Copolymer-drug conjugates for the treatment of tumors - Patent Application 20070122999

Abstract

The present disclosure relates to copolymer-drug conjugates. Specifically disclosed herein are copolymer-drug conjugates with good tissue retention. Also disclosed herein are copolymer-drug conjugates for medical use, such as for the treatment of cancer. Also disclosed herein are methods for producing the copolymer-drug conjugates.

Description

[Technical Field]

[0001] The present invention relates to a conjugate between a copolymer and an anti-cancer drug. The present invention also relates to a method for treating solid cancer tumors and an imaging method using the composition. [Background technology]

[0002] Cancer immunotherapy, which harnesses a patient's own T cells to attack cancer cells, shows promise: the immune system's innate ability to detect and destroy abnormal cells may thwart the development of many cancers.

[0003] However, cancer cells may be able to avoid detection and destruction by the immune system. Cancer cells may reduce the expression of tumor antigens on their surface, making them more difficult for the immune system to detect. This includes expressing proteins on their surface that induce the inactivation of immune cells and / or inducing cells in the microenvironment to release substances that suppress the immune response and promote the growth and survival of tumor cells. Although the characteristics of solid tumors vary considerably, they share several physiological properties that create a distinctive microenvironment for tumor growth and proliferation.

[0004] Toll-like receptors (TLRs) are a class of pattern recognition receptors that play a bridging role in innate and adaptive immunity. TLR activation induces inflammatory responses and contributes to the development of antigen-specific anticancer immunity. TLR7 and TLR8, canonical members of the TLR family, are intracellular receptors expressed on endosomal membranes. TLR7 and TLR8 can be triggered not only by single-stranded RNA during viral infections but also by immune modulators that structurally mimic nucleosides. One example of an artificial TLR agonist is imiquimod, which was first clinically approved for topical administration in 1997. Since then, new iterations of TLR7 and TLR8 agonists, including resiquimod (R848), motolimod (VTX-2337), and others, have been developed, offering improved solubility and more pronounced downstream effects.

[0005] Despite clinical interest and promising results in clinical trials, their clinical application has been limited by narrow therapeutic windows, high systemic toxicity, limited in vivo retention, and difficult manufacturing. Chemical modifications can be used to improve the tolerability and efficacy of such agonists and localize them to the tumor microenvironment, broadening their clinical applicability.

[0006] Developing vaccine strategies that can induce potent antitumor immunity while inducing long-lasting and highly localized tumor-specific effects with minimal systemic side effects is essential for the continued advancement of TLR7 / 8 agonist-based cancer immunotherapy for widespread clinical application. Summary of the Invention

[0007] Disclosed herein is a copolymer-drug conjugate. The copolymer of the present disclosure provides a useful platform for the delivery of drugs to tissues. For example, when administered to tissues by injection, the copolymer-drug conjugate is retained in the tissue for a long period of time. This retention is conferred by the properties of the copolymer.

[0008] Long-term tissue retention is useful for the delivery of drugs that need to act locally on tissues. For example, the drug itself may exhibit systemic toxicity. Therefore, conjugation to the copolymer of the present disclosure reduces systemic exposure of the drug because the drug is retained locally when the conjugate is administered. Improved tissue retention also extends the exposure period of the tissue to the drug, thereby enhancing the effectiveness of the administered drug in the tissue. Improved retention also has the effect that the drug can be metabolized and / or excreted at a much slower rate than if the drug were administered alone.

[0009] The copolymers of the present disclosure are further amphiphilic, which allows for internalization of the conjugates through cell membranes, and thus drug moieties conjugated to the copolymers can exert their effects on either extracellular or intracellular targets.

[0010] In particular, the copolymers of the present disclosure provide a drug delivery platform useful for the treatment of solid tumors through consistent activation of immune cells. This platform is particularly suited for TLR7 / 8 agonists while limiting off-target effects and systemic side effects. Furthermore, by conjugating diagnostic dyes to the copolymers, the copolymer-drug conjugates of the present disclosure enable in vivo imaging, such as imaging of solid tumors.

[0011] One aspect of the present disclosure provides a copolymer-drug conjugate comprising an amphiphilic copolymer and three or more anticancer drug components conjugated to the amphiphilic copolymer. In certain aspects of the present disclosure, the copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer.

[0012] One aspect of the present disclosure provides a copolymer-drug conjugate having the structure of formula (III):

[0013] JPEG2026505989000001.jpg85130 formula: Each R 1 is independently selected from CH3 and H; Each R y are independently H, COOH, and COX-LR 2 is selected from Each R z is independently selected from H and CH3; Each R x are independently selected from:

[0014] JPEG2026505989000002.jpg60123 each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R 2 is independently selected from H or an anticancer drug, 2 at least three of which are anticancer drug moieties, each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

[0015] In one aspect of the disclosure, there is provided a copolymer-drug conjugate having the structure of formula (I):

[0016] JPEG2026505989000003.jpg84132 formula: Each R 1 is independently selected from CH3 and H; each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R 2 is independently selected from H or an anticancer drug, 2 at least three of which are anticancer drug moieties, each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

[0017] The disclosed synthesis method allows for easy functionalization of poly(acrylic acid-co-styrene) (AASTY) copolymers without altering their original properties. Chemical handle moieties are present in the AASTY copolymers, which allows for the functionalization of AASTY copolymers. 9.9Through reaction with the azide moiety on the R terminus of -N3, insertion of, for example, cyanine 7 near-infrared dye for imaging purposes becomes possible. Furthermore, the pendant acrylic acid group of the AASTY copolymer allows for conjugation of a TLR7 / 8 agonist to achieve an antitumor effect in vivo. Thus, one aspect of the present disclosure provides a method for synthesizing the copolymer-drug conjugate of the present disclosure, which method includes conjugating a dye or drug, such as an anticancer drug, to a precursor poly(methacrylic acid-co-styrene) copolymer or precursor poly(acrylic acid-co-styrene) copolymer. Such precursor copolymers may have a (pendant) reactive handle.

[0018] In one aspect of the present disclosure, a pharmaceutical composition is provided comprising the copolymer-drug conjugate of the present disclosure.

[0019] In one aspect of the present disclosure, there is provided a copolymer-drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure for use in medicine.

[0020] The copolymer-drug conjugates of the present disclosure reverse tumor growth and increase overall survival after tumor inoculation. Furthermore, the copolymer-drug conjugates of the present disclosure induce an adaptive response, as reinoculation of the tumor after treatment did not result in tumor growth.

[0021] In one embodiment of the present disclosure, a. styrene and / or diisobutylene, and b. acrylic acid, methacrylic acid, and / or maleic acid The present invention provides the use of a copolymer polymerized from (I) for retaining a drug in tissue. In one embodiment, the copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer.

[0022] The copolymer-drug conjugates of the present disclosure exhibit strong adhesion to the tissue into which they are injected and exhibit reduced diffusion to other tissues over time. [Brief explanation of the drawings]

[0023] [Figure 1] A: General synthetic scheme for the chemical preparation of AASTY copolymers by reaction between styrene and acrylic acid in the presence of a RAFT polymerization agent. The subsequent product is then functionalized with a TLR agonist and / or imaging dye. B: Chart showing the in vitro cellular response to various percentages (0%, 2.5%, 6.5%, and 10%) of conjugated TLR7 / 8a agonist. A direct regression between the observed cellular response and the amount of TLR7 / 8a agonist conjugated was observed, with a maximum signal at 10% of the conjugated agonist. [Figure 2A] Fluorescence imaging studies in two sets of five mouse replicates after subcutaneous injection of free CY7 dye and AASTY-CY7, respectively. For both injections, most of the fluorescent signal remained in the immediate vicinity of the initial injection depot and did not significantly diffuse. Although the integrated signal density slowly decreased over time, the majority of the AASTY-CY7 signal was still clearly visible after two weeks, with no change in distribution. Unlike the free Cy7 control, it was significantly excluded from the injection site during the first few hours. [Figure 2B] Fluorescence signals in isolated organs after intravenous tail vein injection of free CY7 and AASTY-CY7, respectively. For both compounds, as well as the negative control, the signals in the region of interest (ROI) were highest at the injection site (tail) and in the liver, further supporting the slow clearance via the hepatic route. [Figure 3A] Overview of the timeline for tumor inoculation in mice, followed by treatment and tumor re-injection to assess induced resistance. [Figure 3B] Graph showing exponential tumor growth in six mouse replicates without treatment, all of which died within 25 days. [Figure 3C]Graph showing tumor growth timeline for six replicate mice treated with free TLR7 / 8a agonist. A slight improvement in tumor treatment efficacy is observed compared to the control. Two of the six mice recovered, while the other four did not survive. [Figure 3D] Graph showing tumor growth timeline of six mouse replicates treated with AASTY copolymer alone. AASTY delayed tumor growth in one replicate, but none of the mice ultimately achieved complete recovery. [Figure 3E] Graph showing tumor growth timeline for six mouse replicates treated with AAASTY-TRL7 / 8a 1X (low dose). Tumors grew exponentially in five mice, while the remaining mouse in the study experienced a decrease in tumor size and eventually recovered. [Figure 3F] Graph showing tumor growth timeline for six replicate mice treated with AAASTY-TRL7 / 8a 2X (high dose). No tumor growth was observed for the first 20 days, with overall tumor growth rate negative for the first 10 days. Ultimately, four mice recovered from tumors. [Figure 3G] Summary of mouse survival rates after tumor inoculation with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), and negative control. AASTY-TLR7 / 8a 2X achieved the best results, as 80% of the mice survived 60 days after tumor inoculation. [Figure 3H] Time course of mean tumor volume with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), and negative control. AASTY-TLR7 / 8a 2X (high dose) achieves the best outcome, as tumor volume consistently decreases over 10 days. [Figure 3I] Average survival time of mice after tumor inoculation with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), and negative control. The longest survival time, exceeding 100 days, was achieved with AASTY-TLR7 / 8a 2X (high dose). [Figure 3J] Average slope coefficient of tumor growth plot. Meanwhile, tumor growth rate was negative for control, free TLR7 / 8a, AASTY, and AASTY-TLR7 / 8a 1X (low dose); AASTY-TLR7 / 8a 2X (high dose), thus demonstrating antitumor efficacy. [Figure 3K] Graph showing tumor growth timeline of six replicate mice after tumor re-inoculation. After inoculation, no tumor growth was observed in individuals treated with AASTY-TLR7 / 8a 1X (low dose) and AASTY-TLR7 / 8a 2X (high dose). [Figure 3L] Tumor imaging showing the biodistribution of AASTY-TLR7 / 8a-CY7 2X (high dose) after intratumoral administration. The data demonstrate a very long residence time for AASTY-TLR7 / 8a-CY7 2X (high dose), with an estimated half-life of 41 days, and most of the signal was still clearly visible at the end of the study after 117 days (acquired at week 15). [Figure 3M] 3D model showing the biodistribution of AASTY-TLR7 / 8a-CY7 2X (high dose) based on fluorescence tomography scans performed 5-6 days after single-dose administration. AASTY-TLR7 / 8a-CY7 diffuses through the pisiform tumor (signal maximal at the injection point) and accumulates primarily in the liver. [Figure 4] An embodiment of the present disclosure consisting of a copolymer drug conjugate having some styrene moieties, some free acrylic acid moieties, some acrylic acid moieties conjugated to a drug moiety "R", and end groups resulting from RAFT polymerization. [Figure 5]Three-dimensional reconstruction of fluorescence tomography imaging studies in two sets of three mouse replicates after subcutaneous injection of AASTY-Cy7 and MAASTY-Cy7, respectively. Both conjugates exhibited similar biodistribution patterns, with most of the fluorescent signal remaining in the area near the initial injection depot, with only a small proportion spreading into the lymphatic network over time. After 5 days, the majority of the Cy7 signal was still clearly visible for both conjugates, suggesting that the two polymers have similar pharmacokinetic profiles. High fluorescence appears as a solid gray, while low fluorescence appears as a diffuse gray cloud. [Figure 6] Comparison of the structures of AASTY, MAASTY, SMA, and DIBMA. End groups according to the present disclosure are not shown in the structures. All copolymers are composed of both hydrophobic and hydrophilic monomers. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition The term "copolymer" is one of art. It refers to a polymer containing two or more different monomer units that are polymerized in a process called copolymerization. Because copolymers contain at least two different monomer units, they can be classified based on how the monomer units are arranged to form the polymer chain. These classifications include "alternating copolymers" (in which the monomer units repeat in a highly regular alternating pattern), "periodic copolymers" (in which the monomer units are arranged in a repeating sequence), "statistical copolymers" (in which the sequence of the monomer units follows statistical laws), "random copolymers" (in which the monomer units are linked in a random order), and "block copolymers" (in which two or more homopolymer subunits are linked).

