Compounds, compositions and methods for treating inflammatory bowel disease

Abstract

A protein-drug conjugate and its variants for treating inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, is disclosed. The protein-drug conjugate (PDC) comprises a fusion protein having a thioredoxin protein and an IL22 protein or a variant / mutant thereof, wherein the drug laquinimod (or a derivative thereof) is conjugated to the thioredoxin, and laquinimod is cleavable from the fusion protein upon deposition in intestinal epithelial cells or intestinal epithelial stem cells.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. A copy of said XML, created on October 4, 2023, is named 009583_00004_WO.xml and is 22,411 bytes in size.

[0002] Field The present disclosure relates to compounds for treating inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. The present disclosure also relates to compositions containing the compounds, methods of using the compounds or the compositions for the treatment of IBD, and methods for synthesizing the compounds, including intermediates used therein. [Background technology]

[0003] Inflammatory bowel disease (IBD) is a general term used to describe diseases that cause a chronic inflammatory state of the gastrointestinal tract. The two most common forms of the IBD disease group are ulcerative colitis and Crohn's disease. Ulcerative colitis is primarily confined to the colon, whereas Crohn's disease can affect any part of the gastrointestinal (GI) tract.

[0004] Chronic intestinal inflammation resulting from IBD causes a range of symptoms, including abdominal pain, diarrhea, anemia, and weight loss. The clinical course of IBD is highly variable both between individuals and over the course of a given individual's lifetime, with periods of inactivity interspersed with exacerbations, the severity of which may necessitate medication, hospitalization, and even intestinal surgery. The pathogenesis of IBD is driven by an abnormal and prolonged T cell- and NK (natural killer) cell-mediated immune response against the commensal gut microbiota that occurs in genetically susceptible individuals. Most documented IBD risk genes are associated with diverse immune functions, including physical barrier integrity, control of microbiota diversity, and innate immune functions such as autophagy.

[0005] Loss of physical barrier integrity is believed to result from the loss of epithelial cells in the intestinal wall, leading to inflammation / infection, which then triggers the immune response that characterizes IBD. Currently, the mainstay of drug therapy for IBD relies on suppressing the immune system. Four types of medications are used for this purpose: aminosalicylates, corticosteroids, immunomodulators, and biologics. The rationale for applying these medications is that once inflammation is controlled, homeostatic mechanisms for tissue healing and repair are activated, and the system returns to normal. However, this theory does not consider the underlying cause of the disease, and therefore, discontinuing treatment may result in a recurrence of the underlying pathology, potentially requiring lifelong therapy.

[0006] Because most established genetic associations with IBD involve genes responsible for interactions between the intestinal epithelium, immune system, microbiota, and dietary factors, it is not surprising that chronic inflammation develops following molecular changes related to intestinal microbiota dysbiosis, infection, an unbalanced diet, and host metabolism. These molecular changes are primarily caused by disruption of the intestinal epithelial barrier function, which then leads to inflammation. To date, the use of immunosuppressants to treat IBD has not provided satisfactory long-term treatment options for patients with IBD and does not address the underlying cause of IBD: barrier disruption.

[0007] Thus, there continues to be an unmet need to treat IBD by addressing the loss of barrier function in patients that precedes inflammation and immune system involvement. Summary of the Invention

[0008] The present disclosure provides, in part, compounds (drug conjugates or simply conjugates) of IL22-thioredoxin fusion proteins having one or more laquinimod groups attached thereto. The fusion protein comprises an IL22 moiety and a thioredoxin moiety linked by a covalent bond or a first linker. The thioredoxin moiety can comprise two or more thioredoxin proteins, linked with or without a linker, or conjugated to either end of the IL22 moiety. Full sequences of exemplary fusion proteins are provided herein (e.g., SEQ ID NOS: 2, 3, 7, 9, 11, 15, and 17). The IL22 moiety of the protein-drug conjugate provides targeted intracellular delivery to epithelial cells of the gastrointestinal tract, including intestinal epithelial stem cells.

[0009] One or more laquinimod groups or derivatives thereof are then conjugated to the thioredoxin portion of the fusion protein via an enzymatically cleavable linker. Conjugation occurs at distinct amino acid sites available for linker attachment. In one embodiment, free thiol functional groups found only on the side chains of free cysteine ​​residues (to cysteine ​​residues that form disulfide bonds) are used for conjugation via a second linker. Specifically, the thioredoxin portion of the fusion protein contains three unpaired cysteines (free thiols) in the fusion protein available for conjugation using thiol-reactive chemistry, while the IL22 portion does not contain any unpaired cysteine ​​residues.

