic end structure comprises the phenylalanine residue attached to the end group of the branched polymer and has the following formula (I):
   

   

   (In the equation, 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, a heterocycloalkylene group or a phenylene group having 3 to 8 carbon atoms which may have a substituent. Yes,
   Z ₁ is a carboxyl group, a sulfone group, a sulfate group or a salt thereof)
   It is expressed by the following formula (II):
   

   

   (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 maleimide and thiol It is a structure obtained by the reaction,
   R is an alkylene group having 1 to 10 carbon atoms which may have a substituent, or a cycloalkylene group, a heterocycloalkylene group or a phenylene group having 3 to 8 carbon atoms which may have a substituent. Yes,
   Y ₂ is-(C = O) -NH-, -O- (C = O) -NH-, -NH- (C = O) -NH- or -NH- (C = S) -NH- Yes,
   Z ₂ is a carboxyl group or a salt thereof)
   It is expressed by the following formula (III):
   

   

   (In the formula, Z ₃ is a carboxyl group or a salt thereof)
   The carrier according to any one of claims 1 to 5, which is represented by.
7. The carrier according to any one of claims 1 to 6, wherein the immune cell is an immune cell present in a lymph node.
8. The carrier according to any one of claims 1 to 7, wherein the immune cell is at least one selected from T cells, B cells, dendritic cells and macrophages.
9. The carrier according to any one of claims 1 to 8, wherein the branched polymer is a dendrimer.
10. The carrier according to any one of claims 1 to 9, wherein the transferability to T cells changes in response to pH.
11. A reagent for immune cell transfer, which comprises the carrier for immune cell transfer according to any one of claims 1 to 10.
12. Immunize an immune cell transfer carrier, which comprises contacting the immune cell transfer carrier according to any one of claims 1 to 10 in vivo (excluding humans), in vitro or ex vivo. How to transfer to cells.
13. The method according to claim 12, wherein the carrier for transfer of immune cells contains a substance of interest.
14. The method according to claim 13, wherein the substance of interest is at least one selected from a labeling substance, a nucleic acid, a protein and a peptide.
15. The method according to claim 14, wherein the labeling substance is at least one selected from a radioisotope, a low molecular weight organic dye, a fluorescent dye, and a contrast agent for nuclear magnetic resonance imaging.
16. A pharmaceutical composition comprising a drug and the carrier for immune cell transfer according to any one of claims 1 to 10.

Images