[0025] "Amphiphilic copolymer" means a copolymer that includes or consists of both hydrophobic and hydrophilic monomers. The amphiphilic copolymers of the present disclosure are preferably random, alternating, or statistical copolymers with respect to the hydrophobic and hydrophilic monomers. The amphiphilic copolymers of the present disclosure are preferably not block copolymers.

[0026] "Methacrylic acid" refers to the compound having CAS Registry Number 79-41-4.

[0027] "Acrylic acid" refers to the compound having CAS Registry Number 79-10-7.

[0028] "Styrene" means the compound having CAS Registry Number 100-42-5.

[0029] "Maleic acid" refers to the compound having CAS Registry Number 110-16-7.

[0030] "Diisobutylene" means the compound having CAS Registry Number 107-39-1.

[0031] By "copolymer-drug conjugate" is meant a molecular structure consisting of a copolymer chemically attached via a bond, spacer, or linker group to a molecule (other than food) used to prevent, diagnose, treat, or alleviate the symptoms of a disease or abnormal condition (where said condition is cancer). As used herein, a "spacer" or "linker group" is a chemical that connects two or more molecules.

[0032] "Drug" means a compound or moiety used to prevent, diagnose, treat, ameliorate, or alleviate the symptoms of a disease or abnormal condition.

[0033] As used herein, the term "PEG" refers to ethylene glycol polymers of variable length, typically of relative molecular weight between 200 and 8000 Da.

[0034] As used herein, the term "TLR" refers to a single-pass transmembrane receptor that is normally expressed by cells such as macrophages. Activation of TLR receptors activates immune cell responses.

[0035] As used herein, "solid tumor" refers to an abnormally growing mass of cells that affects solid tissues and / or organs, such as the breast or prostate, as opposed to leukemia, a cancer that affects the blood, which is a fluid tissue of the body.

[0036] As used herein, the term "dye" refers to a chemical substance that is colored and chemically bonds to the substrate to which it is applied. As used herein, the term "fluorescent dye" refers to a compound or moiety that can both absorb and emit electromagnetic radiation, for example, in the visible, IR, or UV regions of the electromagnetic radiation spectrum.

[0037] As used herein, "cyanine dye" refers to a closed cyanine dye, i.e., a cyanine dye having a terminal group that is a cyclic moiety, where the cyclic moiety can be aromatic or non-aromatic and can be substituted or unsubstituted at one or more positions. The term "cyanine dye" or "cyanine dyes" refers to any of the cyanine dyes known in the art. Synthetic approaches are disclosed in EP 1,065,250; WO 05 / 014723; WO 99 / 31181; U.S. Patent Nos. 5,268,486, 5,658,751; 5,808,044; 5,981,747; 5,658,751; 4,937,198; 4,937,198; 6,080,868; 6,110,630; 6,225,050; 6,238,838; 6,716,994 and 6,207,464, and U.S. Publication No. 2003 / 0113755.

[0038] As used herein, "triarylmethane dye" refers to a dye incorporated by a scaffold containing three phenyl rings bonded to a common carbon atom, where the phenyl rings are unsubstituted or substituted at one or more positions with the same or different substituents.

[0039] "Indocyanine green" refers to the compound having CAS Registry Number 3599-32-4.

[0040] "Cyanine 7" refers to the compound having CAS Registry Number 477908-53-5.

[0041] "Fluorescein" means the compound having CAS Registry Number 2321-07-5.

[0042] By "thiazine dye" is meant a dye incorporated by at least a thiazine ring, where the thiazine is substituted at one or more positions with the same or different substituents.

[0043] "Methylene blue" refers to the compound having CAS Registry Number 61-73-4.

[0044] As used herein, the term "intratumoral injection" refers to the direct injection of a drug into a tumor, where the drug is a copolymer-drug conjugate such as an AASTY-TLR7 / 8a construct. The intratumoral injection is administered to a living subject in this study, where the subject has colon cancer.

[0045] As used herein, a "reactive handle" is a moiety or functional group on a compound that can react with (e.g., can selectively react with) a moiety or functional group. Examples herein are carboxylic acids or amines (to form amides), or alkynes or azides (to form triazoles), or S N 1 or S N There are haloalkyl groups that can react via two reactions.

[0046] As used herein, the term "immunostimulant" means a substance that stimulates the immune system by inducing or increasing the activity of any of its components.

[0047] Copolymer-drug conjugates of the present disclosure The copolymer of the present disclosure is a synthetic AASTY copolymer. The AASTY copolymer is characterized by increased tissue retention time, for example, after subcutaneous injection. This increased retention time in tissue is observed compared to when a drug is injected / administered without being conjugated to the copolymer of the present disclosure. However, the copolymer-drug conjugate of the present disclosure may also exhibit improved tissue retention compared to other polymer-drug conjugates. Combining the copolymer's good tissue retention properties with a covalently attached therapeutic active moiety, such as an anti-cancer drug, e.g., a TLR7 / 8 agonist, can improve immune cell activation at the injection site, enabling the use of TLR7 / 8 agonists in the treatment of, for example, solid tumors, while limiting off-target effects and side effects associated with systemic exposure.

[0048] In one embodiment of the present disclosure, there is provided a copolymer-drug conjugate comprising an amphiphilic copolymer and one or more anticancer drug moieties conjugated to the amphiphilic copolymer. In a preferred embodiment, the copolymer-drug conjugate comprises two or more anticancer drug moieties. In a more preferred embodiment, the copolymer-drug conjugate comprises three or more anticancer drug moieties. In one embodiment, the anticancer drug moieties are conjugated to the amphiphilic copolymer via a bond or linking group.

[0049] In one embodiment, the amphiphilic copolymer is an anionic amphiphilic copolymer, which may, for example, incorporate carboxylic acid moieties.

[0050] In one embodiment, the amphiphilic copolymer is a copolymer consisting of: 1. styrene and / or diisobutylene, and 2. Acrylic acid, methacrylic acid, and / or maleic acid.

[0051] In preferred embodiments of the present disclosure, the amphiphilic copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer. In further preferred embodiments of the present disclosure, the amphiphilic copolymer is poly(acrylic acid-co-styrene) copolymer or poly(methacrylic acid-co-styrene) copolymer.

[0052] One embodiment of the present disclosure provides a copolymer-drug conjugate having the structure of formula (III):

[0053] JPEG2026505989000004.jpg86132 During the ceremony: Each R 1 is independently selected from CH3 and H; Each R y are independently H, COOH, and COX-LR 2 is selected from Each R z is independently selected from H and CH3; Each R x is, independently, Selected from JPEG2026505989000005.jpg4767, each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R 2 is independently selected from H or an anticancer drug, 2 at least three of which are anticancer drug moieties, each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

[0054] In one embodiment, Rx teeth, JPEG2026505989000006.jpg4634 and R z is H.

[0055] In one embodiment, R x teeth, JPEG2026505989000007.jpg3023 and R z is CH3.

[0056] In one embodiment, R 1 is H and R y COOH or COX-LR 2 is.

[0057] In one embodiment, R 1 is H and R y is H.

[0058] In one embodiment, R 1 is CH3 and R y is H.

[0059] In certain embodiments of the present disclosure, R x teeth JPEG2026505989000008.jpg4434, R z is H and R 1 is H and R y COOH or COX-LR 2 is.

[0060] In certain embodiments of the present disclosure, R x teeth JPEG2026505989000009.jpg4234, R z is H and R 1 is H and R y is H.

[0061] In certain embodiments of the present disclosure, R x teeth JPEG2026505989000010.jpg4132, R zis H and R 1 is CH3 and R y is H.

[0062] In certain embodiments of the present disclosure, R x teeth JPEG2026505989000011.jpg3024, R z is CH3 and R 1 is H and R y COOH or COX-LR 2 is.