[0010] Given that only the thioredoxin portion of the fusion protein has a free cysteine ​​residue, selective attachment of the cleavable linker containing laquinimod or its derivative to the thioredoxin portion of the fusion protein can be achieved, allowing the IL22 portion of the fusion protein to remain intact.

[0011] Thus, in one embodiment, a) a fusion protein comprising an IL22 moiety and a thioredoxin moiety linked by a covalent bond or a first linker; b) comprising one or more laquinimod groups each attached via a separate second linker to separate sulfur atoms of a free cysteine ​​side chain of the thioredoxin portion of the fusion protein; A drug conjugate is provided wherein the first and second linkers contain from 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that the remaining valences of the atoms are satisfied with hydrogen or deuterium atoms.

[0012] In one embodiment, the drug conjugate is represented by Formula A, as well as pharmaceutically acceptable salts thereof: [(LAQ) n -L 2 -S-] b -FP-Y A (wherein n is 1 to 4, Y is hydrogen or L 1 -TR, b is 1 to 3 when Y is hydrogen, and 1 -TR is 1 to 6, L 2 is an enzymatically cleavable linker, L 1 is a linker or covalent bond, FP is the formula TR-L 1 -IL22, wherein TR is thioredoxin or a biologically active fragment thereof, and IL22 is a fusion protein of L 1 or L 1 Interleukin-22 or a biologically active fragment thereof bound to a TR when either or both of the moieties are covalently bound; LAQ is laquinimod or its derivatives, S is the sulfur atom of the thiol group of a free cysteine ​​in the thioredoxin portion of the fusion protein).

[0013] In one embodiment, the second linker L 2comprises a cleavable covalent functional group or group that, when degraded in the presence of intracellular enzymes, releases laquinimod or a derivative thereof in the intracellular environment of intestinal epithelial cells. In some embodiments, the second linker comprises a cleavable covalent functional group or group comprising an amino acid or polypeptide of 2 to about 10 amino acids. In one embodiment, the cleavable linker is a two- or three-amino acid polypeptide that is cleaved in the presence of intracellular endosomal cathepsins or endosomal glycosidases. In another embodiment, the second linker comprises glucuronic acid, which stabilizes the second linker and, when removed by intracellular enzymes, allows for degradation of the cleavable covalent functional group or group and release of free laquinimod or a derivative thereof.

[0014] In one embodiment, each of the second linkers is attached to the sulfur atom of a free cysteine ​​residue found in the thioredoxin portion of the fusion protein to form a sulfide bond. The second linkers include a cleavable covalent functional group or groups that allow cleavage of laquinimod or a derivative thereof from the linker in the presence of an intracellular enzyme, thereby providing intracellular concentrations of both the free laquinimod or a derivative thereof and the fusion protein.

[0015] In another embodiment, when Y is hydrogen, about 1 to about 3 second linkers each having about 1 to about 4 attached laquinimod groups or derivatives thereof are cleavably covalently linked to a free cysteine ​​residue of the thioredoxin portion of the fusion protein, on average, thereby providing up to 12 laquinimod compounds or derivatives thereof that are released by intracellular enzymes in intestinal epithelial cells. 1 In the case of -TR, the number of laquinimod compounds then released by intracellular enzymes in the intestinal epithelial cells can be up to 24.

[0016] In one embodiment, the conjugates described herein can be used in a method for inhibiting de-epithelialization of the intestinal barrier. When used in this manner, the protein-drug conjugate inhibits the onset or further progression of an IBD episode in a patient.

[0017] Thus, in one embodiment, there is provided a method of inhibiting de-epithelialization of the intestinal barrier in a patient suffering from an ongoing episode of inflammatory bowel disease, the method comprising administering to the patient an effective amount of a conjugate (as described herein) or a pharmaceutical composition comprising said conjugate to inhibit de-epithelialization or further de-epithelialization of the intestinal barrier.

[0018] In one embodiment, the conjugates described herein can be used in a method for initiating re-epithelialization of the intestinal barrier. When used in this manner, the conjugates therapeutically treat damage to the epithelium of the intestinal barrier caused by an IBD episode in a patient.

