Lactic acid response system and method

Abstract

Provided are compositions and methods for a lactate-responsive drug delivery system. In some embodiments, there are methods and compositions for treating cancer that utilize the tumor environment to deliver a therapeutic agent to a tumor while reducing problems associated with circulating drugs in a patient's body.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 848,432, filed on May 15, 2019, the entire content of which is incorporated herein by reference.

[0003] Statement of Government Support

[0004] This invention was made with government support under Grant No. R01 OD023700 awarded by the National Institutes of Health. The government has certain rights in this invention. Background of the Invention

[0005] 1. Field of the Invention

[0006] The present invention relates to oncology, biologics, and drug delivery. More specifically, it relates to embodiments involving enzymes that react with lactic acid or other reagents that are present at higher concentrations in the tumor environment than in the normal, non - tumor environment.

[0007] 2. Description of the Related Art

[0008] Lactic acid plays many roles in the body. It can provide many useful functions, such as providing energy for the heart, brain, and skeletal muscles. Cancer cells also produce and release large amounts of lactic acid. In oncology, the well - known "Warburg effect" refers to the phenomenon that cancer cells preferentially use aerobic glycolysis rather than the oxidative phosphorylation pathway to produce energy. In glycolytic tumors, the lactic acid level in cancer cells is significantly increased up to 40 - fold and is highly correlated with cancer aggressiveness and poor survival. 1 The accumulation of lactic acid is a common feature of cancer cells. 2

[0009] Traditional chemotherapy faces many challenges in cancer treatment, such as poor selectivity for targeting tumor cells. Specific - stimulus - triggered drug delivery systems are a promising approach to address this problem and improve treatment efficacy. 3,4 For example, pH - triggered delivery systems can improve selectivity by recognizing the acidic extracellular microenvironment of solid tumors. 5 However, many studies have shown that the accumulation of lactic acid at the cancer site is not necessarily related to tissue acidosis, thus limiting its applicability in the body. 6

[0010] In the present disclosure, there are embodiments of tumor - specific drug delivery systems that can respond to the accumulation of lactic acid in the tumor microenvironment and address problems associated with poor delivery of cancer therapeutics by better targeting cancer cells or tumors. Summary of the Invention

[0011] The present disclosure provides various methods and compositions for achieving lactate-triggered drug delivery. The methods can be used in a series of applications such as cancer treatment, diagnosis, tracking, and biosensors. In particular, lactate-responsive enzymes are introduced into a polymer-based or other chemical-based matrix, which can reversibly or irreversibly change their physicochemical properties in response to changes in the ambient lactate concentration. Different enzymes can catalyze different reactions on lactate, which can be used to stimulate changes in the matrix. Lactate-responsive enzymes include, but are not limited to Figure 1 those described in

[0012] The present disclosure provides compositions and methods. In some embodiments, there is a lactate-triggered beneficial agent release composition, which comprises a chemically responsive matrix, at least one lactate-responsive enzyme provided in the chemically responsive matrix, and at least one beneficial agent provided in the chemically responsive matrix. In some aspects, the lactate-responsive enzyme is capable of converting a lactate substrate into at least one signaling molecule, and at least one signaling molecule is capable of changing at least one physicochemical property of the chemically responsive matrix, which induces the release of at least one beneficial agent. "Beneficial agent" is defined as a compound or component that confers an overall health or therapeutic benefit to a patient. The beneficial agent can be a therapeutic agent or a diagnostic agent. In some aspects, the therapeutic agent is a small molecule, a peptide or polypeptide, a nucleic acid, a microparticle, a nanoparticle, an ion, a salt, a bacterium, a virus, a living cell, a radiopharmaceutical, a chemotherapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a toxin, or a radiotherapy agent. In some aspects, the immunotherapeutic agent can be an antibody-based agent or an immune checkpoint inhibitor. In some embodiments, at least one physicochemical property is a reversible physicochemical property. At least one physicochemical property can be one or more properties selected from protonation, deprotonation, bond cleavage, swelling, deswelling, dissociation, and coalescence. In some embodiments, at least a portion of the lactate-responsive enzyme population is covalently linked to the chemically responsive matrix. In some embodiments, at least a portion of the lactate-responsive enzyme population is conjugated to the chemically responsive matrix by a hydrogen bond. In some embodiments, at least a portion of the lactate-responsive enzyme population is conjugated to the chemically responsive matrix by an ionic bond.

[0013] In some aspects, at least one signaling molecule is selected from O2, CO2, H2O2, and H + . In an embodiment, the lactate-responsive enzyme is selected from lactate oxidase, lactate dehydrogenase, lactate racemase, and lactate 2-monooxygenase. In some aspects, the composition comprises at least a portion of the lactate-responsive enzyme population on the surface of the composition. In some aspects, the composition does not comprise a lactate-responsive enzyme on the surface of the composition. In an embodiment, the composition comprises at least a portion of the lactate-responsive enzyme population embedded in the chemically responsive matrix. In some aspects, the composition does not comprise a lactate-responsive enzyme embedded in the chemically responsive matrix.

[0014] In embodiments, the chemical-responsive matrix comprises at least one functional group capable of reacting with at least one signaling molecule. In some aspects, the chemical-responsive matrix comprises a material selected from chitosan, cyclodextrin, polycyclodextrin, poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine), and poly(2-vinylpyridine). In some embodiments, the composition comprises a structure selected from nanoparticles, microparticles, hydrogels, micelles, monomeric micelles, reverse micelles, nanogels, microgels, wormlike micelles, hollow micelles, reverse hollow micelles, dendrimers, graft polymers, star polymers, branched polymers, and brush polymers.

[0015] The lactate-triggered beneficial agent release composition can be in the form of an AB toxin complex comprising an A component and a B component or a modified B component. In some aspects, the modified B component is at least 50%, 60%, 70%, 80%, 90%, or more than 90% homologous to the B component and comprises a region that interacts with the A component from the AB toxin complex. In some aspects, the B component is from cholera toxin, diphtheria toxin, pertussis toxin, Escherichia coli heat-labile toxin LT, Shiga toxin, Pseudomonas exotoxin A, botulinum toxin, tetanus toxin, anthrax toxin LF, Bordetella pertussis AC, Bacillus anthracis EF, or Staphylococcus aureus exfoliatin B. In some aspects, the B component is PA, LF, or a modified LF from anthrax toxin.

[0016] In some aspects, the amount of component B in the composition is from about 0.001 μg / mg to about 500 μg / mg of the chemical matrix. The composition may comprise component B in an amount of at least about or at most about 0.001 μg, 0.002 μg, 0.003 μg, 0.004 μg, 0.005 μg, 0.006 μg, 0.007 μg, 0.008 μg, 0.009 μg, 0.01 μg, 0.02 μg, 0.03 μg, 0.04 μg, 0.05 μg, 0.06 μg, 0.07 μg, 0.08 μg, 0.09 μg, 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7 μg, 0.8 μg, 0.9 μg, 1.0 μg, 2.0 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6.0 μg, 7.0 μg, 8.0 μg, 9.0 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg to about 500 μg, or any value therebetween. In some embodiments, the chemical response matrix and at least one lactate-reactive enzyme are provided in a mass ratio of 1:1 to 100,000:1. The chemical response matrix and at least one lactate-reactive enzyme may be provided in the following mass ratios: 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 80:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, 100000:1 or any range derivable therefrom. In an embodiment, the chemical response matrix and at least one beneficial agent are provided in a mass ratio of 1:1 to 100,000:1.The chemical-responsive matrix and at least one beneficial agent can be provided in the following mass ratios: 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 80:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, 100000:1, or any range derivable therefrom. In some aspects, the composition comprises 0.001 μmol to 10000 μmol of lactate-responsive enzyme / g of chemical-responsive matrix. The composition can comprise 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000 μmol of lactate-responsive enzyme / g of chemical-responsive matrix, or any value therebetween. In some aspects, the beneficial agent release composition releases at least 0.1×10. -10Beneficial agent / Chemoresponsive matrix. In some embodiments, the composition comprises from 0.001 μg to 500 μg of beneficial agent per mg of chemoresponsive matrix. The composition may comprise 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 μg of beneficial agent per mg of chemoresponsive matrix, or any value between any of the foregoing values. In an embodiment, the composition comprises from 0.001 μg to 500 μg of chemotherapeutic agent per mg of chemoresponsive matrix. The composition may comprise 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 μg of chemotherapeutic agent per mg of chemoresponsive matrix, or any value between any of the foregoing values. In some aspects, the chemoresponsive matrix and at least one chemotherapeutic agent are provided in a mass ratio of 1:1 to 100,000:1. The chemoresponsive matrix and at least one chemotherapeutic agent may be provided in the following mass ratios: 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 80:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, 100000:1 or any range derivable therefrom.

[0017] In some embodiments, there are methods of delivering a beneficial agent to a lactic acid target region of a subject to be treated, the methods comprising: providing a beneficial agent release composition comprising a chemically responsive matrix, at least one lactic acid-responsive enzyme provided in the chemically responsive matrix, and at least one beneficial agent provided in the chemically responsive matrix, wherein the lactic acid-responsive enzyme converts a lactic acid substrate into at least one signaling molecule when the beneficial agent release composition is exposed to a lactic acid concentration that is higher than the ambient physiological lactic acid concentration, and wherein the signaling molecule alters at least one physicochemical property of the chemically responsive matrix and induces the release of at least one beneficial agent, wherein the lactic acid target region has a lactic acid concentration that is higher than the ambient physiological lactic acid concentration.

[0018] In some embodiments, there are methods of treating cancer with a chemotherapeutic agent, the methods comprising: providing a chemotherapeutic agent release composition comprising a chemically responsive matrix, at least one lactic acid-responsive enzyme provided in the chemically responsive matrix, and at least one chemotherapeutic agent provided in the chemically responsive matrix, wherein the lactic acid-responsive enzyme converts a lactic acid substrate into at least one signaling molecule when the chemotherapeutic agent release composition is exposed to a lactic acid concentration that is higher than the ambient physiological lactic acid concentration, and wherein the signaling molecule alters at least one physicochemical property of the chemically responsive matrix and induces the release of at least one chemotherapeutic agent, wherein the lactic acid target region has a lactic acid concentration that is higher than the ambient physiological lactic acid concentration.

[0019] In some embodiments, the release of at least one beneficial agent is targeted such that the beneficial agent is released into a region comprising a lactic acid concentration that is higher than the ambient physiological lactic acid concentration. In some aspects, the region comprising a lactic acid concentration that is higher than the ambient physiological lactic acid concentration is the tumor microenvironment. In some aspects, the chemically responsive matrix functional groups react with at least one signaling molecule. In some embodiments, at least one physicochemical property is at least one of protonation, deprotonation, bond cleavage, swelling, deswelling, dissociation, and coalescence. In some aspects, at least one physicochemical property is a reversible physicochemical property. In embodiments, the chemotherapeutic agent release composition comprises a structure selected from: nanoparticles, microparticles, hydrogels, micelles, monomeric micelles, reverse micelles, nanogels, microgels, wormlike micelles, hollow micelles, reverse hollow micelles, dendrimers, graft polymers or copolymers, star polymers or copolymers, branched polymers or copolymers, and brush polymers or copolymers. In some aspects, the chemically responsive matrix comprises a material selected from: chitosan, cyclodextrin, polycyclodextrin, poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine), and poly(2-vinylpyridine).

