Copolymer-drug conjugates for treatment of tumors

Abstract

The present disclosure relates to a copolymer-drug conjugate. In particular, disclosed herein are copolymer-drug conjugates with good tissue retention. Also disclosed herein are copolymer-drug conjugates for use in medicine, such as in the treatment of cancer. Also disclosed herein are methods for preparing such copolymer-drug conjugates.

Description

TECHNICAL FIELD

[0001] The present invention relates to conjugates between copolymers and anticancer drugs. The present invention also relates to methods of using compositions thereof to treat and image solid cancer tumors. BACKGROUND

[0002] The promise of cancer immunotherapy lies in harnessing the patient’s own T cells to attack cancer cells. The natural ability of the immune system to detect and destroy abnormal cells can prevent the development of many cancers.

[0003] However, cancer cells can sometimes evade detection and destruction by the immune system. Cancer cells can reduce the expression of tumor antigens on their surface, making it more difficult for the immune system to detect them; express proteins on their surface that induce inactivation of immune cells; and / or induce cells in the microenvironment to release substances that suppress the immune response and promote tumor cell proliferation and survival. While the characteristics of solid tumors vary widely, some physiological properties are common and create a characteristic 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. Activation of TLRs induces inflammatory responses and contributes to the development of antigen-specific anticancer immunity. TLR7 and TLR8 are canonical members of the TLR family, which are intracellular receptors expressed on the membrane of endosomes. TLR7 and TLR8 can not only be triggered by single-stranded RNA during viral infection, but also by immunomodulators that structurally mimic nucleosides. An example of an artificial TLR agonist is imiquimod, which was first clinically approved for topical administration in 1997. Since then, new generations of TLR7 and TLR8 agonists have been developed, including resiquimod (R848), motolimod (VTX-2337), etc., which provide improved solubility and more pronounced downstream effects.

[0005] Despite the clinical interest and promising results in clinical studies, their clinical translation has been limited due to narrow therapeutic window, high systemic toxicity, limited in vivo residence time, and production difficulties. Using chemical modifications to localize such agonists in the tumor microenvironment to improve their tolerability and efficacy can expand their clinical applicability.

[0006] Developing a vaccine strategy that can induce robust anti-tumor immunity while producing minimal systemic side effects, while inducing long-term and highly localized tumor-specific effects, is crucial for the continued progress of TLR7 / 8 agonist-based cancer immunotherapy to widespread clinical translation. SUMMARY

[0007] Disclosed herein are copolymer-drug conjugates. The copolymers of the present disclosure provide a useful platform for delivering drugs to tissues. When administered to a tissue, e.g., by injection, the copolymer-drug conjugates remain in the tissue for a long period of time. This retention is conferred by the properties of the copolymer.

[0008] Long retention in a tissue is helpful for delivering drugs that must act locally on the tissue. For example, the drug can exhibit systemic toxicity on its own; conjugation to a copolymer of the present disclosure reduces systemic exposure of the drug because it is retained locally at the site of administration of the conjugate. Improved retention in a tissue also extends the time that the tissue is exposed to the drug, which can enhance the effect of the administered drug on the tissue. Improved retention also results in much slower metabolism and / or excretion of the drug compared to administration of the drug alone.

[0009] In addition, the copolymers of the present disclosure are amphiphilic, which enables the conjugate to be internalized by penetrating a cell membrane. Thus, the drug moiety conjugated to the copolymer can exert its effect on an extracellular or intracellular target.

[0010] In particular, the copolymers of the present disclosure provide a drug delivery platform that can be used to treat solid tumors by sustained activation of immune cells. The platform is particularly suitable for TLR 7 / 8 agonists, while limiting off-target effects and systemic side effects. In addition, by conjugating a diagnostic dye to the copolymer, the copolymer-drug conjugates of the present disclosure allow for 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 moieties conjugated to the amphiphilic copolymer. In one particular aspect of the present disclosure, the copolymer is a poly(acrylic acid-co-styrene) copolymer, a poly(methacrylic acid-co-styrene) copolymer, a poly(maleic acid-co-styrene) copolymer, or a 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]

[0014] wherein:

[0015] each R 1 is independently selected from CH3and H,

[0016] each Ryis independently selected from H, COOH, and COX-L-R 2 ,

[0017] each R z is independently selected from H and CH3,

[0018] each R x is independently selected from the group consisting of:

[0019]

[0020] each X is independently selected from O, S, or NH,

[0021] each L is independently a bond or a linking group,

[0022] each R 2 is independently selected from the group consisting of H or an anticancer drug, wherein at least three R 2 is an anticancer drug moiety,

[0023] each T is the same or different end group,

[0024] n is from 5 to 500,

[0025] m is from 5 to 500,

[0026] wherein the copolymer is a random, alternating, or statistical copolymer.

[0027] One aspect of the present disclosure provides a copolymer-drug conjugate having the structure of Formula (I):

[0028]

[0029] wherein:

[0030] each R 1 is independently selected from the group consisting of CH3and H,

[0031] each X is independently selected from O, S, or NH,

[0032] each L is independently a bond or a linking group,

[0033] each R 2 is independently selected from the group consisting of H or an anticancer drug, wherein at least three R 2 is an anticancer drug moiety,

[0034] each T is the same or different end group,

[0035] n is from 5 to 500,

[0036] m is from 5 to 500,

[0037] wherein the copolymer is a random, alternating, or statistical copolymer.

[0038] The synthetic method of the present disclosure enables poly(acrylic acid-co-styrene) (AASTY) copolymers to be readily functionalized without altering their original properties. The presence of a chemical handle moiety in the AASTY copolymer enables the copolymer to be readily functionalized by reacting the chemical handle moiety with a functionalizing agent to form a functionalized copolymer. 9.9- reaction of the azide moiety on the R-terminus of -N3, insertion into e.g. cyanine 7 near infrared dyes for imaging purposes. In addition, the side groups of the AASTY copolymer (i.e. the acrylic acid groups) enable coupling of TLR7 / 8 agonists to achieve anti-tumor effects in vivo. Accordingly, one aspect of the present disclosure provides a method for synthesizing a copolymer-drug conjugate of the present disclosure, the method comprising coupling a dye or a drug (e.g. an anti-cancer drug) to a precursor poly(methacrylic acid-co-styrene) copolymer or a precursor poly(acrylic acid-co-styrene) copolymer. These precursor copolymers can have (side) reactive handles.

[0039] One aspect of the present disclosure provides a pharmaceutical composition comprising a copolymer-drug conjugate of the present disclosure.

[0040] One aspect of the present disclosure provides a copolymer-drug conjugate of the present disclosure or a pharmaceutical composition of the present disclosure for use in medicine.

[0041] The copolymer-drug conjugates of the present disclosure reverse tumor growth and improve overall survival after tumor inoculation. In addition, the copolymer-drug conjugates of the present disclosure induce an adaptive response, as re-inoculation of tumors after treatment does not result in tumor growth.

[0042] One embodiment of the present disclosure provides use of a copolymer polymerized from:

[0043] a. styrene and / or diisobutylene, and

[0044] b. acrylic acid, methacrylic acid and / or maleic acid.

[0045] In one embodiment, the copolymer is a poly(acrylic acid-co-styrene) copolymer, a poly(methacrylic acid-co-styrene) copolymer, a poly(maleic acid-co-styrene) copolymer or a poly(diisobutylene-co-maleic acid) copolymer.

[0046] The copolymer-drug conjugates of the present disclosure show strong adhesion to the tissue where the injection is performed, and the diffusion to other tissues is lower over time. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1A: General synthetic scheme for the chemical preparation of AASTY copolymers via reaction between styrene and acrylic acid in the presence of a RAFT polymerization agent. Subsequent products are then functionalized with a TLR agonist and / or an imaging dye. B: Graph showing in vitro cellular response with different percentages (0%, 2.5%, 6.5%, and 10%) of coupled TLR7 / 8a agonist. A direct regression was observed between the observed cellular response and the amount of coupled TLR7 / 8a agonist, with the maximum signal seen with 10% coupled agonist.

[0048] Figure 2 A: Fluorescent imaging studies performed after subcutaneous injection of free CY7 dye and AASTY-CY7, respectively, in two groups of five mice. For both injections, the majority of the fluorescent signal remained in the vicinity of the initial injection depot, with no significant spreading. While the integrated signal density slowly decreased over time, the majority of the AASTY-CY7 signal was still clearly visible 2 weeks later, with no change in distribution; this was different from the free Cy7 control, which was significantly cleared from the injection site within the first few hours. B: Fluorescent signal of isolated organs after tail vein injection of free CY7 and AASTY-CY7, respectively. For both compounds and the negative control, the signal in the region of interest (ROI) was highest in the injection site (tail) and the liver, further supporting the slow clearance via the liver route.

