Protonated pH-responsive polymer encapsulation of bispecific antibodies and cytokines

Abstract

Described herein are therapeutic pH-responsive micelle compositions comprising block copolymers and biological therapeutic agents useful in the treatment of cancer, methods for making such micelles, and intermediate compositions useful for making such micelles.

Description

[Background technology]

[0001] Multifunctional nanoparticles have attracted attention for a wide range of applications, including biosensors, diagnostic nanoprobes, and targeted drug delivery systems. These efforts have been driven largely by the need to reduce side effects and improve biological specificity in diagnostics and therapeutics through precise spatiotemporal control of drug delivery in various physiological systems. To achieve this goal, efforts have been made to develop stimuli-responsive nanoplatforms. Environmental stimuli that have been utilized to determine delivery efficiency include pH, temperature, enzyme expression, redox reactions, and light induction. Among these activation signals, pH triggering is one of the most widely studied stimuli based on two types of pH differences: (a) pathological (e.g., tumor) versus normal tissues and (b) acidic intracellular compartments.

[0002] For example, due to the unusual acidity (pH ∼6.5) of the tumor extracellular microenvironment, several pH-responsive nanosystems have been reported to enhance the sensitivity of tumor imaging or the efficacy of therapy. However, in the case of polymeric micelle compositions that release drugs by hydrolysis in an acidic environment, drug release can take several days, during which time the body can excrete or degrade the micelles.

[0003] To target the acidic internal / lysosomal compartments, nanovectors with pH-cleavable linkers have been investigated to improve payload bioavailability. Furthermore, several smart nanovectors with pH-induced charge conversion have been designed to enhance drug efficacy. The endocytic system is composed of a series of compartments with unique roles in sorting, processing, and degradation of internalized cargoes. Selective targeting of different endocytic compartments with pH-sensitive nanoparticles is particularly challenging due to the short nanoparticle residence time (less than a few minutes) and small pH difference (e.g., less than one pH unit between early endosomes and lysosomes) in these compartments. Ultra-pH-sensitive (UPS) nanoparticles remain as intact micelles at physiological pH (7.4) in the blood circulation, but undergo a micelle transition pH (pH ) upon exposure to the acidic tumor environment. t ) and decomposes.

[0004] Bispecific antibodies (BsAbs) are an important class of therapeutic agents for immuno-oncology applications. T cell engagers (TCEs) target tumor-associated antigens and T cells to eradicate antigen-expressing tumor cells. TCEs for solid tumors have also shown promising clinical efficacy but exhibited dose-limiting toxicities due to on-target / off-tumor effects. For example, patients receiving solitomab (EpCAM x CD3 bispecific) experienced severe gastrointestinal toxicity, which hindered further development.

[0005] Cytokines (e.g., IL-12, IL-2) and cytokine fusion proteins (e.g., IL-12Fc, IL-2Fc) can induce anti-tumor immune responses, but their clinical application is limited by unfavorable pharmacokinetic properties and significant dose-limiting toxicities (e.g., cytokine release syndrome, vascular leak syndrome, etc.).

[0006] U.S. Patent No. 9,751,970, entitled "Block Copolymer and Micelle Compositions and Methods of Use Thereof," issued September 5, 2017, by Jinming Gao et al., describes micelle-forming polymers that can be used to entrap therapeutic agents, including chemotherapeutic agents. The '970 patent provides an example of the encapsulation of doxorubicin using PEO-b-PC6A. The method of the '970 patent involves dissolving doxorubicin and PEO-b-PC6A in water and hydrochloric acid. The solution is then added dropwise to a 0.1 M pH 9 buffer solution under sonication. While the encapsulation method disclosed in the '970 patent can encapsulate small molecules and some biological agents, the inventors have found that this method is unable to encapsulate certain biomolecules, particularly cytokines and antibodies.

[0007] There remains a need in the art to provide methods for pH-sensitive targeted delivery of bispecific antibodies and cytokines to the tumor microenvironment. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 9,751,970 Summary of the Invention [Problem to be solved by the invention]

[0009] The polymeric encapsulating agents or micelles described herein are useful therapeutic agents for the treatment of primary and metastatic tumor tissue (including lymph nodes). The block copolymer and micelle compositions presented herein exploit this ubiquitous pH difference between cancerous and normal tissues, providing a highly sensitive and specific response after encountering the acidic pH of the tumor microenvironment, thus enabling the deployment of therapeutic payloads to tumor tissue. pH-sensitive encapsulation minimizes off-tumor effects while providing targeted delivery to the acidic tumor environment. Therapeutic payload encapsulation is achieved through an acidic protonated polymer intermediate, which exhibits an in vitro pH-dependent activation window. Protonation of the polymer generates a strong positive charge on the polymer region. The positively charged regions of the polymer attract the negatively charged regions of the therapeutic payload. The electrostatic interaction between the positively charged polymer and the negatively charged therapeutic payload results in physical proximity between the polymer chain and the biomolecule. Neutralization of the polymer and therapeutic payload results in a sudden increase in hydrophobicity of the positively charged polymer sections which interact with hydrophobic regions in the therapeutic payload to form stable encapsulated structures or micelles. [Means for solving the problem]

[0010] In some embodiments, the block copolymer of Formula (I) comprises poly(ethylene oxide) (PEO) and a hydrophobic polymer segment and has the following structure:

[0011] [ka]

[0012] (wherein n1 is an integer of 40 to 500, x1 is an integer of 4 to 250, y1 is an integer of 0 to 10, X is halogen, —OH, or —C(O)OH, and R 1 and R 2 are each independently hydrogen or optionally substituted C-C alkyl, and R 3 and R 4 are each independently optionally substituted C1-C6 alkyl, C3-C10 cycloalkyl or aryl, or R 3 and R 4 together with the corresponding nitrogen to which they are attached form an optionally substituted 5- to 7-membered ring, and R 5 is hydrogen or —C(O)CH3). 5. In some embodiments, n1 is an integer between 100 and 250, x1 is an integer between 40 and 250, and / or y1 is 0. In some embodiments, n1 is an integer between 100 and 250, x1 is an integer between 100 and 200, and / or y1 is 0. In some embodiments, n1 is an integer between about 114, x1 is about 170, and / or y1 is 0. In some embodiments, n1 is an integer between 114, x1 is 170, and / or y1 is 0. The x1 units are the same as R 3 and R 4 The same units with substituents or different R 3 and R 4 Additionally, it should be understood that the addition of small amounts of other hydrophobic monomeric units that do not significantly affect the ability of the micelle to encapsulate and release biomolecular compositions is encompassed by formula (I).

[0013] In some embodiments, the hydrophobic polymer segment of the block copolymer of Formula (I) is:

[0014] [ka] is selected from.

[0015] In some embodiments, the therapeutic agent is a biomolecule. In some embodiments, the therapeutic agent is a protein. In some embodiments, the therapeutic agent is a bispecific antibody (BsAb). In some embodiments, the therapeutic agent is a cytokine. In some embodiments, the therapeutic agent is a cytokine fusion protein. In some embodiments, the therapeutic agent is human IL-12. In some embodiments, the therapeutic agent is single-chain human IL-12. In some embodiments, the therapeutic agent is monovalent human IL-12 fused to the Fc region of an IgG antibody. In some embodiments, the therapeutic agent is bivalent human IL-12 fused to the Fc region of an IgG antibody. In some embodiments, the therapeutic agent is human IL-2. In some embodiments, the therapeutic agent is bivalent human IL-2 fused to the Fc region of an IgG antibody. In some embodiments, the therapeutic agent is human IL-18. In some embodiments, the therapeutic agent is solitomab bispecific antibody T cell engager (TCE). In some embodiments, the therapeutic agent is lunimotamab bispecific antibody T cell engager. In some embodiments, the therapeutic agent is blinatumomab bispecific antibody T cell engager. In some embodiments, the therapeutic agent is a glofitamab bispecific antibody T cell engager. In some embodiments, the therapeutic agent is an odronextamab bispecific antibody T cell engager.

[0016] In some embodiments, the micelles have a diameter of less than about 1 μm or less than about 50 nm. In some embodiments, the micelles have a diameter of about 25 to about 50 nm. In some embodiments, the micelles have a diameter of about 20 to about 40 nm. In some embodiments, the micelles have a diameter of about 50 to about 70 nm.