[0020] In some embodiments, the lactic acid-responsive enzyme is selected from lactate oxidase, lactate dehydrogenase, lactate racemase, and lactate 2-monooxygenase. In some embodiments, the chemotherapeutic agent-releasing composition comprises at least a portion of the lactic acid-responsive enzyme population on the surface of the composition. In some aspects, the chemotherapeutic agent-releasing composition comprises at least a portion of the lactic acid-responsive enzyme population embedded within a chemically-responsive matrix. In some aspects, the chemotherapeutic agent-releasing composition comprises at least a portion of the lactic acid-responsive enzyme population conjugated to the chemically-responsive matrix via hydrogen bonding. In some aspects, the chemotherapeutic agent-releasing composition comprises at least a portion of the lactic acid-responsive enzyme population conjugated to the chemically-responsive matrix via ionic bonding. In some aspects, the chemotherapeutic agent-releasing composition comprises at least a portion of the lactic acid-responsive enzyme population covalently linked to the chemically-responsive matrix. In some embodiments, the beneficial agent-releasing composition releases at least one beneficial agent in a lactic acid dose-dependent manner. In some aspects, the beneficial agent-releasing composition releases at least about 0.1×10 -4 g of chemotherapeutic agent / g of chemically-responsive matrix. The beneficial agent-releasing composition may release at least about 0.1×10 -4 , 0.2×10 -4 , 0.3×10 -4 , 0.4×10 -4 , 0.5×10 -4 , 0.6×10 -4 , 0.7×10 -4 , 0.8×10 -4 , 0.9×10 -4 , 1×10 -4 , 2×10 -4 , 3×10 -4 , 4×10 -4 , 5×10 -4 , 6×10 -4 , 7×10 -4 , 8×10 -4 , 9×10 -4 , 1×10 -3 , 2×10 -3 , 3×10 -3 , 4×10 -3 , 5×10 -3 , 6×10 -3 , 7×10 -3 , 8×10 -3 , 9×10 -3 or 1×10 -2g chemotherapeutic agent / g chemical response matrix. In an embodiment, the chemotherapeutic agent release composition releases at least 30% more chemotherapeutic agent in the presence of lactate than in the absence of lactate. The chemotherapeutic agent release composition may release at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000% or 10000% more chemotherapeutic agent in the presence of lactate than in the absence of lactate. In some embodiments, the chemical response matrix and at least one lactate-reactive enzyme are provided in a mass ratio of 1:1 to 100000:1. The chemical response matrix and at least one lactate-reactive enzyme may be provided in the following mass ratios: 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 80:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3000:1, 4000:1, 5000:1, 6000:1, 7000:1, 8000:1, 9000:1, 10000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, 100000:1 or any range derivable therefrom. In some aspects, the chemical response matrix and at least one chemotherapeutic agent are provided in a mass ratio of 1:1 to 100000:1. In some embodiments, the composition contains 0.001 μmol to 10000 μmol of lactate-reactive enzyme / g chemical response matrix. The chemical response matrix and at least one chemotherapeutic agent may be provided in the following mass ratios: 1:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 80:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, 1000:1, 2000:1, 3,000:1, 4000:1, 5000:1, 6,000:1, 7000:1, 8000:1, 9000:1, 10000:1, 10000:1, 20000:1, 30000:1, 40000:1, 50000:1, 60000:1, 70000:1, 80000:1, 90000:1, 100000:1 or any range derivable therefrom. In some aspects, 0.001 μg to 500 μg of chemotherapeutic agent / mg chemical response matrix may be used.The method can employ about, at least about, or at most about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 μg chemotherapeutic agent / mg chemically-responsive matrix, or any value between the above values.

[0021] In some aspects, the method includes administering the toxin component A of an AB toxin complex, and wherein at least one chemotherapeutic agent comprises the B component or a modified B component from the AB toxin complex, wherein the modified B component is at least 50%, 60%, 70%, 80%, 90%, or more than 90% homologous to the B component and comprises a region that interacts with the A component from the AB toxin complex. In some embodiments, the toxin component A is injected into the patient's bloodstream. The toxin component A can be administered to the patient at least or at most 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 148 hours, 160 hours, or more than 160 hours after the composition is administered to the patient. In an embodiment, the toxin component A is administered to the patient at least or at most 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 148 hours, 160 hours, or more than 160 hours after the composition is administered to the patient. In some aspects, the toxin component A and the toxin component B bind in or around the patient's tumor.

[0022] In some embodiments, there is a method of preparing such a composition by incorporating one or more lactate-responsive enzymes into the chemically-responsive matrix or combining them with the chemically-responsive matrix. In some embodiments, one or more lactate-responsive enzymes are linked to the chemically-responsive matrix by chemical and / or physical bonds. Examples of physical bonds include ionic bonds and hydrogen bonds.

[0023] The term "treatment" refers to any treatment of a disease or disorder in a mammal, including: preventing or precluding the disease or disorder, i.e., causing the clinical symptoms not to develop; inhibiting the disease or disorder, i.e., arresting or inhibiting the development of clinical symptoms; and / or alleviating the disease or disorder, i.e., causing the clinical symptoms to regress. In certain embodiments, the disease is cancer, which may or may not be characterized by one or more tumors.

[0024] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In several embodiments, these media and agents may be used in combination with the pharmaceutically active substance. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. Any composition or agent discussed herein can be implemented with or in a pharmaceutically acceptable carrier or pharmaceutically acceptable excipient.

[0025] The term "effective amount" or "therapeutically effective amount" refers to the amount of the compositions of the present disclosure that is sufficient to effectuate treatment as defined herein when administered to a mammal in need of such treatment. This amount will vary depending on the subject and disease condition being treated, the body weight and age of the subject, the severity of the disease condition, the particular composition of the present disclosure selected, the dosing regimen to be followed, the timing of administration, the mode of administration, and the like, all of which can be readily determined by one of ordinary skill in the art. In certain embodiments, the effective amount refers to the amount in the case of effectively treating cancer or a tumor.

[0026] As used herein, the terms "or" and "and / or" are used to describe multiple components that are combined with each other or are mutually exclusive. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", "x or (y and z)", or "x or y or z". It is specifically contemplated that x, y, or z can be specifically excluded from the embodiments.

[0027] Throughout this application, the term "about" is used in its plain and ordinary sense in the field of cell biology to indicate that a value includes the standard deviation of the error of the device or method used to determine that value.

[0028] The term "comprising", which is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase "consisting of" excludes any element, step, or ingredient not specified. The phrase "consisting essentially of" limits the scope of the subject matter to the specified materials or steps and those that do not materially affect its essential and novel characteristics. It is contemplated that embodiments described in the context of the term "comprising" may also be implemented in the context of the terms "consisting of" or "consisting essentially of".

[0029] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may be applicable to any other embodiment of the invention. Additionally, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any composition of the invention. Aspects of an embodiment set forth in an example are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different example or elsewhere in the present application, elsewhere in the present application such as in the summary, detailed description, claims, and description of the drawings. Description of the Drawings

[0030] The following drawings form a part of this specification and are included to further illustrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of specific embodiments given herein.

[0031] Figure 1 Multiple lactic acid-related enzymes and the reactions they mediate.

[0032] Figure 2 Composition and design of the lactic acid-responsive system.

[0033] Figure 3 Enzymatic reactions involving lactate oxidase.

[0034] Figure 4A - B Preparation of hydrogels for controlling lactic acid-responsive release.

[0035] Figure 5 Quantitative analysis of BSA release from engineered chitosan materials in water or lactic acid solution.

[0036] Figure 6A-H pH-responsive polymer matrices with different structures: (A) monomer-micelle, (B) micelle-reverse micelle, (C) nanogel or microgel, (D) hollow-reverse hollow, (E) dendrimer, (F) hyperbranched, (G) micelle morphological change (from worm-like to hollow), and (H) polymer brush.

[0037] Figure 7Schematic illustration of lactic acid dissociable vesicles using CO2-responsive polymers and chemical reactions involving lactate 2-monooxygenase, and examples of block copolymer structures.

[0038] Figure 8 Schematic illustration of H2O2 dissociable polymers and examples of block copolymer structures.

[0039] Figure 9A - B Pharmacokinetics of toxin components A and B in normal and tumor environments. When exposed to high lactic acid concentrations in tumors, the particles rapidly release their contents, but retain the toxin inside the particles for a longer time in normal tissues and organs (A). Toxic effects of the drug are observed in the tumor environment (bottom, B), and non-toxic effects are observed in the normal non-tumor environment (top, B).

[0040] Figure 10 AB toxin induces cytotoxicity in vitro. Quantitative analysis of cell viability under different conditions (MTT assay): (1) control; (2) PA alone; (3) LFN-DTA alone; (4) PA + LFN-DTA; (5) free PA + LFN-DTA released from lactate-responsive particles in control solution; (6) free PA + LFN-DTA released from lactate-responsive particles in lactate solution. Cell viability was measured by MTT assay and normalized to control cells (condition 1). LFN = N-terminal region of lethal factor toxin. DTA = diphtheria toxin A.

[0041] Figure 11 Design and performance of a lactate-responsive dual MS trigger magazine system.

[0042] Figure 12A - C Transmission electron microscopy (TEM) results of nanomaterials. Mesoporous silica nanoparticles were used to prepare mesoporous silica (MS)-magazine (A) and MS-trigger (B). Lactate-responsive dual MS trigger magazine nanodevice (C). Arrows pointing outside the nanoparticles are gated integrators. Arrows pointing between the nanoparticles are the junctions between the MS-trigger and MS-magazine. The size of the surface gated integrator and the connection between the spherical MS magazines is approximately 200 nm. The size of the spherical MS trigger is approximately 180 nm.

[0043] Figure 13A - B In vitro release results of doxorubicin (DOX) (A). DMS status 1 hour after cargo release (lactate-responsive dual MS trigger-magazine nanodevice loaded with DOX_(B).

[0044] Figure 14 Mouse experiment results show that the size and weight of breast tumors were significantly reduced 3 days after DMS treatment.