[0049] Figure 3A: Timeline summary of mouse tumor inoculation, subsequent treatment, and tumor re-injection to evaluate induced resistance. B: Plot showing the exponential tumor growth timeline for six mice repeated without treatment. All individuals died in less than 25 days. C: Plot showing the tumor growth timeline for six mice repeated treated with free TLR7 / 8a agonist. Tumor treatment effect was slightly improved compared to control; two of the six mice recovered, but the other four did not survive. D: Plot showing the tumor growth timeline for six mice repeated treated with AASTY copolymer alone. Although AASTY delayed tumor growth in one repetition, no mice achieved ultimate full recovery. E: Plot showing the tumor growth timeline for six mice repeated treated with AASTY-TLR7 / 8a 1X (low dose). Five mice had tumors that grew exponentially, while the remaining one experienced tumor size reduction and ultimately recovered. F: Plot showing the tumor growth timeline for six mice repeated treated with AASTY-TLR7 / 8a 2X (high dose). No tumor growth was observed for the first 20 days, and the overall tumor growth rate was negative for the first 10 days. Ultimately, four mice recovered from the tumor. G: Summary of mouse survival rates following tumor inoculation with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), negative control. AASTY-TLR7 / 8a 2X achieved the best results, as 80% of the mice survived 60 days after tumor inoculation. H: Time evolution of the average tumor volume with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), negative control. AASTY-TLR7 / 8a 2X (high dose) achieved the best results, as the tumor volume steadily decreased within 10 days. I: Average survival time of mice following tumor injection with different treatments: free TLR7 / 8a, AASTY, AASTY-TLR7 / 8a 1X (low dose), AASTY-TLR7 / 8a 2X (high dose), negative control. AASTY-TLR7 / 8a 2X (high dose) had the highest survival time, higher than 100 days. J: Average slope coefficient of the tumor growth plot. However, the tumor growth rate was positive for the control, free TLR7 / 8a, AASTY, and AASTY-TLR7 / 8a 1X (low dose); AASTY-TLR7 / 8a 2X (high dose) reached a negative rate, thus indicating an anti-tumor effect. K: Plot showing the tumor growth timeline for six mice samples following tumor re-inoculation. No tumor growth was observed for those individuals treated with AASTY-TLR7 / 8a 1X (low dose) and AASTY-TLR7 / 8a 2X (high dose) after inoculation.L: Tumor imaging shows the biodistribution of AASTY-TLR7 / 8a-CY72X (high dose) after intratumoral administration. The data show that AASTY-TLR7 / 8a-CY72X (high dose) has a very long residence time, with an estimated half-life of 41 days, and most of the signal is still clearly visible at the end of the study after 117 days (acquisition week 15). M: 3D model shows the biodistribution of AASTY-TLR7 / 8a-CY72X (high dose) based on fluorescence tomography performed 5 to 6 days after a single dose. AASTY-TLR7 / 8a-CY7 diffuses through bean-shaped tumors (with the signal being maximum at the injection point) and accumulates primarily in the liver.

[0050] Figure 4 : The disclosed embodiments consist of a copolymer-drug conjugate having several styrene moieties, several free acrylic moieties, several acrylic moieties coupled to a drug moiety "R", and end groups obtained from RAFT polymerization.

[0051] Figure 5 3D reconstruction of repeated fluorescence tomography studies in two groups of three mice following subcutaneous injection of AASTY-Cy7 and MAASTY-Cy7, respectively. Both conjugates exhibited similar biodistribution patterns, with the majority of the fluorescent signal remaining in the vicinity of the initial injection depot, while a smaller portion diffused into the lymphatic network over time. After five days, the majority of the Cy7 signal remained clearly visible for both conjugates, indicating that the two polymers have similar pharmacokinetic profiles. High fluorescence appears as a solid gray, while low fluorescence appears as a diffuse gray cloud.

[0052] Figure 6 : Comparison of the structures of AASTY, MAASTY, SMA and DIBMA. The end groups according to the present disclosure are not shown in the structures. All copolymers include two monomers, hydrophobic and hydrophilic. Detailed Description of the Invention

[0054] Definitions

[0055] The term "copolymer" is a term of art. It refers to a polymer composed of two or more different monomeric units that are polymerized in a process called copolymerization. Because copolymers contain at least two different monomeric units, they can be classified based on how the monomeric units are arranged to form the polymer chain. Those classifications include "alternating copolymers" (in which the monomeric units repeat in a highly regular alternating pattern), "periodic copolymers" (in which the monomeric units are arranged in a repeating sequence), "statistical copolymers" (in which the sequence of monomeric units follows a statistical rule), "random copolymers" (in which the monomeric units are linked in a random order), and "block copolymers" (in which two or more homopolymer subunits are linked).

[0056] "Amphiphilic copolymer" refers to a copolymer comprising or consisting 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.

[0057] "Methacrylic acid" refers to the compound having CAS Registry Number 79-41-4.

[0058] "Acrylic acid" refers to the compound having CAS Registry Number 79-10-7.

[0059] "Styrene" refers to the compound having CAS Registry Number 100-42-5.

[0060] "Maleic acid" refers to the compound having CAS Registry Number 110-16-7.

[0061] "Diisobutylene" refers to the compound having CAS Registry Number 107-39-1.

[0062] "Copolymer-drug-conjugate" refers to a molecular structure consisting of a copolymer chemically linked to a molecule (other than food) used to prevent, diagnose, treat, or ameliorate the symptoms of a disease or abnormal condition, wherein the condition is cancer. As used herein, "spacer" or "linking group" is a chemical entity that links two or more molecules.

[0063] "Drug" refers to a compound or moiety used to prevent, diagnose, treat, ameliorate, or alleviate the symptoms of a disease or abnormal condition.

[0064] As used herein, the term "PEG" refers to a polyethylene glycol polymer of variable length, typically having a relative molecular weight of 200-8000 Da.

[0065] As used herein, the term "TLR" refers to a single-pass transmembrane receptor typically expressed by cells, such as macrophages. Activation of TLR receptors activates immune cell responses.

[0066] As used herein, "solid tumor" refers to an abnormal growth of cells mass affecting solid tissue and / or organs (such as the breast or prostate), as opposed to leukemia, a cancer affecting the blood, which is a fluid tissue.

[0067] As used herein, the term "dye" refers to a colored chemical substance that chemically bonds with the substrate to which it is applied. As used herein, the term "fluorescent dye" refers to a compound or moiety capable of absorbing and emitting electromagnetic radiation, such as in the visible, IR, or UV regions of the electromagnetic radiation spectrum.

[0068] As used herein, "cyanine dye" refers to a closed cyanine dye, i.e., a cyanine dye having cyclic moieties as end groups, wherein the cyclic moieties can be aromatic or non-aromatic and are substituted or unsubstituted at one or more positions. The term "cyanine dye" or "cyanine" refers to any cyanine dye well known in the art. Synthetic methods have been 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.

[0069] As used herein, "triarylmethane dye" refers to a dye integrated from a backbone containing three benzene rings connected to a common carbon atom, wherein the benzene rings are unsubstituted or substituted at one or more positions with the same or different substituents.

[0070] "Indocyanine green" refers to the compound having CAS Registry Number 3599-32-4.

[0071] "Cyanine 7" refers to the compound having CAS Registry Number 477908-53-5.

[0072] "Fluorescein" refers to the compound having CAS Registry Number 2321-07-5.

[0073] "Thiazine dye" refers to a dye integrated from at least one thiazine ring, wherein the thiazine is substituted at one or more positions with the same or different substituents.

[0074] "Methylene blue" refers to the compound having CAS Registry Number 61-73-4.

[0075] As used herein, the term "intratumoral injection" refers to the direct injection of an agent into a tumor, wherein the agent is a copolymer-drug conjugate, such as an AASTY-TLR7 / 8a construct. The intratumoral injection is performed on a living organism that has been inoculated with colon cancer.

[0076] As used herein, a "reactive handle" is a moiety or functional group on a compound that is capable of reacting, e.g., selectively reacting, with a moiety or functional group. Examples thereof are a carboxylic acid or an amine (forming an amide); or an alkyne or an azide (forming a triazole); or a thiol capable of reacting with a maleimide or haloalkyl group to undergo a S N 1or S N 2haloalkyl group.

[0077] As used herein, the term "immunostimulatory agent" refers to an agent that stimulates the immune system by inducing activation of any component thereof or increasing its activity.

[0078] Copolymer-drug conjugates of the present disclosure

[0079] The copolymers of the present disclosure are synthetic AASTY copolymers. The AASTY copolymers have increased tissue residence time, e.g., after subcutaneous injection. An increased residence time in the tissue can be seen compared to injection / administration of a drug without coupling to a copolymer of the present disclosure. However, the copolymer-drug conjugates of the present disclosure can also exhibit improved tissue retention compared to other polymer-drug conjugates. Combining the good tissue retention properties of the copolymer with a covalently attached therapeutically active moiety, e.g., an anticancer agent, e.g., a TLR 7 / 8 agonist, can, for example, improve the activation of immune cells at the site of injection, thereby enabling the TLR 7 / 8 agonist to be used for the treatment of solid tumors while limiting off-target effects and side effects associated with systemic exposure.