[0017] Another aspect of the invention is a pH-responsive composition comprising one or more micelles described herein. In some embodiments, the pH-responsive composition has a pH transition point. In some embodiments, the pH transition point is between 4 and 8, between 6 and 7.5, or between 4.5 and 6.5. In some embodiments, the composition has a pH response of less than 0.25 or 0.15 pH units.

[0018] Another aspect of the present invention is a method of treating cancer in an individual in need thereof, comprising administering an effective amount of a pH-sensitive micelle composition comprising a chemotherapeutic agent described herein. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is a cancer of the breast, ovary, prostate, peritoneal metastasis, colorectum, bladder, esophagus, head and neck (HNSSC), lung, brain, kidney, renal, or skin (including melanoma and sarcoma). In some embodiments, the tumor is reduced in size by about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, the micelles described herein are administered in conjunction with one further additional therapy. In some embodiments, the additional therapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 therapy, an anti-PD-L1 therapy, or an anti-CTLA-4 therapy.

[0019] Other objects, features, and advantages of the block copolymers, micelle compositions, and methods described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0020] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 depicts a scheme showing bispecific antibody (BsAb) encapsulation and tumor delivery with reduced systemic exposure. [Figure 2A]FIG. 2A shows encapsulation of monovalent murine IL-12-Fc and human IL-2Fc by protonated polymer intermediates and pH-dependent activation in vitro demonstrating a large activation window by reporter cell assay. [Figure 2B] FIG. 2B shows encapsulation of monovalent murine IL-12-Fc and human IL-2Fc without a protonated polymer intermediate and pH-dependent activation in vitro, demonstrating a window of small or no activation by reporter cell assays. [Figure 3A] FIG. 3A shows encapsulation of human IL-12 by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 3B] FIG. 3B shows encapsulation of human IL-12 by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 4A] FIG. 4A shows encapsulation of single-chain human IL-12 by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 4B] FIG. 4B shows encapsulation of single-chain human IL-12 by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 5A] FIG. 5A shows encapsulation of monovalent human IL-12Fc by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 5B] FIG. 5B shows encapsulation of monovalent human IL-12Fc by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 6A]FIG. 6A shows encapsulation of bivalent human IL-12Fc by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 6B] FIG. 6B shows encapsulation of bivalent human IL-12Fc by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 7A] FIG. 7A shows encapsulation of single-chain murine IL-12 by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 7B] FIG. 7B shows encapsulation of single-chain murine IL-12 by a protonated polymer intermediate of PEG-PDBA90 and pH-dependent activation in vitro by reporter cell assay. [Figure 7C] FIG. 7C shows encapsulation of single-chain murine IL-12 by a protonated polymer intermediate of PEG-PDBA120 and pH-dependent activation in vitro by reporter cell assay. [Figure 7D] FIG. 7D shows encapsulation of single-chain murine IL-12 by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 7E] FIG. 7E shows encapsulation of single-chain murine IL-12 by a protonated polymer intermediate of PEG-PDBA200 and pH-dependent activation in vitro by reporter cell assay. [Figure 8A] FIG. 8A shows encapsulation of monovalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 8B]FIG. 8B shows encapsulation of monovalent murine IL-12Fc with a protonated polymer intermediate of PEG-PDBA90 and pH-dependent activation in vitro by reporter cell assay. [Figure 8C] FIG. 8C shows encapsulation of monovalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA120 and pH-dependent activation in vitro by reporter cell assay. [Figure 8D] FIG. 8D shows encapsulation of monovalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 9A] FIG. 9A shows encapsulation of bivalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA60 and pH-dependent activation in vitro by reporter cell assay. [Figure 9B] FIG. 9B shows encapsulation of bivalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA90 and pH-dependent activation in vitro by reporter cell assay. [Figure 9C] FIG. 9C shows encapsulation of bivalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA120 and pH-dependent activation in vitro by reporter cell assay. [Figure 9D] FIG. 9D shows encapsulation of bivalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA140 and pH-dependent activation in vitro by reporter cell assay. [Figure 9E] FIG. 9E shows encapsulation of bivalent murine IL-12Fc by a protonated polymer intermediate of PEG-PDBA170 and pH-dependent activation in vitro by reporter cell assay. [Figure 10] FIG. 10 is a table of the characterization of IL-12 encapsulated formulations. [Figure 11]FIG. 11 shows encapsulation of bivalent human IL-2Fc by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by reporter cell assay. [Figure 12] FIG. 12 shows encapsulation of human IL-18 by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by reporter cell assay. [Figure 13A] FIG. 13A shows encapsulation of solitomab (EPCAMxCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by a T cell-dependent cytotoxicity assay using the SK-CO-1 cell line. [Figure 13B] FIG. 13B shows encapsulation of solitomab (EPCAMxCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by a T-cell dependent cytotoxicity assay using the GSU cell line. [Figure 14A] FIG. 14A shows encapsulation of lunimotamab (a HER2xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and its pH-dependent activation in vitro in a T-cell dependent cytotoxicity assay using the GSU cell line. [Figure 14B] FIG. 14B shows encapsulation of lunimotamab (a HER2xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and its pH-dependent activation in vitro by a T-cell dependent cytotoxicity assay using the HCC827 cell line. [Figure 14C] FIG. 14C shows encapsulation of lunimotamab (a HER2xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and its pH-dependent activation in vitro in a T cell-dependent cytotoxicity assay using the SK-CO-1 cell line. [Figure 15A]FIG. 15A shows encapsulation of blinatumomab (CD19xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by a B cell depletion assay. [Figure 15B] FIG. 15B shows encapsulation of odronextamab (CD20xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by a B cell depletion assay. [Figure 15C] FIG. 15C shows encapsulation of glofitamab (CD20xCD3 bispecific antibody) by a protonated polymer intermediate of PEG-PDBA and pH-dependent activation in vitro by a B cell depletion assay. [Figure 16] FIG. 16 is a table of the characterization of bispecific encapsulation formulations. [Figure 17] FIG. 17 shows systemic cytokine levels in healthy BL6 mice 5 days after intravenous administration of two doses of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on days 0 and 3 of the study. [Figure 18] FIG. 18 shows aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (Cre) levels in healthy BL6 mice 5 days after intravenous administration of two doses of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on days 0 and 3 of the study. [Figure 19] FIG. 19 shows body weight changes in healthy BL6 mice after two doses of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) administered intravenously on days 0 and 3 of the study. [Figure 20A]FIG. 20A shows tumor volume measurements in mice bearing large tumors (approximately 500 mm) after intravenous administration of a single dose of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on day 0 of the study. [Figure 20B] Figure 20B shows the weight change in mice bearing large tumors (approximately 500 mm) after a single intravenous dose of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on day 0 of the study. [Figure 21] FIG. 21 shows tumor volume and weight loss measurements on day 7 of the study in mice bearing large tumors (approximately 500 mm) after intravenous administration of a single dose of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on day 0 of the study. [Figure 22A] Figure 22A shows the increase in CD8-positive T cells and NK cells in tumors of mice on day 2 of the study after intravenous administration of a single dose of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on day 0 of the study. [Figure 22B] Figure 22B shows the increase in CD8-positive T cells and NK cells in tumors of mice on day 2 of the study after intravenous administration of a single dose of PBS, free IL-12Fc protein, or PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) on day 0 of the study. DETAILED DESCRIPTION OF THE INVENTION

[0022] Provided herein are micelle compositions comprising a therapeutic agent. In some embodiments, the micelles comprise a diblock copolymer and a therapeutic agent. In other embodiments, provided herein are micelle compositions comprising a therapeutic agent.

[0023] definition In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the present invention may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments. Unless the context requires otherwise, throughout the following specification and claims, the word "comprises" and variations thereof, such as "includes" and "comprising," should be interpreted in an open and inclusive sense, i.e., "including but not limited to." Furthermore, the headings provided herein are for convenience only and are not intended to interpret the scope or meaning of the claimed invention.

[0024] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Also, it should be noted that the term "or" is used generally in its sense including "and / or" unless the context clearly dictates otherwise.

[0025] As used herein, the following terms have the following meanings unless otherwise indicated.

[0026] "Alkyl" refers to a straight or branched hydrocarbon chain group having from 1 to 20 carbon atoms attached to the rest of the molecule by a single bond. Alkyl containing up to 10 carbon atoms is C1-C 10 Similarly, for example, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl. Alkyl groups containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include, but are not limited to, C1-C 10

[0023] Examples of alkyl include alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethylpropyl, and the like. In some embodiments, alkyl is methyl, ethyl, s-butyl, or 1-ethylpropyl. Unless specifically stated otherwise in the specification, an alkyl group may be optionally substituted as described below. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the remainder of the molecule to a radical group. In some embodiments, alkylene is -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, alkylene is -CH2-. In some embodiments, alkylene is -CH2CH2-. In some embodiments, alkylene is -CH2CH2CH2-. In some embodiments, alkylene is -CH2CH2CH2-.