[0045] Figure 15 Representative images on the 3rd day after treatment confirmed the results of significant reduction in breast tumor size and weight within 3 days after DMS treatment. Detailed implementation

[0046] The present disclosure provides methods and compositions related to lactate-responsive drug delivery systems. The lactate-responsive drug delivery systems disclosed herein include polymeric materials or other chemical matrices, which can be provided in the form of particles, hydrogels, or materials adhered to a substrate such as an electrode substrate. Enzymes can be non-covalently entrapped in the polymeric material or other chemical matrix and / or covalently linked to the polymeric material or other chemical matrix. The drug delivery system responds to lactate by detecting the enzymatic conversion products of lactate, including but not limited to pH changes (H + ), CO2 generation, H2O2 generation, and O2 generation. The methods and compositions can be used to treat cancer by responding to the chemical environment near the tumor. After being triggered by chemical signals in the tumor environment, the composition can deliver a therapeutic agent to the local tumor environment. The methods and compositions address the problems associated with systemic administration of drugs and the systemic circulation of these drugs in patients.

[0047] As used herein, "reversible" is defined as being able to reverse to restore a previous state or condition. "Nanoparticle" is defined as any-shaped particle with a size range from about 1 nm to about 100 nm. "Microparticle" is defined as any-shaped particle with a size range from about 1 μm to about 1000 μm. "Gel" is defined as a non-fluid colloidal network or polymer network that swells by a fluid throughout its volume. "Hydrogel" is defined as a gel in which the swelling agent is water. "Micelle" is defined as an aggregate or assembly of surfactant molecules dispersed in a liquid. "Reverse micelle" is defined as a micelle in which the polar groups of the surfactant are concentrated inside, while the lipophilic groups extend into and into the non-polar solvent. "Reverse hollow micelle" is a reverse micelle with a hollow interior. "Dendrimer" is defined as a molecule with a repeating branched structure. "Graft polymer" is defined as a block copolymer with a linear main chain of one composite material and randomly distributed side chains of another composite material. "Branched polymer" is defined as a polymer with secondary polymer chains attached to the main backbone. "Star polymer" is defined as a branched polymer whose general structure includes multiple (at least three) linear chains connected to a central core. "Brush polymer" is a polymer whose main chain (skeleton) has multiple (at least three) branching points, from which linear side chains emanate.

[0048] I. Antibody

[0049] Aspects of the present disclosure relate to the use of anti-inflammatory antibodies or fragments thereof. The term "antibody" refers to an intact immunoglobulin of any isotype or a fragment thereof that can compete with an intact antibody for specific binding to a target antigen, and includes chimeric, humanized, fully humanized, and bispecific antibodies. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides containing antibody CDR domains that retain antigen-binding activity.

[0050] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent such as an antibody. An antigen can have one or more epitopes that can interact with different antibodies.

[0051] The term "epitope" includes any region or portion of a molecule that can elicit an immune response by binding to an immunoglobulin or a T cell receptor. Epitope determinants can include chemically reactive surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural features and / or specific charge characteristics. Generally, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture.

[0052] Many different epitope mapping techniques well known in the art can be used to identify the epitope regions of a given polypeptide, including: X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y.. Such techniques are known in the art and are described, for example, in the following documents: U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986); see, for example, Epitope Mapping Protocols, supra. Additionally, standard antigenicity and hydrophilicity plots can also be used to predict and identify the antigenic regions of a protein.

[0053] Full antibodies typically consist of two full-length heavy chains and two full-length light chains, but in some cases may include fewer chains. For example, antibodies naturally present in camelids may contain only heavy chains. The antibodies disclosed herein can be derived from a single source only or can be "chimeric", i.e., different parts of the antibody can be derived from two different antibodies. For example, the variable region or CDR region can be derived from a rat or murine source, while the constant region is derived from a different animal source, such as human. Antibodies or binding fragments can be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of full antibodies. Unless otherwise specified, the term "antibody" includes its derivatives, variants, fragments, and mutant proteins, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0054] The term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of approximately 25,000 daltons and include a variable domain (abbreviated herein as VL) and a constant domain (abbreviated herein as CL). Light chains are divided into two classes, designated kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody, including all or part of the variable region of the light chain, including the CDRs. The VL fragment may also include light chain constant region sequence. The variable domain of the light chain is located at the amino terminus of the polypeptide.

[0055] The term "heavy chain" includes full-length heavy chains and fragments thereof having sufficient variable region sequence to confer binding specificity. Full-length heavy chains have a molecular weight of approximately 50,000 daltons and include a variable domain (abbreviated herein as VH) and three constant domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of the heavy chain of a monoclonal antibody, including all or part of the variable region of the heavy chain, including the CDRs. The VH fragment may also include heavy chain constant region sequence. The number of heavy chain constant domains will depend on the isotype. The VH domain is located at the amino terminus of the polypeptide, the CH domains are located at the carboxyl terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the heavy chain present, which are the following five classes respectively: μ chain, δ chain, γ chain, α chain, or ε chain. IgG has multiple subtypes, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0056] Antibodies can be intact immunoglobulins of any isotype or class, chimeric antibodies, or hybrid antibodies specific for two or more antigens. They can also be fragments (e.g., F(ab′)2, Fab′, Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic, or genetically engineered proteins that function as antibodies by binding to specific antigens to form complexes. The term antibody includes genetically engineered or other modified forms of immunoglobulins, such as the following:

[0057] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units that are linked to each other through the constant domain of the antibody heavy chain (Fc, or crystallizable fragment region). The complex can be stabilized by a joining (J) chain protein. The term "polymer" refers to an antibody that contains more than two Ig units that are linked to each other through the constant domain of the antibody heavy chain (Fc region). The complex can be stabilized by a joining (J) chain protein.

[0058] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. The two binding sites can have the same antigen specificity or they can be bispecific, which means that the two antigen-binding sites have different antigen specificities.

[0059] Bispecific antibodies are a class of antibodies that have two paratopes with different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be bisparatopic, where the bispecific antibody can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single-domain antibodies called "nanobodies". Single-domain antibodies are derived from cartilaginous fish and camelids and are modified. Nanobodies can be linked together by a linker using techniques commonly used by those skilled in the art; such methods for selecting and linking antibodies are disclosed in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and et al., Anal Chem. 86:7875-7882 (2014), the entire contents of each of which are specifically incorporated by reference.

[0060] Bispecific antibodies can be constructed as: intact IgG, Fab'2, Fab'PEG, bispecific antibodies or replaced with scFv. Bispecific antibodies and scFv can be constructed using only the variable domains without using the Fc region, thereby potentially reducing the impact of anti-idiotypic responses. Bispecific antibodies can be generated by a variety of methods, including but not limited to the fusion of hybridomas or the ligation of Fab' fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), the entire contents of each of which are specifically incorporated by reference.

[0061] In some aspects, by polymerizing VH and VL domain pairs that bind different antigens, the antigen-binding domain can be multispecific or heterospecific. For example, the antibody can bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, aspects can include but are not limited to bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that target epitopes and other targets, such as Fc receptors on effector cells.

[0062] In some embodiments, multispecific antibodies can be used and directly linked by short flexible polypeptide chains using conventional methods known in the art. One such example is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain and a linker that is too short to pair between domains on the same chain is utilized, thereby forcing the domains to pair with complementary domains on the other chain, generating two antigen-binding sites. The linker functionality is applicable to embodiments of trispecific, tetraspecific, and higher-order antibody multimers. (See, for example, Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0063] In contrast to bispecific full antibodies, bispecific diabodies may also be advantageous because they can be readily constructed and expressed in E. coli. Diabodies (and other polypeptides, such as antibody fragments) with appropriate binding specificities can be readily selected from libraries using phage display (WO94 / 13804). If one arm of the diabody is kept constant, for example, having specificity for a protein, a library can be made in which the other arm varies and antibodies with appropriate specificities are selected. Bispecific full antibodies can be prepared by alternative genetic engineering methods as described by Ridgeway et al. (Protein Eng., 9:616-621, 1996) and Krah et al. (NBiotechnol. 39:167-173, 2017), the entire contents of each of which are incorporated herein by reference.

[0064] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities. See, e.g., U.S. Patent No. 6,010,902, the entire contents of which are incorporated herein by reference.

[0065] The Fv fragment portion of an antibody molecule that binds to an antigenic epitope with high specificity is referred to herein as a "complementary site". The complementary site is composed of amino acid residues that contact the antigenic epitope to facilitate antigen recognition. Each of the two Fv fragments of an antibody consists of two variable domains, VH and VL, in a dimeric configuration. The primary structure of each variable domain includes three hypervariable loops, which are separated by framework regions (FRs) and flank them on either side. The hypervariable loops are the regions of highest primary sequence variability in antibody molecules from any mammalian species. The term hypervariable loop is sometimes used interchangeably with the term "complementary determining region (CDR)". The length of the hypervariable loops (or CDRs) varies among antibody molecules. The framework regions of all antibody molecules from a given mammalian species have a high degree of primary sequence similarity / identity. A person skilled in the art can use the identity of the framework regions to identify both the framework regions and the hypervariable loops (or CDRs) that are interspersed between the framework regions. The hypervariable loops are given identifying names to distinguish their position in the polypeptide and which domain they appear in. The CDRs in the VL domain are designated L1, L2, and L3, with L1 appearing most distally and L3 appearing closest to the CL domain. The CDRs can also be named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the region of highest variability among all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and are separated from each other by the framework regions. The amino-terminal (N-terminal) of the VL chain is called FR1. The region identified as FR2 appears between the L1 and L2 hypervariable loops. FR3 appears between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. The VH chain repeats this structure and nomenclature and includes three CDRs, designated H1, H2, and H3. Most of the amino acid residues in the variable domains or Fv fragments (VH and VL) are part of the framework regions (about 85%). The three-dimensional or tertiary structure of the antibody molecule positions the framework regions more internally in the molecule and provides most of the structure, with the CDRs located on the outer surface of the molecule.

[0066] Several methods have been developed and can be used by those skilled in the art to identify the exact amino acids that make up each of these regions. This can be done using any of a variety of multiple sequence alignment methods and algorithms that identify conserved amino acid residues that make up the framework regions and thus identify the CDRs that may vary in length but are located between the framework regions. Three commonly used methods have been developed to identify the CDRs of antibodies: Kabat (as described in: T.T. Wu and E.A. Kabat, “AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY,” J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in: C. Chothia et al., “Conformations of immunoglobulin hypervariable regions,” Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (as described in: M.-P. Lefranc et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods includes a unique numbering system for identifying the amino acid residues that make up the variable regions. In most antibody molecules, the amino acid residues that actually contact the antigen epitope occur in the CDRs, but in some cases, residues in the framework regions contribute to antigen binding.

[0067] One of a variety of methods can be used by those skilled in the art to determine the complementarity-determining regions of an antibody. These methods include: 1) computational prediction of the tertiary structure of the antibody / epitope binding interaction based on the chemical properties of the amino acid sequence of the antibody variable region and epitope composition; 2) hydrogen-deuterium exchange and mass spectrometry; 3) polypeptide fragmentation and peptide mapping methods, in which multiple overlapping peptide fragments are generated from the full length of the polypeptide and the binding affinity of these peptides for the epitope is evaluated; 4) antibody phage display library analysis, in which antibody Fab fragments encoding mammalian genes are expressed by phages in a manner incorporated into the phage coat. Then the population of phages expressing the Fab is allowed to interact with the immobilized antigen or can be expressed therein by different heterologous expression systems. The unbound Fab fragments are washed away, leaving only the specific binding Fab fragments attached to the antigen. The bound Fab fragments can be easily isolated and the genes encoding them determined. This method can also be used for smaller regions of the Fab fragment, including the Fv fragment or specific VH and VL domains, as appropriate.