[0063] In one embodiment of the present disclosure, there is provided a copolymer-drug conjugate having the structure of formula (I):

[0064] JPEG2026505989000012.jpg81110 is During the ceremony: Each R 1 is independently selected from CH3 and H; each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R 2 is independently selected from H or an anticancer drug, 2 at least three of which are anticancer drug moieties, each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

[0065] In one embodiment, R 1 is CH3. In one embodiment, R 1 is H. In one embodiment, the copolymer drug conjugate of the present disclosure is a poly(methacrylic acid-co-styrene) copolymer or a poly(acrylic acid-co-styrene) copolymer.

[0066] In one embodiment, the copolymer-drug conjugate has the structure of Formula (II):

[0067] JPEG2026505989000013.jpg80114.

[0068] In one embodiment, X is selected from O, S, or NH, for example, wherein X is O, for example, wherein X is NH. When X corresponds to O, the functional group linking the copolymer to the anticancer drug is an ester. When X corresponds to NH, the functional group linking the copolymer to the anticancer drug is an amide.

[0069] In one embodiment, L is a bond. In one embodiment, L is a linking group selected from PEG, alkylene, triazole, and a group formed from the reaction between a haloalkyl and a nucleophile.

[0070] In one embodiment, n and m are each 5 to 10, 10 to 15, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, and / or 450 to 500. In one embodiment, n is 5 to 10, 10 to 15, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500. In one embodiment, m is 5 to 10, 10 to 15, 15 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 120, 120 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, or 450 to 500. In one embodiment of the present disclosure, the ratio n:m is 10:1 to 1:10. In one embodiment of the present disclosure, the ratio n:m is between 10:1 and 9:1, between 9:1 and 8:1, between 8:1 and 7:1, between 7:1 and 6:1, between 6:1 and 5:1, between 5:1 and 4:1, between 4:1 and 3:1, between 3:1 and 2:1, between 2:1 and 3:2, between 3:2 and 1:1, between 1:1 and 2:3, between 2:3 and 1:2, between 1:2 and 1:3, between 1:3 and 1:4, between 1:4 and 1:5, between 1:5 and 1:6, between 1:6 and 1:7, between 1:7 and 1:8, between 1:8 and 1:9, and / or between 1:9 and 1:10.

[0071] In one embodiment of the present disclosure, the copolymer has a molecular weight of less than 100 kDa, for example, less than 90 kDa, for example, less than 80 kDa, for example, less than 70 kDa, for example, less than 60 kDa, for example, less than 50 kDa, for example, less than 40 kDa, for example, less than 30 kDa, for example, less than 20 kDa, for example, less than 15 kDa, for example, less than 14 kDa, for example, less than 13 kDa. In one embodiment, the copolymer has a molecular weight of at least 1 kDa, for example, at least 3 kDa, for example, at least 5 kDa, for example, at least 7 kDa, for example, at least 9 kDa. In one embodiment of the present disclosure, the copolymer has a molecular weight of 1 to 100 kDa, for example, 1 to 5 kDa, for example, 5 to 9 kDa, for example, 9 to 13 kDa, for example, 13 to 17 kDa, for example, 17 kDa to 20 kDa, for example, 20 to 30 kDa, for example, 30 to 40 kDa, for example, 40 to 50 kDa, for example, 50 to 60 kDa, for example, 60 to 70 kDa, for example, 70 to 80 kDa, for example, 80 to 90 kDa, for example, 90 to 100 kDa. In one embodiment, the copolymer has a molecular weight of 1-5 kDa, 5-9 kDa, 9-13 kDa, 13-17 kDa, 17-20 kDa, 20-30 kDa, 30-40 kDa, 40-50 kDa, 50-60 kDa, 60-70 kDa, 70-80 kDa, 80-90 kDa, and / or 90-100 kDa. The mass of the copolymer is preferably evaluated without considering the mass of any drug and / or end groups conjugated thereto. Specifically, when referring to the mass of the copolymer, the moieties T, L, and / or R 2 Refers to the structure of formula (I) except for:

[0072] In one embodiment of the present disclosure, the anti-cancer drug is an anti-tumor drug. As shown in the examples herein, the carboxylic acid groups of the copolymers disclosed herein were used to conjugate TLR7 / 8 agonists and induce an immune response in vivo. The copolymer-drug conjugates shown in the examples remained water-soluble, and a significant amount of their carboxylic acid groups was conjugated to the test drug. This was observed despite the high styrene content in the copolymer. In one embodiment of the present disclosure, the anti-tumor drug is an anti-tumor drug that is effective against solid tumors. In one embodiment of the present disclosure, the anti-cancer drug is a Toll-like receptor (TLR) agonist, such as a TLR7 agonist, a TLR8 agonist, or a TLR7 / 8 agonist. Toll-like receptor agonists, such as TLR7 / 8, stimulate an immune response against cancer cells.

[0073] In one embodiment, R 2 at least three of which are anticancer drug moieties, e.g., R 2 At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 of the monomer units of the copolymer are conjugated to an anticancer drug moiety, e.g., at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%.

[0074] In one embodiment of the present disclosure, the anticancer agent has the structure of Formula (AI), Formula (A-II), Formula (A-III), or Formula (A-IV): JPEG2026505989000014.jpg93163, JPEG2026505989000015.jpg121160, JPEG2026505989000016.jpg63116, JPEG2026505989000017.jpg83156; During the ceremony, X 1a is -O-, -S-, or -NR Ca and R 1ais hydrogen, (C 1-10 ) alkyl, substituted (C 1-10 ) Alkyl, C 6-10 Aryl or substituted C 6-10 Aryl, C 5-9 Heterocyclic, substituted C 5-9 is heterocyclic, R Ca is hydrogen, (C 1-10 ) Alkyl or substituted C 1-10 alkyl, or R Ca and R 1a together with the nitrogen atom to which they are attached form a heterocyclic ring or a substituted heterocyclic ring, Each R 2a are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 ) Aryl (aroyl), substituted -C(O)-(C6-C 10 )aryl, -C(O)OH (carboxyl), -C(O)O(C1-C6)alkyl (alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -NR aa R ba , -C(O)NR aa R ba (carbamoyl), halo, nitro, or cyano, or R 2a does not exist; Each R aa and R ba are independently hydrogen, (C1-C6)alkyl, substituted (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted (C3-C8)cycloalkyl, (C1-C6)alkoxy, substituted (C1-C6)alkoxy, (C1-C6)alkanoyl, substituted (C1-C6)alkanoyl, aryl, aryl(C1-C6)alkyl, Het, Het(C1-C6)alkyl, or (C1-C6)alkoxycarbonyl; wherein the substituents on any alkyl, aryl, or heterocyclic group are hydroxy, C 1-6 Alkyl, hydroxy C1-6 Alkylene, C 1-6 Alkoxy, C 3-6 Cycloalkyl, C 1-6 Alkoxy C 1-6 alkylene, amino, cyano, halo, or aryl; j is 0, 1, 2, 3 or 4; X 3a is -N- or -CH-, R 4a is -CH2- or -CH(R 2a )- and k is 0 or 1, X 4a -O-, -S-, -NH-, -N(R da )-, -CH2-, or -CH(R 2a )- and Each R da are independently -OH, (C1-C6) alkyl, substituted (C1-C6) alkyl, (C1-C6) alkoxy, substituted (C1-C6) alkoxy, -C(O)-(C1-C6) alkyl (alkanoyl), substituted -C(O)-(C1-C6) alkyl, -C(O)-(C6-C 10 ) Aryl (aroyl), substituted -C(O)-(C6-C 10 )Aryl, -C(O)O(C1-C6)alkyl(alkoxycarbonyl), substituted -C(O)O(C1-C6)alkyl, -C(O)NR aa R ba (carbamoyl), or a tautomer thereof.

[0075] In one embodiment of the present disclosure, the anticancer agent has the structure of formula (BI): JPEG2026505989000018.jpg57132, During the ceremony, R 1b is (C 1-8 ) alkyl, (C 3-8 ) cycloalkyl or a 3- to 8-membered saturated heterocyclic group containing an O atom, 1b is halogen, cyano, hydroxyl, and (C 1-3) optionally substituted with one or more substituents independently selected from alkoxy; Z 1b is (C 2-6 ) alkylene group, wherein Z is not adjacent to a nitrogen atom 1b may be replaced by an oxygen atom; X 1b is NR 5b ,>N-COR 5b ,>N-CONR 5b R 5ab ,CONR 5b , N.R. 5b CO, NR 5b CONR 6b , or NR 6b CONR 5b represents Y 1b is a single bond or (C 1-6 ) alkylene; Each R 2b is halogen, cyano, hydroxy, thiol, (C 1-3 ) alkyl, (C 1-3 ) hydroxyalkyl, (C 1-3 ) haloalkyl, (C 1-3 ) alkoxy, (C 1-3 ) haloalkoxy, (C 1-3 ) alkylthio, (C 1-3 ) alkylsulfonyl, and (C 1-3 ) alkylsulfinyl; R 3b is (C 1-6 ) C optionally substituted with alkoxy 1-6 represents alkyl, Each R ab is halogen, cyano, hydroxy, thiol, (C 1-3 ) alkyl, (C 1-3 ) hydroxyalkyl, (C 1-3 ) haloalkyl, (C 1-3 ) alkoxy, (C 1-3 ) haloalkoxy, (C 1-3 ) alkylthio, (C 1-3 ) alkylsulfonyl, and (C 1-3) alkylsulfinyl; R 5b and R 5ab are each independently hydrogen, a ring group O, S(O) p Or NR 10b 3-8 membered saturated heterocyclic rings containing (C 1-6 ) alkyl group or (C 3-6 ) cycloalkyl groups, the latter two groups being NR 7b R 8b or R 9b and optionally substituted by one or more substituents independently selected from R 7b and R 8b each independently represents hydrogen, and the 3- to 8-membered saturated heterocycle is a ring group O, S(O) p or NR 10ab , (C 1-6 ) alkyl or (C 3-6 ) cycloalkyl, the latter two groups being halogen, cyano, S(O) q R 11b , OR 12b , CO2R 12b ,OC(O)R 12b , SO2NR 12b R 13b CONR 12b R 13b , N.R. 12b R 13b , N.R. 12b SO2R 14b , N.R. 12b COR 13b , or ring group O, S(O) p or NR 10bb and optionally substituted with one or more groups independently selected from a 3- to 8-membered saturated heterocycle comprising or R 7b and R 8b together with the nitrogen atom to which they are attached form a 3-8 membered saturated heterocycle containing a ring nitrogen atom and optionally one or more additional heteroatoms independently selected from nitrogen, oxygen, sulfur, and sulfonyl, wherein the heterocycle is substituted with any of halogen, cyano, S(O) q R 15b , OR 15b , CO2R 15b , C.O.R.15b ,OC(O)R 15b , SO2NR 15b R 16b ,CONR 15b R 16b , N.R. 15b R 16b , N.R. 15b SO2R 17b , N.R. 15b COR 16b , N.R. 15b CO2R 16b , heteroaryl, (C 1-6 ) haloalkyl, (C 3-8 ) cycloalkyl, and (C 1-6 ) alkyl, the latter two groups being optionally substituted by one or more substituents independently selected from cyano, S(O) q R 18b , OR 18b , CO2R 18b , SO2NR 18b R 19b ,CONR 18b R 19b or NR 18b R 19b optionally substituted with one or more groups independently selected from R Qb are halogen, cyano, CO2R 20b , S(O) q R 20b , OR 20b , SO2NR 20b R 22b ,CONR 20b R 22b , N.R. 20b SO2R 21b , N.R. 20b CO2R 21b , N.R. 20b COR 22b , or the ring group NR 10cb represents a 3- to 8-membered saturated heterocycle containing R 10b , R 10ab , R 10bb and R 10cb are independently hydrogen, CO2R 23b , S(O) q R 23b , C.O.R. 24b , or (C1-6 ) alkyl, (C 2-6 ) alkenyl, (C 2-6 ) alkynyl or (C 3-8 ) cycloalkyl groups, each of which is selected from halogen, cyano, OR 25b or NR 25b R 26b and optionally substituted by one or more substituents independently selected from R 6b , R 11b , R 12b , R 13b , R 15b , R 16b , R 18b , R 19b , R 20b , R 22b , R 24b , R 25b and R 26b are each independently hydrogen, (C 1-6 ) alkyl or (C 3-6 ) represents cycloalkyl, R 14b , R 17b , R 21b and R 23b are each independently (C 1-6 ) alkyl or (C 3-6 ) represents cycloalkyl, h, i, p, and q each independently represent the integer 0, 1, or 2; and Ab is a monocyclic or bicyclic (C 6-10 ) aryl, or monocyclic or bicyclic rings containing 1 to 3 heteroatoms (C 5-12 ) represents a heteroaryl group, and R bb and R cb are independently hydrogen or (C 1-6 ) alkyl, or R bb and R cb are bonded together (C 3-8 ) forming a cycloalkyl.

