Cytokine-encapsulated polymer micelles

Abstract

To provide a polymeric complex.SOLUTION: A polymeric complex contains a cytokine and a block copolymer comprising a PEG portion and a polycation portion. The block copolymer contains the following substituent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to cytokine-encapsulating polymer micelles, particularly micelles encapsulating interleukin-12, which can improve stability in harsh in vivo environments by using block copolymers. All disclosures of the cited literature are incorporated herein by reference in their entirety. [Background technology]

[0002] Proteins are physiologically active substances present throughout the body and are therefore used in the treatment of various intractable diseases such as cancer, autoimmune diseases, and metabolic disorders. However, systemic administration of proteins alone is subject to enzymatic degradation and renal excretion, and they also possess immunogenicity, making the development of delivery carriers necessary for the biomedical application of proteins. Therefore, the development of protein-PEG conjugates, in which the biocompatible polymer poly(ethylene glycol) (PEG) is introduced into proteins, is progressing. This approach aims to address the challenges of protein metabolism by suppressing interactions with proteolytic enzymes and immune cells, and by increasing protein size. [1-4] It is possible to overcome this. In fact, many protein-PEG conjugates are approved by the FDA and have a multi-billion dollar market as protein formulations. [5,6] It has the properties of PEGylation of proteins, which leads to enzymatic degradation, renal excretion, and immunogenicity. [7,8] While suppressing this process, it also inhibits the inactivation of proteins due to irreversible chemical modification of proteins, and the function of proteins. [6,9] Issues include insufficient spatiotemporal control. Therefore, by fabricating proteins via reversible chemical bonding, it is possible to suppress protein expression in normal tissue while targeting the protein.

[10] Development is underway to create delivery carriers that can release substances specifically.

[0003] Stimulus-responsive nanocarriers target tissue [4,11]By sensing a physiologically active substance, it is possible to release it specifically to a target tissue while maintaining the activity of the protein. Among such nanocarriers, core-shell type polymer micelles formed by the self-association of a block copolymer and a protein can induce the release of the protein in response to an external stimulus by introducing an environmentally responsive site into the core-forming chain of the block copolymer. [4] As an external stimulus to which the polymer micelles can respond, pH can be mentioned. For example, many diseases (e.g., cancer or autoimmune diseases) exhibit a lower pH (pH 6.5 - 7.2) than normal tissues (pH 7.4). [12,13]

[0004] On the other hand, the present inventors have previously shown that polyion complex (PIC) type polymer micelles can be prepared by adding a PEG-polycation to a protein in which an amino group is converted to a carboxyl group by a pH-responsive maleic anhydride derivative. These micelles stably encapsulate the protein in the core at the pH of normal tissues (pH 7.4), but have succeeded in releasing the protein by cleavage of the pH-responsive maleic anhydride derivative at the acidic pH (pH 6.5 - 7.2) within the target tissue. [14-16] However, for medical applications, an increase in the accumulation in the target tissue due to an improvement in blood retention is important.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, for increasing the therapeutic effect of therapeutic proteins, it is important to develop micelles that enable an increase in blood retention and efficient release of proteins under acidic conditions.

Means for Solving the Problems

[0006] This invention aims to increase micelle stability and efficiently release proteins under acidic conditions by introducing a pH-responsive maleic anhydride derivative into the core-forming chain of a block copolymer and forming a reversible covalent bond with the amino group of the protein. Furthermore, it aims to further stabilize the micelles by forming a PIC (polycrystalline symmetry) between the amino group of the core-forming chain of the block copolymer and the carboxyl group of the protein. The objective is to stabilize the micelle structure through covalent bonding and PIC formation and increase its retention in the bloodstream. The invention was completed by successfully encapsulating interleukin-12 (IL-12) as the target protein.

[0007] In other words, the present invention is as follows: [1] A polymeric complex comprising cytokines and a block copolymer represented by the following formula (1). [ka] [In the formula, R 1 and R 2 Each of these independently represents a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, ligand, or labeling agent. R 3 This represents the compound shown by the following formula (I), [ka] (In the formula, R a and R b Each of these independently represents a hydrogen atom, or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group, heterocyclic alkyl group, hydroxyl group, alkoxy group, or aryloxy group. a and R b The two atoms may be bonded to each other, and together with the carbon atoms to which they are bonded, they may form an aromatic ring or a cycloalkyl ring. a and R b The bonds between the carbon atoms to which each atom is bonded may be single bonds or double bonds. L1 is NH, CO, or the following formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer from 1 to 6.) The base shown by, or formula (12) below: -L 2a -(CH2) q1 -L 3a - (12) (In the formula, L 2a This represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH, or COO, L 3a (where 'h' represents NH or CO, and 'q1' represents an integer from 1 to 6.) It represents the base indicated by, m1 and m2 each independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500), m3, m4, and m5 each independently represent integers from 1 to 5, and n represents an integer from 0 to 500. The notation " / " indicates that the arrangement order of the (m1+m2) monomer units shown to the left and right of it is arbitrary. [2] The complex according to [1], wherein the compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig). [ka] [3] The complex according to [2], wherein the compound represented by formula (I) is the compound represented by the following formula (Ia) or (Ib). [ka] [4] The complex according to [1], wherein the block copolymer represented by formula 1 is represented by the following formula (2). [ka] [5] The complex according to [1], wherein a cytokine is covalently bonded to a block copolymer represented by formula 1. [6] The complex according to [5], wherein the covalent bond is cleaved in a pH-dependent manner. [7] The complex described in any one of [1] to [6], wherein the cytokine is interleukin-12. A cytokine delivery device comprising a polymer complex described in any one of the items [8] [1] to [7], to the cell surface, intracellular, or extracellular, selected from within. [9] A cytokine delivery kit comprising a block copolymer represented by the following formula (1), to a selected location on the cell surface, inside or outside the cell. [ka] [In the formula, R 1 and R 2 Each of these independently represents a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms, or an azide, amine, maleimide, ligand, or labeling agent. R 3 This represents the compound shown by the following formula (I), [ka] (In the formula, R a and R b Each of these independently represents a hydrogen atom, or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group, heterocyclic alkyl group, hydroxyl group, alkoxy group, or aryloxy group. a and R b The two atoms may be bonded to each other, and together with the carbon atoms to which they are bonded, they may form an aromatic ring or a cycloalkyl ring. a and R b The bonds between the carbon atoms to which each atom is bonded may be single bonds or double bonds. L 1 is NH, CO, or the following formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer from 1 to 6.) The base shown by, or formula (12) below: -L 2a -(CH2)q1 -L 3a - (12) (In the formula, L 2a This represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH, or COO, L 3a (where 'h' represents NH or CO, and 'q1' represents an integer from 1 to 6.) It represents the base indicated by, m1 and m2 each independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500), m3, m4, and m5 each independently represent integers from 1 to 5, and n represents an integer from 0 to 500. The notation " / " indicates that the arrangement order of the (m1+m2) monomer units shown to the left and right of it is arbitrary.

[10] The kit according to [9], wherein the compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig). [ka]

[11] The kit according to [9], wherein the compound represented by formula (I) is the compound represented by formula (Ia) or (Ib) below. [ka]

[12] The kit described in [9], wherein the block copolymer represented by formula 1 is represented by the following formula (2). [ka]

[13] The complex according to any one of claims 9 to 12, wherein the cytokine is interleukin-12.