[0027] "Aryl" refers to an aromatic ring in which each of the atoms forming the ring is a carbon atom. An aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthalenyl. In some embodiments, an aryl is phenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Unless specifically stated otherwise in this specification, the term "aryl" or the prefix "ar-" (e.g., "aralkyl") is meant to include aryl groups that are optionally substituted.

[0028] "Cycloalkyl" refers to a monocyclic or polycyclic non-aromatic group in which each of the atoms forming the ring (i.e., the skeletal atoms) is a carbon atom. Cycloalkyls can be saturated or partially unsaturated. Cycloalkyls can be fused to an aromatic ring (in which case the cycloalkyl is attached via a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. In some embodiments, a cycloalkyl is a C3-C6 cycloalkyl. In some embodiments, a cycloalkyl is a 3- to 6-membered cycloalkyl. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, a monocyclic cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Polycyclic groups include, for example, adamantyl, norbornyl, decalinyl, and 3,4-dihydronaphthalen-1(2H)-one. Unless stated otherwise specifically in the specification, cycloalkyl groups may be optionally substituted.

[0029] The term "optionally substituted" or "substituted" means that the referenced group may be substituted with one or more additional groups individually and independently selected from alkyl, haloalkyl, cycloalkyl, aryl, heteroaryl, heterocycloalkyl, -OH, alkoxy, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone, -CN, alkyne, C-C alkylalkyne, halogen, acyl, acyloxy, -COH, -COalkyl, nitro, and amino, including mono- and di-substituted amino groups (e.g., -NH, -NHR, -N(R)), and protected derivatives thereof. In some embodiments, the optional substituents are independently selected from alkyl, alkoxy, haloalkyl, cycloalkyl, halogen, -CN, -NH, -NH(CH), -N(CH), -OH, -COH, and -COalkyl. In some embodiments, optional substituents are independently selected from fluoro, chloro, bromo, iodo, -CH, -CHCH, -CF, -OCH, and -OCF. In some embodiments, optional substituents are independently selected from fluoro, chloro, -CH, -CF, -OCH, and -OCF. In some embodiments, substituted groups are substituted with one or two of the foregoing groups. In some embodiments, optional substituents on aliphatic carbon atoms (acyclic or cyclic, saturated or unsaturated carbon atoms, excluding aromatic carbon atoms) include oxo (=O).

[0030] As used herein, terms such as "co-administration" are meant to encompass the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which agents are administered by the same or different routes of administration or at the same or different times.

[0031] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the quantity of a composition comprising a compound disclosed herein that is required to provide a clinically significant reduction in a disease symptom. An appropriate "effective" amount in any individual case can be determined using techniques, such as a dose escalation study.

[0032] Unless otherwise stated, the following terms used in this application have the definitions set forth below. The use of the term "including" and other forms such as "include," "includes," and "included" is not limiting. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0033] As used herein, "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the block copolymer and that is relatively non-toxic, i.e., a material that may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0034] The term "pharmaceutically acceptable salt" refers to a form of a therapeutically active agent consisting of the cationic form of the therapeutically active agent combined with a suitable anion, or in an alternative embodiment, the anionic form of the therapeutically active agent combined with a suitable cation. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. SM Berge, LD Bighley, DC Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. PH Stahl and CG Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich: Wiley-VCH / VHCA, 2002. Pharmaceutical salts are typically more soluble in gastric and intestinal fluids and more rapidly dissolve than non-ionic species, making them useful in solid dosage forms. Furthermore, their solubility is often a function of pH, allowing for selective dissolution in one or another part of the gastrointestinal tract, an ability that can be manipulated as an aspect of delayed- and sustained-release behavior. Also, salt-forming molecules can be in equilibrium with neutral forms, allowing for tailored passage through biological membranes.

[0035] As used herein, the terms "pH-responsive system," "pH-responsive composition," "micelle," "pH-responsive micelle," "pH-sensitive micelle," "pH-activatable micelle," "pH-sensitive encapsulant," "pH-activatable encapsulant," "pH-responsive encapsulant," and "pH-activatable micellar (pHAM) nanoparticles" are used interchangeably herein to refer to micelles containing one or more compounds that dissociate in response to pH (e.g., above or below a particular pH). As a non-limiting example, at a particular pH, the block copolymer of Formula (I) is substantially in micellar form. As the pH changes (e.g., decreases), the micelles begin to dissociate, and as the pH changes further (e.g., decreases further), the block copolymer of Formula (I) exists in a substantially dissociated (non-micellar) form.

[0036] As used herein, "encapsulation" or "encapsulation process" are used interchangeably herein to refer to the formation of micelles.

[0037] As used herein, "pH transition range" refers to the pH range at which micelles dissociate.

[0038] As used herein, "pH transition value" (pH) refers to the pH at which half of the micelles dissociate.

[0039] "Nanoprobe" is used herein to refer to a pH-sensitive micelle that includes an imaging label moiety. In some embodiments, the label moiety is a fluorescent dye. In some embodiments, the fluorescent dye is indocyanine green dye.

[0040] As used herein, the terms "administer," "administering," "administration," and the like refer to methods that can be used to enable delivery of a compound or composition to a desired site of biological action. These methods include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, or infusion), topical, and rectal administration. Those of skill in the art are familiar with administration techniques that can be used with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally. In some embodiments, the compositions described herein are administered intravenously.

[0041] As used herein, terms such as "co-administration" are meant to encompass the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens in which agents are administered by the same or different routes of administration or at the same or different times.

[0042] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient quantity of an agent or compound being administered to relieve to some extent one or more of the symptoms of the disease or condition being treated. Results include reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the quantity of a composition comprising a compound disclosed herein that is required to provide a clinically significant reduction in a disease symptom. An appropriate "effective" amount in any individual case is optionally determined using techniques, such as a dose escalation study.

[0043] The terms "enhance" or "enhancing," as used herein, means to increase or prolong either in potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term "enhancing" refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system. An "enhancing-effective amount," as used herein, refers to an amount sufficient to enhance the effect of another therapeutic agent in a desired system.

[0044] The term "subject" or "patient" includes mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees and other ape and monkey species; domestic animals such as cows, horses, sheep, goats, pigs, and the like; domestic animals such as rabbits, dogs, and cats; and laboratory animals, including rodents such as rats, mice, and guinea pigs. In one aspect, the mammal is a human.

[0045] As used herein, the terms "treat," "treating," or "treatment" include alleviating, attenuating, or ameliorating at least one symptom of a disease or condition, preventing further symptoms, inhibiting a disease or condition, e.g., halting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating symptoms caused by a disease or condition, or prophylactically and / or therapeutically arresting symptoms of a disease or condition.

[0046] The use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or unless the alternatives are mutually exclusive, although the present disclosure supports the definition referring to alternatives only and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In accordance with long-standing patent law, the words "a" and "an," when used in conjunction with the word "comprising" in the claims or specification, refer to one or more unless otherwise stated.

[0047] I. Micelles One or more block copolymers described herein can be used to form pH-sensitive micelles or encapsulating agents. In some embodiments, the composition comprises a single type of micelle. In some embodiments, two or more different types of micelles can be combined to form a mixed micelle composition. In some embodiments, the micelle comprises one or more block copolymers that non-covalently encapsulate a therapeutic agent.

[0048] In certain embodiments, provided herein are micelles comprising: (i) Block copolymer In some embodiments, the block copolymer of Formula (I) comprises poly(ethylene oxide) (PEO) and a hydrophobic polymer segment and has the following structure:

[0049] [ka]

[0050] (wherein n1 is an integer of 40 to 500, x1 is an integer of 4 to 250, y1 is an integer of 0 to 10, X is halogen, —OH, or —C(O)OH, and R 1 and R 2are each independently hydrogen or optionally substituted C-C alkyl, and R 3 and R 4 are each independently optionally substituted C1-C6 alkyl, C3-C 10 cycloalkyl or aryl, or R 3 and R 4 together with the corresponding nitrogen to which they are attached form an optionally substituted 5- to 7-membered ring, and R 5 is hydrogen or —C(O)CH3). 5. In some embodiments, n1 is an integer between 100 and 250, x1 is an integer between 40 and 250, and / or y1 is 0. In some embodiments, n1 is an integer between 100 and 250, x1 is an integer between 100 and 200, and / or y1 is 0. In some embodiments, n1 is an integer between about 114, x1 is about 170, and / or y1 is 0. In some embodiments, n1 is an integer between 114, x1 is 170, and / or y1 is 0. The x1 units are the same as R 3 and R 4 The same units with substituents or different R 3 and R 4 Additionally, it should be understood that the addition of small amounts of other hydrophobic monomeric units that do not significantly affect the ability of the micelle to encapsulate and release biomolecular compositions is encompassed by formula (I).