[0076] In one embodiment of the present disclosure, the anticancer agent has the structure of formula (CI): JPEG2026505989000019.jpg85127, During the ceremony, R 1c is -(C 2-6 ) alkyl-N(R 3c )2, -(C 2-6 )Alkyl-NR 3c -SO2-X c -R 4c , and -(C 2-6 )Alkyl-NR 6c -SO2-R 7c , X c is a bond or -NR 5c - and R 4c is alkyl, aryl, or heteroaryl; R 2c is hydrogen, alkyl, alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl-O-aryl, alkyl-O-alkyl, alkyl-O-alkenyl, and OH, halogen, -N(R 3c )2, -CO-N(R 3c )2, -CO-(C 1-10 ) alkyl, -CO-O-(C 1-10 ) selected from the group consisting of alkyl, -N3, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, -CO-aryl, -CO-(substituted aryl), -CO-heteroaryl, and -CO-(substituted heteroaryl); Each R 3c is hydrogen and (C 1-10 ) alkyl; R 5c is hydrogen and (C 1-10 ) alkyl, or R 4c and R 5c can be combined to form a 3- to 7-membered heterocyclic ring or substituted heterocyclic ring, R 6c is hydrogen and (C 1-10 ) alkyl; R 7c is hydrogen and (C1-10 ) alkyl, or R 6c and R 7c can be combined to form a 3- to 7-membered heterocyclic ring or substituted heterocyclic ring, r is 0 to 4, and each R c is (C 1-10 ) alkyl, (C 1-10 ) independently selected from the group consisting of alkoxy, halogen, and trifluoromethyl.

[0077] In one embodiment of the present disclosure, the anti-cancer drug is TLR7, TLR8, or the TLR7 / 8 agonist is imiquimod, resiquimod, gardikimod, 852A, loxoribine, bropirimine, 3M-011 (CAS No. 642473-62-9), 3M-052 (CAS No. 1359993-59-1), DSR-6434 (CAS No. 1059070-10-8). The TLR7 agonist is selected from the list consisting of DSR-29133, SZU-101, SM-360320 (CAS No. 226907-52-4), SM-276001 (CAS No. 473930-22-2), VTX-2337 (CAS No. 926927-61-9), and 1-(3-aminopropyl)-2-(ethoxymethyl)imidazo[4,5-c]quinolin-4-amine. Shukla et al. (2011) describes a SAR test for TLR7 agonists that can be used to predict where binding can occur without inhibiting agonist activity.

[0078] In one embodiment of the present disclosure, the anti-cancer agent is a stimulator of interferon genes (STING) agonist. In one embodiment, the anti-cancer agent is IL-2, IL-12, or IL-15, or a fragment thereof.

[0079] In one embodiment of the present disclosure, the anti-cancer agent is a radiopharmaceutical or radioactive drug.

[0080] In one embodiment, the copolymer drug conjugate comprises one type of anticancer drug. In one embodiment, the copolymer drug conjugate comprises two or more different anticancer drugs.

[0081] In one embodiment of the present disclosure, R 2 is an anticancer drug, L is a linking group, and 2 is a carbon atom, or R 2 In one embodiment of the present disclosure, R 2 is an anticancer drug, L is a linking group, and 2 is a carbon atom, or R 2 The heteroatom, such as N, O, or S, in the aryl group is conjugated to L through a bond to X.

[0082] In one embodiment, T is independently selected from the group consisting of H, alkyl, substituted alkyl, nitrile, hydroxy, carboxyl, halogen, thiol, substituted thiol, acyl, substituted acyl, a group of formula XI, a group of formula YI, a group of formula Y-II, a fluorescent dye, and a diagnostic agent.

[0083] wherein the group of formula XI has the following structure: JPEG2026505989000020.jpg2435 In the formula, R 3 and R 4 are each independently selected from hydrogen, alkyl, and substituted alkyl; wherein the group of formula YI has the following structure: JPEG2026505989000021.jpg2725 In the formula, R 5 is selected from hydrogen, alkyl, and substituted alkyl; wherein the group of formula Y-II has the following structure: JPEG2026505989000022.jpg3050 wherein R5 is selected from hydrogen, alkyl, and substituted alkyl; X 2 and X 3 are each independently selected from S and O.

[0084] In one embodiment of the present disclosure, the fluorescent dye is selected from cyanine dyes such as indocyanine green or cyanine 7, triarylmethane dyes such as fluorescein, methylene blue, LUM015, VGT-309, AVB-620, C-Dots, BLZ-100, and other triarylmethane dyes.

[0085] In one embodiment of the present disclosure, there is provided a pharmaceutical composition comprising the copolymer-drug conjugate of the present disclosure. In one embodiment of the present disclosure, the pharmaceutical composition is formulated for administration by injection, such as intratumoral injection.

[0086] Synthesis method In one embodiment of the present disclosure, the copolymer drug conjugate is synthesized by conjugating an anticancer drug to a precursor copolymer, such as, for example, a precursor poly(methacrylic acid-co-styrene) copolymer, a precursor poly(acrylic acid-co-styrene) copolymer, a precursor poly(maleic acid-co-styrene) copolymer, or a precursor poly(diisobutylene-co-maleic acid) copolymer.

[0087] In one embodiment of the present disclosure, the precursor copolymer has the structure of formula (III-P): JPEG2026505989000023.jpg77132, In the formula, R x’ teeth, JPEG2026505989000024.jpg4635 or JPEG2026505989000025.jpg3323, wherein each L' is independently selected from H and a reactive handle; T, n, m, R 1 , X, R z , and R y is as defined herein.

[0088] In one embodiment of the present disclosure, the precursor poly(methacrylic acid-co-styrene) copolymer or the precursor poly(acrylic acid-co-styrene) copolymer has a structure of formula (IP):

[0089] JPEG2026505989000026.jpg78121, wherein each L' is independently selected from H and a reactive handle; T, n, m, R 1 and X is as defined herein.

[0090] In one embodiment of the present disclosure, in the formula, each L' is independently selected from the group consisting of H, CH2Cl, CH2Br, or CH2I.

[0091] treatment In one embodiment of the present disclosure, a copolymer-drug conjugate or a pharmaceutical composition of the present disclosure is provided for use in medical treatment. The copolymer of the present disclosure is amphiphilic, which allows the conjugate to be internalized by permeation of the cell membrane. Thus, the drug moiety conjugated to the copolymer can exert its effect on either extracellular or intracellular targets. In one embodiment of the present disclosure, a copolymer-drug conjugate or a pharmaceutical composition of the present disclosure is provided for use in the treatment of cancer.

[0092] In one embodiment, the present disclosure provides a copolymer-drug conjugate or a pharmaceutical composition for use in treating cancer. In a preferred embodiment, the cancer is characterized by the presence of a tumor. In one embodiment, the tumor is a solid tumor. In one embodiment, the tumor, such as a solid tumor, is a sarcoma. In one embodiment, the tumor, such as a solid tumor, is a carcinoma. In one embodiment, the solid tumor is a primary or metastatic tumor, or a secondary metastatic tumor.

[0093] In one embodiment, the cancer is liver cancer or spleen cancer.

[0094] In one embodiment, the copolymer drug conjugate or pharmaceutical composition is administered intratumorally, such as by injection.

[0095] One embodiment of the present disclosure provides a method of treating cancer, such as a cancer characterized by a tumor, such as a solid tumor, comprising administering to a subject in need thereof a copolymer drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure.

[0096] One embodiment of the present disclosure provides a method of eliciting an immune response in a subject, the method comprising administering to the subject a copolymer drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure.

[0097] One embodiment of the present disclosure provides a method of immunization in a subject, the method comprising administering to the subject a copolymer drug conjugate described herein or a pharmaceutical composition of the present disclosure.