[14] A pharmaceutical composition comprising the complex described in any one of items [1] to [8].

[15] The pharmaceutical composition according to

[14] for use in immunotherapy or antitumor therapy. [Brief explanation of the drawing]

[0008] [Figure 1]This figure shows the synthesis scheme for PEG-p(Lys-CDM). PEG-pLys(TFA) was synthesized by ring-opening polymerization of Lys(TFA)-NCA initiated with MeO-PEG-NH2, followed by deprotection of the TFA group to obtain PEG-pLys. CDM was reacted with oxalyl chloride to produce CDM-Cl, and CDM was bonded to PEG-pLys via the reaction of CDM-Cl with lysineamine. [Figure 2] This figure shows the characterization of PEG-pLys(TFA). a) 1H-NMR result of the product dissolved in DMSO. It was determined to be PEG12k-pLys(TFA)40. b) GPC result of PEG12k-pLys(TFA)40 dissolved in DMF. Mw / Mn = 1.029 [Figure 3] This figure shows the characterization of PEG-pLys. a) 1H-NMR result of the product dissolved in D2O. The polymer formula was determined to be PEG12k-pLys40. b) GPC result of PEG12k-pLys40 dissolved in PBS. [Figure 4] This figure shows the characterization of PEG-p(Lys-CDM). a) 1H-NMR result of the product dissolved in DMSO. The formula of this polymer was determined to be PEG12k-p(Lys20-CDM20). b) GPC result of PEG12k-p(Lys20-CDM20) dissolved in acetate buffer at pH 3.3. [Figure 5] This figure shows the micelle preparation method. a) Scheme for visualizing the preparation protocol. b) HPLC results suggest successful encapsulation due to peak shift, and encapsulation efficiency is shown by comparing peak areas. c) The encapsulation efficiency of the system containing unlabeled IL-12 was determined using ELISA measurement. After dilution, the total IL-12 supply to the system was 500 pg / mL, and the red dots indicate the measurement results of free IL-12 in the system. The encapsulation efficiency was determined to be approximately 68%. [Figure 6]This figure shows the characterization of IL-12 / m. a) Micelle formation was visualized with Alexa647-IL-12 after ultrafiltration through a 300,000 MWCO membrane. b) Representative DLS results. c) Size and zeta potential of IL-12 and IL-12 / m. d) TEM image of IL-12 / m. Scale bar = 100 nm [Figure 7] This figure shows the pH-sensitive stability and cargo release of IL-12 / m. a) Normalized count rates of the solution after different incubation times. Count rates were normalized to the corresponding initial values. b) Average size change during incubation time. c) PDI varies with incubation time. d) IL-12 release kinetics at pH 6.5 and 7.4. n=3 [Figure 8] This figure shows an in vitro assay to test the bioactivity of IL-12 / m and released IL-12. Splenocytes were treated with samples of different IL-12 equivalent concentrations. Natural IL-12 from the same batch was used for reference (black line). n=4 [Figure 9] This figure shows the size distribution and bioactivity of lyo-IL-12 / m. a) DLS results showed that lyo-IL-12 / m has a similar size distribution to the original IL-12 / m. b) In vitro splenocyte assay confirmed the same bioactivity of lyo-IL-12 / m and the original IL-12 / m (n=3). [Figure 10] This figure shows the blood circulation of IL-12 / m at different injection doses. a) 10 μg IL-12 equivalent; b) 1 μg IL-12 equivalent. Half-lives were calculated using a single-phase decay model. n=3, AUC was calculated using a single-phase decay model. [Figure 11] This figure shows representative IVCLM results. IVCLM occurs in the earlobes of mice after intravenous injection of 10 μg of fluorescently labeled IL-12 or an equivalent IL-12 / m. Clear fluorescence could be detected in the tissue area (blue) 20 minutes after injection in IL-12-treated mice, but minimal fluorescence was detected in the tissue of IL-12 / m-treated mice. Scale bar = 100 μm. [Figure 12]This figure shows the accumulation of IL-12 / m at the tumor site 24 hours after injection. a) IVIS image of melanoma after injection of 10 μg of Alexa647-IL-12 or equivalent micelles. b) Quantification of fluorescence intensity from melanoma. c) Values ​​were standardized for ELISA measurements of homogenates from TNBC tumors injected with 10 μg of unlabeled IL-12 or equivalent micelles. d) ELISA measurement of homogenates from melanoma tumors injected with 1 μg of unlabeled IL-12 or equivalent micelles. n=3, p-values ​​were analyzed by unpaired t-test. [Figure 13] This is a representative IVCLSM observation of organs 3 hours after injection. Tumor-free mice were injected with 10 μg of fluorescently labeled IL-12 or an equivalent IL-12 / m³. Two hours and thirty minutes after injection, 30 μL of Hoechst 33342 solution was intravenously injected. Three hours after injection, organ samples were collected from the mice and observed by IVCLSM. Scale bar = 100 μm. [Figure 14] This figure shows the in vivo distribution of IL-12 / m² 24 hours after injection of a high dose (10 μg IL-12 equivalent). a) IVIS images of major organs and melanoma tumors. Fluorescence intensity indicates the accumulation level of Alexa647-IL-12. b) Quantitative results obtained from ELISA measurements of mice with TNBC tumors. The level of encapsulated IL-12 (green column) was calculated by subtracting the value of released IL-12 (blue column) from the total IL-12 delivery level (red column). Values ​​were normalized to the initial dose. (n=3) [Figure 15] This figure shows systemic IFN-γ and IL-10 secretion after low-dose therapy. 1 μg of IL-12 or IL-12 / m² equivalent was administered intravenously on day 0. Plasma IFN-γ and IL-10 concentrations were measured daily. IFN-γ (a) and IL-10 (c) concentrations were plotted against time, and the AUC was calculated. Individual values ​​of IFN-γ (b) and IL-10 (d) concentrations are also shown. n=5, p-values ​​were analyzed by unpaired t-test. [Figure 16]This figure shows the intratumoral concentrations of IFN-γ and IL-10. For high-dose treatment, tumor samples were collected on day 7 for ELISA analysis, and IFN-γ(a) and IL-10(b) levels were detected. For low-dose treatment, tumors were collected on days 1, 2, and 3 after treatment for ELISA analysis, and IFN-γ(c) and IL-10(d) levels were detected. n=5, p-values ​​were analyzed by unpaired t-test. [Figure 17] This figure shows systemic toxicity indicated by blood biomarkers and body weight changes. a) TP concentration and b) ALT concentration indicate liver damage. c) BUN levels indicate kidney damage. d) Lipase concentration indicates pancreatic damage. e) Relative body weight of mice during the administration period (red: IL-12 group, blue: IL-12 / m group). The individual body weight of each mouse on day 0 was set as the zero point. n=6, p-values ​​were analyzed by unpaired t-test. [Figure 18] This figure shows typical H&E staining results for the liver and kidneys. No macroscopic pathological changes were observed in any of the samples. Scale bar = 100 μm. [Figure 19] This figure shows the antitumor activity of IL-12 / m in a melanoma model. Mice were intravenously administered 10 μg of IL-12 or an equivalent amount of IL-12 / m on day 8. a) Individual tumor growth curves. b) Mean tumor size for each treatment group. c) Survival curves for each group. d) Mean change in body weight of mice during treatment. n=5, tumor size was analyzed by unpaired t-test, and survival rate was analyzed by log-rank. [Figure 20] This figure shows the antitumor activity of IL-12 / m in a TNBC model. 10 μg of IL-12 or an equivalent dose of IL-12 / m was administered intravenously on day 7. a) Individual tumor growth curves. b) Mean tumor size for each treatment group. c) Survival curves for each group. d) Mean change in body weight of mice during treatment. n=5, tumor size was analyzed using an unpaired t-test, and survival rates were analyzed using log-rank. [Figure 21]This figure shows the antitumor activity of IL-12 / m under a low-dose multiple-injection treatment schedule in a melanoma model. a) Scheme showing the treatment schedule in the experiment. Treatment was repeated twice. The first treatment was performed 8 days after tumor inoculation. The second treatment was administered intravenously with 1 μg of IL-12 or an equivalent dose of IL-12 / m, and anti-PD-1 was administered intraperitoneally 3 days after the first administration (Day 11). b) Individual tumor growth curves. c) Mean tumor size for each treatment group. d) Change in body weight of mice during treatment. e) Survival curves for each group. n=6, survival was analyzed by log rank. [Figure 22] This figure shows the antitumor activity of IL-12 / m under a low-dose (1 μg) multiple injection treatment schedule in TNBC. [Figure 23] This figure shows the antitumor activity of IL-12 / m under a high-dose, multiple-injection treatment schedule combined with anti-PD-1 in a melanoma model. a) Scheme showing the experimental treatment schedule. IL-12 / m or IL-12 treatment was repeated three times. The first treatment was performed 8 days after tumor inoculation. The following treatments were performed every 4 days. Anti-PD-1 was injected three times each on Day 10, Day 14, and Day 18. b) Individual tumor growth curves. c) Mean tumor size for each treatment group. d) Change in body weight of mice during treatment. e) Survival curves for each group. n=6, survival was analyzed by log-rank. [Figure 24] This figure shows the antimetastatic activity of IL-12 or IL-12 / m in combination with anti-PD-1. a) Scheme showing the treatment schedule. IL-12 and IL-12 / m were administered by five low-dose injections (equivalent to 1 μg of IL-12 per injection). The first injection was given on postoperative day 4 (Day 29). Subsequent injections were given every two days. Anti-PD-1 was administered twice on Day 31 and Day 35. b) Photograph of a lung specimen taken on Day 35 (postoperative day 10). Metastatic sites are indicated by arrows. c) H&E stained sections show the size of metastatic spots (indicated by arrows). Scale bar = 100 μm, n = 3. d) Statistical results of the number of lung metastases on Day 35, n = 8. e) Statistical results of lung metastasis size on H&E sections. p-values ​​were analyzed by unpaired t-test. [Figure 25]This figure shows typical IHC results for TNBC tumor sections. Mice were treated with 10 μg of IL-12 or an equivalent IL-12 / m³ when the tumor had grown to 50 mm³, and tumor tissue samples were collected from the mice 5 days after treatment. Scale bar = 100 μm. [Figure 26] Figure 25 shows the quantitative analysis results of the IHC results. Analysis was performed on five different fields within each group. n=5, and p-values ​​were analyzed using unpaired t-tests. [Figure 27] This figure shows the IHC results of low-dose multiple therapy in TNBC tumors. a) Scheme showing detailed treatment schedule. b) Representative results in the corresponding group. Scale bar = 100 μm. [Figure 28] Figure 27 shows the quantitative analysis results of the IHC (Intra-Cognitive Hypothesis Control) results. Five different fields were analyzed in each group. n=5, and p-values ​​were analyzed using unpaired t-tests. [Figure 29] This figure shows the FCM results of tumor samples after multiple low-dose treatments. In this study, mice with melanoma (average diameter 50 mm3) were intravenously injected twice with 1 μg of IL-12 or equivalent micelles. The second injection was administered two days after the first injection. Five days after the second injection, tumors were collected from the mice. Single-cell suspensions of tumor samples were prepared, stained with the corresponding antibodies, and then detected by flow cytometry. a) Representative scatter plot of CD3 / CD8 double staining. b) Representative scatter plot of CD4 / CD8 double staining. c) Quantified statistical results from FCM measurements shown in (a) and (b). n=6, p-values ​​were analyzed by unpaired t-test. [Modes for carrying out the invention]