[0051] In some embodiments, the hydrophobic polymer segment of the block copolymer of Formula (I) is:

[0052] [ka] is selected from.

[0053] (ii) Therapeutic Agent Bispecific antibodies (BsAbs) are an important class of therapeutic agents for immuno-oncology applications. T cell engagers (TCEs) target tumor-associated antigens and T cells to eradicate antigen-expressing tumor cells. TCEs for solid tumors have also shown promising clinical efficacy but have shown dose-limiting toxicity due to on-target / off-tumor effects.

[0054] Cytokines (e.g., IL-12, IL-2) and cytokine fusion proteins (e.g., IL-12Fc, IL-2Fc) can induce anti-tumor immune responses, but their clinical application is limited by unfavorable pharmacokinetic properties and significant dose-limiting toxicities (e.g., cytokine release syndrome, vascular leak syndrome, etc.).

[0055] In some embodiments, the therapeutic agent is a biomolecule. In some embodiments, the therapeutic agent is a protein. In some embodiments, the therapeutic agent is a bispecific antibody (BsAb). In some embodiments, the therapeutic agent is a bispecific antibody having a TAA targeting domain and a T cell targeting domain. In some embodiments, the therapeutic agent is a cytokine. In some embodiments, the therapeutic agent is an asymmetric 1+1 IgG bispecific antibody. In some embodiments, the therapeutic agent is an asymmetric 2+1 IgG bispecific antibody. In some embodiments, the therapeutic agent is an HLE-BiTE bispecific antibody. In some embodiments, the therapeutic agent is a tandem scFv-scFv bispecific antibody. In some embodiments, the therapeutic agent is a protein having a molecular weight of at least 6 kDa.

[0056] In some embodiments, the Therapeutic Agent is human IL-12. In some embodiments, the Therapeutic Agent is single-chain human IL-12. In some embodiments, the Therapeutic Agent is monovalent human IL-12 fused to the Fc region of an IgG antibody. In some embodiments, the Therapeutic Agent is bivalent human IL-12 fused to the Fc region of an IgG antibody. In some embodiments, the Therapeutic Agent is human IL-2. In some embodiments, the Therapeutic Agent is bivalent human IL-2 fused to the Fc region of an IgG antibody. In some embodiments, the Therapeutic Agent is human IL-18. In some embodiments, the Therapeutic Agent is solitomab bispecific antibody T cell engager (TCE). In some embodiments, the Therapeutic Agent is lunimotamab bispecific antibody T cell engager. In some embodiments, the Therapeutic Agent is blinatumomab bispecific antibody T cell engager. In some embodiments, the Therapeutic Agent is glofitamab bispecific antibody T cell engager. In some embodiments, the Therapeutic Agent is odronextamab bispecific antibody T cell engager.

[0057] Monovalent human IL12(p35 / p40)-hIgG1-Fc-LALA / PG heterodimer sequence

[0058] SEQ ID NO: 1 human IL12-p35-hIgG1-Fc-knob (L234A / L235A / P329G / S354C / T366W)

[0059]

number

[0060] SEQ ID NO: 2 human IL12-p40-hIgG1-Fc-hole

[0061] (L234A / L235A / P329G / Y349C / T366S / L368A / Y407V)

[0062]

number

[0063] Bivalent human IL12(p40 / p35)-hIgG1-Fc-LALA / PG homodimer sequence

[0064] SEQ ID NO: 3 Human IL12-p40-G4S linker-P35-hIgG1-Fc (L234A / L235A / P329G)

[0065]

number

[0066] Single-chain human IL12 heterodimer sequence

[0067] SEQ ID NO: 4 Human IL12-p40-(G4S)5-P35-HIS tag

[0068]

number

[0069] Wild-type human IL-12 sequence

[0070] SEQ ID NO: 5 IL-12 p35 (Arg23-Ser219) Accession No. P29459

[0071]

number

[0072] SEQ ID NO: 6 IL-12 p40 (Ile23-Ser328) Accession No. P29460

[0073]

number

[0074] IL-2Fc homodimer sequence

[0075] SEQ ID NO: 7 IL2-(G4S)1-Fc

[0076]

number

[0077] Wild-type human IL-18 sequence

[0078] SEQ ID NO: 8 NCBI accession nNP_001553.1 (predicted N-terminal AA Y37)

[0079]

number

[0080] II. Encapsulation Method To formulate drug-loaded micelles, the polymer is dissolved in an organic solvent and the polymer is protonated with acid. After protonation, the organic solvent and excess acid are removed. The therapeutic agent is dispensed into an aqueous buffer and mixed with the protonated polymer. The mixture is then dialyzed against a neutral buffer to complete the encapsulation process.

[0081] Encapsulation of the therapeutic payload is achieved using an acid-protonated polymer intermediate. Protonation of the polymer generates a strong positive charge in regions of the polymer. The positively charged regions of the polymer attract the negatively charged regions in the therapeutic payload. Electrostatic interactions between the positively charged polymer and the negatively charged therapeutic payload result in physical proximity between the polymer chain and the biomolecule. Neutralization of the polymer and therapeutic payload results in a sudden increase in the hydrophobicity of the positively charged polymer sections, which interact with the hydrophobic regions in the therapeutic payload, forming stable encapsulation structures or micelles that exhibit a pH-dependent activation window in vitro.

[0082] In one embodiment, the step of encapsulating the therapeutic agent comprises: (i) Dissolving the polymer in an organic solvent (ii) protonating the polymer of (i) with an acid; (iii) removing excess organic solvent and the acid of (ii); (iv) adding a therapeutic agent to the polymer of (iii); (v) Dialyzing the mixture of (iv) against a neutral buffer solution. Includes.

[0083] In one embodiment, the polymer is a PEG-PDBA polymer, which is dissolved in methanol and protonated with acetic acid. An Amicon Ultra 10k MWCO device is used to remove the organic solvent and excess acetic acid. The therapeutic protein is dispensed into 1x PBS or 10 mM sodium phosphate buffer, mixed, and incubated overnight with gentle rocking. The mixture is then dialyzed against 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process. The step of protonating the polymer intermediate allows for the large activation window seen in Figure 2A. The same method without the step of protonating the polymer results in a small or no activation window, as seen in Figure 2B.

[0084] In some embodiments, the therapeutic agent is about 0.1 wt% of the micelle. In some embodiments, the therapeutic agent is about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1% of the micelle. In some embodiments, the therapeutic agent is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the micelle. In some embodiments, the therapeutic agent is about 12%, about 14%, about 16%, about 18%, or about 20% of the micelle.

[0085] III. pH-Responsive Compositions In another aspect, pH-responsive compositions are provided herein. The pH-responsive compositions disclosed herein include one or more pH-responsive micelles and / or nanoparticles comprising a block copolymer and a therapeutic agent. Each block copolymer comprises a hydrophilic polymer segment and a hydrophobic polymer segment, and the hydrophobic polymer segment comprises an ionizable amine group to confer pH sensitivity. This pH sensitivity can be utilized to provide compositions suitable as drug-encapsulated therapeutic agents.

[0086] Micelles can have different pH transition values ​​within the physiological range to target specific cells or microenvironments. In some embodiments, micelles have a pH transition value of about 5 to about 8. In some embodiments, micelles have a pH transition value of about 5 to about 6. In some embodiments, micelles have a pH transition value of about 6 to about 7. In some embodiments, micelles have a pH transition value of about 7 to about 8. In some embodiments, micelles have a pH transition value of about 6.3 to about 6.9. In some embodiments, micelles have a pH transition value of about 5.0 to about 6.2. In some embodiments, micelles have a pH transition value of about 5.9 to about 6.2. In some embodiments, micelles have a pH transition value of about 5.0 to about 5.5. In some embodiments, the pH transition point is 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5.