[0080] One embodiment of the present disclosure provides a copolymer-drug conjugate comprising an amphiphilic copolymer and one or more anticancer drug moieties coupled to the amphiphilic polymer. In a preferred embodiment, the copolymer-drug conjugate comprises two or more anticancer drug moieties. In an even more preferred embodiment, the copolymer-drug conjugate comprises three or more anticancer drug moieties. In one embodiment, the anticancer drug moieties are coupled to the amphiphilic copolymer via a bond or a linking group.

[0081] In one embodiment, the amphiphilic copolymer is an anionic amphiphilic copolymer. The anionic amphiphilic copolymer can be, for example, those incorporating a carboxylic acid moiety.

[0082] In one embodiment, the amphiphilic copolymer is a copolymer of:

[0083] 1. styrene and / or diisobutylene, and

[0084] 2. acrylic acid, methacrylic acid and / or maleic acid.

[0085] In one preferred embodiment of the disclosure, the amphiphilic copolymer is a poly(acrylic acid-co-styrene) copolymer, a poly(methacrylic acid-co-styrene) copolymer, a poly(maleic acid-co-styrene) copolymer, or a poly(diisobutylene-co-maleic acid) copolymer. In one even more preferred embodiment of the disclosure, the amphiphilic copolymer is a poly(acrylic acid-co-styrene) copolymer or a poly(methacrylic acid-co-styrene) copolymer.

[0086] One embodiment of the disclosure provides a copolymer-drug conjugate having the structure of Formula (III):

[0087]

[0088] wherein:

[0089] each R 1 is independently selected from CH3and H,

[0090] each Ryis independently selected from H, COOH, and COX-L-R 2 ,

[0091] each R z is independently selected from H and CH3,

[0092] each R x is independently selected from

[0093] each X is independently selected from O, S, or NH,

[0094] each L is independently a bond or a linking group,

[0095] each R 2 is independently selected from H or an anticancer drug, wherein at least three R 2 are anticancer drug moieties,

[0096] each T is the same or different end group,

[0097] n is 5 to 500,

[0098] m is 5 to 500,

[0099] wherein the copolymer is a random, alternating, or statistical copolymer.

[0100] In one embodiment, R x is and R z is H.

[0101] In one embodiment, R x is and R z is CH3.

[0102] In one embodiment, R 1 is H and Ry is COOH or COX-L-R 2 .

[0103] In one embodiment, R 1 is H and Ry is H.

[0104] In one embodiment, R 1 is CH3and Ry is H.

[0105] In one specific embodiment of the present disclosure, R x is R z is H, R 1 is H, and Ry is COOH or COX-L-R 2 .

[0106] In one specific embodiment of the present disclosure, R x is R z is H, R 1 is H, and Ry is H.

[0107] In one specific embodiment of the present disclosure, R x is R z is H, R 1 is CH3, and Ry is H.

[0108] In one specific embodiment of the present disclosure, R x is R z is CH3, R 1 is H, and Ry is COOH or COX-L-R 2 .

[0109] One embodiment of the present disclosure provides a copolymer-drug conjugate having a structure of Formula (I):

[0110]

[0111] wherein:

[0112] each R 1 is independently selected from CH3and H,

[0113] each X is independently selected from O, S, or NH,

[0114] each L is independently a bond or a linking group,

[0115] each R 2 is independently selected from H or an anti-cancer drug, wherein at least three R2 is an anticancer drug moiety,

[0116] each T is the same or different end group,

[0117] n is 5 to 500,

[0118] m is 5 to 500,

[0119] wherein the copolymer is a random, alternating, or statistical copolymer.

[0120] 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.

[0121] In one embodiment, the copolymer-drug conjugate has the structure of Formula (II):

[0122]

[0123] 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 and the anticancer drug is an ester. When X corresponds to NH, the functional group linking the copolymer and the anticancer drug is an amide.

[0124] In one embodiment, L is a bond. In one embodiment, L is a linking group selected from PEG, alkylene, triazole, and a group formed by a reaction between a haloalkyl and a nucleophile.

[0125] 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, e.g., 90 to 100, e.g., 100 to 120, e.g., 120 to 150, e.g., 150 to 200, e.g., 200 to 250, e.g., 250 to 300, e.g., 300 to 350, e.g., 350 to 400, e.g., 400 to 450, e.g., 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, e.g., 90 to 100, e.g., 100 to 120, e.g., 120 to 150, e.g., 150 to 200, e.g., 200 to 250, e.g., 250 to 300, e.g., 300 to 350, e.g., 350 to 400, e.g., 400 to 450, e.g., 450 to 500. In one embodiment of the disclosure, the ratio n:m is 10:1 to 1:10. In one embodiment of the disclosure, the ratio n:m is 10:1 to 9:1, 9:1 to 8:1, 8:1 to 7:1, 7:1 to 6:1, 6:1 to 5:1, 5:1 to 4:1, 4:1 to 3:1, 3:1 to 2:1, 2:1 to 3:2, 3:2 to 1:1, 1:1 to 2:3, 2:3 to 1:2, 1:2 to 1:3, 1:3 to 1:4, 1:4 to 1:5, 1:5 to 1:6, 1:6 to 1:7, 1:7 to 1:8, 1:8 to 1:9, and / or 1:9 to 1:10.

[0126] In one embodiment of the disclosure, the copolymer has a molecular mass of less than 100 kDa, such as less than 90 kDa, such as less than 80 kDa, such as less than 70 kDa, such as less than 60 kDa, such as less than 50 kDa, such as less than 40 kDa, such as less than 30 kDa, such as less than 20 kDa, such as less than 15 kDa, such as less than 14 kDa, such as less than 13 kDa. In one embodiment, the copolymer has a molecular mass of at least 1 kDa, such as at least 3 kDa, such as at least 5 kDa, such as at least 7 kDa, such as at least 9 kDa. In one embodiment of the disclosure, the copolymer has a molecular mass of 1 to 100 kDa, such as 1 to 5 kDa, such as 5 to 9 kDa, such as 9 to 13 kDa, such as 13 to 17 kDa, such as 17 to 20 kDa, such as 20 to 30 kDa, such as 30 to 40 kDa, such as 40 to 50 kDa, such as 50 to 60 kDa, such as 60 to 70 kDa, such as 70 to 80 kDa, such as 80 to 90 kDa, such as 90 to 100 kDa. In one embodiment, the copolymer has a molecular mass of 1 to 5 kDa, 5 to 9 kDa, 9 to 13 kDa, 13 to 17 kDa, 17 to 20 kDa, 20 to 30 kDa, 30 to 40 kDa, 40 to 50 kDa, 50 to 60 kDa, 60 to 70 kDa, 70 to 80 kDa, 80 to 90 kDa, and / or 90 to 100 kDa. The mass of the copolymer is preferably assessed without taking into account the mass of any drug and / or end group coupled thereto. In particular, when referring to the mass of the copolymer, reference is made to the structure of formula (I) but excluding moieties T, L and / or R 2 .

[0127] In one embodiment of the disclosure, the anticancer drug is an antitumor drug. As shown in the examples herein, the carboxylic acid groups of the copolymers of the disclosure are used to couple TLR7 / 8 agonists to trigger an immune response in vivo. The copolymer-drug conjugates shown in the examples remain soluble in water, with a large number of carboxylic acid groups coupled to the test drug. This phenomenon is observed despite the high content of styrene in the copolymer. In one embodiment of the disclosure, the antitumor drug is an antitumor drug having an effect against solid tumors. In one embodiment of the disclosure, the anticancer 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.

[0128] In one embodiment, at least 3 R 2 are anticancer drug moieties, such as at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 R 2In one embodiment, 2% of the monomer units in the copolymer are coupled to an anticancer drug moiety, such as 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%.

[0129] In one embodiment of the disclosure, the anticancer drug has the structure of Formula (A-I), Formula (A-II), Formula (A-III), or Formula (A-IV):

[0130]

[0131]

[0132] wherein

[0133] X 1a is -O-, -S-, or -NR Ca ;

[0134] 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 heterocycle, or substituted C 5-9 heterocycle;

[0135] 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;

[0136] each R 2a is 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 (carboxy), -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 is absent;

[0137] each R aa and R ba is 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;

[0138] wherein any substituents on the alkyl, aryl, or heterocyclyl groups are hydroxy, C 1-6 alkyl, hydroxyC 1-6 alkylene, C 1-6 alkoxy, C 3-6 cycloalkyl, C 1-6 alkoxyC 1-6 alkylene, amino, cyano, halo, or aryl;

[0139] j is 0, 1, 2, 3, or 4;

[0140] X 3a is -N- or -CH-;

[0141] R 4a is -CH2- or -CH(R 2a )-;

[0142] k is 0 or 1;

[0143] X 4a is -O-, -S-, -NH-, -N(R da )-, -CH2-, or -CH(R 2a )-;

[0144] each R da is 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);

[0145] or a tautomer thereof.