[0009] Therapeutic proteins hold promise for treating intractable diseases, but their systemic administration presents various challenges, including instability, short half-lives, and nonspecific immune responses. Therefore, an approach that delivers proteins using stimulus-responsive nanocarriers can be an effective strategy for tissue-selectively enhancing protein activity in target tissues. In this invention, we developed a polymer micelle that uses the loaded protein as a cytokine and forms a polyionic complex between the cytokine and a block copolymer to release the cytokine in a pH-dependent manner, thereby encapsulating the cytokine via a covalent bond that can be cleaved under predetermined pH conditions.

[0010] The carboxydimethylmaleic anhydride (CDM)-amide bond is stable at physiological pH (pH 7.4), but is cleaved at pH 6.5, the pathophysiological pH of tumors and inflammatory tissues. Therefore, CDM was selected as a pH-responsive functional group. In this invention, interleukin-12 (IL-12) was encapsulated with an efficiency of over 50% by using a poly(ethylene glycol)-poly(L-lysine) block copolymer having 45% CDM addition. By using IL-12-encapsulated micelles (IL-12 / m) as a model, the stability of the micelles under physiological conditions, as well as the disintegration of the micelles and the release of functional IL-12 at pH 6.5, were confirmed. Furthermore, IL-12 / m showed an improved blood half-life compared to IL-12 alone and micelles associated only by electrostatic interactions without covalent bonds. Thus, the usefulness of the system for in vivo delivery of cytokines was demonstrated using the above model.

[0011] CDM-amide bond at pH 6.5 [17-19]Because it is unstable at physiological pH, and this allows for the release of conjugated amino compounds at pathological pH, the CDM was selected as the pH-responsive site in this invention. Consequently, the resulting cytokine-encapsulating micelles form a stable cross-linked core at physiological pH, but degrade into free block copolymers and active cytokines at pH 6.5. Therefore, in this invention, the ability of these micelles to encapsulate cytokines, particularly IL-12, was evaluated. Furthermore, the inventors used micelles encapsulating IL-12 as a model to evaluate the in vitro stability of the micelles, the release of IL-12 at different pH levels, and the in vivo blood retention after systemic administration.

[0012] 1. Polymer composite of the present invention The polymer composite of the present invention is a protein-encapsulated polymer micelle complex (polyion complex: PIC), and comprises a specific cationic polymer (block copolymer, graft copolymer, etc.) and a protein (details of the protein will be described later).

[0013] (1) Cationic polymers The specific cationic polymer that is a component of the PIC of the present invention is a cationic polymer having at least a polycationic moiety. This cationic polymer may, for example, be a block copolymer or graft polymer having a polyethylene glycol (PEG) moiety and a polycationic moiety, but is not limited to these. A suitable and preferred embodiment can be selected depending on the application of the PIC of the present invention.

[0014] The structure (e.g., degree of polymerization) of the above-mentioned PEG and polycation is not limited, and any structure can be selected. In particular, the polycation is preferably a polypeptide having a cationic group in its side chain. The term "cationic group" here includes not only groups that are already cations due to coordination with a hydrogen ion, but also groups that become cations when a hydrogen ion is coordinated to them. All known cationic groups are included. Polypeptides having a cationic group in their side chain include not only those formed by peptide bonds of known amino acids having basic side chains (lysine, arginine, histidine, etc.), but also those in which various amino acids are peptide-bonded and their side chains (e.g., the side chains of aspartic acid or glutamic acid) are substituted to have a cationic group.