[0087] The pH-sensitive micelle compositions of the present invention can advantageously have a narrow pH transition range, in contrast to other pH-sensitive compositions that have a very broad pH response (i.e., 2 pH units). This pH transition is the transition point at which the micelles dissociate, release the payload, or activate the photophore (i.e., indocyanine green dye). In some embodiments, the micelles have a pH transition range of less than about 1 pH unit. In various embodiments, the micelles have a pH transition range of less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, or less than about 0.1 pH units. In some embodiments, the micelles have a pH transition range of less than about 0.5 pH units. In some embodiments, the micelles have a pH transition range of less than about 0.25 pH units. A narrow pH transition range advantageously provides a sharper pH response that can result in complete turn-on of the fluorophore or release the therapeutic payload with small pH changes.

[0088] In some embodiments, the pH-responsive composition has an emission spectrum. In some embodiments, the emission spectrum is 600-800 nm. In some embodiments, the emission spectrum is 700-800 nm.

[0089] IV.How to use Aerobic glycolysis, known as the Warburg effect, occurs in all solid cancers, in which cancer cells preferentially take up glucose and convert it to lactate or other acids. Lactate or other acids preferentially accumulate in the extracellular space via monocarboxylate transporters or other transporters. The resulting acidification of the extracellular space promotes extracellular matrix remodeling for further tumor invasion and metastasis.

[0090] Some embodiments provided herein describe compounds that form micelles at physiological pH (7.35-7.45). In some embodiments, the compounds described herein are non-covalently incorporated into a therapeutic agent. In some embodiments, the therapeutic agent is sequestered within the micelle core at physiological pH (7.35-7.45) (e.g., during blood circulation). In some embodiments, when the micelles encounter an acidic environment (e.g., tumor tissue), the micelles dissociate into individual compounds, allowing the therapeutic agent to be released. In some embodiments, the micelles dissociate at a pH below the pH transition point (e.g., the acidic conditions of the tumor microenvironment).

[0091] In some embodiments, therapeutic agents can be incorporated into the interior of micelles. Certain pH conditions (e.g., the acidic pH present in tumors and endocytic compartments) can result in rapid protonation and dissociation of the micelles into unimers, thereby releasing the therapeutic agent (e.g., drug). In some embodiments, micelles provide stable drug encapsulation at physiological pH (pH 7.4) but can rapidly release the drug in acidic environments.

[0092] In some examples, the pH-sensitive micelle compositions described herein have a narrow pH transition range. In some embodiments, the micelles described herein have a pH transition range (ΔpH 10-90% In various embodiments, the micelles have a pH transition range of less than about 0.9, less than about 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, or less than about 0.1 pH units. In some embodiments, the micelles have a pH transition range of less than about 0.5 pH units. In some embodiments, the pH transition range is less than about 0.25 pH units. In some embodiments, the pH transition range is less than about 0.15 pH units. The sharp transition point allows the micelles to dissociate with the acidic tumor microenvironment.

[0093] These micelles can be used as drug delivery agents. Drug-containing micelles can be used, for example, to treat cancer or other diseases, and the drug can be delivered to the appropriate location due to a localized pH difference (e.g., a pH different from physiological pH (7.4)). In some embodiments, the disorder being treated is cancer. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the tumor is a secondary tumor resulting from metastasis of one or more primary tumors. In some embodiments, drug delivery can be to lymph nodes or the pleural surface.

[0094] In some embodiments of the methods disclosed herein, the tumor originates from a cancer. In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), lung cancer, ovarian cancer, prostate cancer, bladder cancer, urethral cancer, esophageal cancer, colorectal cancer, peritoneal metastasis, renal cancer, or brain, skin (including melanoma and sarcoma). In some embodiments, the cancer is breast cancer, head and neck squamous cell carcinoma (NHSCC), esophageal cancer, colorectal cancer, or renal cancer.

[0095] In some embodiments, the tumor is reduced by about 5%, about 10%, about 15%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the tumor is reduced by about 50%. In some embodiments, the tumor is reduced by about 60%. In some embodiments, the tumor is reduced by about 70%. In some embodiments, the tumor is reduced by about 75%. In some embodiments, the tumor is reduced by about 80%. In some embodiments, the tumor is reduced by about 85%. In some embodiments, the tumor is reduced by about 90%. In some embodiments, the tumor is reduced by about 95%. In some embodiments, the tumor is reduced by about 99%.

[0096] V. Combination Therapy In another aspect, the micelle comprises a PEG-PDBA block copolymer, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, and the therapeutic agent further comprises administering one or more additional therapies. In some embodiments, the additional therapy is a checkpoint inhibitor. Checkpoint inhibitor therapy is a form of cancer immunotherapy. The therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can dampen the immune response to immunological stimulation. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints and restore immune system function. Examples of checkpoint proteins found on T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. Some immune checkpoint inhibitors are used to treat cancer.

[0097] PD-1 and PD-L1 inhibitors are a group of checkpoint inhibitor anticancer drugs that block the activity of the PD-1 and PDL1 immune checkpoint proteins present on the surface of cells. Immune checkpoint inhibitors have emerged as frontline treatments for several types of cancer.

[0098] In some embodiments, the additional therapy is a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 therapy, an anti-PD-L1 therapy, or an anti-CTLA-4 therapy. In some embodiments, the checkpoint inhibitor is an anti-PD-1 therapy.

[0099] In some embodiments, the additional therapy is selected from pembrolizumab (Keytruda), nivolumab (Opdivo), cemiplimab (Libtayo), and durvalumab (Imfinzi), or any combination thereof. In some embodiments, the additional therapy is pembrolizumab or Keytruda. In some embodiments, the additional therapy is nivolumab or Opdivo. In some embodiments, the additional therapy is durvalumab or Imfinzi. In some embodiments, the additional therapy is cemiplimab or Libtayo.

[0100] The additional therapy can be administered simultaneously with or sequentially to the pH-responsive compositions described herein. [Example]

[0101] The block copolymers and micelles described herein are synthesized using standard synthetic techniques or methods known in the art.

[0102] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are employed.

[0103] Block copolymers are described, for example, in March's Advanced Organic Chemistry, 6 th They are prepared using standard organic chemistry techniques, such as those described in The Journal of Organic Chemistry, Vol. 1, No. 1, pp. 111-115, 1997, Edition, John Wiley and Sons, Inc.

[0104] Some abbreviations used herein are as follows: I. MeOH: Methanol II. PEG (Polyethylene Glycol) III. PEO Polyethylene Oxide IV. PDBA Poly(2-(dibutylamino)ethyl methacrylate) V.Hr time VI. ISR Reanalysis of Real Samples VII. kg Kilogram VIII. mg milligram IX. mL Milliliters X. NP Nanoparticles XI. μg microgram XII. μm micron XIII. UPS Ultra pH Sensitive XIV. BsAb Bispecific Antibodies XV. TCE T Cell Engagers XVI. IL Interleukin XVII. PDI Polydispersity Index XVIII. WFI Water for Injection

[0105] Suitable PEG polymers may be purchased commercially (e.g., from Sigma Aldrich) or synthesized according to methods known in the art. In some embodiments, hydrophilic polymers can be used as initiators for polymerizing hydrophobic monomers to form block copolymers.

[0106] [Example 1] Encapsulation of single-chain murine IL-12 using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was first dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 10 mg / mL at the end of the process. The desired amount (0.1%–20% of the polymer weight) of single-chain murine IL-12 at a concentration of 14.22 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0107] [Example 2] Encapsulation of monovalent murine IL-12Fc using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of the process. The desired amount (0.1%–20% of the polymer weight) of monovalent murine IL-12Fc at a concentration of 16.4 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0108] [Example 3] Encapsulation of bivalent murine IL-12Fc using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 16 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 2.5 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of bivalent murine IL-12Fc at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0109] [Example 4] Encapsulation of human IL-12 using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of human IL-12 at a concentration of 2 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0110] [Example 5] Encapsulation of single-chain human IL-12 using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of single-chain human IL-12 was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0111] [Example 6] Encapsulation of monovalent human IL-12Fc using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted with WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of monovalent human IL-12Fc was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0112] [Example 7] Encapsulation of bivalent human IL-12Fc using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of bivalent human IL-12Fc was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0113] [Example 8] Encapsulation of human IL-18 using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 5 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of human IL-18 at a concentration of 3.0 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0114] [Example 9] Encapsulation of human IL-2Fc using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. Protonation was achieved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer, dissolving the polymer. The organic solvent and excess acid were removed from the protonated polymer using an Amicon Ultra 10k MWCO device. This was accomplished by diluting the polymer solution in WFI to the volume of the Amicon Ultra device, centrifuging, and discarding the permeate. This process was repeated seven times. The polymer was concentrated to 15 mg / mL at the end of this step. The desired amount of human IL-2Fc (0.1%–20% of the polymer weight) at a concentration of 17.01 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0115] Figure 10 shows a table of IL-12 encapsulated formulations described in Examples 1 to 4. The size (nm) and PDI of the encapsulated agent are reported.