[0098] One embodiment of the present disclosure provides a method for reducing tumor volume in a subject, the method comprising administering to the subject a copolymer drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure.

[0099] One embodiment of the present disclosure provides the use of the disclosed copolymer-drug conjugate or the disclosed pharmaceutical composition as an immunostimulant.

[0100] One embodiment of the present disclosure provides the use of a copolymer drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for use in the treatment of cancer.

[0101] One embodiment of the present disclosure provides the use of poly(acrylic acid-co-styrene) copolymer or poly(methacrylic acid-co-styrene) copolymer to retain a drug. By "retaining a drug" is meant increasing tissue retention of the compound / drug when administered to a subject as a result of mixing, formulating, or conjugating the compound / drug with the poly(acrylic acid-co-styrene) copolymer or poly(methacrylic acid-co-styrene) copolymer. In one embodiment, the drug is an anti-cancer drug, diagnostic agent, and / or radiopharmaceutical. In one embodiment, the drug is an anti-cancer drug disclosed herein. In one embodiment, the drug is an agent capable of inducing an immune response.

[0102] One embodiment provides a method for increasing the residence time of a drug in a tissue, the method comprising administering the drug together with a copolymer of the present disclosure. The tissue can be cancer tissue or tissue surrounding cancer tissue. In one embodiment of the method, the drug is conjugated to the copolymer of the present disclosure, such as by conjugation via a bond or a linking group. [Example]

[0103] Example 1: Synthesis of copolymers and copolymer-dye conjugates Materials and Methods The copolymer was prepared using the RAFT agent 2-cyano-2-propyldodecyltrithiocarbonate (copolymer AASTY 12.5 ), or 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester (copolymer AASTY 9.9 The synthesis was carried out using either acrylic acid (AA) or styrene (STY) (N3), the initiator azobisisobutylnitrile (AIBN), and the monomers acrylic acid (AA) and styrene (STY) in an initial molar ratio of 45:55 (AA:STY). The reagents were placed in a Schlenk flask, and oxygen was removed via four freeze-pump-thaw cycles. The mixture was heated to 60 or 70 °C for 9 to 12 hours, and the AA:STY was obtained. 12.5 At 95% monomer conversion, AASTY 9.9-N3 reached 69% monomer conversion. The product was dissolved in diethyl ether, precipitated in heptane, and dried in vacuo to give a yellow crispy solid. 12.5 For the polymers, the polymers were solubilized in 30% H2O2 (water:ethanol) (1:3) and incubated overnight at 70 °C to remove the dodecyltrithiocarbonate (ttc) end groups, resulting in a colorless solution. The polymers were precipitated in deionized water and collected by centrifugation. All final polymer products were converted to partial sodium salts by solubilizing in deionized water with the addition of NaOH (1 M) until the pH reached 7-7.5. The opaque mixtures were filtered and lyophilized. The number-average molecular weight (M n ), weight average molecular weight (M w ), and dispersity (D=M n / M w ) was measured using a Dionex Ultimate 3000 system. Detection was performed with a Dawn Heleos II multi-angle light scattering detector and an Optilab rEX refractive index detector. Gel permeation chromatography (GPC) was performed on a Superose 6 Increase column (10 / 300, Cytiva). Data were analyzed on an Astra 7.0 using a dn / dc of 0.170 mL / g.

[0104] The polymer was dissolved in methanol at 50 mg / mL, and an equimolar amount of cyanine 7-DBCO (a NIR fluorescent dye containing a cycloalkyne moiety for conjugation with azides—Lumiprobe GmbH) was added, and the reaction was left overnight at room temperature. The methanol was evaporated using a nitrogen stream, and the solid was dissolved in water. The dye-conjugated polymer was purified by size-exclusion column chromatography and lyophilized to yield a deep green solid.

[0105] result All products were analyzed by SEC-MALS coupled to a UV detector. Although the Cy7 dye absorbs the MALS laser, preventing mass determination, the elution time of the polymer is identical to that of the unconjugated polymer. The copolymers and conjugates produced are listed in Table 1.

[0106] JPEG2026505989000027.jpg119159Conclusion AASTY polymers were fabricated using azide RAFT agents, allowing for "click" conjugation to the R terminus of the RAFT agent.

[0107] Example 2: Synthesis of copolymers conjugated to TLR agonists Materials and Methods AASTY copolymer was added to the TLR7 / 8 agonist 4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-propanamine (referred to as "TLR7 / 8a") in DMF at 5%, 10%, or 15% molar equivalents of AA content. The majority of the agonist was solubilized by gentle heating, and 2 molar equivalents of NHS (relative to TLR7 / 8a) and 1 molar equivalent of N,N'-diisopropylcarbodiimide were added to the sample. The reaction was left at room temperature overnight, and the next day the product was purified by size-exclusion chromatography and lyophilized to yield a white solid.

[0108] A fraction of the conjugate solubilized in methanol with 1 molar equivalent of cyanine 7-DBCO. The reaction was left at room temperature overnight, and the product was purified by size exclusion chromatography and lyophilized to give a dark green solid.

[0109] result 1 H NMR characterization confirmed and quantified the conjugation of TLR7 / 8a via its aminopropyl group to the AA carboxylic acid of the copolymer.

[0110] Example 3: Copolymer constructs persist in tissues for exceptional duration in vivo Materials and Methods A fluorescent dye-containing AASTY construct (AASTY-Cy7) was administered to healthy mice via different routes of administration, and biodistribution was monitored over a two-week period.

[0111] result AASTY of Example 1 9.9After subcutaneous (SC) injection of AASTY-Cy7, most of the fluorescent signal remained in the area near the initial injection depot and did not appear to significantly diffuse after the formulation was absorbed by SC tissue (Figure 2A). Although the integrated signal density slowly decreased over time, the majority of the signal was still clearly visible after 2 weeks, with no change in distribution. Unlike the free dye control, this was largely eliminated by renal excretion over the first few hours after administration. This suggests that the AASTY-Cy7 conjugate binds strongly and rapidly to local tissues for an extended period of time. Image analysis estimated the compound's half-life to be 58 hours, which is remarkable for a water-soluble material. To rule out the possibility that the Cy7 dye could be cleaved from the AASTY conjugate and mislead our interpretation, an equivalent dose of the DBCO-Cy7 dye in the form used for conjugation was solubilized in 5% DMSO-DPBS and injected SC in a similar manner. The control experiment showed that the free dye, despite not being eliminated by renal excretion, was rapidly metabolized at the injection site, with most of the signal disappearing after one week, confirming that the conjugate exhibited a different PK profile from the free form of the dye. Given that no significant signal was detected outside the injection area in vivo, mice were dissected at the end of two weeks to extract organs and analyze signal distribution with greater sensitivity. Images of isolated organs revealed that the signal emanated from a spot on the penile lymph node on the injection side, with residual signal also visible within the liver. This suggests that the compound was slowly eliminated via the liver and lymphatic pathways. No signs of toxicity or inflammation were observed during the study, suggesting that the copolymer itself (i.e., not conjugated to any drug) was not significantly toxic or immunogenic. Following intravenous (IV) injection into the tail vein, the compound rapidly reached the systemic circulation, as evidenced by signals emanating from the paws, nose, and eyes during the first few hours after injection. As with SC injection, a significant portion of the signal remained near the injection point (tail) and, after stabilization, did not diffuse for the entire duration of the study, where significant signal was visible in the liver from the initial acquisition time through the end of the 2-week period.Imaging analysis of the liver region revealed a short half-life of the compound, approximately 17 hours, although the signal was still visible after 2 weeks. Post-mortem organ extraction and analysis (including the mouse tail) confirmed significant accumulation of the compound in the liver and also demonstrated a small but significant accumulation in the spleen, which was not observed with SC administration (Figure 2B). However, no signal was reported in the inguinal lymph nodes or kidneys. Again, no signs of toxicity or inflammation were reported.

[0112] These two studies combined suggest that AASTY has the ability to deeply adhere to the local tissues into which it is injected and stain them for a surprisingly long period of time. Furthermore, the data also suggest that a portion of it is slowly eliminated over time via the liver and lymphatic pathways. Control experiments also indicate that the dye may not be cleaved from AASTY during its in vivo retention. Furthermore, given the compound's good tolerance in mice and its apparent lack of immunogenicity, AASTY conjugates are ideal candidates for tissue staining or local drug exposure.

[0113] Example 4: Copolymer conjugates show good retention within solid tumors in mouse models Materials and Methods Healthy Balb / cJRj mice were subcutaneously injected with 100 μL of mouse colon cancer CT26 cancer cells (3 × 10 cells) in RPMI medium into the right flank. The volume was 50–200 mm. 3 Tumor-bearing mice were randomized and relocated into new cages by blocking the initial tumor volume, and each cage received one of the four treatments listed in Table 2, administered intratumorally (50 μL). For each treatment, a total of three injections were administered, 7 days apart. Table 2 provides an overview of the treatments tested, and Figure 3A provides an overview of the study.

[0114] Eighty-three days after the first tumor inoculation, surviving mice were re-immunized with a second inoculation of CT26 cancer cells (at the same dose) in the left flank, along with the control group receiving the first inoculation. Mice were monitored for weight, their tumors were measured twice weekly using electronic calipers, and fluorescence imaging was performed periodically for all formulations containing Cy7 moieties. Necropsies were performed within 1 week after euthanasia (stored at -20°C during this time), and tumors were excised, including both negative lymph nodes. The mean signal for each treatment group for each organ was aligned, corrected for first-order decay based on the half-life estimated from in vivo scans (taking into account that all mice were euthanized at different times during the study). Compound biodistribution was further assessed by injecting a single dose of AASTY-TLR7 / 8a 2X (50 μL, intratumorally) and scanning mice by fluorescence tomography (MILabs U-CT in FLT mode). Scans were performed 5-6 days after euthanasia with mice stored at -20°C between euthanasia and scanning. Reconstructed images were analyzed and 3D rendered using Imalytics Preclinical 2.1. Tumor volume was measured (length x width) throughout the study. 2 / 2, and the tumor volume is 2000 mm 3 Mice were euthanized when they reached 1. Humane endpoints included general signs of failure, significant weight loss (15% of initial weight or 10% weight loss overnight), or the presence of a tumor wound greater than 8 mm.