[0015] As for the specific cationic polymers mentioned above, for example, block copolymers represented by the following general formula (1) are preferred. [ka]

[0016] Here, in the structural formula of general formula (1), the block portion with n repeating units (degree of polymerization) is the PEG portion, and the block portion formed by combining the m1 portion and the m2 portion (the portion shown in [ ] in general formula (1)) is the polycation portion. Furthermore, the notation " / " in the structural formula of the polycation portion means that the order of the monomer units shown to the left and right of it is arbitrary. For example, if a block portion composed of monomer units A and B is written as [-(A)a- / -(B)b-], it means that the (a+b) monomer units, consisting of a A and b B, can be linked together in any random order (however, all A and B are linked in a linear chain).

[0017] In general formula (1), R 1 and R 2Each of these independently represents a hydrogen atom, or a linear or branched alkyl group having 1 to 12 carbon atoms, which may be substituted, or a functional group such as an azide, amine, maleimide, ligand, or labeling agent. Examples of the linear or branched alkyl groups having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, decyl, and undecyl groups. Examples of substituents on the alkyl groups include acetalized formyl, cyano, formyl, carboxyl, amino, alkoxycarbonyl groups having 1 to 6 carbon atoms, acylamide groups having 2 to 7 carbon atoms, siloxy, silylamino, and trialkylsiloxy groups (each alkylsiloxy group independently has 1 to 6 carbon atoms).

[0018] Ligand molecules refer to compounds used to target specific biomolecules, such as antibodies, aptamers, proteins, amino acids, small molecules, and monomers of biomacromolecules. Labeling agents include, but are not limited to, rare earth fluorescent labeling agents, coumarin, dimethylaminosulfonylbenzoxadiazole (DBD), dansyl, nitrobenzoxadiazole (NBD), pyrene, fluorescein, and fluorescent proteins.

[0019] If the substituent is an acetal-protected formyl group, this substituent can be converted to another substituent, a formyl group (or aldehyde group; -CHO), when hydrolyzed under mildly acidic conditions. Also, the substituent (especially R 1 If the substituent in is a formyl group, a carboxyl group, or an amino group, for example, an antibody or a fragment thereof, or other functional or target-directed proteins, can be attached via these groups.

[0020] In general formula (1), R 3 This represents a compound represented by the following general formula (I). [ka] In the above equation (I), R a and R b Each of these independently represents a hydrogen atom, or an optionally substituted alkyl group, alkenyl group, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclic group, heterocyclic alkyl group, hydroxyl group, alkoxy group, or aryloxy group. a and R b The two atoms may be bonded together, and together with the carbon atoms to which they are bonded, they may form an aromatic ring or a cycloalkyl ring. Also, in formula (I), R a and R b The bonds between the carbon atoms to which each atom is bonded may be single bonds or double bonds; they are not limited to either. In formula (I), both types of bonds are shown together, represented by one solid line and another dashed line.

[0021] L 1 NH, CO, and the following general formula (11): -(CH2) p1 -NH- (11) (In the formula, p1 represents an integer from 1 to 6.) The base shown by, or the following general formula (12): -L 2a -(CH2) q1 -L 3a - (12) (In the formula, L 2a This represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH, or COO, L 3a (where 'h' represents NH or CO, and 'q1' represents an integer from 1 to 6.) It represents the base indicated by .

[0022] In formula (1) above, m1 and m2 each independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500), and m3, m4, and m5 each independently represent integers from 1 to 5. In formula (1) above, n represents the number of repeating units (degree of polymerization) of the PEG portion, and more specifically, an integer from 1 to 500 (preferably 100 to 400, more preferably 200 to 300).

[0023] The molecular weight (Mn) of the cationic polymer represented by general formula (1) is not limited, but is preferably 23,000 to 45,000, and more preferably 28,000 to 34,000. Furthermore, for each block portion, the molecular weight (Mw) of the PEG portion is preferably 8,000 to 15,000, and more preferably 10,000 to 12,000, and the molecular weight (Mn) of the polycation portion as a whole is preferably 15,000 to 30,000, and more preferably 18,000 to 22,000.

[0024] The method for producing the cationic polymer represented by general formula (1) is not limited, but for example, R 1 A segment (PEG segment) containing the PEG chain block is synthesized in advance, and one end of this PEG segment (R 1 Examples of methods include sequentially polymerizing predetermined monomers to the opposite end and then substituting or converting the side chains to include cationic groups as needed, or pre-synthesizing the PEG segment and a block portion having a side chain containing a cationic group, and then linking them together. The methods and conditions of the various reactions in this manufacturing method can be appropriately selected or set considering conventional methods.

[0025] In one embodiment of the present invention, the compound represented by formula (I) is at least one of the compounds represented by the following formulas (Ia) to (Ig). [ka]

[0026] In a preferred embodiment of the present invention, the compound represented by formula (I) is the compound represented by the following formula (Ia) or (Ib). [ka]

[0027] In formula (I), the substituents are saturated or unsaturated acyclic or cyclic hydrocarbon groups. In the case of acyclic hydrocarbon groups, they may be linear or branched. Examples of hydrocarbon groups include C1-C 20 Alkyl alkyl group, C2-C 20 Alkenyl group, C4-C 20 Cycloalkyl groups, C6-C 18 Aryl group, C6-C 20 Aralkyl group, C1-C 20 Alkoxy group, C6-C 18 An example is the aryloxy group.

[0028] The compound represented by formula (I) is used as a charge regulator. The compound represented by formula (I) converts the overall charge of a basic or neutral protein to the charge of an acidic protein. In other words, the charge regulator of the present invention controls the amount of charge so that a protein with a positive (+) or neutral total charge becomes a protein with a negative (-) total charge, thereby converting the total charge. Specifically, this conversion of total charge is achieved when the compound represented by formula (I) or its derivatives binds to an amino group (a positively charged group) contained in the protein, making the entire protein negatively charged. For this purpose, the binding is achieved, for example, by the compound represented by formula (I) binding (covalently) to an amino group in the protein, taking on a structure as shown in formula (I') below.

[0029] [ka]

[0030] Regarding the above bond, for example, if the compound represented by formula (I) is the same as the compounds represented by formulas (Ib) and (Ic), the structure represented by formula (I') after the bond will be as follows. [ka]

[0031] In a further embodiment of the present invention, the block copolymer represented by formula 1 is represented by the following formula 2. [ka]

[0032] (2) Cytokines In the PIC of the present invention, the protein that constitutes the core portion can be any protein whose overall charge has been converted by the compound represented by formula (I) described above (charge-converting protein). Specifically, it can be any protein whose total charge has been converted from the total charge of a basic or neutral protein (positive or neutral state) to the negative side, similar to the total charge of an acidic protein. A protein whose total charge has been converted to the negative side can be said to be an anionic substance (polyanion) as a whole. Therefore, through electrostatic interaction with the polycation portion in the cationic polymer, micelle-like complexes, which are difficult to form with basic or neutral proteins, can be easily formed.