[0019] Thus, in one embodiment, there is provided a method for initiating re-epithelialization of the intestinal barrier in a patient suffering from an inflammatory bowel disease episode, the method comprising administering to the patient an effective amount of a conjugate (as described herein) or a pharmaceutical composition comprising said conjugate to initiate re-epithelialization of the intestinal barrier.

[0020] Without being limited by any theory, the IL22 portion of the fusion protein forming part of the conjugates described herein provides directional guidance and intracellular delivery to intestinal epithelial cells, including epithelial stem cells, resulting in targeting specificity for the conjugates. This is because intracellular delivery of the conjugates is restricted to cells expressing the heterodimeric receptor for IL22, composed of the IL22R1 and IL10R2 pair. These receptor pairs are continuously expressed in intestinal epithelial cells, such as intestinal epithelial stem cells, but are not expressed in, for example, uninjured lung or liver tissue. Thus, the conjugates described herein target intestinal epithelial cells even in the absence of lung or liver tissue injury. In one embodiment, patients suffering from a disease or condition that causes damage to lung or liver tissue are preferably evaluated for use with the conjugates and methods described herein. Such lung diseases include, by way of example only, chronic obstructive pulmonary disease (COPD), lung cancer, pulmonary hypertension, pneumonia, smoking-related diseases, and the like. Such liver diseases include, by way of example only, hepatitis (e.g., hepatitis A, B, C), cirrhosis, and non-alcoholic steatohepatitis (NASH). At the discretion of the clinician, these patients may also be treated for IBD as described herein or excluded from such treatment.

[0021] In one embodiment, the second linker connecting laquinimod or a derivative thereof to the fusion protein contains a cleavable covalent functional group or group that is selectively cleaved upon intracellular absorption of the conjugate into intestinal epithelial cells, including intestinal epithelial stem cells. When cleaved in this manner, the degraded conjugate provides free laquinimod or a derivative thereof and free fusion protein. In this embodiment, the IL22 portion of the free fusion protein stimulates the proliferation of intestinal epithelial stem cells. Similarly, the free laquinimod or a derivative thereof initiates the differentiation and maturation of stem cells into epithelial cells. Thus, the IL22 portion of the fusion protein and laquinimod or a derivative thereof act synergistically and cooperatively to induce stem cell proliferation coupled with epithelial cell differentiation and maturation, leading to intestinal barrier repair.

[0022] Thus, in one embodiment, laquinimod or a derivative thereof is represented by formula I:

[0023] [ka] (wherein R is hydrogen or L 2 -X, R 1 and R 2 is hydrogen, chloro, bromo, iodo, hydroxyl, C1-C4 alkyl and L 2 - is selected independently from X, L 2 is a monovalent or polyvalent linker, X is a reactive functional group capable of forming a covalent sulfide bond with a thiol (SH) group).

[0024] In one preferred embodiment, the conjugates described herein are represented by the following formula IA:

[0025] [ka] where b is 1 to 3, FP is a fusion protein (e.g., as defined herein), and S and L 2 is as defined above). In the above formula, if only a single laquinimod compound or derivative thereof is attached to the linker, then the linker is monovalent. In such a case, only one laquinimod compound or derivative thereof is provided for each free cysteine ​​residue on the protein-drug conjugate.

[0026] In one embodiment, a laquinimod monovalent linker compound is provided for use in covalently attaching to a fusion protein, the compound having the following formula IB:

[0027] [ka] wherein q is from 1 to about 10. In this embodiment, the linker has the formula:

[0028] [ka] where q is defined above and the wavy line represents the point where the linker is attached to laquinimod.

[0029] In one embodiment, a drug conjugate is provided, wherein the conjugate comprises a monovalent linker represented by the following formula IC:

[0030] [ka] (where b, q, and FP are as defined herein). In this case, b is 1, so only a single laquinimod compound or derivative thereof is attached to the fusion protein. However, as previously described, free cysteine ​​residues in the thioredoxin portion of the fusion protein can be used to attach up to three linkers (b is 1-3) to the fusion protein without disrupting any disulfide bridges in the fusion protein. In this case, reaction of the linker with the cysteine ​​thiol group converts the maleimide to a succinimide, and the linker is defined as follows:

[0031] [ka] (where q is defined above and the wavy line on the bond projecting from the succinimide group represents the site of attachment to the sulfide formed by reaction of the maleimide group with the SH group of the cysteine ​​amino acid found in the thioredoxin portion of the fusion protein.) Note that in this case, the two methylene groups on the succinimide are equivalent, so the point of attachment can be either methylene group.