[0146] In one embodiment of the present disclosure, the anticancer drug has the structure of Formula (BI):

[0147]

[0148] in

[0149] R 1b Indicates (C 1-8 )alkyl, (C 3-8 ) cycloalkyl or a 3 to 8 membered saturated heterocyclic ring group containing an O atom, wherein R 1b Optionally, one or more independently selected from halogen, cyano, hydroxy and (C 1-3 ) substituted with a substituent of the alkoxy group;

[0150] Z 1b Indicates (C 2-6 )alkylene, wherein Z 1b The carbon atoms that are not adjacent to the nitrogen atoms can be replaced by oxygen atoms;

[0151] X 1b Indicates NR 5b 、>N-COR 5b 、>N-CONR 5b R 5ab 、CONR 5b NR 5b CO,

[0152] NR 5b CONR 6b or NR 6b CONR 5b ;

[0153] Y 1b Represents a single bond or (C 1-6 )alkylene;

[0154] Each R 2b are independently selected from halogen, cyano, hydroxyl, 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;

[0155] R 3b Indicates that (C 1-6 ) Alkoxy-substituted C 1-6 alkyl;

[0156] each R ab is independently selected from the group consisting of 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;

[0157] R 5b and R 5ab each independently represent hydrogen, a 3- to 8-membered saturated heterocyclic ring comprising a ring member O, S(O) p or NR 10b , (C 1-6 )alkyl or (C 3-6 )cycloalkyl, the latter two groups being optionally substituted by one or more substituents independently selected from NR 7b R 8b or R 9b , R 7b and R 8b each independently represent hydrogen, a 3- to 8-membered saturated heterocyclic ring comprising a ring member O, S(O) p or NR 10ab , (C 1-6 )alkyl or (C 3-6 )cycloalkyl, the latter two groups being optionally substituted by one or more substituents independently selected from halogen, cyano, S(O) q R 11b , OR 12b , CO2R 12b , OC(O)R 12b , SO2NR 12b R 13b CONR 12b R 13b , NR 12b R 13b , NR 12b SO2R 14b , NR 12b COR 13b or a 3- to 8-membered saturated heterocyclic ring comprising a ring member O, S(O) p or NR 10bb ,

[0158] or R 7b and R 8bwith the nitrogen atom to which they are attached form a 3- to 8-membered saturated heterocyclic ring containing a ring nitrogen atom and optionally one or more additional heteroatoms independently selected from the group consisting of nitrogen, oxygen, sulfur, and sulfonyl, the heterocyclic ring being optionally substituted with one or more substituents independently selected from the group consisting of halogen, cyano, S(O) q R 15b , OR 15b , CO2R 15b , COR 15b , OC(O)R 15b , SO2NR 15b R 16b , CONR 15b R 16b , NR 15b R 16b , NR 15b SO2R 17b , NR 15b COR 16b , NR 15b CO2R 16b , heteroaryl, (C 1-6 )haloalkyl, (C 3-8 )cycloalkyl, and (C 1-6 )alkyl, the last two groups being optionally substituted with one or more groups 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 ;

[0159] R Qb represents halogen, cyano, CO2R 20b , S(O) q R 20b , OR 20b , SO2NR 20b R 22b , CONR 20b R 22b , NR 20b SO2R 21b , NR 20b CO2R 21b , NR 20b COR 22b , or a 3- to 8-membered saturated heterocyclic ring containing a ring group NR 10cb ;

[0160] R 10b , R 10ab, R 10bb and R 10cb independently represent hydrogen, CO2R 23b , S(O) q R 23b , COR 24b , or (C 1-6 )alkyl, (C 2-6 )alkenyl, (C 2-6 )alkynyl or (C 3-8 )cycloalkyl, each of which can be optionally substituted with one or more substituents independently selected from halogen, cyano, OR 25b or NR 25b R 26b ;

[0161] 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 each independently represent hydrogen, (C 1-6 )alkyl or (C 3-6 )cycloalkyl;

[0162] R 14b , R 17b , R 21b and R 23b each independently represent (C 1-6 )alkyl or (C 3-6 )cycloalkyl;

[0163] h, i, p and q each independently represent an integer of 0, 1 or 2; and

[0164] Ab represents a monocyclic or bicyclic (C 6-10 )aryl or a monocyclic or bicyclic (C 5-12 )heteroaryl containing 1 to 3 heteroatoms; and

[0165] R bb and R cb independently represent hydrogen or (C 1-6 )alkyl, or R bb and R cb together form a (C 3-8 )cycloalkyl.

[0166] In one embodiment of the present disclosure, the anticancer drug has a structure of Formula (C-I):

[0167]

[0168] wherein

[0169] R 1c is selected from -(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 ;

[0170] X c is a bond or -NR 5c -;

[0171] R 4c is alkyl, aryl or heteroaryl;

[0172] R 2c is selected from hydrogen, alkyl, alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl-O-aryl, alkyl-O-alkyl, alkyl-O-alkenyl and alkyl or alkenyl substituted with one or more substituents selected from OH, halogen, -N(R 3c )2, -CO-N(R 3c )2, -CO-(C 1-10 )alkyl, -CO-O-(C 1-10 )alkyl, -N3, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, -CO-aryl, -CO-(substituted aryl), -CO-heteroaryl and -CO-(substituted heteroaryl);

[0173] each R 3c is independently selected from hydrogen and (C 1-10 )alkyl,

[0174] R 5c is selected from hydrogen and (C 1-10 )alkyl, or R 4c and R 5c may combine to form a 3- to 7-membered heterocyclic or substituted heterocyclic ring;

[0175] R 6c is selected from hydrogen and (C 1-10 )alkyl;

[0176] R 7c is selected from hydrogen and (C 1-10 )alkyl, or R 6c and R7c may combine to form a 3- to 7-membered heterocyclic ring or a substituted heterocyclic ring;

[0177] r is 0 to 4 and each R c is independently selected from the group consisting of (C 1-10 )alkyl, (C 1-10 )alkoxy, halogen, and trifluoromethyl.

[0178] In one embodiment of the disclosure, the anticancer drug is a TLR7, TLR8, or TLR7 / 8 agonist selected from the group consisting of: Imiquimod, Resiquimod, Gardiquimod, 852A, Loxoribine, Bropirimine, 3M-011 (CAS Registry No. 642473-62-9), 3M-052 (CAS Registry No. 1359993-59-1), DSR-6434 (CAS Registry No. 1059070-10-8), DSR-29133, SZU-101, SM-360320 (CAS Registry No. 226907-52-4), SM-276001 (CAS Registry No. 473930-22-2), VTX-2337 (CAS Registry No. 926927-61-9), and 1-(3-aminopropyl)-2-(ethoxymethyl)imidazolyl[4,5-c]quinolin-4-amine. Shukla et al., 2011 is a SAR study of TLR7 agonists that can be used to predict where coupling can be made without abrogating the activity of the agonist.

[0179] In one embodiment of the disclosure, the anticancer drug is a stimulator of interferon genes (STING) agonist. In one embodiment, the anticancer drug is IL-2, IL-12, or IL-15 or a fragment thereof.

[0180] In one embodiment of the disclosure, the anticancer drug is a radiopharmaceutical drug or a radioactive agent.

[0181] 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.

[0182] In one embodiment of the disclosure, when R 2 is an anticancer drug, L is a linking group, wherein R 2 moiety is coupled to L at a carbon atom or a heteroatom such as N, O, or S in R 2 In one embodiment of the disclosure, when R 2 is an anticancer drug, L is a bond, wherein R2 R is coupled to X at a carbon atom or heteroatom such as N, O, or S in R 2

[0183] In one embodiment, T is independently selected from the group consisting of H, alkyl, substituted alkyl, nitrile, hydroxyl, carboxyl, halogen, thiol, substituted thiol, acyl, substituted acyl, a group of Formula X-I, a group of Formula Y-I, a group of Formula Y-II, a fluorescent dye, a diagnostic agent;

[0184] wherein the group of Formula X-I has the structure:

[0185]

[0186] wherein R 3 and R 4 are each independently selected from the group consisting of hydrogen, alkyl, and substituted alkyl;

[0187] wherein the group of Formula Y-I has the structure:

[0188]

[0189] wherein R 5 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl; and

[0190] wherein the group of Formula Y-II has the structure:

[0191]

[0192] wherein R 5 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl, and

[0193] X 2 and X 3 are each independently selected from the group consisting of S and O.