[0115] result The intratumoral administration data demonstrated a very long residence time for the AASTY conjugate, with an estimated half-life of 41 days, and most of the signal was still clearly visible at the end of the study after 117 days (acquired at week 15) (Figure 2L). A large amount of the compound was retained in the tumor (where it was injected) and distributed throughout most of its volume. In cured mice, the signal remained in the area adjacent to the tumor scarring, suggesting that the compound redistributed to surrounding tissues as tumor cells were eliminated. As with IV administration, corrected data from autopsy revealed that a large amount of signal was also observed in the liver, and that a small amount of the AASTY-TLR7 / 8a conjugate reached the spleen. While none was detected in the kidney or left lymph node (on the opposite flank from the tumor), a significant signal was detected at a very high AASTY-TLR7 / 8a dose in the right lymph node (on the same flank as the tumor) with the AASTY formulation.

[0116] conclusion AASTY conjugates exhibit a high residence time after intratumoral injection, exceeding the half-life of both SC and IV injections. The low biodistribution to the kidney indicates that the disclosed conjugates may have fewer kidney-related adverse effects than other chemotherapeutics that can damage the kidneys, ureters, and bladder.

[0117] Example 5: TLR7 / 8a-copolymer conjugates reduce solid tumor volume and improve survival in mouse models Materials and Methods Materials and methods were as outlined for Example 3.

[0118] result Tumor kinetics monitoring showed that most tumors rapidly grew exponentially after the start of the study in all mouse groups (Figures 2B-2E) except for the high AASTY-TLR7 / 8a dose treatment group (Figure 2F). Here, we evaluated the effects of two parameters: TLR7 / 8a dose (including non-lethal high doses within the range expected to induce an immune response and half doses below that range) and AASTY dose (maintained at a constant ratio to TLR7 / 8a). First, the results for the AASTY and AASTY-TLR7 / 8a 1X treatment groups suggest that AASTY itself does not exhibit antitumor activity at both doses. TLR7 / 8a alone injected intratumorally at a high dose showed a potential slight improvement in tumor treatment compared to the control, considering that two of six mice recovered, while the remaining four mice did not respond to treatment. These results are interpreted as an effect of the extremely high potency of these compounds, as opposed to the rapid diffusion of the compounds from the tumor microenvironment after injection. The copolymer-drug conjugates of the present disclosure were designed to provide sustained local retention. No significant improvement was observed with AASTY-TLR7 / 8a 1X. Meanwhile, with AASTY-TLR7 / 8a 2X, no tumor growth was observed during the first 20 days, and the overall tumor growth rate was negative during the first 10 days (Figures 2F, 2H, and 2J). Overall survival was generally higher compared to the low-dose treatment, with only two of six mice showing tumor growth after 20 days. The remaining four mice recovered completely (Figures 2G and 2I).

[0119] conclusion TLR7 / 8a-copolymer conjugates reduce solid tumor volume in mouse models and improve survival at appropriate doses.

[0120] Example 6: TLR7 / 8a-copolymer conjugates prevent tumor growth in a mouse model tumor challenge study Materials and Methods Eighty-three days after the first tumor inoculation, surviving mice were re-immunized with a second inoculation of CT26 cancer cells (same dose) in the left flank along with the control group receiving the first inoculation. Mice were monitored for weight, their tumors were measured twice weekly using electronic calipers, and fluorescence imaging was performed periodically for all formulations containing the Cy7 moiety.

[0121] result When surviving mice were re-immunized with tumor cells 83 days after the initial inoculation, no tumor growth was observed (Fig. 2K).

[0122] conclusion Mice that survive this study have an adaptive response and are resistant to CT26.

[0123] Example 7: Copolymers are retained in injected tissues and are slowly distributed in small amounts through the lymphatic system in a mouse model Materials and Methods To gain better insight into the biodistribution kinetics after intratumoral administration, fluorescence tomography scans were performed 5–6 days after a single dose of AASTY-TLR7 / 8a 2X was administered to a small group of mice.

[0124] result The compound diffused throughout all bean-shaped tumors, with the signal maximal at the injection point and mostly accumulated in the liver (Fig. 2M). The new information provided by this study is that the compound was clearly distributed throughout the lymphatic system, with signals detectable in nodes of higher intensity (especially the mandible, neck, axilla, upper arm, lumbar, and coccyx) that more or less corresponded to known lymph node locations.

[0125] conclusion This and other examples disclosed herein suggest the following possible mechanism of biodistribution: after injection, the copolymer / copolymer conjugate rapidly adheres to local tissues, possibly by incorporating itself into lipid membranes or cells, possibly exploiting the amphiphilic properties of the copolymer. When antibodies are injected IV or into highly vascularized tissues, some of them enter the systemic circulation and rapidly accumulate, primarily in the liver and, to a lesser extent, in the spleen.

[0126] Example 8: Copolymer MAASTY exhibits a similar biodistribution profile to AASTY Materials and Methods Poly(methacrylic acid-co-styrene) (MAASTY) was synthesized using the RAFT agent 2-cyano-2-propyldodecyltrithiocarbonate 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester, the initiator azobisisobutylnitrile (AIBN), and the monomers methacrylic acid (MAA) and styrene (STY) in an initial molar ratio of 50:50 (MAA:STY) as described in Example 1. As an alternative to using a Schlenk flask, the reagents were filled into a glass ampoule, oxygen was removed via four freeze-pump-thaw cycles, and the ampoule was sealed under vacuum with a torch. The mixture was heated at 55 °C for 16 h, reaching an estimated monomer conversion of 85% (estimated based on viscosity). The product was dissolved in diethyl ether, precipitated into heptane, and dried in vacuo to yield a yellow, crispy solid.

[0127] The copolymer was then dissolved in methanol at 25 mg / mL, an equimolar amount of cyanine 7-DBCO (same as in Example 1) was added, and the reaction was left at room temperature overnight. The methanol was evaporated using a stream of nitrogen, and the solid was dissolved in water. The dye-conjugated polymer was purified by size-exclusion column chromatography and lyophilized to yield a deep green solid.

[0128] As a reference, a new batch of AASTY-Cy7 was prepared using the RAFT agent 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid 3-azido-1-propanol ester (45:55) AA:STY) following a similar protocol as in Example 1, except that AASTY was prepared using a sealed ampoule instead of a Schlenk flask. After heating the mixture at 60°C for several hours, 85% conversion was achieved ( 1 H-NMR measurement), copolymer AASTY 7.1 (M n =7.1kDa, 1 For MAASTY, the copolymer was then conjugated to Cy7 at 25 mg / mL of copolymer, and the AASTY 7.1 -Cy7 was obtained.

[0129] The two Cy7 copolymer constructs were administered subcutaneously to mice. All mice bearing palpable CT26 tumors were injected into the lateral flank, immediately adjacent to the tumor. Biodistribution was monitored over a 5-day period by fluorescence tomography.

[0130] result AASTY 7.1 The biodistribution of the -Cy7 construct (Figure 5) was consistent with the previous biodistribution data from Examples 3 and 7. (Note that in Example 7, the copolymer was administered intratumorally rather than subcutaneously, which explains the stronger liver accumulation compared to that new data set.) Most of the fluorescent signal remained in the area near the initial injection depot, with some spreading to the lymphatic network over time, and the majority of the signal was still measurable 5 days after injection.

[0131] The MAASTY-Cy7 conjugate appeared to distribute according to the same pattern, closely mimicking the results of AASTY-Cy7 in terms of tissue retention and biodistribution, although a slightly higher signal remained after 5 days. These results demonstrate that both polymers share a common pharmacokinetic profile, due to their structural and physicochemical similarities (Figure 6), suggesting that they exhibit very similar types of interactions with biological tissues. Therefore, it is assumed that the copolymers SMA and DIBMA will also be useful for retaining cargo within tissues. It should be noted that although the mice in this biodistribution study were tumor-bearing, based on the consistency of AASTY-Cy7 with the results in healthy mice in Example 3 and the similarity of the biodistribution profiles of both AASTY and MAASTY in Figure 5, this assumption can be reasonably extrapolated to healthy mice.

[0132] conclusion Due to their structural similarities, AASTY and MAASTY exhibited similar pharmacokinetic profiles and both showed excellent tissue retention characteristics. When conjugated to a TLR7 / 8 agonist as in Examples 5 and 6, both copolymers are expected to perform at similar levels in cancer therapy. Based on these findings, it is also anticipated that other polymers similar to poly(styrene-co-maleic acid) (SMA) and poly(diisobutylene-co-maleic acid) (DIBMA), which, like AASTY and MAASTY, are amphiphilic copolymers containing both hydrophobic and hydrophilic subunits, will behave similarly in vivo and exhibit the same type of pharmacokinetic profile with respect to tissue retention (see Figure 6).

[0133] References Slezak et al., Tumor Cell-Surface Binding of Immune Stimulating Polymeric Glyco-Adjuvant via Cysteine-Reactive Pyridyl Disulfide Promotes Antitumor Immunity, ACS Cent.Sci. 2022, 8, 1435-1446. Sci. 2022, 8, 1435-1446. Shukla et al., Structure-Activity Relationships in Human Toll-like Receptor 7-Active Imidazoquinoline Analogues, J Med Chem. 2010 Jun 10;53(11):4450‐4465. WO 2022 / 226032 A1.

Claims

1. A copolymer drug conjugate comprising an amphiphilic copolymer and three or more anticancer drug moieties conjugated to said amphiphilic copolymer.

2. The copolymer-drug conjugate of claim 1 , wherein the amphiphilic copolymer is an anionic amphiphilic copolymer.

3. 10. The copolymer-drug conjugate of any one of the preceding claims, wherein the amphiphilic copolymer is polymerized from: a. styrene and / or diisobutylene, and b. Acrylic acid, methacrylic acid, and / or maleic acid.

4. 10. The copolymer-drug conjugate of any one of the preceding claims, wherein the amphiphilic copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer.

5. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anticancer drug moiety is conjugated to the amphiphilic copolymer via a bond or a linking group.

6. A copolymer-drug conjugate having the structure of formula (III): 、 During the ceremony: Each R １ are independently CH ３ and H; Each R ｙ are independently H, COOH, and COX-L-R ２ is selected from Each R ｚ are independently H and CH ３ is selected from Each R ｘ is, independently, is selected from each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R ２ is independently selected from H or an anticancer drug, ２ at least three of which are anticancer drug moieties; each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

7. The copolymer-drug conjugate has the structure of formula (I): 、 During the ceremony: Each R １ But independently, CH ３ and H; each X is independently selected from O, S, or NH; each L is independently a bond or a linking group; Each R ２ is independently selected from H or an anticancer drug, ２ at least three of which are anticancer drug moieties; each T is the same or different terminal group; n is 5 to 500; m is 5 to 500; wherein the copolymer is a random, alternating, or statistical copolymer.