[0032] In one embodiment, the conjugate is represented by formula ID:

[0033] [ka] (wherein b and FP are as defined herein), where b is 1, 2, or 3, and when b is 2 or 3, multiple linkers, each comprising a single laquinimod compound or a derivative thereof, are attached to the fusion protein via sulfide bonds using free cysteine ​​residues in the thioredoxin portion of the fusion protein without disrupting disulfide bridges on the fusion protein.

[0034] In one embodiment, a laquinimod (or laquinimod derivative) bivalent linker compound is provided for use in covalently linking a fusion protein, the compound having the following formula IE:

[0035] [ka] Here, the maleimide linker group attached to the fusion protein FP is represented as follows:

[0036] [ka] wherein q is 1 to about 10. In this embodiment, the plurality of laquinimod compounds (and / or derivatives thereof) are linked by a L-type linker group containing a maleimide linker group. 2 Attached to the linker, L 2 The linker is referred to as multivalent (in this example, bivalent), in which case multiple laquinimod compounds or derivatives thereof are provided for each free cysteine ​​residue on the protein-drug conjugate.

[0037] In one embodiment, the protein-drug conjugate described herein comprises a bivalent linker, and the conjugate is represented by the following formula IF:

[0038] [ka] where n and FP are as defined above, and the succinimide linker attached to FP is represented as follows:

[0039] [ka] (where q is 1 to about 10). Note that in Formula IF and Formula IE above, only one conjugate is attached to the thioredoxin portion of the fusion protein. This is shown for illustrative purposes only, given that up to three such bivalent linkers can be attached. If all three linkers are used, the total number of laquinimod molecules that can be released into the intracellular environment is six.

[0040] In one embodiment, there is provided a method for re-epithelialization of the intestinal wall of a mammal, comprising administering to said mammal an effective amount of a conjugate of any one of formulas A, I, IA, IC, ID, or IF above, or a mixture thereof.

[0041] In one embodiment, there is provided a method for treating inflammatory bowel disease in a patient, comprising administering to said patient an effective amount of a conjugate of any one of formulas A, I, IA, IC, ID, or IF above, or a mixture thereof.

[0042] In one embodiment, L 2 is connected to one end by L 2 and a single laquinimod or derivative thereof attached at its other end to a sulfur atom of the thioredoxin portion of the fusion protein. 2 is connected to one end by L 2 and a polyvalent linker capable of linking 2 to about 10 units of laquinimod or a derivative thereof, which are bound at the other end to a sulfur atom of the thioredoxin portion of the fusion protein.

[0043] Suitable polyvalent linkers are described herein and are well known in the art, and include dendrimers, polyols, polycarboxylates, and the like.

[0044] In one embodiment, the linker comprises a cleavable element that is enzymatically degraded in the presence of glucuronidase.

[0045] In one embodiment, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and an effective amount of any one of the conjugates of formula A, I, IA, IC, ID, or IF above, or a mixture thereof.

[0046] In one embodiment, an intermediate of formula II is provided:

[0047] [ka] (In the formula, R 3 is hydrogen or L 3 -maleimide, L 3 -maleimide is L together with -NHC(O)CH2CH2NH- 2 It is understood that the maleimide group, when conjugated with a thiol group, is converted to a succinimide group, which then becomes part of the linker.

[0048] Another embodiment describes a protein-drug conjugate comprising a thioredoxin polypeptide linked to an interleukin-22 (IL22) polypeptide via a peptide linker, wherein one or more laquinimod molecules or derivatives are conjugated to one or more cysteine ​​residues present in the thioredoxin polypeptide, and the thioredoxin polypeptide is attached to the amino terminus of the IL22 polypeptide. The protein-drug conjugate may be glycosylated or non-glycosylated.

[0049] The protein-drug conjugate comprises one or more laquinimod molecules joined by linkers, each of which comprises one or more laquinimod molecules, and the linkers comprise from 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that the valences are satisfied with hydrogen or deuterium atoms.

[0050] The contemplated protein-drug conjugates described above and herein include a cleavable linker that cleaves in the presence of intracellular enzymes located in intestinal epithelial cells and intestinal epithelial stem cells, cleaving laquinimod or a derivative thereof from the fusion protein, thereby releasing laquinimod into the intracellular environment.