[0194] In one embodiment of the present disclosure, the fluorescent dye is selected from the group consisting of a cyanine dye such as indocyanine green or cyanine 7; a triarylmethane dye such as fluorescein; a thiazine dye such as methylene blue, LUM015, VGT-309, AVB-620, C-Dots, BLZ-100.

[0195] One embodiment of the present disclosure provides a pharmaceutical composition comprising a 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.

[0196] Synthesis methods

[0197] ​In one embodiment of the present disclosure, copolymer-drug conjugates are synthesized by coupling an anticancer 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.

[0198] In one embodiment of the present disclosure, the precursor copolymer has the structure of Formula (III-P):

[0199]

[0200] where R x’ yes

[0201] wherein each L' is independently selected from H and a reactive handle, and

[0202] T, n, m, R 1 , X, R z and Ry are as defined herein.

[0203] In one embodiment of the present disclosure, the precursor poly(methacrylic acid-co-styrene) copolymer or precursor poly(acrylic acid-co-styrene) copolymer has the structure of Formula (IP):

[0204]

[0205] wherein each L' is independently selected from H and a reactive handle, and

[0206] T, n, m, R 1 , and X are as defined herein.

[0207] In one embodiment of the present disclosure, wherein each L' is independently selected from H, CH2Cl, CH2Br or CH2I.

[0208] Treatment

[0209] One embodiment of the present disclosure provides a copolymer-drug conjugate or a pharmaceutical composition of the present disclosure for use in medicine. The copolymer of the present disclosure is amphiphilic, which enables the conjugate to be internalized by disrupting cell membranes. Therefore, the drug moiety coupled to the copolymer can exert its effect on an extracellular or intracellular target. One embodiment of the present disclosure provides a copolymer-drug conjugate or a pharmaceutical composition of the present disclosure for use in treating cancer.

[0210] One embodiment provides the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure for use in the treatment of 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.

[0211] In one embodiment, the cancer is a liver cancer or a spleen cancer.

[0212] In one embodiment, the copolymer-drug conjugate or the pharmaceutical composition is administered intratumorally, for example by injection.

[0213] One embodiment of the disclosure provides a method of treating cancer, for example cancer characterized by a tumor (such as a solid tumor), the method comprising administering to a subject in need thereof the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure.

[0214] One embodiment of the disclosure provides a method of inducing an immune response in a subject, the method comprising administering to the subject the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure.

[0215] One embodiment of the disclosure provides a method of immunizing a subject, the method comprising administering to the subject the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure.

[0216] One embodiment of the disclosure provides a method of reducing tumor volume in a subject, the method comprising administering to the subject the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure.

[0217] One embodiment of the disclosure provides the use of the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure as an immune stimulant.

[0218] One embodiment of the disclosure provides the use of the copolymer-drug conjugate of the disclosure or the pharmaceutical composition of the disclosure for the manufacture of a medicament for the treatment of cancer.

[0219] One embodiment of the present disclosure provides the use of a poly(acrylic acid-co-styrene) or poly(methacrylic acid-co-styrene) copolymer for retaining an agent. "Retaining an agent" refers to increasing tissue retention of the compound / agent upon administration to a subject by mixing, formulating, or conjugating the compound / agent with the poly(acrylic acid-co-styrene) copolymer or the poly(methacrylic acid-co-styrene) copolymer. In one embodiment, the agent is an anti-cancer agent, a diagnostic agent, and / or a radiopharmaceutical agent. In one embodiment, the agent is an anti-cancer drug disclosed herein. In one embodiment, the agent is an agent capable of inducing an immune response.

[0220] One embodiment provides a method of increasing the residence time of an agent in a tissue, the method comprising administering the agent with a copolymer of the present disclosure. The tissue can be a cancerous tissue or a tissue surrounding a cancerous tissue. In one embodiment of the method, the agent is conjugated to a copolymer of the present disclosure, for example, by a bond or a linking group. Examples

[0221] Example 1: Synthesis of copolymer and copolymer-dye conjugates

[0222] Materials and Methods

[0223] Copolymer was synthesized using the RAFT agent 2-cyano-2-propyl dodecyl trithiocarbonate (resulting in copolymer AASTY 12.5 ) or 2-(dodecylsulfanylthiocarbonylsulfanyl)-2-methylpropanoic acid 3-azido-1-propanol ester (resulting in copolymer AASTY 9.9 -N3), the initiator azobisisobutyronitrile (AIBN), and the monomers acrylic acid (AA) and styrene (STY) with an initial molar ratio of (45:55) (AA:STY). The reagents were charged to a Schlenk flask and oxygen was removed by 4 freeze-pump-thaw cycles. The mixture was heated to 60 °C or 70 °C for 9 hours to 12 hours, reaching a monomer conversion of 95% for AASTY 12.5 and 69% for AASTY 9.9 -N3. The product was dissolved in diethyl ether, precipitated in heptane, and vacuum dried to yield a yellow brittle solid. For AASTY 12.5The polymer was dissolved in (water:ethanol) (1 :3) containing 30% H2O2 and incubated at 70 °C overnight to remove dodecyltrithiocarbonate (ttc) end groups, resulting in a colourless solution. The polymer was precipitated into deionised water and collected by centrifugation. All final polymer products were converted to partial sodium salts by dissolution in deionised water and addition of NaOH (1 M) until pH 7-7.5, the opaque mixture was filtered and lyophilised. Number average molecular weight (Mn), weight average molecular weight (Mw) and dispersity (D = Mw / Mn) were measured using a Dionex Ultimate 3000 system. Detection was carried out by a Dawn Heleos II multi-angle light scattering detector and Optilab rEX refractive index detector. Gel permeation chromatography (GPC) was carried out in a Superose 6-Increase column (10 / 300, Cytiva). Data were analysed using Astra 7.0, using a dn / dc of 0.170 mL / g. n w n w

[0224] The polymer was dissolved in methanol at 50 mg / mL, an equimolar amount of Cyanine 7-DBCO (NIR fluorescent dye with a cycloalkyne moiety for coupling to azides - Lumiprobe GmbH) was added and the reaction left at room temperature overnight. The methanol was evaporated by a stream of nitrogen and the solid dissolved in water. The dye conjugated polymer was purified by size exclusion column chromatography and lyophilised to give a dark green solid.

[0225] Results

[0226] All products were analysed by SEC-MALS coupled with a UV detector. The Cy7 dye absorbs the MALS laser, hindering mass determination, although the elution time of the polymer was the same as the non-conjugated polymer. The copolymers and conjugates produced are shown in Table 1.

[0227] Table 1: Summary of conjugates produced.

[0228]

[0229] Conclusion

[0230] Azide RAFT agent was used to make AASTY polymers and enabled “click” conjugation to the R terminus of the RAFT agent.

[0231] Example 2: Synthesis of copolymer conjugated with TLR agonist

[0232] Materials and methods

[0233] ​​​​AASTY copolymers were added to DMF at 5%, 10%, or 15% molar equivalents of AA relative to the TLR7 / 8 agonist 4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinoline-1-propylamine (referred to as "TLR7 / 8a"). Most agonists were dissolved by gentle heating, and 2 molar equivalents (relative to TLR7 / 8a) of NHS and 1 molar equivalent of N,N-diisopropylcarbodiimide were added to the sample. The reaction was left at room temperature overnight, and the product was purified by size exclusion chromatography the next day and lyophilized to obtain a white solid.

[0234] A portion of the conjugate was dissolved in methanol containing 1 molar equivalent of cyanine 7-DBCO. The reaction was allowed to stand at room temperature overnight, purified by size exclusion chromatography, and lyophilized to yield a dark green solid.

[0235] result

[0236] pass 1 H NMR characterization confirmed and quantified the coupling of TLR7 / 8a to the AA carboxylic acid of the copolymer via its aminopropyl group.

[0237] Example 3: Copolymer constructs reside in tissues for very long duration in vivo

[0238] Materials and methods

[0239] AASTY constructs (AASTY-Cy7) with fluorescent dye were administered to healthy mice via different routes of administration. Biodistribution was monitored over a 2-week period.