[0116] [Example 10] Encapsulation of the solitomab bispecific antibody T cell engager using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 1.25 mg / mL at the end of this process. The desired amount of solitomab (0.1%–20% of the polymer weight) at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0117] [Example 11] Encapsulation of lunimotamab bispecific antibody T cell engager using PEG-PDBA polymer via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer was added to protonate and dissolve the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 2.5 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of lunimotamab at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0118] [Example 12] Encapsulation of the Blinatumomab Bispecific Antibody T Cell Engager Using PEG-PDBA Polymers via a Protonated Polymer Intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. The polymer was protonated and dissolved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer. Using an Amicon Ultra 10k MWCO device, the polymer solution was diluted in WFI to the volume of the Amicon Ultra device, centrifuged, and the permeate was discarded to remove the organic solvent and excess acid from the protonated polymer. This process was repeated seven times. The polymer was concentrated to 2.5 mg / mL at the end of this process. The desired amount (0.1%–20% of the polymer weight) of blinatumomab at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0119] [Example 13] Encapsulation of glofitamab bispecific antibody T cell engager using PEG-PDBA polymers via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. Protonation was achieved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer, dissolving the polymer. The organic solvent and excess acid were removed from the protonated polymer using an Amicon Ultra 10k MWCO device. This was accomplished by diluting the polymer solution in WFI to the volume of the Amicon Ultra device, centrifuging, and discarding the permeate. This process was repeated seven times. The polymer was concentrated to 6.25 mg / mL at the end of this process. The desired amount of glofitamab (0.1%–20% of the polymer weight) at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0120] [Example 14] Encapsulation of odronextamab bispecific antibody T cell engager using PEG-PDBA polymer via a protonated polymer intermediate To formulate drug-loaded micelles, PEG-PDBA polymer was dispersed in methanol at a concentration of 10 mg / mL. Protonation was achieved by adding 1.05 equivalents of acetic acid per ionizable amine moiety on the polymer, dissolving the polymer. The organic solvent and excess acid were removed from the protonated polymer using an Amicon Ultra 10k MWCO device. This was accomplished by diluting the polymer solution in WFI to the volume of the Amicon Ultra device, centrifuging, and discarding the permeate. This process was repeated seven times. The polymer was concentrated to 2.5 mg / mL at the end of this process. The desired amount of odronextamab (0.1%–20% of the polymer weight) at a concentration of 0.125 mg / mL was then added from above to the protonated polymer intermediate, gently mixed, and incubated overnight with gentle rocking. The mixture was then dialyzed against a neutral buffer, such as 10 mM sodium phosphate buffer, pH 7.4, to complete the encapsulation process.

[0121] Figure 16 shows a table characterizing the bispecific encapsulation formulations described in Examples 7 to 11. The BsAb, TTA target, T cell target, BsAb structure, numerical size (nm) and PDI of the encapsulated agents are reported.

[0122] [Example 15] In vitro characterization of the pH-dependent activation window of murine or human IL-12 preparations by reporter cell assay To characterize the pH-dependent activation window, IL-12 formulations were serially diluted in cell culture medium (RPMI 1640 10% HI-FBS), and the resulting formulation / media mixture was acidified to a pH below the formulation's pH transition point by adding an equal volume of acidic cell culture medium. The formulation / media mixture was then neutralized by adding basic cell culture medium. Appropriate controls included parallel addition of neutral medium and addition of payload protein as a sample. The above-treated formulations were then added to IL-12 reporter HEK293 cells. IL-12 bioactivity was then assayed by adding substrate followed by absorbance measurement (Figures 2A-B, 3A-B, 4A-B, 5A-B, 6A-B, 7A-E, 8A-D, and 9A-E).

[0123] [Example 16] In vitro characterization of the pH-dependent activation window of human IL-18 formulations by reporter cell assay To characterize the pH-dependent activation window, IL-18 formulations were serially diluted in cell culture medium (RPMI1640 10% HI-FBS), and the resulting formulation / medium mixture was acidified to a pH below the formulation's pH transition by adding an equal volume of acidic cell culture medium. The formulation / medium mixture was then neutralized by adding basic cell culture medium. Appropriate controls included parallel addition of neutral medium and addition of payload protein as a sample. The above-treated formulations were then added to IL-18 reporter HEK293 cells. IL-18 bioactivity was then assayed by adding substrate followed by absorbance measurement (Figure 12).

[0124] [Example 17] In vitro characterization of the pH-dependent activation window of human IL-2Fc formulations by reporter cell assay To characterize the pH-dependent activation window, IL-2 formulations were serially diluted in cell culture medium (RPMI 1640 10% HI-FBS), and the resulting formulation / medium mixture was acidified to a pH below the formulation's pH transition point by adding an equal volume of acidic cell culture medium. The formulation / medium mixture was then neutralized by adding basic cell culture medium. Appropriate controls included parallel addition of neutral medium and addition of payload protein as a sample. The above-treated formulations were then added to IL-2 reporter HEK293 cells. IL-2 bioactivity was then assayed by adding substrate followed by absorbance measurement (Figures 2A-B, 11).

[0125] [Example 18] In vitro characterization of the pH-dependent activation window of bispecific antibody T cell engager formulations using a T cell-dependent cytotoxicity assay To characterize the pH-dependent activation window, bispecific antibody formulations were serially diluted in cell culture medium (RPMI 1640 10% HI-FBS). The resulting formulation / medium mixture was acidified to a pH below the formulation's pH transition by adding an equal volume of acidic cell culture medium. The formulation / medium mixture was then neutralized by adding basic cell culture medium. Appropriate controls included parallel addition of neutral medium and addition of the payload protein as a sample. The above-treated formulations were added to cancer cell lines expressing antigen and firefly luciferase and human PBMCs (or human pan T cells). The tumor-killing ability of the bispecific antibodies was assessed by measuring bioluminescence from residual tumor cells after 2 days of incubation (Figures 13A-B, 14A-C).

[0126] [Example 19] In vitro characterization of the pH-dependent activation window of bispecific antibody T cell engager formulations using a B cell depletion assay To characterize the pH-dependent activation window, bispecific antibody formulations were serially diluted in cell culture medium (RPMI 1640 10% HI-FBS), and the resulting formulation / media mixture was acidified to a pH below the formulation's pH transition by adding an equal volume of acidic cell culture medium. The formulation / media mixture was then neutralized by adding basic cell culture medium. Appropriate controls included parallel addition of neutral medium and sample addition of payload protein. The above-treated formulations were added to human PBMCs (final density 2 million / mL) supplemented with 10 ng / mL recombinant human IL-2. B cell depletion was assessed by flow cytometry measurement of the percentage of remaining B cells within the CD45+ population after 4 days of incubation. B cells were stained with CD19 / CD20 antibodies (Figures 15A-C).

[0127] [Example 20] pH-sensitive micelle-encapsulated mIL-12Fc significantly reduces systemic cytokine levels, prevents liver toxicity, and prevents weight loss To characterize systemic cytokine levels in subjects injected with PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc, three separate groups of healthy BL6 mice were studied. Each group received intravenous administration of control PBS, 1 μg of free IL12-Fc per injection, or 5 μg of PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc) per injection. Mice were administered injections on days 0 and 3 of the study. Plasma samples were collected on day 5 of the study. Figure 17 shows sampled analytes from the three groups demonstrating significant decreases in IFNγ, IL-6, IL-10, TNFα, and MCP-1 in mice administered PDBA-IL-12Fc compared to mice administered IL-12Fc.

[0128] Figure 18 shows measurements of aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (Cre). Mice treated with PDBA-IL-12Fc showed significantly reduced levels of AST, ALT, and BUN compared to mice administered IL-12Fc.

[0129] Figure 19 shows the weight changes of mice in each individual group. The weight of mice treated with free IL-12Fc was significantly reduced by day 5 of the study, while the weight of mice treated with PDBA-IL-12Fc remained relatively unchanged.