[0068] In certain aspects, affinity matured antibodies are enhanced by one or more modifications in one or more of their CDRs, which results in an increased affinity of the antibody for the target antigen as compared to the parental antibody that does not have those alterations. Certain affinity matured antibodies will have a nanomolar or picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art, such as those described by Marks et al., Bio / Technology 10:779 (1992), affinity maturation by VH and VL domain shuffling, random mutagenesis of CDR and / or framework residues used in phage display is described by Rajpal et al., PNAS. 24:8466 - 8471 (2005) and Thie et al., Methods Mol Biol. 525:309 - 22 (2009) in combination with the computational methods demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0069] Chimeric immunoglobulins are products of fusion genes from different species; "humanized" chimeras generally have framework regions (FRs) from human immunoglobulins and one or more CDRs from non - human sources.

[0070] In some aspects, portions of the heavy and / or light chains are identical or homologous to the corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequences in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods related to chimeric antibodies, see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), the entire contents of each of which are specifically incorporated herein by reference. CDR grafting is described, e.g., in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are hereby incorporated by reference for all purposes.

[0071] In some embodiments, minimizing antibody polypeptide sequences from non-human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues in the non-antigen recognition regions of non-human antibodies are modified to be homologous to the corresponding residues in human antibodies or isotypes. An example is a "CDR-grafted" antibody, in which the antibody contains one or more CDRs from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass. For use in humans, the V region consisting of CDR1, CDR2, and part of CDR3 of the light and heavy chain variable regions of non-human immunoglobulins is grafted to the human antibody framework region, replacing the antigen receptor of the naturally occurring human antibody with non-human CDRs. In some cases, the corresponding non-human residues replace framework region residues of the human immunoglobulin. In addition, a humanized antibody may contain residues not found in the recipient or donor antibody to further improve performance. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0072] Intracellular antibodies are immunoglobulins that are intracellularly localized and bind to intracellular antigens, as opposed to secreted antibodies, which bind antigens in the extracellular space.

[0073] Polyclonal antibody preparations generally include different antibodies directed against different determinants (epitopes). To produce polyclonal antibodies, a host such as a rabbit or goat is immunized with an antigen or antigen fragment, usually with an adjuvant and, if desired, conjugated to a carrier. Subsequently, the antibodies directed against the antigen are collected from the host serum. Polyclonal antibodies can be affinity purified against the antigen to render them monospecific.

[0074] A monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies from a dedicated parent cell, e.g., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0075] 2. Functional antibody fragments and antigen-binding fragments

[0076] a. Antigen-binding fragments

[0077] Certain aspects relate to antibody fragments, such as antibody fragments that bind and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of an antibody that retain the ability to specifically bind an antigen. These fragments are composed of various arrangements of the variable heavy chain (VH) and / or light chain (VL); and in some embodiments, include the constant heavy chain 1 (CH1) and light chain (CL). In some embodiments, they do not contain the Fc region composed of the heavy chain 2 (CH2) and heavy chain 3 (CH3) domains. Embodiments of antigen-binding fragments and their modifications can include: (i) the Fab fragment type composed of VL, VH, CL, and CHl domains; (ii) the Fd fragment type composed of VH and CHl domains; (iii) the Fv fragment type composed of VH and VL domains; (iv) the single-domain fragment type, dAb (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003), which is composed of a single VH or VL domain; (v) isolated complementarity-determining region (CDR) regions. These terms are described, for example, in: Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, R.A. (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and Day, E.D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015). The citations in this paragraph are hereby incorporated by reference.

[0078] Antigen-binding fragments also include antibody fragments that precisely retain at least or at most 1, 2, or 3 complementarity-determining regions (CDRs) from the variable region of the light chain. The fusion of sequences containing CDRs with the Fc region (or its CH2 or CH3 region) is included within this definition, including, for example, scFvs that are directly or indirectly fused to the Fc region, which are also included herein.

[0079] The term Fab fragment refers to the monovalent antigen-binding fragment of an antibody that contains the VL, VH, CL, and CH1 domains. The term Fab' fragment refers to the monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, the Fab' fragment includes the VL, VH, CL, and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment refers to the bivalent antigen-binding fragment of a monoclonal antibody that contains two Fab' fragments linked by a disulfide bond in the hinge region. The F(ab')2 fragment includes, for example, all or part of two VH and VL domains and may also include all or part of two CL and CH1 domains.

[0080] The term Fd fragment refers to the heavy chain fragment of a monoclonal antibody that includes all or part of the VH, including the CDRs. The Fd fragment may also include the CH1 region sequence.

[0081] The term Fv fragment refers to the monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of the VL and VH and lacks the CL and CH1 domains. The VL and VH include, for example, the CDRs. A single-chain antibody (sFv or scFv) is an Fv molecule in which the VL and VH regions have been linked by a flexible linker to form a single polypeptide chain, which forms the antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain that includes an oligomerization domain at its C-terminus, separated from the sFv by a hinge region (Pack et al., 1992). The oligomerization domain contains self-associating α-helices, such as leucine zippers, which can be further stabilized by additional disulfide bonds. The (scFv)2 fragment is also referred to as a "minibody" or "miniantibody".

[0082] A single-domain antibody is an antigen-binding fragment that contains only the VH or VL domain. In some cases, two or more VH regions are covalently linked to a peptide linker to produce a bivalent domain antibody. The two VH regions of the bivalent domain antibody can target the same or different antigens.

[0083] b. Fragment crystallizable region, Fc

[0084] The Fc region contains two heavy chain fragments, including the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. As used herein, the term "Fc polypeptide" includes native and mutant protein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides that include a hinge region that promotes dimerization are included.

[0085] 3. A polypeptide having an antibody CDR and a scaffold domain displaying the CDR

[0086] According to an embodiment, antigen-binding peptide scaffolds such as complementarity determining regions (CDRs) are used to generate protein-binding molecules. Generally, one of ordinary skill in the art can determine the type of protein scaffold onto which at least one CDR is grafted. The best scaffolds known must meet a number of criteria, such as: good phylogenetic conservation; known three-dimensional structure; small size; few or no post-transcriptional modifications; and / or ease of production, expression, and purification. Skerra, J Mol Recognit, 13:167-87 (2000).

[0087] Protein scaffolds can be derived from, but are not limited to: fibronectin type III FN3 domains (referred to as "monomers"), fibronectin type III domain 10, lipocalin, anticalin, the Z domain of protein A of Staphylococcus aureus, thioredoxin A, or proteins having repeating motifs such as "ankyrin repeat motifs", "armadillo protein repeat motifs", "leucine-rich repeat motifs", and "three-four amino acid repeat motifs". Such proteins are described in U.S. Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. WO2006 / 056464, the entire contents of each of which are specifically incorporated herein by reference. Scaffolds derived from toxins of scorpions, insects, plants, mollusks, etc., as well as protein inhibitors of neuronal nitric oxide synthase (PIN) can also be used.

[0088] II. Methods of Treatment

[0089] The compositions of the present disclosure can be used for in vivo, in vitro, or ex vivo administration. The route of administration of the composition can be, for example, intradermal, subcutaneous, intravenous, topical, and intraperitoneal administration.

[0090] Autoimmune or inflammatory diseases suitable for treatment may include, but are not limited to, for example, the following diseases: diabetes (e.g., type 1 diabetes), transplant rejection, arthritis (rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, osteoarthritis and systemic juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, degenerative arthritis, chronic primary polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, guttate psoriasis, pustular psoriasis and nail psoriasis, atopy, including atopic diseases such as hay fever and Job's syndrome, dermatitis, including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, X-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis such as spinal-ocular multiple sclerosis, primary progressive multiple sclerosis (PPMS) and relapsing-remitting multiple sclerosis (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polypous colitis, necrotizing enterocolitis, transmural colitis and autoimmune inflammatory bowel disease), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, all or part of uveal inflammation, iritis, choroiditis, autoimmune blood diseases, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage such as meningitis, herpes gestationis, pemphigoid gestationis, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss caused by autoimmune diseases, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis and marginal and / or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens antigenic uveitis,Posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome such as chronic or acute glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN), including type I and type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyendocrine failure, balanitis, including plasma cell circumscribed balanitis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, erythema multiforme, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, precancerous keratosis, pyoderma gangrenosum, allergic conditions and reactions, anaphylaxis, eczema, including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema and vesicular palmar and plantar eczema, asthma such as bronchial asthma, allergic asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, immune responses against foreign antigens (such as the fetus) such as fetal ABO blood group during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus encephalitis, juvenile lupus, non-renal lupus, extrarenal lupus, discoid lupus and discoid lupus erythematosus, alopecia areata, systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, juvenile-onset (type I) diabetes, including childhood insulin-dependent diabetes mellitus (IDDM) and adult-onset diabetes (type II diabetes) and autoimmune diabetes. Also contemplated are immune responses associated with acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases, including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and giant cell (temporal) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa / perarteritis nodosa), microscopic polyangiitis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, allergic vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, ANCA-associated vasculitis such as allergic granulomatous angiitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs'-positive anemia, congenital pure red cell aplasia, hemolytic anemia or immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory diseases, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes such as those secondary to sepsis, trauma or hemorrhage, antigen-antibody complex-mediated diseases,Anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's disease / syndrome, Castleman's syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as bullous pemphigoid and cicatricial pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucosae, pemphigus membranaceus and pemphigus erythematosus), autoimmune polyendocrine diseases, Reiter's disease or syndrome, thermal injury, pre-eclampsia, immune complex diseases such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy, autoimmune or immune-mediated thrombocytopenia, such as idiopathic thrombocytopenic purpura (ITP), including chronic or acute ITP, scleritis, such as idiopathic keratosing scleritis, episcleritis, autoimmune diseases of the testis and ovary, including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases, including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes such as autoimmune polyglandular syndrome (or polyglandular endocrinopathy syndrome), paraneoplastic syndromes, including neurological paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff person or stiff person syndrome, encephalomyelitis such as allergic encephalomyelitis or allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS) and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, cytomegalic hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) and NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac or celiac disease, celiac sprue, refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease) with migraine, coronary artery disease, autoimmune ear diseases such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or recurrent or relapsing polychondritis, alveolar proteinosis, Cogan's syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, herpes zoster-associated pain,Amyloidosis, non-cancerous lymphocytosis, primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndromes, ion channelopathies such as epilepsy, migraine, arrhythmia, muscle diseases, deafness, blindness, periodic paralysis and CNS channelopathies, autism, inflammatory myopathies, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, choroidoretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine failure, Schmidt syndrome, adrenalitis, gastric atrophy, Alzheimer's disease, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, post-myocardial infarction syndrome, nausea alopecia, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma) and telangiectasia), male and female autoimmune infertility (e.g., due to anti-sperm antibodies), mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent miscarriage, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing syndrome, bird fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport syndrome, alveolitis such as allergic alveolitis and fibrotic alveolitis, interstitial pneumonia, transfusion reaction, leprosy, malaria, parasitic diseases such as leishmaniasis, trypanosomiasis (kypanosomiasis), schistosomiasis, ascariasis, aspergillosis, Sampter syndrome, Kaplan syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum diutinum, fetal erythroblastosis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic) or Fuch's cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immunodeficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham chorea, post-streptococcal glomerulonephritis, thromboangitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, chronic allergic pneumonia, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephrotic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, organ transplant reperfusion, retinal autoimmunity, joint inflammation,Bronchitis, chronic obstructive airway / lung diseases, silicosis, aphthae, aphthous stomatitis, arteriosclerotic diseases, asperniogenese, autoimmune hemolysis, Berk's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia phacoanaphylactica, allergic enteritis, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich disease, sensorineural deafness, paroxysmal nocturnal hemoglobinuria, hypogonadism, regional enteritis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyroiditis, acquired spenic atrophy, non-malignant thymoma, leukoplakia, toxic shock syndrome, food poisoning, conditions involving T cell infiltration, leukocyte adhesion deficiency, immune responses related to cytokine- and T lymphocyte-mediated acute and delayed hypersensitivity, diseases involving leukocyte extravasation, multiple organ damage syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, ophthalmia symphatica, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, multiple endocrine failure, autoimmune polyendocrine syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary or sphenoid sinusitis, eosinophil-related diseases such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loeffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma or eosinophil-containing granuloma, allergic reactions, seronegative spondyloarthritides, polyendocrine autoimmunity, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndromeAtaxia-telangiectasia syndrome, telangiectasia, autoimmune diseases associated with collagen diseases, rheumatism, nervous system diseases, lymphadenitis, reduced blood pressure response, vascular dysfunction, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, diseases associated with angiogenesis, allergic hypersensitivity diseases, glomerulonephritis, reperfusion injury, ischemic reperfusion disorder, reperfusion injury of the myocardium or other tissues, lymphomatoid tracheobronchitis, inflammatory skin diseases, skin diseases with an acute inflammatory component, multiple organ failure, bullous diseases, renal cortical necrosis, acute suppurative meningitis or other central nervous system inflammatory diseases, orbital inflammatory diseases, granulocyte transfusion-related syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelonephritis, intimal hyperplasia of arteries, digestive system ulcers, valvulitis, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness and endometriosis.