[0240] result

[0241] Subcutaneous (SC) injection of AASTY of Example 1 9.9 After Cy7 injection, most of the fluorescent signal remained in the vicinity of the initial injection depot and did not appear to diffuse significantly after the formulation was taken up by the SC tissue ( Figure 2A). While the integrated signal density slowly decreased over time, most of the signal remained clearly visible after 2 weeks and was distributed similarly, unlike the free dye control, which was mostly cleared by the kidneys within the first few hours after administration. This indicates that the AASTY-Cy7 conjugate strongly and rapidly adhered to the local tissue for an extended period of time. Image analysis estimated the half-life of the compound to be 58 hours, which is notable for a water-soluble substance. To rule out the possibility that the Cy7 dye was cleaved from the AASTY conjugate and misleading our interpretation, an equivalent dose of the DBCO-Cy7 dye in the form used for conjugation was dissolved in 5% DMSO-DPBS and injected SC in a similar manner. This control experiment showed that, although not cleared by the kidneys, the free dye was rapidly metabolized at its injection site, with most of the signal disappearing after 1 week, which confirmed that the conjugate exhibited different PK characteristics than the free form of the dye. Considering that no significant signal was detected outside the injection area in vivo, the mice were necropsied at the end of 2 weeks to extract the organs and analyze the signal distribution with higher sensitivity. Ex vivo organ images showed that the signal came from a single point on the inguinal lymph node above the injection site, and residual signal was also visible in the liver. This indicates that the compound was slowly cleared through the liver and lymphatic pathways. No signs of toxicity or inflammation were observed during the study, which indicates that the copolymer itself (i.e., not conjugated to any drug) was neither significantly toxic nor immunogenic. After intravenous (IV) injection in the tail vein, the compound rapidly reached the systemic circulation within the first few hours after injection, as can be seen from the signal emitted by the paws, nose, and eyes. As with the SC injection, a significant portion of the signal remained close to the injection site (tail), and did not spread during the entire duration of the study after deposition. A significant signal was visible in the liver from the first acquisition time to the end of 2 weeks. Image analysis of the liver area showed that the half-life of the compound was smaller, at approximately 17 hours, but the signal was still visible after 2 weeks. The organ extraction and analysis after necropsy, which included the tail of the mouse, confirmed the significant accumulation of the compound in the liver and also showed a small but significant accumulation in the spleen, which was not observed with SC administration Figure 2 B). However, no signal was reported in the inguinal lymph nodes or the kidneys. Again, no signs of toxicity or inflammation were reported.

[0242] These two studies collectively indicate that AASTY is able to strongly adhere to the local tissue where it is injected and stain these tissues for a long period of time. However, the data also indicate that a portion of it is slowly cleared through the liver and lymphatic pathways over time. The control experiment also pointed out that the dye can not be cleaved from AASTY during its residence in vivo. In addition to this, the good tolerance of the compound by the mice and the apparent lack of immunogenicity make AASTY conjugates ideal candidates for tissue staining or local drug exposure.

[0243] Example 4: Copolymer conjugates show good retention in solid tumors in mouse models

[0244] Materials and methods

[0245] Healthy Balb / cJ Rjj mice were injected subcutaneously in the right flank with 100 pL of murine colon carcinoma CT26 cancer cells (3 x 105) in RPMI medium. Mice bearing tumors with a volume of 50 mm 3 to 200 mm 3 were randomly grouped and rearranged to new cages by blocking the initial tumor volume, each cage receiving one of the four treatments described in Table 2 (50 pL) administered intratumorally. For each treatment, a total of 3 injections were performed, with 7 days interval between each injection. Table 2 shows a summary of the treatments tested, and Figure 3 A shows a summary of the study.

[0246] Table 2: Summary of the treatments tested.

[0247]

[0248] Surviving mice were re-challenged 83 days after the first tumor inoculation by a second inoculation of CT26 cancer cells (same dose) in the left flank of the mice, while setting a control group that received its first inoculation. Mice were monitored for body weight, tumor measurements using an electronic caliper, twice a week, and fluorescent imaging of all formulations containing the Cy7 moiety was performed periodically. Necropsy was performed within one week after euthanasia (period of storage at -20°C), including excised tumors and bilateral inguinal lymph nodes. The average signal of each organ was corrected and calibrated for each treatment group, according to the half-life estimated by in vivo scans, assuming first-order decay and taking into account that all mice were euthanized at different moments of the study. The biodistribution of the compound was further evaluated by injecting a single dose of AASTY-TLR7 / 8a2X and (50 pL, intratumorally) and scanning the mice by fluorescence tomography (MILabs U-CT, in FLT mode). Scans were performed 5 to 6 days after mice euthanasia, with the mice stored at -20°C between euthanasia and scanning. Reconstructed images were analyzed and 3D rendered using Imalytics Preclinical 2.1. During the study, tumor volume was calculated as (length x width) 2 / 2, and mice were euthanized when tumor volume reached 2000 mm 3 . Humane endpoints included mice presenting clear signs of weakness, significant weight loss (15% of initial body weight or 10% overnight weight loss), or presence of tumor wounds greater than 8 mm.

[0249] Results

[0250] Intratumoral administration data show that the residence time of the AASTY conjugate is extremely long, with a half-life estimated at 41 days, and most of the signal is still clearly visible at the end of the study (week 15) after 117 days Figure 2 A large amount of compound remains in the tumor (its injection site) and is distributed in most of its volume. For the cured mice, the signal is still present in the area near the scar of the tumor, which suggests a redistribution of the compound in the surrounding tissues as the tumor cells are eliminated. As after intravenous administration, the exhumation-corrected data show that a large amount of signal is also observed in the liver and a small amount of AASTY-TLR7 / 8a conjugate also reached the spleen. While no signal is still detected in the kidneys and in the left lymph node (contralateral to the tumor), a significant signal of the AASTY formulation is detected in the right lymph node (ipsilateral to the tumor) and the amount of signal is much higher for the high AASTY-TLR7 / 8a dose.

[0251] Conclusions

[0252] The conjugate of AASTY shows a very long residence time after intratumoral injection, exceeding the half-life of both SC and IV injections. The low biodistribution in the kidneys suggests that the disclosed conjugate can produce fewer kidney-related adverse effects compared to other chemotherapies that can damage the kidneys, ureters, and bladder.

[0253] Example 5: TLR7 / 8a-copolymer conjugates reduce solid tumor volume and improve survival in murine models

[0254] Materials and methods

[0255] Materials and methods are as indicated in Example 3.

[0256] Results

[0257] Tumor kinetics monitoring shows that most of the tumors of each mouse group grew exponentially rapidly after the start of the study, except for the high-dose AASTY-TLR7 / 8a treatment group Figure 2 (F). Figure 2 B to Figure 2E). Here, the influence of two parameters was evaluated: the dose of TLR7 / 8a (non-lethal high dose in the range expected to elicit an immune response, and half dose below this range), and the dose of AASTY (in a constant ratio to TLR7 / 8a). First, the results of the AASTY and AASTY-TLR7 / 8a 1X treatment groups showed that AASTY itself had no anti-tumor activity at both doses. In comparison to the control group, intratumoral injection of TLR7 / 8a alone at high dose showed a potential minor improvement in tumor treatment, considering that two out of six mice recovered, while the remaining four mice did not respond to the treatment. An explanation for these results is that these compounds have very high potency, on the other hand they diffuse rapidly out of the tumor microenvironment after injection. The co-polymer-drug conjugates of the present disclosure are designed to provide sustained local residence. For AASTY-TLR7 / 8a 1X, no significant improvement was observed. In contrast, for AASTY-TLR7 / 8a 2X, there was no tumor growth at all in the first 20 days, and the overall tumor growth rate was negative in the first 10 days Figure 2 F, Figure 2 H and Figure 2 J). In comparison to the low dose treatment, the overall survival rate was higher, with tumor growth in only two out of six mice after 20 days; the remaining four mice recovered completely Figure 2 G and Figure 2 I).

[0258] Conclusions

[0259] TLR7 / 8a-co-polymer conjugates reduced solid tumor volume in a murine model and improved survival at an appropriate dose.

[0260] Example 6: TLR7 / 8a-copolymer conjugates prevent tumor growth in tumor inoculation studies in murine models

[0261] Materials and Methods

[0262] At 83 days after the first tumor inoculation, surviving mice were re-challenged by a second inoculation of CT26 cancer cells (same dose) in the left flank of the mice, while a control group that received its first inoculation was set. Mice were monitored for body weight, tumor measurements using electronic calipers twice a week, and fluorescent imaging of all formulations containing the Cy7 moiety was performed periodically.

[0263] Results

[0264] At 83 days after the first inoculation, surviving mice were re-challenged with tumor cells, and no tumor growth was observed Figure 2 K).

[0265] Conclusions

[0266] Surviving mice in the study had an adaptive response and were resistant to CT26.

[0267] Example 7: Copolymer retention in injected tissues; slow distribution through lymphatic system in murine models

[0268] Materials and Methods

[0269] To better understand the biodistribution kinetics after intratumoral administration, a small group of mice was subjected to fluorescence tomography 5 to 6 days after a single dose of AASTY-TLR7 / 8a 2X.

[0270] Results

[0271] The compound diffused through all the bean-shaped tumors, with the signal being maximal at the injection point, and mainly accumulated in the liver ( Figure 2 M). The new information brought by this study is that the compound is apparently distributed throughout the lymphatic system, since the signal can be detected in a globally symmetrical network of tubes connected by nodes that more or less align with known lymph node locations (mandible, neck, axillary, arm, lumbar, and codal, among others).