[0033] In this invention, cytokines are used as proteins. Cytokines are small proteins secreted by cells that participate in intercellular interactions and influence surrounding cells. As a result, immune and antitumor effects are exerted by cells. Examples of cytokines include interleukins (IL) (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, etc.), hematopoietic factors (CSF, EPO, TPO), interferons (IFN), tumor necrosis factor (TNF), and growth factors (EGF, FGF, PDGF). In particular, IL-12 can be used in this invention.

[0034] Furthermore, the cytokines used in the present invention are not limited to those consisting of full-length amino acid sequences, but also include partial fragments and peptides thereof. Moreover, the cytokines used in the present invention are not limited to those composed of natural amino acids, but also include modified cytokines that contain at least some non-natural amino acids as constituent components. Furthermore, the cytokines used in the present invention may, as needed, include those to which various labeling substances have been appropriately attached.

[0035] (3) Polyion complex (PIC) The PIC of the present invention can be described as a core-shell type micelle-like complex in which cytokines and a portion of the cationic polymer (polycation portion) described above interact electrostatically to form a core portion, and other portions of the cationic polymer (including the PEG portion) form a shell portion around the core portion.

[0036] The PIC of the present invention can be easily prepared by mixing a cytokine (e.g., IL-12) and a cationic polymer in any buffer (e.g., Tris buffer). The mixing ratio of the cationic polymer to the cytokine is not limited, but in the present invention, for example, the ratio (N / C ratio) of the total number of cationic groups (e.g., amino groups) in the block copolymer to the total number of carboxyl groups (C) in the cytokine can be 0.1 to 200, or 0.5 to 100, or even 1 to 50. When the N / C ratio is within the above range, it is preferable in that the amount of free cationic polymer can be reduced. The cationic group (N) refers to a group that can form an ionic bond with the carboxyl group in the cytokine encapsulated in the micelle through electrostatic interaction.

[0037] The size of the PIC in the present invention is not limited, but is preferably 5 to 200 nm, and more preferably 10 to 100 nm, based on a particle size determined by dynamic light scattering (DLS).

[0038] The PIC of the present invention, after being introduced into a cell, releases the cytokines it contained. At this time, a change in the pH environment within the cytoplasm (a change to a weakly acidic environment (for example, pH 5.5)) causes the compound represented by formula (I) to dissociate from the cytokine (the bond is broken). As a result, the overall charge (total charge) of the cytokine is restored to the original intrinsic charge (total charge) of the cytokine, allowing the cytokine to exist in the introduced cell in a state where its structure and activity have been regenerated.

[0039] 2. Cytokine delivery devices The present invention provides a cytokine delivery device comprising the polyion complex (PIC) described above. The cytokine delivery device of the present invention can be used as a means to efficiently introduce cytokines encapsulated in the core portion of the PIC into the cell surface, inside, or outside of target cells, by utilizing changes in the redox environment inside and outside the cell.

[0040] Specifically, a solution containing PIC encapsulating cytokines is administered to test animals, allowing it to be taken up by target cells in the body. Once the PIC reaches the endosomes, the compound shown in equation (I) detaches from the cytokines, altering the charge balance within the PIC and causing it to disintegrate. Upon disintegration, cytokines are released, and simultaneously, the polymers dissociated from the PIC damage the endosomal membrane. This disruption of the endosomes allows for the release of the cytokines into the cytoplasm. In micelles containing cytokines, the cytokines are released extracellularly, allowing them to bind to receptors on the cell surface, thus enabling delivery to the cell surface.

[0041] The cytokine delivery device of the present invention can be applied to various mammals such as humans, mice, rats, rabbits, pigs, dogs, and cats, but is not limited to them. The method of administration to test animals is usually parenteral administration such as intravenous infusion, and each condition such as the dosage, number of administrations, and duration of administration can be appropriately set according to the type and condition of the test animal.

[0042] 3. Pharmaceutical Compositions The complex and cytokine delivery device of the present invention can be used in therapies that introduce cytokines into cells causing various diseases (for example, in the case of a complex containing IL-12, antitumor therapy, etc.). Therefore, the present invention can also provide a pharmaceutical composition containing the aforementioned PIC (for example, a pharmaceutical composition for antitumor therapy), and a method for treating various diseases (for example, tumors) using the aforementioned PIC. The method and conditions of administration are the same as described above.

[0043] The target tumors in the pharmaceutical composition of the present invention are not particularly limited. For example, brain tumors (pituitary adenoma, glioma), head and neck cancer, cervical cancer, jaw cancer, oral cancer, salivary gland cancer, sublingual gland cancer, parotid gland cancer, nasal cavity cancer, paranasal sinus cancer, laryngeal cancer, esophageal cancer, lung cancer, breast cancer, pancreatic cancer, stomach cancer, biliary tract cancer (bile duct cancer, gallbladder cancer), small intestine or duodenal cancer, colorectal cancer, bladder cancer, kidney cancer, liver cancer, prostate cancer, uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, thyroid cancer, pharyngeal cancer, sarcomas (e.g., osteosarcoma, chondrosarcoma, Kaposi's sarcoma). Examples include tumors, myosarcomas, angiosarcomas, fibrosarcomas, etc., malignant lymphomas (Hodgkin lymphoma, non-Hodgkin lymphoma), leukemias (including, for example, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL), lymphomas, multiple myeloma (MM), myelodysplastic syndromes, etc.), skin cancers, melanomas, and others.

[0044] Furthermore, when using cytokines such as IL-10, IL-4, IL-6, IL-11, IL-13, IL-1R, IL-18R, TNF-R1, TNF-R2, TGF-β, and CXCL12 in the present invention, inflammatory diseases and immune system disorders can be targeted. Inflammatory diseases include rheumatoid arthritis, psoriasis, multiple sclerosis, Crohn's disease, myocarditis, type 1 diabetes, systemic lupus erythematosus, inflammatory bowel disease, ulcerative colitis, pharyngitis, cystitis, hepatitis, pneumonia, pancreatitis, enteritis, polymyositis, dermatomyositis, scleroderma, Sjögren's syndrome, Crohn's disease, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, rapidly progressive glomerulonephritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (immune thrombocytopenia), Graves' disease, and pemphigus.

[0045] The above-mentioned pharmaceutical compositions can be prepared by conventional methods using appropriately selected excipients, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, antiseptics, flavoring and odor-masking agents, analgesics, stabilizers, and isotonic agents, etc., which are commonly used in pharmaceutical manufacturing.

[0046] The pharmaceutical composition of the present invention can be administered by injection, and in addition to systemic administration such as intravenous and intra-arterial administration, it can be administered locally to muscles, joints, subcutaneously, intradermally, etc. In this case, it is usually provided in the form of a unit dose ampoule or a multi-dose container, and may be a suitable carrier, such as a powder that is redissolved in sterile water, when used. Furthermore, these dosage forms may contain additives commonly used in pharmaceutical formulations. The dosage can be varied over a wide range depending on the therapeutic purpose, micelle form, age of the recipient, route of administration, and number of administrations, and the amount of cytokine contained in the pharmaceutical composition of the present invention can be appropriately set by those skilled in the art. For example, the effective dose administered as a combination of the effective amount of the pharmaceutical composition of the present invention, a suitable diluent, and a pharmacologically usable carrier is 1 μg to 1000 μg per kg of body weight per dose, administered at intervals of 1 day to 6 weeks.

[0047] 4. Cytokine delivery kit The cytokine delivery kit of the present invention is characterized by containing the block copolymer. This kit can be preferably used, for example, in immunotherapy methods, antitumor therapy, and the like.