[0130] [Example 22] Large tumors (approximately 500 mm) after a single injection of the pH-sensitive micelle formulation without weight loss 3 ) involution To observe the effect of PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc on the size and weight loss of large MC38 tumors in subjects, three separate groups of healthy BL6 mice were studied. Each group received intravenous administration of control PBS, 5 μg of free IL12-Fc, or 5 μg of PEG-PDBA pH-sensitive micelle-encapsulated IL-12Fc (PDBA-IL-12Fc). Mice were administered a single injection on day 0 of the study. Figure 20A shows measurements of tumor size in each group of mice over the course of the study. Both groups treated with IL-12Fc showed a reduction in tumor size, while the control group showed an increase in tumor size. Figure 20B shows the percent change in body weight compared to day 0 for each group over the course of the study.

[0131] Figure 21 shows the tumor volume and percent weight loss for the three groups on day 7 of the study. Both groups treated with IL-12Fc showed similar tumor volumes, demonstrating tumor regression after a single injection. This indicates that PDBA-IL-12Fc has similar antitumor efficacy as free IL-12Fc. However, the group treated with free IL-12Fc experienced a significant weight loss of approximately 10% compared to day 0. The control and PDBA-IL-12Fc groups were comparable and showed no weight loss. Figures 22A and 22B show an increase in activated CD8 T cells and NK cells in tumors in both the free IL-12-treated and PDBA-IL-12Fc-treated groups compared to controls.

[0132] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method of making an encapsulated biomolecule, comprising: providing an encapsulation composition comprising an organic solvent, a plurality of protonated block copolymer units, and a biomolecule; encapsulating the biomolecule within a micelle formed from the plurality of protonated block copolymer units; Including, The block copolymer comprises poly(ethylene oxide) (PEO) and a hydrophobic polymer segment and has the following structure: 【Chemistry 1】 (In the formula, n １ is an integer from 40 to 500; x １ is an integer from 4 to 250; y １ is an integer from 0 to about 10; X is halogen, —OH, or —C(O)OH; R １ and R ２ are each independently hydrogen or optionally substituted C １ -C ６ is alkyl; R ３ and R ４ each independently represents an optionally substituted C １ -C ６ Alkyl, C ３ -C １０ is cycloalkyl or aryl; or R ３ and R ４ taken together with the corresponding nitrogen to which they are attached form an optionally substituted 5- to 7-membered ring; and R ５ is hydrogen or -C(O)CH ３ is) having method.

2. The method of claim 1 , wherein the encapsulation composition is prepared by dissolving a block copolymer in an organic solvent and adding at least one molar equivalent of an acid relative to the block copolymer.

3. The method of any of claims 1 to 2, wherein the step of encapsulating biomolecules in micelles comprises removing organic solvents and acids and adding a therapeutic agent to the polymer.

4. The method of any one of claims 1 to 3, wherein the step of encapsulating the biomolecule in the micelle comprises dialyzing the mixture of the therapeutic agent and the block copolymer against a neutral buffer.

5. The hydrophobic polymer segment may be: 【Chemistry 2】 The method according to any one of claims 1 to 5, selected from the group consisting of

6. The hydrophobic polymer segment may be: 【Transformation 3】 The method according to any one of claims 1 to 5, selected from the group consisting of

7. n １ is an integer from 100 to 250, and x １ is an integer from 40 to 200, and / or y １ The method according to any one of claims 1 to 6, wherein is 0.

8. n １ is an integer from 100 to 250, and x １ is an integer from 100 to 200, and / or y １ The method according to any one of claims 1 to 7, wherein is 0.

9. n １ is an integer of about 114, and x １ is about 170, and / or y １ The method according to any one of claims 1 to 8, wherein is 0.

10. n １ is 114, and x １ is 170, and y １ The method according to any one of claims 1 to 9, wherein is 0.

11. The method according to any one of claims 1 to 10, wherein the step of encapsulating the biomolecule further comprises neutralization with a neutral buffer by dialysis or mixing.

12. 12. The method of claim 11, wherein the neutral buffer has a pH of 7.

4.

13. 13. The method of claim 12, wherein the neutral buffer is sodium phosphate.

14. The method of any one of claims 1 to 13, wherein the acidified block copolymer units are positively charged block copolymer units.

15. The method of any one of claims 1 to 14, wherein the step of encapsulating a biomolecule further comprises a non-covalent bond between the biomolecule and the positively charged block copolymer.

16. The method of any one of claims 1 to 15, wherein the acidified plurality of block copolymer units are acidified with acetic acid.

17. The method according to any one of claims 1 to 16, wherein the biomolecule is a protein.

18. The method according to any one of claims 1 to 16, wherein the biomolecule is a bispecific antibody.

19. The method of any one of claims 1 to 16, wherein the biomolecule is solitomab.

20. The method of any one of claims 1 to 16, wherein the biological molecule is lunimotamab.

21. The method of any one of claims 1 to 16, wherein the biomolecule is blinatumomab.

22. The method of any one of claims 1 to 16, wherein the biological molecule is odronextamab.

23. The method of any one of claims 1 to 16, wherein the biological molecule is glofitamab.

24. The method according to any one of claims 1 to 16, wherein the biomolecule is a cytokine.

25. The method of any one of claims 1 to 16, wherein the biological molecule is interleukin-12 (IL-12).

26. The method of any one of claims 1 to 16, wherein the biomolecule is single-chain interleukin-12 (IL-12).

27. The method of any one of claims 1 to 16, wherein the biomolecule is monovalent interleukin-12 (IL-12) fused to Fc from IgG.

28. The method of any one of claims 1 to 16, wherein the biomolecule is a bivalent interleukin-12 (IL-12) fused to an Fc derived from IgG.

29. The method of any one of claims 1 to 16, wherein the biological molecule is interleukin-2 (IL-2).

30. The method according to any one of claims 1 to 16, wherein the biomolecule is interleukin-2 (IL-2) fused with Fc derived from IgG.

31. The method of any one of claims 1 to 16, wherein the biological molecule is interleukin-18 (IL-18).

32. 32. The method of any one of claims 1 to 31, wherein the micelles comprise from about 0.1 wt% to about 20 wt% of the biomolecule.

33. 32. The method of any one of claims 1 to 31, wherein the micelles comprise from about 0.1 wt% to about 1 wt% of the biomolecule.

34. 32. The method of any one of claims 1 to 31, wherein the micelles comprise from about 1 wt% to about 5 wt% of the biomolecule.

35. 32. The method of any one of claims 1 to 31, wherein the micelles comprise about 5 wt% to about 10 wt% of the biomolecule.

36. 32. The method of any one of claims 1 to 31, wherein the micelles comprise from about 10 wt% to about 15 wt% of the biomolecule.

37. 32. The method of any one of claims 1 to 31, wherein the micelles comprise about 15 wt% to about 20 wt% of the biomolecule.

38. 38. The method of any one of claims 1 to 37, wherein the micelles have a diameter of less than about 1 μm or less than about 50 nm.

39. 39. The method of any one of claims 1 to 38, wherein the micelles have a diameter of about 25 to about 50 nm.

40. 40. The method of any one of claims 1 to 39, wherein the micelles are pH responsive.

41. 41. The method of any one of claims 1 to 40, wherein the micelles have a pH transition point.

42. 42. The method of claim 41, wherein the pH transition point of the micelles is between 4 and 8, 6 and 7.5, or 4.5 and 6.

5.

43. 43. The method of any one of claims 1 to 42, wherein the composition has a pH response of less than 0.25 or 0.15 pH units.

44. 1. An intermediate composition for making an encapsulated biomolecule, comprising: biomolecules; and a plurality of protonated block copolymer units, the block copolymer comprising poly(ethylene oxide) (PEO) and a hydrophobic polymer segment, having the following structure: 【Chemistry 4】 (In the formula, n １ is an integer from about 40 to about 500; x １ is an integer from about 4 to about 250; y １ is an integer from 0 to about 10; X is halogen, —OH, or —C(O)OH; R １ and R ２ are each independently hydrogen or optionally substituted C １ -C ６ is alkyl; R ３ and R ４ each independently represents an optionally substituted C １ -C ６ Alkyl, C ３ -C １０ is cycloalkyl or aryl; or R ３ and R ４ taken together with the corresponding nitrogen to which they are attached form an optionally substituted 5- to 7-membered ring; and R ５ is hydrogen or -C(O)CH ３ is) have] An intermediate composition comprising:

45. 45. The intermediate composition of claim 44, wherein the encapsulating composition further comprises an organic solvent and at least one molar equivalent of an acid relative to the block copolymer.