[0272] Conclusions

[0273] From this example and the other examples disclosed herein, a possible mechanism of action of the biodistribution can be as follows: upon injection, the copolymer / copolymer conjugate will rapidly adhere to the local tissue, possibly by using the amphiphilic nature of the copolymer to incorporate itself into lipid membranes or even inside cells. If it is injected intravenously (IV) or in a highly vascularized tissue, a portion of it will enter the systemic circulation and rapidly accumulate mainly in the liver, and secondarily in the spleen.

[0274] Example 8: Copolymer MAASTY shows similar biodistribution profile as AASTY

[0275] Materials and Methods

[0276] Poly(methacrylic acid-co-styrene) (MAASTY) was synthesized analogously to Example 1 using the RAFT agent 2-cyano-2-propyl dodecyl trithiocarbonate 2-(dodecylsulfanylthiocarbonylsulfanyl)-2-methylpropanoic acid 3-azido-1-propanol ester, the initiator azobisisobutyronitrile (AIBN) and the monomers methacrylic acid (MAA) and styrene (STY) in an initial molar ratio of (50:50) (MAA:STY). As an alternative to the use of a Schlenk flask, here the reagents were charged to a glass ampoule, the oxygen was removed by 4 freeze-pump-thaw cycles and the ampoule was sealed under vacuum using a torch lighter. The mixture was heated to 55 °C for 16 hours, with an estimated monomer conversion of 85% (estimated from viscosity). The product was dissolved in diethyl ether, precipitated in heptane and vacuum dried to yield a yellow brittle solid.

[0277] 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 overnight at room temperature. The methanol was evaporated by a stream of nitrogen, and the solid was dissolved in water. The dye-coupled polymer was purified by size exclusion column chromatography and lyophilized to obtain a dark green solid.

[0278] For reference, a new batch of AASTY-Cy7 was prepared using the RAFT agent 2-(dodecylthiothiocarbonylthio)-2-methylpropanoate (45:55) (AA:STY) following a similar protocol as in Example 1, but using sealed ampoules instead of Schlenk flasks for MAASTY. The mixture was heated to 60°C for several hours to achieve 85% conversion (via 1 H-NMR measurement), the copolymer AASTY 7.1 (M n =7.1kDa, only through 1 H-NMR estimation). For MAASTY, the copolymer was then coupled with Cy7 at 25 mg / mL copolymer to give AASTY 7.1 -Cy7.

[0279] The two Cy7 copolymer constructs were administered to mice subcutaneously. All mice had palpable CT26 tumors, but were injected in the flank just outside the tumor area. Biodistribution was monitored by fluorescence tomography over the course of 5 days.

[0280] result

[0281] AASTY 7.1 -Biodistribution of Cy7 constructs ( Figure 5 ) is consistent with 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 the new dataset). The majority of the fluorescent signal remained in the vicinity of the initial injection depot, with a small portion diffusing into the lymphatic network over time, and the majority of the signal still measurable 5 days after injection.

[0282] The MAASTY-Cy7 conjugate appears to distribute according to the same pattern, with a slightly higher signal remaining after 5 days, but mimics the results of AASTY-Cy7 very well in terms of tissue retention and biodistribution. These results demonstrate that the two polymers share common pharmacokinetic characteristics, which is attributed to their structural and physicochemical similarities ( Figure 6), indicating that their types of interaction with biological tissues are very similar. Therefore, copolymers SMA and DIBMA are also envisioned to be useful for retaining cargo in tissues. Notably, the mice in this biodistribution study carried tumors, but this assumption can be reasonably extrapolated to healthy mice based on the consistency of the results of AASTY-Cy7 with healthy mice in Example 3 and the similarity of the biodistribution profiles of AASTY and MAASTY in Figure 5 Example 4.

[0283] Conclusion

[0284] Due to their structural similarity, AASTY and MAASTY exhibit similar pharmacokinetic profiles, both exhibiting excellent tissue-residence properties. It can be expected that these two copolymers will reach similar levels in cancer treatment when coupled with TLR7 / 8 agonists as in Examples 5 and 6. Based on these findings, it can also be envisioned that other polymers such as poly(styrene-co-maleic acid) (SMA) and poly(diisobutylene-co-maleic acid) (DIBMA), exhibit similar behavior in vivo and exhibit the same type of pharmacokinetic profiles in tissue residence because they, like AASTY and MAASTY, are amphiphilic copolymers containing both hydrophobic and hydrophilic subunits (see Figure 6 ).

[0285] References

[0286] 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.

[0287] 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.

[0288] WO 2022 / 226032 A1.

Claims

1. A copolymer-drug conjugate comprising an amphiphilic copolymer and three or more anticancer drug moieties coupled to the amphiphilic copolymer. The copolymer-drug conjugate according to claim 1 , wherein the amphiphilic copolymer is an anionic amphiphilic copolymer.

3. The copolymer-drug conjugate according to 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. The copolymer-drug conjugate of any preceding claim, 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. The copolymer-drug conjugate according to any one of the preceding claims, wherein the anticancer drug moiety is coupled to the amphiphilic copolymer via a bond or a linking group.

6. A copolymer-drug conjugate having the structure of formula (III): in: Each R 1 independently selected from CH3 and H, Each R y Independently selected from H, COOH and COX-LR 2 , Each R z independently selected from H and CH3, Each R x Independently 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 are independently selected from H or anticancer drugs, wherein at least three R 2 It is the anticancer drug part, Each T is the same or different terminal group, n is 5 to 500, m is 5 to 500, The copolymers are random, alternating or statistical copolymers.

7. The copolymer-drug conjugate according to any one of the preceding claims, wherein the copolymer-drug conjugate has the structure of formula (I): in: Each R 1 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 are independently selected from H or anticancer drugs, wherein at least three R 2 It is the anticancer drug part, Each T is the same or different terminal group, n is 5 to 500, m is 5 to 500, The copolymers are random, alternating or statistical copolymers.

8. The copolymer-drug conjugate according to any one of the preceding claims, wherein R 1 For CH3.

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

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

11. The copolymer-drug conjugate according to any one of the preceding claims, wherein X is selected from O, S or NH, for example wherein X is O.

12. The copolymer-drug conjugate of any preceding claim, wherein L is a bond.

13. The copolymer-drug conjugate of any preceding claim, wherein L is a linking group selected from the group consisting of PEG, an alkylene group, a triazole, and a group formed by the reaction between a haloalkyl group and a nucleophile.

14. The copolymer-drug conjugate of any preceding claim, wherein the copolymer of the copolymer-drug conjugate has a molecular mass of less than 20 kDa, such as less than 15 kDa, such as less than 14 kDa, such as less than 13 kDa, and / or wherein the copolymer has a molecular mass of at least 1 kDa, such as at least 3 kDa, such as at least 5 kDa, such as at least 7 kDa, such as at least 9 kDa.

15. The copolymer-drug conjugate of any preceding claim, wherein the anticancer drug is an antitumor drug. 16 . The copolymer-drug conjugate according to claim 1 , wherein the anti-tumor drug is an anti-tumor drug having an anti-solid tumor effect.

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

18. The copolymer-drug conjugate of any one of the preceding claims, wherein the anticancer drug has a structure of Formula (AI), Formula (A-II), Formula (A-III), or Formula (A-IV): in X 1a Is -O-, -S-, or -NR Ca ; 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 heterocyclic ring; 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)-(C1-C6) 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 is 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 any substituent on the alkyl, aryl or heterocyclic group is hydroxyl, C 1-6 Alkyl, hydroxyl 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 -CH2- or -CH(R 2a )-; k is 0 or 1; X 4a Is -O-, -S-, -NH-, -N(R da )-、-CH2-、or -CH(R 2a )-; 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)-(C1-C6) 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.