[0091] A. Checkpoint inhibitors and combination therapies

[0092] Embodiments of the present disclosure may include administering immune checkpoint inhibitors, which are further described below.

[0093] 1. PD-1, PDL1, and PDL2 inhibitors

[0094] PD-1 can act in the tumor microenvironment where T cells encounter infection or tumors. Activated T cells upregulate PD-1 and continue to express it in peripheral tissues. Cytokines such as IFN-γ induce the expression of PDL1 on epithelial cells and tumor cells. PDL2 is expressed on macrophages and dendritic cells. The main role of PD-1 is to limit the activity of peripheral effector T cells and prevent excessive damage to tissues during the immune response. The inhibitors of the present disclosure can block one or more functions of PD-1 and / or PDL1 activity.

[0095] Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0096] In some embodiments, a PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The inhibitor can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, the entire contents of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, such as those described in U.S. Patent Application Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, the entire contents of which are incorporated herein by reference.

[0097] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PDL1 inhibitor comprises AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and is the anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, pembrolizumab, and SCH-900475, is the anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is the anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is the PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680 (also known as AMP-514) and REGN2810.

[0098] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor, such as durvalumab (also known as MEDI4736), atezolizumab (also known as MPDL3280A), avelumab (also known as MSB00010118C), MDX-1105, BMS-936559, or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, such as rHIgM12B7.

[0099] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the above antibodies. In another embodiment, the antibody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any derivable range therein) variable region amino acid sequence identity to the above antibodies.

[0100] 2. CTLA-4, B7-1, and B7-2

[0101] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The Genbank accession number for the complete cDNA sequence of human CTLA-4 is L15006. CTLA-4 is present on the surface of T cells and acts as a "shut-off" switch when bound to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily, is expressed on the surface of helper T cells, and transmits an inhibitory signal to the T cell. CTLA4 is similar to the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to the T cell, whereas CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also present in regulatory T cells and may be important for their function. Activation of T cells through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules. The inhibitors of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the CTLA-4 and B7-2 interaction.

[0102] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0103] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the present disclosure can be generated using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, anti-CTLA-4 antibodies disclosed in the following: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; previously known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998; can be used in the methods disclosed herein. The teachings of each of the foregoing publications are hereby incorporated by reference. Antibodies that compete with any of these art-recognized antibodies that bind CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114; all of which are incorporated herein by reference.

[0104] Other anti-CTLA-4 antibodies used as checkpoint inhibitors in the methods and compositions of the present disclosure are ipilimumab (also known as 10D1, MDX-010, MDX-101, and ) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0105] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding with and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the above antibodies. In another embodiment, the antibody has at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (or any range derivable therein) variable region amino acid sequence identity with the above antibodies.

[0106] III. Immunotherapy

[0107] In some embodiments, the method includes utilizing cancer immunotherapy. Cancer immunotherapy (sometimes referred to as immuno - oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive, or combination (active and passive). These methods utilize the fact that cancer cells typically have molecules on their surface that can be detected by the immune system, called tumor - associated antigens (TAAs); they are usually proteins or other macromolecules (such as carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti - tumor responses, including the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapy is known in the art, and some are described below.

[0108] A. Inhibiting co - stimulatory molecules

[0109] In some embodiments, the immunotherapy comprises an inhibitor of a co - stimulatory molecule. In some embodiments, the inhibitor includes inhibitors of B7 - 1 (CD80), B7 - 2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4 - 1BB (CD137, TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. The inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0110] B. Dendritic cell therapy

[0111] Dendritic cell therapy stimulates an anti - tumor response by enabling dendritic cells to present tumor antigens to lymphocytes, which activate them and cause them to kill other antigen - presenting cells. Dendritic cells are antigen - presenting cells (APCs) in the mammalian immune system. In cancer treatment, they contribute to cancer antigen targeting. An example of a dendritic cell - based cellular cancer therapy is sipuleucel - T.

[0112] One way to induce dendritic cells to present tumor antigens is to vaccinate with autologous tumor lysates or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are usually used in combination with adjuvants (highly immunogenic substances) to increase the immune and anti - tumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte - macrophage colony - stimulating factor (GM - CSF).

[0113] Dendritic cells can also be activated in vivo by causing tumor cells to express GM - CSF. This can be achieved by genetically engineering tumor cells to produce GM - CSF or by infecting tumor cells with oncolytic viruses that express GM - CSF.

[0114] Another strategy is to remove dendritic cells from the patient's blood and activate them in vitro. Dendritic cells are activated in the presence of tumor antigens, which can be a single tumor-specific peptide / protein or a tumor cell lysate (a solution of lysed tumor cells). These cells (with optional adjuvants) are injected and an immune response is elicited.

[0115] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, and dendritic cell maturation can be induced and immunity against tumors can be provided. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 have been used as antibody targets.

[0116] C. CAR-T Cell Therapy

[0117] A chimeric antigen receptor (CAR, also known as a chimeric immune receptor, chimeric T cell receptor, or artificial T cell receptor) is an engineered receptor that confers new specificity to immune cells to target cancer cells. Typically, these receptors transplant the specificity of a monoclonal antibody onto a T cell. These receptors are called chimeras because they are formed by the fusion of parts from different sources. CAR-T cell therapy refers to a treatment method using such transformed cells for cancer treatment.

[0118] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T cell activation functions. The general premise of CAR-T cells is to artificially generate T cells that target markers found on cancer cells. Scientists can take T cells from a person, genetically modify them, and then put them back into the patient so that they attack cancer cells. Once a T cell is engineered into a CAR-T cell, it acts as a "living drug". CAR-T cells establish a connection between an extracellular ligand recognition domain and an intracellular signaling molecule, which in turn activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, rather than normal cells. The specificity of CAR-T cells depends on the choice of targeting molecule.

[0119] Exemplary CAR-T therapies include Tisagenlecleucel (Kymriah) and Axicabtageneciloleucel (Yescarta). In some embodiments, CAR-T therapy targets CD19.

[0120] D. Cytokine Therapy

[0121] Cytokines are proteins produced by a variety of cells present within tumors. They can regulate immune responses. Tumors often use them to enable their growth and reduce immune responses. These immunomodulatory effects enable them to be used as drugs to trigger immune responses. Two commonly used cytokines are interferons and interleukins.

[0122] Interferons are produced by the immune system. They are typically involved in antiviral responses but can also be used to treat cancer. They are divided into three groups: type I (IFNα and IFNβ), type II (IFNγ), and type III (IFNλ).

[0123] Interleukins have a range of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0124] E. Adoptive T cell therapy

[0125] Adoptive T cell therapy is a form of passive immunization by infusion of T cells (adoptive cell transfer). They are present in the blood and tissues and are usually activated when they detect foreign pathogens. Specifically, they are activated when the surface receptors of T cells encounter cells that display part of a foreign protein on their surface antigens. These can be infected cells or antigen-presenting cells (APCs). They are present in normal and tumor tissues, where they are called tumor-infiltrating lymphocytes (TILs). They are activated by APCs such as dendritic cells that present tumor antigens. Although these cells can attack tumors, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0126] Multiple methods have been developed to generate and obtain tumor-targeted T cells. T cells specific for tumor antigens can be removed from tumor samples (TILs) or filtered from the blood. Subsequent activation and culturing are performed ex vivo and the results are re-infused. Activation can occur through gene therapy or by exposing the T cells to tumor antigens.

[0127] It is contemplated that cancer treatment can exclude any cancer treatment described herein. In addition, embodiments of the present disclosure include patients who have previously been treated with the therapies described herein, patients who are currently being treated with the therapies described herein, or patients who have not yet been treated with the therapies described herein. In some embodiments, the patient is a patient who has been determined to be resistant to the therapies described herein. In some embodiments, the patient is a patient who has been determined to be sensitive to the therapies described herein.

[0128] IV. Administration of Therapeutic Compositions

[0129] The therapies provided herein may include administering a combination of therapeutic agents, such as a first cancer therapy and a second cancer therapy. The therapies can be administered in any suitable manner known in the art. For example, the first and second cancer treatments can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second cancer treatments are administered in separate compositions. In some embodiments, the first and second cancer treatments are administered in the same composition.