46. The hydrophobic polymer segment may be: 【Transformation 5】 The intermediate composition according to any one of claims 44 to 45, selected from:

47. The hydrophobic polymer segment may be: 【Transformation 6】 The intermediate composition according to any one of claims 44 to 46, selected from:

48. n １ is an integer from 100 to 250, and x １ is an integer from 40 to 200, and / or y １ The intermediate composition of any one of claims 44 to 47, wherein is 0.

49. n １ is an integer from 100 to 250, and x １ is an integer from 100 to 200, and / or y １ The intermediate composition of any one of claims 44 to 48, wherein is 0.

50. n １ is an integer of about 114, and x １ is about 170, and / or y １ 50. The intermediate composition of any one of claims 44 to 49, wherein:

51. n １ is 114, and x １ is 170, and y １ The intermediate composition of any one of claims 44 to 50, wherein is 0.

52. 52. The intermediate composition of any one of claims 44 to 51, wherein the block copolymer is positively charged with an acid.

53. 53. The intermediate composition of any one of claims 44 to 52, wherein there is a non-covalent bond between the biomolecule and the positively charged block copolymer.

54. 54. The intermediate composition of any one of claims 44 to 53, wherein the acid comprises acetic acid.

55. 55. The intermediate composition of any one of claims 44 to 54, wherein the biomolecule is a protein.

56. 56. The intermediate composition of any one of claims 44 to 55, wherein the biomolecule is a bispecific antibody.

57. 57. The intermediate composition of any one of claims 44 to 56, wherein the biomolecule is solitomab.

58. 58. The intermediate composition of any one of claims 44 to 57, wherein the biomolecule is lunimotamab.

59. 59. The intermediate composition of any one of claims 44 to 58, wherein the biomolecule is blinatumomab.

60. 60. The intermediate composition of any one of claims 44 to 59, wherein the biomolecule is odronextamab.

61. 61. The intermediate composition of any one of claims 44 to 60, wherein the biomolecule is glofitamab.

62. 62. The intermediate composition of any one of claims 44 to 61, wherein the biomolecule is a cytokine.

63. 63. The intermediate composition of any one of claims 44 to 62, wherein the biomolecule is interleukin-12 (IL-12).

64. 64. The intermediate composition of any one of claims 44 to 63, wherein the biomolecule is single-chain interleukin-12 (IL-12).

65. 65. The intermediate composition of any one of claims 44 to 64, wherein the biomolecule is monovalent interleukin-12 (IL-12) fused to an Fc derived from IgG.

66. 66. The intermediate composition of any one of claims 44 to 65, wherein the biomolecule is a bivalent interleukin-12 (IL-12) fused to an Fc derived from IgG.

67. 67. The intermediate composition of any one of claims 44 to 66, wherein the biomolecule is interleukin-2 (IL-2).

68. 68. The intermediate composition of any one of claims 44 to 67, wherein the biomolecule is interleukin-2 (IL-2) fused with Fc from IgG.

69. 69. The intermediate composition of any one of claims 44 to 68, wherein the biomolecule is interleukin-18 (IL-18).

70. 70. The intermediate composition of any one of claims 44 to 69, comprising from about 0.1 wt% to about 20 wt% of the biomolecule.

71. 71. The intermediate composition of any one of claims 44 to 70, comprising about 0.1 wt% to about 1 wt% of the biomolecule.

72. 72. The intermediate composition of any one of claims 44 to 71, comprising about 1 wt% to about 5 wt% of the biomolecule.

73. 73. The intermediate composition of any one of claims 44 to 72, comprising about 5 wt% to about 10 wt% of the biomolecule.

74. 74. The intermediate composition of any one of claims 44 to 73, comprising about 10 wt% to about 15 wt% of the biomolecule.

75. 75. The intermediate composition of any one of claims 44 to 74, comprising about 15 wt% to about 20 wt% of the biomolecule.

76. 1. A micelle encapsulating a biomolecule, comprising: biomolecules; and a plurality of protonated block copolymer units, the block copolymer comprising poly(ethylene oxide) (PEO) and a hydrophobic polymer segment, having the following structure: 【Transformation 7】 (In the formula, n １ is an integer from about 40 to about 500; x １ is an integer from about 4 to about 250; y １ is an integer from 0 to about 10; X is halogen, —OH, or —C(O)OH; R １ and R ２ are each independently hydrogen or optionally substituted C １ -C ６ is alkyl; R ３ and R ４ each independently represents an optionally substituted C １ -C ６ Alkyl, C ３ -C １０ is cycloalkyl or aryl; or R ３ and R ４ taken together with the corresponding nitrogen to which they are attached form an optionally substituted 5- to 7-membered ring; and R ５ is hydrogen or -C(O)CH ３ is) have] 1. A micelle-encapsulated biomolecule comprising:

77. 77. The micelle-encapsulated biomolecule of claim 76, wherein the biomolecule is a protein having a molecular weight of at least 6 kDa.

78. 78. The micelle-encapsulated biomolecule of any one of claims 76 to 77, wherein the biomolecule is a cytokine or a bispecific antibody.

79. 79. The micelle-encapsulated biomolecule of any one of claims 76 to 78, wherein the biomolecule is selected from solitomab, lunimotamab, blinatumomab, odronextamab, or glofitamab.

80. 79. The micelle-encapsulated biomolecule of any one of claims 76 to 78, wherein the biomolecule is selected from interleukin-12 (IL-12), single chain interleukin-12 (IL-12), monovalent interleukin-12 (IL-12) fused to Fc from IgG, bivalent interleukin-12 (IL-12) fused to Fc from IgG, interleukin-2 (IL-2), interleukin-2 (IL-2) fused to Fc from IgG, or interleukin-18 (IL-18).

81. The hydrophobic polymer segment may be: 【Transformation 8】 81. The micelle-encapsulated biomolecule of any one of claims 76 to 80, selected from:

82. The hydrophobic polymer segment may be: 【Chemistry 9】 82. The micelle-encapsulated biomolecule of any one of claims 76 to 81, selected from:

83. n １ is an integer from 100 to 250, and x １ is an integer from 40 to 200, and / or y １ 83. The micelle-encapsulated biomolecule of any one of claims 76 to 82, wherein is 0.

84. n １ is an integer from 100 to 250, and x １ is an integer from 100 to 200, and / or y １ 84. The micelle-encapsulated biomolecule of any one of claims 76 to 83, wherein is 0.

85. n １ is an integer of about 114, and x １ is about 170, and / or y １ 85. The micelle-encapsulated biomolecule of any one of claims 76 to 84, wherein is 0.

86. n １ is 114, and x １ is 170, and y １ 86. The micelle-encapsulated biomolecule of any one of claims 76 to 85, wherein is 0.

87. 87. The micelle-encapsulated biomolecule of any one of claims 76 to 86, wherein the block copolymer is positively charged with an acid.

88. 88. The micelle encapsulated biomolecule of any one of claims 76 to 87, wherein there is a non-covalent bond between the biomolecule and the positively charged block copolymer.

89. 89. The micelle-encapsulated biomolecule of any one of claims 76 to 88, wherein the acid comprises acetic acid.

90. 90. The micelle-encapsulated biomolecule of any one of claims 76-89, wherein the intermediate composition comprises from about 0.1 wt% to about 20 wt% of the biomolecule.

91. 91. The micelle-encapsulated biomolecule of any one of claims 76-90, wherein the intermediate composition comprises from about 0.1 wt% to about 1 wt% of the biomolecule.

92. 92. The micelle-encapsulated biomolecule of any one of claims 76-91, wherein the intermediate composition comprises from about 1 wt% to about 5 wt% of the biomolecule.

93. 93. The micelle-encapsulated biomolecule of any one of claims 76 to 92, wherein the intermediate composition comprises from about 5 wt% to about 10 wt% of the biomolecule.

94. 94. The micelle-encapsulated biomolecule of any one of claims 76-93, wherein the intermediate composition comprises from about 10 wt% to about 15 wt% of the biomolecule.

95. 95. The micelle-encapsulated biomolecule of any one of claims 76-94, wherein the intermediate composition comprises about 15 wt% to about 20 wt% of the biomolecule.

96. 96. A method of treating cancer, comprising administering to a patient in need thereof a composition according to any one of claims 76 to 95.

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