[0048] In the kit of the present invention, the storage state of the cationic polymer is not limited, and a state such as solution or powder can be selected considering its stability (storability) and ease of use. The kit of the present invention may contain other components in addition to the block copolymer. Examples of other components include various buffers, various proteins to be introduced into cells (charge-converting proteins), lysis buffers, and instructions for use (user manual). The kit of the present invention is used to prepare a polyion complex (PIC) with cytokines to be introduced into target cells as the core component, and the prepared PIC can be effectively used as a cytokine delivery device to target cells (e.g., an IL-12 delivery device).

[0049] Examples The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0050] 1. Synthesis of PEG-p (Lys-CDM) (Figure 1-4) Poly(ethylene glycol)-poly(L-lysine) (PEG-p(Lys)) was synthesized by ring-opening polymerization. Thiourea (1500 mg) was dissolved in 20 mL of anhydrous DMF. MeO-PEG-NH2 (Mw=12k, 500 mg) was dissolved in 5 mL of the prepared thiourea solution, and Lys(TFA)-NCA (500 mg) was dissolved in another 5 mL of thiourea solution. Next, the Lys(TFA)-NCA solution was added to the MeO-PEG-NH2 solution in an argon atmosphere. The mixture was reacted at 35°C for 3 days, followed by precipitation in cold ether. The resulting polymer PEG-pLys(TFA) was d 6 -In DMSO, GPC (TOSOH HLC-8220 system, TOSOH, Japan; Column: TSK Gel G4000HHR; Mobile phase: DMF; Flow rate: 0.75 mL / min; Detector: UV 220 nm) and 1 The samples were characterized by 1H-NMR (400MHz, JASCO, Japan).

[0051] Deprotection of the TFA group was performed by dissolving PEG-p(Lys)(TFA) in a 1M NaOH-containing methanol solution and reacting it at 35°C for 12 hours. The mixture was dialyzed against DI water in a 6-8k MWCO membrane for 2 days and then freeze-dried to obtain PEG-p(Lys). PEG-p(Lys) was then analyzed in GPC (JASCXO LC-EXTREMA, JASCO, Japan; column: Superdex 200-10 / 300GL; mobile phase: D-PBS; flow rate: 0.75 mL / min; detector: UV 220 nm) and D2O. 1 The compounds were characterized by 1H-NMR. Next, 100 mg of CDM was dissolved in 8 mL of anhydrous CH2Cl2, and 2 mL of oxalyl chloride was added and the mixture was reacted overnight at room temperature. To remove the remaining oxalyl chloride and CH2Cl2, the mixture was dried under reduced pressure to obtain oily CDM-Cl. Subsequently, CDM-Cl was dissolved again in 2 mL of anhydrous CH2Cl2, and 8 mL of CH2Cl2 containing PEG-p(Lys) (100 mg) was added and the mixture was reacted overnight at room temperature. The synthesis scheme for PEG-p(Lys-CDM) is shown in Figure 1.

[0052] The final product, PEG-p(Lys-CDM), was recovered by precipitation of the mixture in cold ether. PEG-p(Lys-CDM) was analyzed using GPC (JASCXO LC-EXTREMA, JASCO, Japan; column: Superdex 200-10 / 300GL; mobile phase: acetic acid-buffered saline with 10 mM acetic acid and 500 mM NaCl at pH 3.3; flow rate: 0.75 mL / min; detector: UV 220 nm) and d 6 -During DMSO 1 The data was characterized by 1H-NMR. The results are shown in Figures 2-4. Figures 2-4 show that the polymer has a narrow molecular weight distribution and CDM is introduced into the p(Lys) side chain structure.

[0053] 2. Preparation of IL-12-encapsulated micelles (IL-12 / m) (Figure 5-6) IL-12 / m was prepared by pH-controlled titration. To prevent the formation of empty micelles, 10 mg / mL of PEG-p(Lys-CDM) was prepared in pH 5.0 phosphate buffer (20 mM). 40 μg / mL of IL-12 was prepared in pH 8.0 phosphate buffer (20 mM). A 250:1 mass ratio between PEG-p(Lys-CDM) and IL-12 was used to prepare IL-12 / m. The PEG-p(Lys-CDM) solution was added to the IL-12 solution at a strictly controlled rate (2 μL / min). The mixture was then titrated to pH 7.4 by adding pH 8.0 PBS buffer, followed by incubation overnight at 4°C with continuous shaking.

[0054] To determine encapsulation efficiency, a titrated mixture using Alexa-647-labeled IL-12 was loaded into an HPLC (JASCXO LC-EXTREMA, JASCO, Japan) (column: Superdex 200-10 / 300GL, GE Healthcare, USA; mobile phase: pH 7.4 PBS; flow rate: 0.75 mL / min; detector: fluorescence 650 / 665 nm). Encapsulation efficiency was determined by the area ratio between the peak referenced to IL-12 / m and free IL-12. In biological experiments using unlabeled IL-12, encapsulation efficiency was determined by ELISA measurement to detect unencapsulated IL-12 in the mixture.

[0055] Next, this mixture was purified by ultrafiltration using a centrifugal filter (300,000 MWCO) to remove free IL-12. After filtration through a 0.22 μm syringe-driven filtration membrane, the size distribution of IL-12 / m was characterized by dynamic laser scattering (DLS), and the surface charge was determined by zeta potential measurement (Zetasizer Nano-ZS, Malvern, UK). For TEM imaging, IL-12 / m was first dialyzed overnight in DI water (100,000 MWCO) to remove phosphates and NaCl, then stained with phosphotungstic acid (PTA) (2%, w / v), and placed on a 400-mesh copper grid for TEM observation (JEM-1400, JEOL). The results are shown in Figures 5-6. Figures 5-6 show that the prepared micelles encapsulate IL-12 at all concentrations with a high encapsulation rate and are 40 nm sized particles with neutralized surface potential.

[0056] 3. In vitro stability and pH-controlled drug release profile (Figure 7) To measure the in vitro stability of IL-12 / m, micelle samples were prepared and concentrated to contain 100 μg / mL of PEG-p(Lys-CDM) in pH 7.4 PBS buffer. The micelle solutions were then diluted 10-fold in pH 7.4 and pH 6.5 PBS buffers in tubes, respectively. The diluted solutions were monitored at predetermined time points using the DLS method to record the micelle derivative count rate, average size, and polydispersity changes. In the drug release test, IL-12 / m samples containing 10 μg of Alexa-647-labeled IL-12 in 200 μL of PBS (pH 7.4) were dialyzed in 1 L of pH 6.5 or pH 7.4 PBS at room temperature in a dialysis cassette (100,000 MWCO). Samples were taken from the cassette at predetermined time points, and fluorescence intensity was measured using a nanodrop spectrometer to calculate the cumulative release ratio. The results are shown in Figure 7. Figure 7 shows that the prepared micelles are stable at pH 7.4, but unstable at pH 6.5.

[0057] 4. In vitro biological activity assay (Figure 8) In an in vitro bioactivity assay, splenocytes (9 weeks) collected from BALB / c mice were plated in 100 μL of RPMI medium per well (containing 10% FBS, 1× penicillin-streptomycin, 2 mM L-glutamine, and 50 μM 2-mercaptoethanol) in a 96-well plate (10 5 Cells were seeded in wells. Released IL-12 was prepared by diluting 10 μg / mL of IL-12 / m 10-fold in pH 5.0 PBS, incubating overnight, and then further diluting to determine the IL-12 equivalent concentration in pH 7.4 PBS buffer. Cells were then incubated for 24 hours with either natural IL-12, released IL-12, or IL-12 / m (IL-12 equivalent concentration of 0-100 ng / mL). After centrifugation at 500 g × 5 minutes, the culture supernatant was collected, and the IFN-γ concentration was measured by an ELISA kit. The results are shown in Figure 8. Figure 8 shows that the IL-12 released from the micelles retains its activity.