[0130] Embodiments of the present disclosure relate to compositions and methods comprising therapeutic compositions. Different therapies can be administered in one composition or more than one composition, such as 2 compositions, 3 compositions, or 4 compositions. Various combinations of agents can be used.

[0131] The therapeutic agents of the present disclosure can be administered by the same administration route or different administration routes. In some embodiments, the cancer therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly / intracerebrally, or intranasally. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly / intracerebrally, or intranasally. Appropriate dosages can be determined according to the type of disease to be treated, the severity and course of the disease, the clinical condition of the individual, the clinical history of the individual and the response to the treatment, and the judgment of the attending physician.

[0132] Treatment can include various "unit doses". A unit dose is defined as containing a predetermined amount of the therapeutic composition. The amount to be administered, the particular route, and the formulation are within the capabilities of a person skilled in the clinical art. A unit dose does not need to be administered as a single injection, but can include a continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0133] Depending on the number of treatments and the unit dose, the amount to be administered depends on the desired therapeutic effect. The effective dose should be understood to mean the amount required to achieve a particular effect. In the practice of certain embodiments, doses in the range of 10 mg / kg to 200 mg / kg are contemplated to affect the protective ability of these agents. Thus, contemplated doses include doses such as approximately 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day or mg / day or any range derivable therefrom. In addition, such doses may be administered multiple times within a day and / or over multiple days, weeks or months.

[0134] In certain embodiments, an effective dose of the pharmaceutical composition is a dose that provides a blood level of from about 1 μM to 150 μM. In another embodiment, the effective dose provides a blood level of: from about 4 μM to 100 μM or from about 1 μM to 100 μM; or from about 1 μM to 50 μM; or from about 1 μM to 40 μM; or from about 1 μM to 30 μM; or from about 1 μM to 20 μM; or from about 1 μM to 10 μM; or from about 10 μM to 150 μM; or from about 10 μM to 100 μM; or from about 10 μM to 50 μM; or from about 25 μM to 150 μM; or from about 25 μM to 100 μM; or from about 25 μM to 50 μM; or from about 50 μM to 150 μM; or from about 50 μM to 100 μM (or any range derivable therefrom). In other embodiments, the dose provides an agent that produces the following blood levels upon administration of the therapeutic agent to a subject: about, at least about, or at most about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, 60 μM, 61 μM, 62 μM, 63 μM, 64 μM, 65 μM, 66 μM, 67 μM, 68 μM, 69 μM, 70 μM, 71 μM, 72 μM, 73 μM, 74 μM, 75 μM, 76 μM, 77 μM, 78 μM, 79 μM, 80 μM, 81 μM, 82 μM, 83 μM, 84 μM, 85 μM, 86 μM, 87 μM, 88 μM, 89 μM, 90 μM, 91 μM, 92 μM, 93 μM, 94 μM, 95 μM, 96 μM, 97 μM, 98 μM, 99 μM or 100 μM or any range derivable therefrom. In certain embodiments, the therapeutic agent to be administered to the subject is metabolized in vivo to a metabolized therapeutic agent, in which case the blood level may refer to the amount of this agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0135] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and varies from individual to individual. Factors influencing the dosage include the physical and clinical state of the patient, the route of administration, the intended goal of the treatment (symptom relief versus cure), and the potency, stability, and toxicity of the particular therapeutic substance or other treatments the subject may be receiving.

[0136] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg body weight can be converted and expressed as comparable concentration units of μg / ml or mM (blood levels), e.g., 4 μM to 100 μM. It should also be understood that absorption depends on the species and the organ / tissue. Applicable conversion factors and physiological assumptions for uptake and concentration measurements are well known and will allow those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the dosages, efficacy, and outcomes described herein.

[0137] A. Kits

[0138] Certain aspects of the invention also relate to kits comprising the compositions of the invention or compositions for practicing the methods of the invention. In some embodiments, the kits can be used to evaluate one or more biomarkers. In certain embodiments, the kit comprises, consists of, or consists essentially of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000 or more than 1000 probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therefrom. In some embodiments, there are kits for evaluating biomarker activity in cells.

[0139] The kit can include components that can be individually packaged or placed in a container, such as tubes, bottles, vials, syringes, or other suitable container devices.

[0140] Individual components can also be provided in the kit in concentrated amounts; in some embodiments, the components are provided individually at the same concentration as in a solution with the other components. The concentration of the components can be provided as 1x, 2x, 5x, 10x, or 20x or more.

[0141] Kits for using the probes, synthetic nucleic acids, non-synthetic nucleic acids, and / or inhibitors of the present disclosure for prognostic or diagnostic applications are included as part of the present disclosure. Any such molecule corresponding to any biomarker identified herein is specifically contemplated, which includes nucleic acid primers / primer sets and probes that are identical or complementary to all or part of the biomarker, which may include non-coding sequences of the biomarker as well as coding sequences of the biomarker.

[0142] In some aspects, some kit embodiments include negative and / or positive control nucleic acids, probes, and inhibitors. Additionally, the kit may include samples that serve as negative or positive controls for methylation of one or more biomarkers. In some embodiments, the control includes a nucleic acid containing at least one CpG or capable of identifying CpG methylation sites.

[0143] Any method or composition described herein is contemplated to be implemented in relation to any other method or composition described herein, and different embodiments may be combined. The originally filed claims are intended to cover claims that are multiply dependent on any filed claim or combination of filed claims.

[0144] Any embodiment of the present disclosure that refers by name to a specific biomarker also encompasses embodiments of biomarkers whose sequences are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the mature sequence of the specific nucleic acid.

[0145] Embodiments of the present disclosure include kits for analyzing a pathological sample by assessing the biomarker profile of a sample, which contain two or more biomarker probes in a suitable container device, wherein the biomarker probes detect one or more biomarkers identified herein. The kit may also include reagents for labeling nucleic acids in the sample. The kit may also include labeling reagents, including at least one of amine-modified nucleotides, poly(A) polymerase, and poly(A) polymerase buffer. The labeling reagent may include an amine-reactive dye.

[0146] V. Cancer Treatment

[0147] In some embodiments, the method includes administering a cancer therapy to a patient. The cancer therapy can be selected based on expression levels measured alone or in combination with a clinical risk score calculated for the patient. In some embodiments, the cancer therapy includes a local cancer therapy. In some embodiments, the cancer therapy does not include a systemic cancer therapy. In some embodiments, the cancer therapy does not include a local therapy. In some embodiments, the cancer therapy includes a local cancer therapy without administering a systemic cancer therapy. In some embodiments, the cancer therapy includes an immunotherapy, which can be an immune checkpoint therapy. Any of these cancer therapies can also be excluded. Combinations of these therapies can also be administered. In some embodiments, gene or miRNA expression measurements and analysis can indicate that one or more cancer therapies may be effective or ineffective. A particular advantage of the methods disclosed herein is that they allow a physician to make a treatment decision based on the molecular subtype of the metastasis for the first time.

[0148] VI. Diagnosis and Treatment of Cancer

[0149] Also disclosed are methods for diagnosing and treating a patient having a cancerous tumor, the method comprising: (a) obtaining a tissue sample from a metastasis; (b) measuring the expression of one or more genes and / or miRNAs in the sample; (c) comparing the measured expression level of each gene or miRNA with a reference expression level of that gene or miRNA; (d) identifying the metastasis as an SNF1, SNF2, or SNF3 type metastasis based on the measured expression levels; (e) administering an appropriate therapy to the patient based on the type of metastasis determined in step (d).

[0150] VII. Examples

[0151] The following examples are included to demonstrate preferred embodiments of the disclosure. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those skilled in the art will, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the disclosure.

[0152] Example 1

[0153] Lactate-responsive drug delivery systems represent a novel approach to tumor therapy. In a first example, the inventors developed and tested a hydrogel for the controlled lactate-responsive release of an anti-tumor drug. In this specific example, the matrix consisted of a pH-responsive chitosan matrix that encapsulated lactate oxidase and the drug internally. The chitosan hydrogel swelled with increasing environmental lactate. The increased lactate concentration led to additional enzymatic conversion of lactate to pyruvate and hydrogen peroxide ( Figure 3 ). The increased peroxide level led to free protons (H+ ) The availability of which increases, which in turn increases the protonation of the chitosan network. As a self-regulating valve system, this hydrogel matrix is capable of releasing drugs in response to a high lactic acid concentration.

[0154] The hydrogel was prepared by dissolving chitosan together with BSA (bovine serum albumin), BSA-alex488, and an enzyme in an acetic acid solution. The homogeneous mixture was transferred to a syringe and sprayed into a sodium tripolyphosphate solution. The collected hydrogel particles were washed and evenly divided into two groups (Figure 4). The prepared materials were exposed to water or a 35 mM sodium lactate solution at 37 °C. The supernatant was collected and the released BSA-alex488 was quantified by a fluorescence reader. Control experiments were performed using the same hydrogel in the absence of the enzyme. The present inventors successfully detected a significant level of BSA-alex488 released from the hydrogel particles in the lactate-containing solution ( Figure 5 ).

[0155] Example 2

[0156] Similar to Example 1, the present inventors can employ other pH-responsive polymer matrices, such as poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine), and poly(2-vinylpyridine). A large number of pH-responsive polymers can be designed using various electrolyte groups, and the pH-responsive polymers are divided into two categories. One group includes polymers having acidic groups, and the other group includes polymers having basic groups. 7 When used in combination with lactic acid-related enzymes such as lactate oxidase or lactate dehydrogenase, the polymer matrix has the ability to indirectly respond to lactic acid, resulting in physicochemical changes in the polymer and achieving drug release.

[0157] Example 3

[0158] Lactate 2-monooxygenase is used in a pH-responsive matrix, and different structures are used to prepare a lactate-responsive system. Lactate 2-monooxygenase produces CO2, which can be used as a signaling molecule for the stimulus-responsive polymer. These CO2-responsive polymers can be constructed from different types of functional groups, including amidine, amine, or carboxyl. 8,9 When lactate 2-monooxygenase produces CO2, the polymer matrix will respond to lactic acid, resulting in physicochemical changes in the polymer and subsequent drug release.

[0159] Example 4

[0160] The product of lactate enzymatic conversion can also be H2O2, which can be the target of a stimulus-responsive matrix. The H2O2-responsive platform can be constructed with multiple functions, including drug release, targeted imaging, diagnosis, and treatment. 10,11When combined with lactate-related enzymes such as lactate oxidase, the matrix has the ability to respond to lactate. Based on the high reactivity of MnO2 with H2O2, hybrid nanoparticles that generate O2 can be used to enhance drug delivery.