10. The copolymer drug conjugate of any one of the preceding claims.

8. In the formula, R １ is CH ３ 10. The copolymer drug conjugate of any one of the preceding claims, wherein

9. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the copolymer according to formula (I) is poly(methacrylic acid-co-styrene) copolymer or poly(acrylic acid-co-styrene) copolymer.

10. wherein the copolymer-drug conjugate has the structure of formula (II): 、 10. The copolymer drug conjugate of any one of the preceding claims, comprising:

11. 10. The copolymer drug conjugate of any one of the preceding claims, wherein X is selected from O, S, or NH, e.g., X is O.

12. 10. The copolymer-drug conjugate of any one of the preceding claims, wherein L is a bond.

13. 10. The copolymer drug conjugate of any one of the preceding claims, wherein L is a linking group selected from PEG, alkylene, triazole, and a group formed from the reaction of a haloalkyl with a nucleophile.

14. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the copolymer of the copolymer drug conjugate has a molecular weight of less than 20 kDa, such as less than 15 kDa, for example less than 14 kDa, such as less than 13 kDa, and / or the copolymer has a molecular weight of at least 1 kDa, such as at least 3 kDa, for example at least 5 kDa, for example at least 7 kDa, for example at least 9 kDa.

15. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anti-cancer drug is an anti-tumor drug.

16. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anti-cancer drug is an anti-cancer drug effective against solid tumors.

17. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anti-cancer drug is a Toll-like receptor (TLR) agonist, such as a TLR7 agonist, a TLR8 agonist, or a TLR7 / 8 agonist.

18. the anticancer agent has the structure of formula (AI), (A-II), (A-III), or (A-IV); 、 、 、 ； During the ceremony, X １ａ is —O—, —S—, or —NR Ｃａ and R １ａ is hydrogen, (C １－１０ ) alkyl, substituted (C １－１０ ) alkyl, C ６－１０ Aryl, or substituted C ６－１０ Aryl, C ５－９ Heterocyclic, substituted C ５－９ is heterocyclic, R Ｃａ is hydrogen, (C １－１０ ） Alkyl, or substituted C １－１０ alkyl, or R Ｃａ and R １ａ together with the nitrogen atom to which they are attached form a heterocyclic ring or a substituted heterocyclic ring, Each R ２ａ are independently —OH, (C １ ~C ６ ) alkyl, substituted (C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkoxy, substituted (C １ ~C ６ ) alkoxy, —C(O)—(C １ ~C ６ ) alkyl (alkanoyl), substituted —C(O)—(C １ ~C ６ ) alkyl, —C(O)—(C ６ ~C １０ ) aryl (aroyl), substituted —C(O)—(C ６ ~C １０ ) aryl, —C(O)OH (carboxyl), —C(O)O(C １ ~C ６ ) alkyl (alkoxycarbonyl), substituted —C(O)O(C １ ~C ６ ) alkyl, —NR ａａ R ｂａ , —C(O)NR ａａ R ｂａ (carbamoyl), halo, nitro, or cyano, or R ２ａ does not exist; Each R ａａ and R ｂａ are independently hydrogen, (C １ ~C ６ ) alkyl, substituted (C １ ~C ６ ) alkyl, (C ３ ~C ８ ) cycloalkyl, substituted (C ３ ~C ８ ) cycloalkyl, (C １ ~C ６ ) alkoxy, substituted (C １ ~C ６ ) alkoxy, (C １ ~C ６ ) alkanoyl, substituted (C １ ~C ６ ) alkanoyl, aryl, aryl (C １ ~C ６ ) alkyl, Het, Het(C １ ~C ６ ) alkyl, or (C １ ~C ６ ) alkoxycarbonyl, wherein the substituents on any alkyl, aryl, or heterocyclic group are hydroxy, C １－６ Alkyl, hydroxy C １－６ Alkylene, C １－６ Alkoxy, C ３－６ Cycloalkyl, C １－６ Alkoxy C １－６ alkylene, amino, cyano, halo, or aryl; j is 0, 1, 2, 3 or 4; X ３ａ is —N— or —CH—, R ４ａ is -CH ２ - or -CH(R ２ａ ) - and k is 0 or 1; X ４ａ -O-, -S-, -NH-, -N(R ｄａ ) -, -CH ２ -, or -CH(R ２ａ ) - and Each R ｄａ are independently —OH, (C １ ~C ６ ) alkyl, substituted (C １ ~C ６ ) alkyl, (C １ ~C ６ ) alkoxy, substituted (C １ ~C ６ ) alkoxy, —C(O)—(C １ ~C ６ ) alkyl (alkanoyl), substituted —C(O)—(C １ ~C ６ ) alkyl, —C(O)—(C ６ ~C １０ ) aryl (aroyl), substituted —C(O)—(C ６ ~C １０ ) aryl, —C(O)O(C １ ~C ６ ) alkyl (alkoxycarbonyl), substituted —C(O)O(C １ ~C ６ ) alkyl, —C(O)NR ａａ R ｂａ (carbamoyl), 10. The copolymer drug conjugate of any one of the preceding claims, which is:

19. the anticancer agent has the structure of formula (BI), 、 During the ceremony, R １ｂ is (C １－８ ) alkyl, (C ３－８ ) cycloalkyl or a 3- to 8-membered saturated heterocyclic group containing an O atom, １ｂ is halogen, cyano, hydroxyl, and (C １－３ ) optionally substituted with one or more substituents independently selected from alkoxy; Z １ｂ is (C ２－６ ) represents an alkylene group, wherein Z is not adjacent to a nitrogen atom. １ｂ may be replaced by an oxygen atom; X １ｂ is NR ５ｂ , >N-COR ５ｂ , >N-CONR ５ｂ R ５ａｂ , C.O.R. ５ｂ , N.R. ５ｂ CO, NR ５ｂ CONR ６ｂ , or NR ６ｂ CONR ５ｂ represents Y １ｂ is a single bond or (C １－６ ) represents alkylene; Each R ２ｂ is halogen, cyano, hydroxy, thiol, (C １－３ ) alkyl, (C １－３ ) hydroxyalkyl, (C １－３ ) haloalkyl, (C １－３ ) alkoxy, (C １－３ ) haloalkoxy, (C １－３ ) alkylthio, (C １－３ ) alkylsulfonyl, and (C １－３ ) alkylsulfinyl; R ３ｂ is (C １－６ ) C optionally substituted with alkoxy １－６ represents alkyl, Each R ａｂ is halogen, cyano, hydroxy, thiol, (C １－３ ) alkyl, (C １－３ ) hydroxyalkyl, (C １－３ ) haloalkyl, (C １－３ ) alkoxy, (C １－３ ) haloalkoxy, (C １－３ ) alkylthio, (C １－３ ) alkylsulfonyl, and (C １－３ ) alkylsulfinyl; R ５ｂ and R ５ａｂ are each independently hydrogen, a ring group O, S(O) ｐ Or NR １０ｂ 3-8 membered saturated heterocycles containing (C １－６ ) alkyl group or (C ３－６ ) cycloalkyl groups, the latter two groups being NR ７ｂ R ８ｂ or R ９ｂ and optionally substituted with one or more substituents independently selected from R ７ｂ and R ８ｂ each independently represents hydrogen, and the 3- to 8-membered saturated heterocycle is a ring group O, S(O) ｐ or NR １０ａｂ , (C １－６ ) alkyl or (C ３－６ ) cycloalkyl, the latter two groups being halogen, cyano, S(O) ｑ R １１ｂ , OR １２ｂ , CO ２ R １２ｂ , O.C.(O.)R １２ｂ , S.O. ２ NR １２ｂ R １３ｂ CONR １２ｂ R １３ｂ , N.R. １２ｂ R １３ｂ , N.R. １２ｂ SO ２ R １４ｂ , N.R. １２ｂ COR １３ｂ , or a ring group O, S(O) ｐ or NR １０ｂｂ optionally substituted with one or more groups independently selected from a 3- to 8-membered saturated heterocycle comprising or R ７ｂ and R ８ｂ together with the nitrogen atom to which they are attached form a 3-8 membered saturated heterocycle containing a ring nitrogen atom and optionally one or more additional heteroatoms independently selected from nitrogen, oxygen, sulfur, and sulfonyl, wherein the heterocycle is substituted with any of halogen, cyano, S(O) ｑ R １５ｂ , OR １５ｂ , CO ２ R １５ｂ , C.O.R. １５ｂ , O.C.(O.)R １５ｂ , S.O. ２ NR １５ｂ R １６ｂ , C.O.R. １５ｂ R １６ｂ , N.R. １５ｂ R １６ｂ , N.R. １５ｂ SO ２ R １７ｂ , N.R. １５ｂ COR １６ｂ , N.R. １５ｂ CO ２ R １６ｂ , heteroaryl, (C １－６ ) haloalkyl, (C ３－８ ) cycloalkyl, and (C １－６ ) alkyl, the latter two groups being optionally substituted by one or more substituents independently selected from cyano, S(O) ｑ R １８ｂ , OR １８ｂ , CO ２ R １８ｂ , S.O. ２ NR １８ｂ R １９ｂ , C.O.R. １８ｂ R １９ｂ or NR １８ｂ R １９ｂ optionally substituted with one or more groups independently selected from R Ｑｂ is halogen, cyano, CO ２ R ２０ｂ , S(O) ｑ R ２０ｂ , OR ２０ｂ , S.O. ２ NR ２０ｂ R ２２ｂ , C.O.R. ２０ｂ R ２２ｂ , N.R. ２０ｂ SO ２ R ２１ｂ , N.R. ２０ｂ CO ２ R ２１ｂ , N.R. ２０ｂ COR ２２ｂ or a ring group NR １０ｃｂ represents a 3- to 8-membered saturated heterocycle containing R １０ｂ , R １０ａｂ , R １０ｂｂ and R １０ｃｂ are independently hydrogen, CO ２ R ２３ｂ , S(O) ｑ R ２３ｂ , C.O.R. ２４ｂ , or (C １－６ ) alkyl, (C ２－６ ) alkenyl, (C ２－６ ) alkynyl or (C ３－８ ) cycloalkyl groups, each of which is independently selected from halogen, cyano, OR ２５ｂ or NR ２５ｂ R ２６ｂ and optionally substituted by one or more substituents independently selected from R ６ｂ , R １１ｂ , R １２ｂ , R １３ｂ , R １５ｂ , R １６ｂ , R １８ｂ , R １９ｂ , R ２０ｂ , R ２２ｂ , R ２４ｂ , R ２５ｂ and R ２６ｂ are each independently hydrogen, (C １－６ ) alkyl or (C ３－６ ) represents cycloalkyl; R １４ｂ , R １７ｂ , R ２１ｂ and R ２３ｂ are each independently １－６ ) alkyl or (C ３－６ ) represents cycloalkyl; h, i, p, and q each independently represent the integer 0, 1, or 2; and Ab is a monocyclic or bicyclic (C ６－１０ ) aryl, or monocyclic or bicyclic rings containing 1 to 3 heteroatoms (C ５－１２ ) represents a heteroaryl group, and R ｂｂ and R ｃｂ are independently hydrogen or (C １－６ ) alkyl, or R ｂｂ and R ｃｂ are bonded together (C ３－８ ) forming a cycloalkyl.