[0058] 5. Preparation of freeze-dried IL-12 / m (Figure 9 and Table 1) For lyophilization, IL-12 / m was first prepared and purified according to the protocol described above. Trehalose (5% of the total weight of the IL-12 / m solution) was added to the final purified solution as a lyophilization protective agent. This mixture was frozen with liquid nitrogen and lyophilized. The lyophilized powder was reconstituted by adding DI water to restore the original solution volume, and then allowed to stand at 4°C for 5 hours. The reconstituted IL-12 / m was loaded into a Zetasizer for DLS and zeta potential measurement, and the free IL-12 concentration in the IL-12 / m solution was detected by ELISA.

[0059] To measure the biological activity of lyophilized IL-12 / m, an in vitro assay using splenocytes was used again. After reconstitution, lyophilized IL-12 / m and released IL-12 were prepared according to previously reported protocols. For comparison, the original, unlyophilized IL-12 / m was prepared. Samples were diluted to the specified equivalent concentrations (0.1–10 ng / mL) and incubated with splenocytes for 24 hours. The supernatant was then collected for ELISA analysis, and the IFN-γ concentration was measured. The results are shown in Figure 9 and Table 1. Figure 9 and Table 1 show that the micelles after freeze-drying have the same physicochemical properties and activity as the micelles before freeze-drying.

[0060] [Table 1]

[0061] 6. Blood circulation (Figure 10) Blood flow profiles were examined under both high-dose (10 μg IL-12 equivalent) and low-dose (1 μg IL-12 equivalent) conditions. The corresponding drug was dispersed in 200 μL of PBS and intravenously (iv) injected into the tail vein of mice. After injection, blood samples were collected from the abdominal aorta of mice (n=3 per group) at predetermined time points and stored in heparinized tubes. The blood was then centrifuged at 1000 g for 10 minutes, and the supernatant was collected as a plasma sample. The plasma sample was diluted to an appropriate concentration range for ELISA measurement (in PBS at pH 7.4), and the released IL-12 concentration was measured.

[0062] To measure the total amount of IL-12 delivered by micelles, the micelles were first disrupted by diluting the sample five-fold in pH 5.0 PBS, incubated overnight at 4°C, and then further diluted in the dilution buffer of the ELISA kit to a concentration range suitable for ELISA measurement. Endogenous IL-12 levels in PBS-injected mice were undetectable by the kit. Plasma half-lives were estimated assuming a one-phase decay model, and the area under the curve (AUC) was calculated using GraphPad Prism software. The results are shown in Figure 10. Figure 10 shows that micelle formation increases the retention of IL-12 in the bloodstream and suppresses the release of IL-12 in the blood.

[0063] 7. In vivo confocal laser scattering microscope (Figure 11) The circulating status of IL-12 / m was tracked by in vivo confocal laser scanning microscopy (IVCLSM) observation of the right earlobe of 8-week-old C57BL / 6 mice. Mice were anesthetized with 2.5% isoflurane and fixed. 10 μg of Alexa647-IL-12 or Alexa647-IL-12 / m was dispersed in 100 μL of PBS and intravenously injected into the mice. Continuous IVCLSM observation was started immediately after injection and continued for 3 hours. After 3 hours, tissue samples were collected from the mice. The tissue samples were observed by IVCLSM to investigate the microdistribution of fluorescence. The results are shown in Figure 11. Figure 11 shows that while IL-12 alone causes migration to normal tissue, which is the cause of side effects, IL-12 / m can suppress extravasation of IL-12.

[0064] 8. Biological distribution (Figure 12-14) The biodistribution profiles were investigated under both high-dose (10 μg IL-12 equivalent) and low-dose (1 μg IL-12 equivalent) conditions. The experiment was conducted in an average volume of 200 mm³. 3 The study was conducted in mice carrying melanoma and TBNC tumors. The drug for injection was dispersed in 200 μL of PBS.

[0065] For high-dose experiments, Alexa Fluor-647-labeled IL-12 was first used in melanoma-bearing mice for fluorescence-based quantification. 10 μg of IL-12 or IL-12 / m equivalent was administered intravenously. Tissue samples (heart, liver, spleen, lung, kidney, and tumor) were collected from the mice 24 hours after injection. The tissue samples were first imaged using the IVIS imaging system (PerkinElmer, USA) and then homogenized using a Dounce tissue homogenizer in pH 7.4 PBS. After centrifugation at 2000 g for 15 minutes, the supernatant was collected, and fluorescence intensity was quantified using a multi-well plate reader (Tecan, Switzerland). Furthermore, to directly quantify the IL-12 distribution, unlabeled IL-12 and IL-12 / m were intravenously injected into TNBC tumor-bearing mice. Tissue samples were collected 24 hours after injection. The tissues were homogenized for protein extraction in a tissue extraction reagent containing a proteinase inhibitor. The sample extracts were centrifuged at 2000 g for 15 minutes, and the supernatant was collected. The released IL-12 and total IL-12 concentrations in the supernatant were detected by ELISA after the corresponding dilution protocol.

[0066] For the low-dose experiment, melanoma-carrying mice were intravenously injected with either unlabeled IL-12 or IL-12 / m (equivalent to 1 μg of IL-12 per injection). Tissue samples were collected from the mice at predetermined time points (1, 4, 8, 24, and 48 hours after injection). The samples were isolated and measured by ELISA according to the same protocol used in the high-dose experiment. The results are shown in Figures 12-14. Figures 12-14 show that IL-12 / m has a higher tumor accumulation rate than IL-12 alone. In addition, accumulation of IL-12 / m was observed in normal tissues such as the liver, but the amount of IL-12 released was limited because the pH within the tissue was normal.

[0067] 9. Systemic and intratumor cytokine secretion (Figures 15-16) Post-treatment systemic and intratumor IFN-γ and IL-10 levels were detected to evaluate systemic and intratumor immune responses. Experiments were also conducted under both high-dose (10 μg IL-12 equivalent) and low-dose (1 μg IL-12 equivalent) conditions.

[0068] For the high-dose experiment, mice with TNBC tumors were intravenously injected with 10 μg of IL-12 or IL-12 / m equivalent on days 0 and 3. 100 μL of blood was collected daily from the orbital vein using heparinized capillaries, and plasma samples were separated after centrifugation at 1000 g for 10 minutes (samples on days 0 and 3 were collected before IL-12 or IL-12 / m injection). On day 7, tumors were collected from the mice. The tumors were then homogenized in tissue protein extraction buffer containing proteinase inhibitor using a Dounce homogenizer. After centrifugation at 2000 g for 15 minutes, the supernatant was collected. IFN-γ and IL-10 concentrations in plasma and tumor samples were measured using an ELISA kit.