[0161] Example 5

[0162] In the experiment shown in Figure 9, the A component of the toxin (such as PA of anthrax toxin) will be directly injected into the blood. Free PA in the blood will undergo rapid degradation and excretion in the body (Figure 9A, left panel). The B component of the toxin (such as LF, or modified LF, such as LFN-DTA) will be packaged in tumor-responsive delivery particles, such as lactate-responsive particles. These particles will rapidly release their contents when exposed to high concentrations of lactate in the tumor (Figure 9A, upper right panel), but will keep the toxin inside the particles for a longer time in normal tissues and organs (Figure 9A, lower right panel). Thus, with this design, toxin components A and B can only bind in the tumor and induce cytotoxicity in tumor cells (Figure 9B, lower panel). When component B is released in normal tissues, component A has degraded and been excreted from the blood, and the separate B component will not cause significant side effects in normal tissues (Figure 9B, upper panel). LFN = N-terminal region of lethal factor toxin. DTA = diphtheria toxin A.

[0163] Example 6

[0164] Figure 10 The graphs depicted represent the experimental results of the cytotoxicity induced by AB toxin in vitro. MTT cell viability assays were performed under different conditions: (1) control; (2) protective antigen (PA) alone; (3) N-terminal region of lethal factor toxin combined with diphtheria toxin A alone (LFN-DTA); (4) protective antigen (PA) + LFN-DTA; (5) free PA + LFN-DTA released from lactate-responsive particles in the control solution; (6) free PA + LFN-DTA released from lactate-responsive particles in lactate solution. Cell viability was normalized to control cells, condition (1). Low cell viability was observed in the PA + LFN-DTA experiment (condition 4), which was caused by the co-administration of A and B toxin components. Low cell viability was also observed in response to free PA and LFN-DTA released from lactate-responsive particles in lactate solution (condition 6). The similar results observed in condition 6 and condition 4 indicate that lactate-responsive particles release their LFN-DTA payload in a high-lactate environment (condition 6). Similar results are expected to be observed in the tumor microenvironment.

[0165] Example 7

[0166] The outer surface of the nanoparticles was functionalized with H2O2-sensitive self-immolative arylboronate derivatives. The MS magazine was first loaded with selected "bullets" (i.e., payload drugs) and then capped by host-guest complexation with α-cyclodextrin (α-CD) and poly-β-CD. Poly-β-CD also served as a linker between the MS magazine and the MS trigger. The MS trigger was constructed by anchoring the enzyme lactate oxidase (Lox) in the MS. Lox served as: (i) a recognition agent for detecting the presence of lactate; (ii) a mediator that generates hydrogen peroxide (H2O2) molecules and induces the self-immolation reaction of the arylboronate derivative, resulting in the opening of the MS pores and triggering the release of the bullet from the MS magazine. After the MS trigger detected lactate and sent the chemical messenger (H2O2), the MS magazine received the messenger and then released the payload drug as a bullet.

[0167] Example 8

[0168] To test the ability of the nanodevice to recognize lactate, a release experiment was conducted in which the concentration of the nanodevice DMS in physiological buffer (PBS, pH 7.5) was set to 2.0 mg / m in the absence and presence of low and high concentrations of lactate (1 mM and 10 mM). The samples were rotated at room temperature, and aliquots were taken at predetermined times and centrifuged to remove the nanoparticles. The release of the cargo was evaluated by measuring the fluorescence of the cargo. As shown in Figure 13A, in the absence of lactate and in the presence of low concentrations of lactate, the DMS was capped and the cargo release was negligible. In contrast, in the presence of high lactate concentrations, significant payload release was observed in less than one hour. The observed cargo delivery was attributed to the recognition of lactate and its conversion to H2O2 by the Lox enzyme. Subsequently, H2O2 served as a chemical messenger sent from the enzyme control unit (MS trigger) to the MS magazine. H2O2 induced the cleavage of the self-immolative arylboronate derivative, resulting in the delivery of the payload from the MS magazine. By using DMS with a blank MS magazine, in the absence of lactate and in the presence of low concentrations of lactate, the DMS suspension appeared as a stable white colloid within 1 hour. In contrast, in the presence of high concentrations of lactate, the DMS precipitated to the bottom (Figure 13B). This result could be attributed to the uncapping of DMS and the aggregation of the uncapped silica nanoparticles.

[0169] Example 9

[0170] Mice with approximately 1 cm mammary tumors were injected with DMS and monitored for three days. Two doses of 100 μl were injected into the tail vein within 24 hours. The concentration of DMS in PBS was 40 mg / ml (approximately 70 μg Dox was loaded into 40 mg DMS). The free Dox control sample included a Dox concentration of 70 μg / ml in PBS. The results showed that within 3 days after DMS treatment, the size and weight of the mammary tumors were significantly reduced ( Figure 14 ).Figure 15 Representative pictures at day 3 after treatment are shown. Tumor weight confirmed that DMS treatment led to a significant reduction in tumor size and weight 3 days after treatment.

[0171] ***

[0172] According to the present disclosure, all methods disclosed and claimed herein can be made and executed without undue experimentation. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or the order of the steps of such methods without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents can be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications which are apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0173] References

[0174] The following references and publications mentioned throughout the specification are specifically incorporated herein by reference to the extent that they provide exemplary procedures or other details to supplement those set forth herein.

[0175] 1. San-Millán, I. & Brooks, G. A. Reexamining cancer metabolism: lactate production for carcinogenesis could be the purpose and explanation of the Warburg Effect. Carcinogenesis 38, 119 - 133 (2017).

[0176] 2. Hirschhaeuser, F., Sattler, U. G. & Mueller-Klieser, W. Lactate: a metabolic key player in cancer. Cancer research 71, 6921 - 6925 (2011).

[0177] 3. Mura, S., Nicolas, J. & Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nature materials 12, 991 (2013).

[0178] 4. Lu, Y., Sun, W. & Gu, Z. Stimuli-responsive nanomaterials for therapeutic protein delivery. Journal of controlled release 194, 1-19 (2014).

[0179] 5. Kanamala, M., Wilson, W. R., Yang, M., Palmer, B. D. & Wu, Z. M. Mechanisms and biomaterials in pH-responsive tumour targeted drug delivery: a review. Biomaterials 85, 152-167 (2016).

[0180] 6. Marchiq, I. & Pouysségur, J. Hypoxia, cancer metabolism and the therapeutic benefit of targeting lactate / H+ symporters. Journal of Molecular Medicine 94, 155-171, doi:10.1007 / s00109-015-1307-x (2016).

[0181] 7. Kocak, G., Tuncer, C. & Bütün, V. pH-Responsive polymers. Polymer Chemistry 8, 144-176 (2017).

[0182] 8. Lin, S. & Theato, P. CO2-Responsive Polymers. Macromolecular rapid communications 34, 1118-1133 (2013).

[0183] 9. Yan, B., Han, D., Boissière, O., Ayotte, P. & Zhao, Y. Manipulation of block copolymer vesicles using CO 2: dissociation or “breathing”. Soft Matter 9, 2011-2016 (2013).

[0184] 10. Yu, L., Chen, Y. & Chen, H. H2O2-responsive theranostic nanomedicine. Chinese Chemical Letters 28, 1841-1850 (2017).

[0185] 11. Cui, Y., Zhang, M., Du, F.-S. & Li, Z.-C. Facile Synthesis of H2O2-Cleavable Poly(ester-amide)s by Passerini Multicomponent Polymerization. ACS Macro Letters 6, 11-15 (2016).

[0186] 12. De Jong, W. H. et al. Particle size-dependent organ distribution of gold nanoparticles after intravenous administration. Biomaterials 29, 1912-1919, doi:10.1016 / j.biomaterials.2007.12.037 (2008).

[0187] 13. Huang, X. et al. The shape effect of mesoporous silica nanoparticles on biodistribution, clearance, and biocompatibility in vivo. ACS nano 5, 5390-5399, doi:10.1021 / nn200365a (2011).

[0188] 14. Minchin, R. Nanomedicine: sizing up targets with nanoparticles. Nature nanotechnology 3, 12-13, doi:10.1038 / nnano.2007.433 (2008).

[0189] 15. Semete, B. et al. In vivo evaluation of the biodistribution and safety of PLGA nanoparticles as drug delivery systems. Nanomedicine: nanotechnology, biology, and medicine 6, 662 - 671, doi:10.1016 / j.nano.2010.02.002 (2010).

[0190] 16. van der Zande, M. et al. Distribution, elimination, and toxicity of silver nanoparticles and silver ions in rats after 28 - day oral exposure. ACS nano 6, 7427 - 7442, doi:10.1021 / nn302649p (2012).

[0191] 17. Zhang, X. D. et al. Size - dependent in vivo toxicity of PEG - coated gold nanoparticles. International journal of nanomedicine 6, 2071 - 2081, doi:10.2147 / IJN.S21657 (2011).

[0192] 18. Ffrench - Constant, R. & Waterfield, N. An ABC guide to the bacterial toxin complexes. Advances in applied microbiology 58, 169 - 183 (2006).

[0193] 19. Schiavo, G. & van der Goot, F. G. The bacterial toxin toolkit. Nat Rev Mol Cell Biol 2, 530 - 537, doi:10.1038 / 35080089 (2001).

Claims

1. A lactate-triggered beneficial agent release composition, comprising: A chemical-responsive matrix comprising mesoporous silica nanoparticles, wherein the mesoporous silica nanoparticles are functionalized with an aryl borate ester derivative; At least one lactate-reactive enzyme provided in the chemical-responsive matrix; and At least one beneficial agent provided within the chemical-responsive matrix; Wherein the at least one lactate-reactive enzyme comprises lactate oxidase, which is capable of converting a lactate substrate into at least one signaling molecule, Wherein the chemical-responsive matrix comprises at least one functional group capable of reacting with the at least one signaling molecule; Wherein the at least one signaling molecule comprises H2O2, which is capable of inducing the cleavage of the aryl borate ester derivative, thereby releasing the at least one beneficial agent.

2. The lactate-triggered beneficial agent release composition according to claim 1, wherein the at least one signaling molecule further comprises O2, CO2 or H + .

3. The lactate-triggered beneficial agent release composition according to any one of claims 1-2, wherein the composition further comprises a structure selected from the group consisting of microparticles, hydrogels, micelles, nanogels, microgels and branched polymers.

4. The lactate-triggered beneficial agent release composition according to claim 1, wherein the chemical-responsive matrix comprises a material selected from the group consisting of chitosan, cyclodextrin, polycyclodextrin, poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine) and poly(2-vinylpyridine).

5. The lactate-triggered beneficial agent release composition according to claim 1, wherein the at least one lactate-reactive enzyme further comprises lactate dehydrogenase, lactate racemase or lactate 2-monooxygenase.

6. The lactate-triggered beneficial agent release composition according to claim 1, wherein the composition comprises at least a portion of the lactate-reactive enzyme population on the surface of the composition.

7. The lactate-triggered beneficial agent release composition according to claim 1, wherein the composition comprises at least a portion of the lactate-reactive enzyme population embedded in the chemical-responsive matrix.

8. The lactate-triggered beneficial agent release composition according to claim 1, wherein the composition comprises at least a portion of the lactate-reactive enzyme population covalently linked to the chemical-responsive matrix.