20. the anticancer agent has the structure of formula (CI), 、 During the ceremony, R １ｃ is -(C ２－６ ) alkyl-N(R ３ｃ ) ２ , -(C ２－６ ) alkyl-NR ３ｃ -SO ２ -X ｃ -R ４ｃ , and -(C ２－６ ) alkyl-NR ６ｃ -SO ２ -R ７ｃ , X ｃ is a bond or -NR ５ｃ - and R ４ｃ is alkyl, aryl, or heteroaryl; R ２ｃ is hydrogen, alkyl, alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl-O-aryl, alkyl-O-alkyl, alkyl-O-alkenyl, and OH, halogen, —N(R ３ｃ ) ２ , —CO—N(R ３ｃ ) ２ , —CO—(C １－１０ ) alkyl, —CO—O—(C １－１０ ) alkyl, —N ３ , aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, —CO-aryl, —CO-(substituted aryl), —CO-heteroaryl, and —CO-(substituted heteroaryl); Each R ３ｃ is hydrogen and (C １－１０ ) alkyl; R ５ｃ is hydrogen and (C １－１０ ) alkyl, or R ４ｃ and R ５ｃ can combine to form a 3- to 7-membered heterocyclic or substituted heterocyclic ring; R ６ｃ is hydrogen and (C １－１０ ) alkyl; R ７ｃ is hydrogen and (C １－１０ ) alkyl, or R ６ｃ and R ７ｃ can combine to form a 3- to 7-membered heterocyclic or substituted heterocyclic ring; r is 0 to 4, and each R ｃ is (C １－１０ ) alkyl, (C １－１０ 2. The copolymer-drug conjugate of claim 1, wherein: R 1 is independently selected from the group consisting of alkoxy, halogen, and trifluoromethyl.

21. The TLR7, TLR8, or TLR7 / 8 agonist may be imiquimod, resiquimod, gardikimod, 852A, loxoribine, bropirimine, 3M-011 (CAS No. 642473-62-9), 3M-052 (CAS No. 1359993-59-1), DSR-6434 (CAS No. 1059070-10-8), DSR-29133, SZU-101, SM-3 60320 (CAS No. 226907-52-4), SM-276001 (CAS No. 473930-22-2), VTX-2337 (CAS No. 926927-61-9), and 1-(3-aminopropyl)-2-(ethoxymethyl)imidazo[4,5-c]quinolin-4-amine.

22. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anti-cancer drug is a stimulator of interferon genes (STING) agonist.

23. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anti-cancer drug is IL-2, IL-12, or IL-15, or a fragment thereof.

24. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the anticancer drug is a radiopharmaceutical or radioactive substance.

25. The copolymer drug conjugate of any one of the preceding claims, wherein the copolymer drug conjugate comprises an anti-cancer drug.

26. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the copolymer drug conjugate comprises two or more different anti-cancer drugs.

27. L is a linking group, and the moiety R ２ is a carbon atom, or R ２ 10. The copolymer of claim 1, wherein L is conjugated to L at a heteroatom such as N, O, or S in

28. L is a bond and the moiety R ２ is a carbon atom, or R ２ 10. The copolymer of claim 1, wherein X is conjugated to X at a heteroatom such as N, O, or S in

29. T is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, nitrile, hydroxy, carboxyl, halogen, thiol, substituted thiol, acyl, substituted acyl, a group of formula (XI), a group of formula (Y-I), a group of formula (Y-II), a fluorescent dye, and a diagnostic agent; wherein the group of formula XI has the following structure: In the formula, R ３ and R ４ are each independently selected from hydrogen, alkyl, and substituted alkyl; wherein the group of formula Y-I has the following structure: In the formula, R ５ is selected from hydrogen, alkyl, and substituted alkyl; wherein the group of formula Y-II has the following structure: wherein R5 is selected from hydrogen, alkyl, and substituted alkyl; X ２ and X ３ 4. The copolymer drug conjugate of claim 1, wherein each is independently selected from S and O.

30. 10. The copolymer drug conjugate of any one of the preceding claims, wherein the fluorescent dye is selected from the group consisting of cyanine dyes (e.g., indocyanine green or cyanine 7), triarylmethane dyes (e.g., fluorescein), thiazine dyes (e.g., methylene blue), LUM015, VGT-309, AVB-620, C-Dots, BLZ-100.

31. A pharmaceutical composition comprising the copolymer drug conjugate of any one of the preceding claims.

32. 32. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition is formulated for administration by injection, e.g., intratumoral injection.

33. 10. The method of synthesizing a copolymer drug conjugate of any one of the preceding claims, wherein the method comprises conjugating an anti-cancer drug to a precursor copolymer, such as a precursor poly(methacrylic acid-co-styrene) copolymer, a precursor poly(acrylic acid-co-styrene) copolymer, a precursor poly(maleic acid-co-styrene) copolymer, or a precursor poly(diisobutylene-co-maleic acid) copolymer.

34. the precursor copolymer has the structure of formula (III-P), 、 In the formula, R ｘ’ teeth, or and wherein each L' is independently selected from H and a reactive handle; T, n, m, R １ , X, R ｚ , and R ｙ The method according to claim 33, wherein said first and second hydroxyl groups are as defined in any one of claims 6 to 11 or 27 to 28.

35. the precursor poly(methacrylic acid-co-styrene) copolymer or the precursor poly(acrylic acid-co-styrene) copolymer has the structure of formula (IP), 、 wherein each L' is independently selected from H and a reactive handle; T, n, m, R １ and X is as defined in any one of claims 6 to 11 or 27 to 28.

36. Each L' is H, CH ２ Cl, CH ２ Br, or CH ２ 36. The method of any one of claims 34 and 35, wherein each of the compounds is independently selected from the group consisting of I.

37. A copolymer-drug conjugate according to any one of claims 1 to 30 or a pharmaceutical composition according to any one of claims 31 to 32 for use in medicine.

38. The copolymer drug conjugate of any one of claims 1 to 30 or the pharmaceutical composition of any one of claims 31 to 32 for use in the treatment of cancer.

39. 39. The copolymer drug conjugate or pharmaceutical composition for use of claim 38, wherein the cancer is characterized by the presence of a tumor.

40. 40. The copolymer drug conjugate or pharmaceutical composition for use according to claim 39, wherein the tumor is a solid tumor.

41. 41. The copolymer drug conjugate or pharmaceutical composition for use according to claim 39 or 40, wherein the tumor, such as the solid tumor, is a sarcoma.

42. 41. The copolymer drug conjugate or pharmaceutical composition for use according to claim 39 or 40, wherein the tumor, such as the solid tumor, is a carcinoma.

43. The copolymer drug conjugate or pharmaceutical composition for use according to any one of claims 40 to 42, wherein the solid tumor is a primary tumor or a metastatic tumor.

44. The copolymer-drug conjugate or pharmaceutical composition for use according to any one of claims 39 to 43, wherein the cancer is liver cancer or spleen cancer.

45. The copolymer drug conjugate or pharmaceutical composition for use according to any of claims 38 to 44, wherein the copolymer drug conjugate or pharmaceutical composition is administered intratumorally, such as by injection.

46. 32. A method of treating cancer, e.g., a cancer characterized by a tumor, such as a solid tumor, comprising administering to a subject in need thereof the copolymer drug conjugate of any one of claims 1-30 or the pharmaceutical composition of any one of claims 31-32.

47. 33. A method of inducing an immune response in a subject, comprising administering to the subject the copolymer-drug conjugate of any one of claims 1 to 30 or the pharmaceutical composition of any one of claims 31 to 32.

48. 33. A method of immunizing a subject, comprising administering to the subject the copolymer drug conjugate of any one of claims 1-30 or the pharmaceutical composition of any one of claims 31-32.

49. 33. A method of reducing tumor volume in a subject, comprising administering to the subject the copolymer drug conjugate of any one of claims 1-30 or the pharmaceutical composition of any one of claims 31-32.

50. Use of the copolymer-drug conjugate according to any one of claims 1 to 30 or the pharmaceutical composition according to any one of claims 31 to 32 as an immunostimulant.

51. 33. Use of the copolymer drug conjugate of any one of claims 1 to 30 or the pharmaceutical composition of any one of claims 31 to 32 for the manufacture of a medicament for use in the treatment of cancer.

52. below, a. styrene and / or diisobutylene, and b. Acrylic acid, methacrylic acid, and / or maleic acid 10. Use of copolymers polymerized from to retain drugs in tissue.

53. 53. The use of claim 52, wherein the copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer.

54. For example, a method for increasing the residence time of a drug in a tissue, such as a cancerous tissue, comprising administering the drug to: a. styrene and / or diisobutylene, and b. acrylic acid, methacrylic acid, and / or maleic acid; The method comprises administering to a subject a copolymer polymerized from

55. 55. The method of claim 54, wherein the copolymer is poly(acrylic acid-co-styrene) copolymer, poly(methacrylic acid-co-styrene) copolymer, poly(maleic acid-co-styrene) copolymer, or poly(diisobutylene-co-maleic acid) copolymer.

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