[0069] For the low-dose experiment, mice with melanoma were intravenously injected with 1 μg of IL-12 or IL-12 / m² equivalent on day 0. Blood, spleen, and tumor samples were collected from the mice on days 1, 2, and 3. Blood samples were centrifuged at 1000 g for 10 minutes, and the plasma was separated. Tumors and spleens were homogenized in tissue protein extraction buffer containing proteinase inhibitor using a Dounce homogenizer. The supernatant was collected after centrifugation at 2000 g for 15 minutes. IFN-γ and IL-10 concentrations in the samples were measured using an ELISA kit. The results are shown in Figures 15-16. Figures 15-16 show that IL-12 alone increases the expression of the anti-inflammatory cytokine IL-10, while IL-12 / m increases the expression of inflammatory cytokines.

[0070] 10. Systemic toxicity (Figures 17-18) The systemic toxicity of IL-12 and IL-12 / m was evaluated using the same dose schedule as in high-dose experiments for systemic and intratumor cytokine secretion. Five-week-old, tumor-free Balb / c mice were intravenously injected with 10 μg equivalent of IL-12 or IL-12 / m on days 0 and 3. Body weight change curves were plotted against time, and the body weight of each mouse was monitored individually. On day 7, blood and tissue samples were collected from the mice. Blood was centrifuged at 1000 g for 10 minutes, and then the plasma was separated. Total protein (TP) concentration, alanine transaminase (ALT), blood urea nitrogen (BUN), and lipase in the plasma were detected using a DRI-CHEM NX500 blood analyzer (Fujifilm, Japan). TP and ALT were used to evaluate liver damage. BUN was used to evaluate kidney damage, and lipase was used to evaluate pancreatic damage. Fresh liver and kidney samples were collected, and tissue sections were stained with H&E. The results are shown in Figures 17-18. Figures 17-18 show that IL-12 alone is toxic to the liver, pancreas, and kidneys, but its toxicity can be reduced by micellization.

[0071] 11. Treatment of mice (Figures 19-24) In the antitumor activity experiments, different dose schedules were used. IL-12 and IL-12 / m were dispersed in 200 μL of PBS and intravenously injected into mice via the tail vein. 100 μg of anti-PD-1 antibody was dissolved in 100 μL of PBS and intraperitoneally (ip) injected into the right lower abdomen of mice. The results are shown in Figures 19-24. Figures 19-24 show that IL-12 / m has higher antitumor activity than IL-12 alone, and that its combination with immune checkpoint inhibitors can enhance therapeutic efficacy. Furthermore, IL-12 / m exhibits a level of safety comparable to the untreated group.

[0072] 12. Histology (Figures 25-28) Fresh tumor and organ samples were collected from mice, and tissue sections were prepared. For lung samples, the lungs were first immersed overnight in 4% paraformaldehyde (PFA), and then immersed in 30% sucrose solution until the samples were completely submerged. Next, the lung samples were washed twice with an optimal cutting temperature compound (OCT compound) to remove the sucrose solution, then completely immersed in the OCT compound, and subsequently rapidly frozen in liquid nitrogen. For tumors and other organs, fresh samples were directly immersed in the OCT compound and frozen with liquid nitrogen. The frozen tissue samples were sectioned to 10 μm thickness at -30°C using a cryostat (Leica, German).

[0073] For immunohistochemistry (IHC) staining, fresh tissue sections were first washed with PBS to remove OCT compounds and then fixed with 4% PFA for 5 minutes. The nuclei were stained with a 100-fold dilution of Hoechst 33342 solution for 15 minutes, followed by antibody staining. To stain membrane antigens (CD8, NKg2D, PD-L1), antibodies were diluted 50-fold in PBS as a working solution, incubated for 30 minutes, and then added to the tissue sections.

[0074] For staining of intracellular antigens (Tbet and Foxp3), sections were first made permeable using a Foxp3 / transcription factor staining buffer set according to the manufacturer's protocol, and then stained with antibodies using the same method as described above. After staining, sections were washed with DI water for 3 × 5 minutes, dried, and mounted in mounting medium. Sections were observed using a CLSM (LSM-700, Zeiss, German) with a 10x eyepiece. The fluorescence intensity from the labeled antibody in each file was measured using ZEN lite software (v3.3, Zeiss, German), and the number of antibody-positive cells was calculated by dividing the total intensity by the average fluorescence intensity of 10 individual positive cells.

[0075] For histopathological examination, H&E staining was performed. Tissue sections were prepared using the same protocol as described above. The sections were stained with an H&E staining kit according to the manufacturer's instructions. The sections were observed under a light microscope (Zeiss, German). The results are shown in Figures 25-28. Figures 25-28 show that administration of IL-12 / m improved the infiltration of killer T cells, helper T cells, and NK cells, while suppressing the increase of regulatory T cells. Furthermore, PD-L1 expression increased after IL-12 / m administration, revealing the mechanism of synergistic effect with anti-PD-1 antibodies.

[0076] 13. Flow cytometry (Figure 29) Flow cytometry experiments were performed to quantitatively analyze lymphocyte infiltration within tumors. Fresh tumors were collected from mice to prepare the samples. A single-cell suspension was prepared by homogenizing the tumors in PBS (liquid buffer) supplemented with 2% FBS, followed by removal of RBCs via RBC lysis buffer. The cells were resuspended in liquid buffer and filtered through a 100 μm cell strainer. The cell concentration of the suspension was reduced to 10 6The solution was adjusted to 1 / mL and subjected to antibody staining in liquid buffer. The amount of antibody for staining was calculated according to the manufacturer's instructions. Cells were incubated with antibody for 30 minutes, and washed twice by centrifugation at 500g for 5 minutes using liquid buffer, followed by resuspension. Finally, the sample was 10 6 The solution was adjusted to cells / mL and loaded into a flow cytometer (LSR II, BD Bioscience, USA). The results are shown in Figure 29. Figure 29 shows that various T cells, including killer T cells and helper T cells, infiltrate the tumor after administration of IL-12 / m.

Claims

1. A pharmaceutical composition for use in immunotherapy or antitumor therapy, comprising a polymer complex containing a cytokine and a block copolymer represented by the following formula (1), 【Chemistry 1】 [In the formula, R １ R represents a linear or branched alkyl group having 1 to 12 carbon atoms, which may be substituted, and R2 represents a hydrogen atom. R ３ This represents a compound represented by the following formula (Ia) or (Ib), 【Chemistry 2】 L １ is NH or the following formula (II): -(CH) ２ ) ｐ１ -NHH- (II) (In the formula, p1 represents an integer from 1 to 6.) It represents the base indicated by, m1 and m2 each independently represent integers from 0 to 500 (however, the sum of m1 and m2 represents an integer from 10 to 500, excluding m1=0 and m2=0). m3, m4, and m5 each independently represent integers from 1 to 5, and n represents an integer from 1 to 500. The notation " / " indicates that the arrangement order of the (m1 + m2) monomer units shown to the left and right of it is arbitrary. The pharmaceutical composition wherein the cytokine is interleukin 12.

2. The pharmaceutical composition according to claim 1, wherein the cytokine is covalently bonded to a block copolymer represented by formula (1).

3. The pharmaceutical composition according to claim 2, wherein the covalent bond is cleaved in a pH-dependent manner.

4. A freeze-dried composition comprising interleukin 12, a block copolymer represented by formula (1) as defined in claim 1, and an excipient.

5. The freeze-drying composition according to claim 4, further comprising trehalose as a freeze-drying protective agent.

6. The freeze-dried composition according to claim 5, wherein the amount of trehalose is 5% of the total weight of the freeze-dried composition.

Images