9. The lactate-triggered beneficial agent release composition according to claim 1, wherein the beneficial agent is a therapeutic agent or a diagnostic agent.

10. The lactic acid-triggered beneficial agent release composition according to claim 9, wherein the beneficial agent is a therapeutic agent.

11. The lactic acid-triggered beneficial agent release composition according to claim 10, wherein the therapeutic agent is a small molecule, a peptide or polypeptide, a nucleic acid, a nanoparticle, a microparticle, an ion, a salt, a bacterium, a virus, a live cell, or a radiopharmaceutical.

12. The lactic acid-triggered beneficial agent release composition according to claim 10, wherein the therapeutic agent is a chemotherapeutic agent, an immunotherapeutic agent, a gene therapeutic agent, a toxin, or a radiotherapeutic agent.

13. The lactic acid-triggered beneficial agent release composition according to claim 12, wherein the immunotherapeutic agent is an antibody-based agent or an immune checkpoint inhibitor.

14. The lactic acid-triggered beneficial agent release composition according to claim 1, further comprising the B component or a modified B component from an AB toxin complex, wherein the modified B component is at least 50%, 60%, 70%, 80%, 90%, or more than 90% homologous to the B component and comprises a region that interacts with the A component from the AB toxin complex.

15. The lactic acid-triggered beneficial agent release composition according to claim 14, wherein the B component is from cholera toxin, diphtheria toxin, pertussis toxin, Escherichia coli heat-labile toxin LT, Shiga toxin, Pseudomonas aeruginosa exotoxin A, botulinum toxin, tetanus toxin, anthrax toxin LF, Bordetella pertussis AC, Bacillus anthracis EF, or Staphylococcus aureus exfoliatin B.

16. The lactic acid-triggered beneficial agent release composition according to claim 14, wherein the B component is PA from anthrax toxin.

17. The lactic acid-triggered beneficial agent release composition according to claim 14, wherein the B component or the modified B component is LF from anthrax toxin or a modified LF.

18. The lactic acid-triggered beneficial agent release composition according to claim 14, wherein the amount of the B component in the composition is from 0.001 μg / mg to 500 μg / mg of the chemical response matrix.

19. The lactic acid-triggered beneficial agent release composition according to claim 1, wherein the chemical response matrix and the at least one lactic acid-reactive enzyme are provided in a ratio of 1:1 to 100,000:

1.

20. The lactic acid-triggered beneficial agent release composition according to claim 1, wherein the chemical response matrix and the at least one beneficial agent are provided in a ratio of 1:1 to 100,000:

1.

21. The lactic acid-triggered beneficial agent release composition according to claim 1, wherein the composition comprises 0.001 μmol to 10,000 μmol of lactic acid-responsive enzyme per g of the chemical-responsive matrix.

22. The lactic acid-triggered beneficial agent release composition according to claim 1, wherein the composition comprises 0.001 μg to 500 μg of beneficial agent per mg of the chemical-responsive matrix.

23. Use of a beneficial agent release composition in the preparation of a medicament for delivering a beneficial agent to a lactic acid target region of a subject to be treated, wherein the beneficial agent release composition comprises a chemical-responsive matrix, at least one lactic acid-responsive enzyme provided in the chemical-responsive matrix, and at least one beneficial agent provided in the chemical-responsive matrix; wherein the chemical-responsive matrix comprises mesoporous silica nanoparticles functionalized with an aryl borate derivative; wherein the at least one lactic acid-responsive enzyme comprises lactate oxidase, and when the beneficial agent release composition is exposed to a lactic acid concentration higher than the ambient physiological lactic acid concentration, the at least one lactic acid-responsive enzyme converts a lactic acid substrate into at least one signaling molecule; wherein the at least one signaling molecule comprises H2O2, which induces the cleavage of the aryl borate derivative, thereby releasing the at least one beneficial agent, and wherein the lactic acid target region has a lactic acid concentration higher than the ambient physiological lactic acid concentration.

24. The use according to claim 23, wherein the release of the at least one beneficial agent is targeted to release the beneficial agent to a region comprising a lactic acid concentration higher than the ambient physiological lactic acid concentration.

25. The use according to claim 24, wherein the region comprising a lactic acid concentration higher than the ambient physiological lactic acid concentration is a tumor microenvironment.

26. The use according to claim 23, wherein the signaling molecule further comprises O2, CO2 or H + .

27. The use according to claim 23, wherein the beneficial agent release composition further comprises a structure selected from the following: microparticles, hydrogels, micelles, nanogels, microgels, and branched polymers or copolymers.

28. The use according to claim 23, wherein the chemical-responsive matrix comprises a material selected from the following: chitosan, cyclodextrin, polycyclodextrin, poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine), and poly(2-vinylpyridine).

29. The use according to claim 23, wherein the at least one lactic acid-responsive enzyme further comprises lactate dehydrogenase, lactate racemase, or lactate 2-monooxygenase.

30. Use according to claim 23, wherein the beneficial agent release composition comprises at least a portion of the lactate-responsive enzyme population on the surface of the composition.

31. Use according to claim 23, wherein the beneficial agent release composition comprises at least a portion of the lactate-responsive enzyme population embedded in the chemically responsive matrix.

32. Use according to claim 23, wherein the beneficial agent release composition comprises at least a portion of the lactate-responsive enzyme population covalently linked to the chemically responsive matrix.

33. Use according to claim 23, wherein the beneficial agent is a therapeutic agent or a diagnostic agent.

34. Use according to claim 23, wherein the beneficial agent release composition releases the at least one beneficial agent in a lactate dose-dependent manner.

35. Use according to claim 23, wherein the beneficial agent release composition releases at least 0.1×10 -10 g of beneficial agent / g of chemically responsive matrix.

36. Use according to claim 23, wherein the beneficial agent release composition releases at least 30% more of the beneficial agent in the presence of lactate than in the absence of lactate.

37. Use according to claim 23, which is for targeted release of a beneficial agent for cancer treatment, cancer diagnosis, cancer tracking, or for a biosensor.

38. Use according to claim 23, wherein the subject has cancer or is at risk of cancer or cancer recurrence.

39. Use according to claim 23, wherein the chemically responsive matrix and the at least one lactate-responsive enzyme are provided in a ratio of 1:1 to 100,000:

1.

40. Use according to claim 23, wherein the chemically responsive matrix and the at least one beneficial agent are provided in a ratio of 1:1 to 100,000:

1.

41. Use according to claim 23, wherein the composition comprises 0.001 μmol to 10,000 μmol of lactate-responsive enzyme / g of chemically responsive matrix.

42. Use according to claim 23, wherein the composition comprises 0.001 μg to 500 μg of beneficial agent / mg of chemically responsive matrix.

43. Use of a chemotherapeutic agent release composition in the preparation of a medicament for treating cancer with a chemotherapeutic agent, wherein the chemotherapeutic agent release composition comprises a chemically responsive matrix, at least one lactate-responsive enzyme provided in the chemically responsive matrix, and at least one chemotherapeutic agent provided in the chemically responsive matrix; wherein the chemical response matrix comprises mesoporous silica nanoparticles functionalized with aryl borate derivatives; wherein the at least one lactate-responsive enzyme comprises lactate oxidase, which converts a lactate substrate into at least one signaling molecule when the chemotherapeutic agent release composition is exposed to a lactate concentration higher than the ambient physiological lactate concentration; and wherein the at least one signaling molecule comprises H2O2, which induces cleavage of the aryl borate derivative, thereby releasing at least one chemotherapeutic agent, wherein the lactate target region has a lactate concentration higher than the ambient physiological lactate concentration.

44. The use according to claim 43, wherein release of the at least one chemotherapeutic agent is targeted such that the chemotherapeutic agent is released into a region comprising a lactate concentration higher than the ambient physiological lactate concentration.

45. The use according to claim 44, wherein the region comprising a lactate concentration higher than the ambient physiological lactate concentration is the tumor microenvironment.

46. The use according to claim 43, wherein the at least one signaling molecule further comprises O2, CO2, or H + 。 47. The use according to claim 43, wherein the chemotherapeutic agent release composition further comprises a structure selected from the group consisting of microparticles, hydrogels, micelles, nanogels, microgels, and branched polymers or copolymers.

48. The use according to claim 43, wherein the chemical response matrix comprises a material selected from the group consisting of chitosan, cyclodextrin, polycyclodextrin, poly[(2-dimethylamino)ethyl methacrylate], poly[(2-diethylamino)ethyl methacrylate], poly[(2-diisopropylamino)ethyl methacrylate], poly(4-vinylpyridine), and poly(2-vinylpyridine).

49. The use according to claim 43, wherein the at least one lactate-responsive enzyme further comprises lactate dehydrogenase, lactate racemase, or lactate 2-monooxygenase.

50. The use according to claim 43, wherein the chemotherapeutic agent release composition comprises at least a portion of the lactate-responsive enzyme population on the surface of the composition.

51. The use according to claim 43, wherein the chemotherapeutic agent release composition comprises at least a portion of the lactate-responsive enzyme population embedded in the chemical response matrix.

52. The use according to claim 43, wherein the chemotherapeutic agent release composition comprises at least a portion of the lactate-responsive enzyme population covalently linked to the chemical response matrix.

53. The use according to claim 43, wherein the chemotherapeutic agent release composition releases the at least one beneficial agent in a lactate dose-dependent manner.

54. The use according to claim 43, wherein the chemotherapeutic agent releasing composition releases at least 0.1×10 -4 g of chemotherapeutic agent / g of chemically responsive matrix.

55. The use according to claim 43, wherein the chemotherapeutic agent releasing composition releases at least 30% more chemotherapeutic agent in the presence of lactic acid than in the absence of lactic acid.

56. The use according to claim 43, wherein the chemically responsive matrix and the at least one lactic acid-reacting enzyme are provided in a ratio of 1:1 to 100,000:

1.

57. The use according to claim 43, wherein the chemically responsive matrix and the at least one chemotherapeutic agent are provided in a ratio of 1:1 to 100,000:

1.

58. The use according to claim 43, wherein the composition comprises 0.001 μmol to 10,000 μmol of lactic acid-reacting enzyme / g of chemically responsive matrix.

59. The use according to claim 43, wherein the composition comprises 0.001 μg to 500 μg of chemotherapeutic agent / mg of chemically responsive matrix.

60. The use according to claim 43, which comprises administering the toxin component A of an AB toxin complex, and wherein the at least one chemotherapeutic agent comprises the B component or a modified B component of the AB toxin complex, wherein the modified B component is at least 50%, 60%, 70%, 80%, 90% or more than 90% homologous to the B component and comprises a region that interacts with the A component from the AB toxin complex.

61. The use according to claim 60, wherein the toxin component A is injected into the blood.

62. The use according to claim 60, wherein the toxin component A is administered at least or at most 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 148 hours, 160 hours or more than 160 hours after administering the composition.

63. The use according to claim 60, wherein the toxin component A and the toxin component B bind in or around the tumor.

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