19. The copolymer-drug conjugate of any one of the preceding claims, wherein the anticancer drug has the structure of formula (BI): in R 1b Indicates (C 1-8 )alkyl, (C 3-8 ) cycloalkyl or a 3 to 8 membered saturated heterocyclic ring group containing an O atom, wherein R 1b Optionally, one or more independently selected from halogen, cyano, hydroxy and (C 1-3 ) substituted with a substituent of the alkoxy group; Z 1b Indicates (C 2-6 ) alkylene, wherein Z not adjacent to the nitrogen atom 1b The carbon atoms in can be replaced by oxygen atoms; X 1b Indicates NR 5b 、>N-COR 5b 、>N-CONR 5b R 5ab 、CONR 5b NR 5b CO、NR 5b CONR 6b or NR 6b CONR 5b ; Y 1b Represents a single bond or (C 1-6 )alkylene; Each R 2b are independently selected from halogen, cyano, hydroxyl, 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 Indicates that (C 1-6 ) Alkoxy-substituted C 1-6 alkyl; Each R ab are independently selected from halogen, cyano, hydroxyl, 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 Each independently represents hydrogen, including cyclic groups O, S(O) p or NR 10b 3 to 8 membered saturated heterocyclic ring, (C 1-6 )alkyl or (C 3-6 ) cycloalkyl, the latter two groups are optionally substituted by one or more independently selected from NR 7b R 8b or R 9b Substituents substituted, R 7b and R 8b Each independently represents hydrogen, including cyclic groups O, S(O) p or NR 10ab 3 to 8 membered saturated heterocyclic ring, (C 1-6 )alkyl or (C 3-6 )cycloalkyl, the latter two groups are optionally substituted by one or more independently selected from halogen, cyano, S(O) q R 11b , OR 12b 、CO2R 12b 、OC(O)R 12b 、SO2NR 12b R 13b CONR 12b R 13b NR 12b R 13b NR 12b SO2R 14b NR 12b COR 13b , or containing a ring group O, S(O) p or NR 10bb substituted with a 3- to 8-membered saturated heterocyclic ring group, or R 7b and R 8b Together with the nitrogen atom to which they are attached, they form a 3- to 8-membered saturated heterocyclic ring comprising a ring nitrogen atom and optionally one or more other heteroatoms independently selected from nitrogen, oxygen, sulfur and sulfonyl, the heterocyclic ring being optionally substituted by one or more independently selected from halogen, cyano, S(O) q R 15b , OR 15b 、CO2R 15b 、COR 15b 、OC(O)R 15b 、SO2NR 15b R 16b 、CONR 15b R 16b NR 15b R 16b NR 15b SO2R 17b NR 15b COR 16b NR 15b CO2R 16b , heteroaryl, (C 1-6 ) haloalkyl, (C 3-8 )cycloalkyl and (C 1-6 )alkyl, the latter two groups are optionally substituted by one or more 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 The group substitution; R Qb Indicates halogen, cyano, CO2R 20b 、S(O) q R 20b , OR 20b 、SO2NR 20b R 22b 、CONR 20b R 22b NR 20b SO2R 21b NR 20b CO2R 21b NR 20b COR 22b or containing a ring group NR 10cb A 3- to 8-membered saturated heterocyclic ring; R 10b 、R 10ab 、R 10bb and R 10cb Independently represent hydrogen, CO2R 23b 、S(O) q R 23b 、COR 24b , or (C 1-6 )alkyl, (C 2-6 ) alkenyl, (C 2-6 ) alkynyl or (C 3-8 ) cycloalkyl, each of which may be optionally substituted by one or more independently selected from halogen, cyano, OR 25b or NR 25b R 26b Substituents substituted; 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 Each independently represents hydrogen, (C 1-6 )alkyl or (C 3-6 )cycloalkyl; R 14b 、R 17b 、R 21b and R 23b Each independently represents (C 1-6 )alkyl or (C 3-6 )cycloalkyl; h, i, p, and q each independently represent an integer 0, 1, or 2; and Ab represents a monocyclic or bicyclic ring (C 6-10 ) aryl or a monocyclic or bicyclic ring containing 1-3 heteroatoms (C 5-12 ) heteroaryl; and R bb and R cb independently represents hydrogen or (C 1-6 ) alkyl, or R bb and R cb Combined together to form (C 3-8 )cycloalkyl.

20. The copolymer-drug conjugate of any one of the preceding claims, wherein the anticancer drug has the structure of formula (CI): in R 1c Selected from -(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 -; R 4c is alkyl, aryl or heteroaryl; R 2c is selected from hydrogen, alkyl, alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl-O-aryl, alkyl-O-alkyl, alkyl-O-alkenyl, and alkyl or alkenyl substituted with one or more substituents selected from OH, halogen, -N(R 3c )2、-CO-N(R 3c )2、-CO-(C 1-10 )alkyl, -CO-O-(C 1-10 )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 are independently selected from hydrogen and (C 1-10 )alkyl, R 5c Selected from hydrogen and (C 1-10 ) alkyl, or R 4c and R 5c may combine to form a 3- to 7-membered heterocyclic ring or a substituted heterocyclic ring; R 6c Selected from hydrogen and (C 1-10 )alkyl; R 7c Selected from hydrogen and (C 1-10 ) alkyl, or R 6c and R 7c may combine to form a 3- to 7-membered heterocyclic ring or a substituted heterocyclic ring; r is 0 to 4 and each R present c Independently selected from (C 1-10 )alkyl, (C 1-10 )alkoxy, halogen and trifluoromethyl.

21. The copolymer-drug conjugate of any of the preceding claims, wherein the TLR7, TLR8 or TLR7 / 8 agonist is selected from the list consisting of: imiquimod, resiquimod, gademod, 852A, loxoribine, bropirimine, 3M-011 (CAS Reg. No. 642473-62-9), 3M-052 (CAS Reg. No. 1359993-59-1), DSR-6434 (CAS No. 1059070-10-8), DSR-29133, SZU-101, SM-360320 (CAS Reg. No. 226907-52-4), SM-276001 (CAS Reg. No. 473930-22-2), VTX-2337 (CAS Reg. No. 926927-61-9), and 1-(3-aminopropyl)-2-(ethoxymethyl)imidazo[4,5-c]quinolin-4-amine.

22. The copolymer-drug conjugate of any preceding claim, wherein the anticancer drug is a stimulator of interferon genes (STING) agonist.

23. The copolymer-drug conjugate of any preceding claim, wherein the anticancer drug is IL-2, IL-12, or IL-15, or a fragment thereof.

24. The copolymer-drug conjugate of any preceding claim, wherein the anticancer drug is a radiopharmaceutical or radiopharmaceutical agent.

25. The copolymer-drug conjugate of any preceding claim, wherein the copolymer-drug conjugate comprises one type of anticancer drug.

26. The copolymer-drug conjugate of any preceding claim, wherein the copolymer-drug conjugate comprises two or more different anticancer drugs.

27. The copolymer according to any one of the preceding claims, wherein L is a linking group, and wherein R 2 Part in R 2 The carbon atom or heteroatom such as N, O or S in the reaction mixture is coupled to L.

28. The copolymer of any one of the preceding claims, wherein L is a bond, and wherein R 2 Part in R 2 The carbon atom or heteroatom such as N, O or S in the alkyl group is coupled to X.

29. The copolymer-drug conjugate of any preceding claim, wherein T is independently selected from 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, a diagnostic agent; The group of formula XI has the structure: where R 3 and R 4 are each independently selected from hydrogen, alkyl, and substituted alkyl; The group of formula YI has the structure: where R 5 is selected from hydrogen, alkyl, and substituted alkyl; and The group of formula Y-II has the structure: where R 5 is selected from hydrogen, alkyl and substituted alkyl, and X 2 and X 3 are each independently selected from S and O.

30. The copolymer-drug conjugate of any preceding claim, wherein the fluorescent dye is selected from cyanine dyes such as indocyanine green or cyanine 7; triarylmethane dyes such as fluorescein; thiazine dyes such as methylene blue, LUM015, VGT-309, AVB-620, C-Dots, BLZ-100.

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

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

33. A method for synthesizing a copolymer-drug conjugate according to any one of the preceding claims, the method comprising conjugating an anticancer 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 method of claim 33, wherein the precursor copolymer has the structure of formula (III-P): where R x’ yes wherein each L' is independently selected from H and a reactive handle, and T, n, m, R 1 , X, R z and Ry as described in any one of claims 6 to 11 or 27 to 28.

35. The method of any one of claims 33 and 34, wherein the precursor poly(methacrylic acid-co-styrene) copolymer or the precursor poly(acrylic acid-co-styrene) copolymer has a structure of formula (IP): wherein each L' is independently selected from H and a reactive handle, and T, n, m, R 1 and X as described in any one of claims 6 to 11 or 27 to 28.

36. The method of any one of claims 34 and 35, wherein each L' is independently selected from H, CH2Cl, CH2Br, or CH2I.

37. 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, for use in medicine.

38. 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 for use in treating cancer.

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

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

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. 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 cancer.

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 or 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 one of claims 38 to 44, wherein the copolymer-drug conjugate or the pharmaceutical composition is administered intratumorally, such as by injection.

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

47. A method of inducing an immune response in a subject, the method 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. A method of immunizing a subject, the method 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.

49. A method of reducing tumor volume in a subject, the method 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.

50. 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 as an immunostimulant.

51. 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 in the preparation of a medicament for treating cancer.

52. Use of a copolymer produced by polymerization of the following substances for retaining a pharmaceutical agent in tissue: a. styrene and / or diisobutylene, and b. Acrylic acid, methacrylic acid and / or maleic acid.

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

54. A method of increasing the residence time of an agent in a tissue, such as a cancerous tissue, comprising administering the agent together with a copolymer produced by polymerization of: a. styrene and / or diisobutylene, and b. Acrylic acid, methacrylic acid and / or maleic acid.

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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