[0051] The protein-drug conjugates described herein comprise a thioredoxin polypeptide linked to an IL22 polypeptide. The linkage between the two polypeptides can be via a peptide linker, chemical conjugation, or no linker. The peptide linker in the protein-drug conjugate can contain 1 to 100 amino acids and any integer number therebetween. An exemplary peptide linker is Gly-Ser-Ala-Met (SEQ ID NO: 4). For example, in some embodiments, the peptide linker can contain 2 to 4 amino acids, 4 to 8, 8 to 16, 8 to 15, 3 to 8, and 10 amino acids.

[0052] The protein-drug conjugate can comprise a mammalian thioredoxin, or a primate thioredoxin (e.g., monkey, chimpanzee, or other primate thioredoxin), or a human thioredoxin. The protein-drug conjugate can comprise a mammalian IL22, a primate IL22, or a human IL22. The IL22 can be a mature protein lacking or including a signal peptide.

[0053] In another embodiment, the IL22 portion of the protein-drug conjugate comprises cysteine ​​to serine mutations at positions 32 and 35 of the mature IL22 sequence.

[0054] Another embodiment describes a protein-drug conjugate in which 0-2 free cysteine ​​groups in the thioredoxin portion of the fusion protein lack a laquinimod molecule (or a derivative thereof) and are not conjugated to cysteines in the IL22 polypeptide.

[0055] In another embodiment, a pharmaceutical composition is contemplated comprising any of the above protein-drug conjugates and a pharmaceutically acceptable carrier or excipient.

[0056] Also described is a method of treating a subject with inflammatory bowel disease (IBD), comprising administering to the subject a protein-drug conjugate described herein, the method comprising administering an amount of the drug sufficient to clinically improve one or more of the following conditions: i) inhibiting de-epithelialization of the intestinal epithelial barrier; ii) inhibiting intestinal microbial infections; iii) protecting intestinal goblet cells during infection; iv) enhancing epithelial cell integrity; v) increasing epithelial cell proliferation; vi) enhancing epithelial cell differentiation; and vii) Initiating re-epithelialization of compromised areas of the intestinal epithelial barrier.

[0057] This protein-drug conjugate and its method of use are for treating inflammatory bowel diseases, including ulcerative colitis and Crohn's disease.The protein-drug conjugate is manufactured for use in the treatment of inflammatory bowel diseases, for example, as a drug.Treatment can be performed for moderate and severe inflammatory bowel diseases. [Brief explanation of the drawings]

[0058] [Figure 1] FIG. 1 shows that both thioredoxin-IL22 fusion proteins analyzed (i.e., Thio-IL22 and Thio-C3235S-IL22) were expressed at high levels in the E. coli BL21(DE3) host. [Figure 2] Figure 1 shows the results of a reporter bioassay of samples analyzed using human Thio-IL22 and human Thio-C3235S-IL22 constructs, where Thio is an abbreviation for thioredoxin. [Figure 3] FIG. 1 shows the mechanism underlying the design of an IL22 receptor cell line that allows for independent measurement of the IL22 bioactivity of protein-drug conjugates (PDCs) and the activity of the drug payload released intracellularly after IL22 reporter-mediated endocytosis. [Figure 4] Figures 4A and 4B show the ability of IL22-fused PDC to activate both pathways as expected when IL22 is internalized and releases laquinimod intracellularly. DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed Description The present disclosure provides compounds / conjugates for treating inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis. However, before discussing this disclosure in detail, the following terms will first be defined.

[0060] definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting or restrictive of the disclosure. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0061] Wavy or dashed lines drawn through the structures indicate specific points of attachment of groups. No directionality or stereoselectivity is indicated or implied by the order of depiction or naming of chemical groups unless chemically or structurally required.

[0062] Prefix “C” u~v " indicates that the following group has u to v carbon atoms. For example, "C 1~6 "Alkyl" indicates that the alkyl group has from 1 to 6 carbon atoms.

[0063] The term "about," when used before other numerical designations, including, for example, temperature, time, amount, concentration, and ranges, indicates an approximation that can vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. In one embodiment, when used in reference to a dose, the term "about" means that the dose can vary by + / - 10%.

[0064] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements.

[0065] "Consisting essentially of," when used to define compositions and methods, means excluding other elements that are of any essential importance to the combination for the stated purpose, thereby materially altering the stated purpose of the composition or method. A composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure.

[0066] "Consisting of" shall mean excluding other components and substantial method steps beyond trace elements. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0067] "Alkyl" refers to an unbranched or branched saturated hydrocarbon chain. As used herein, alkyl refers to an alkyl group having 1 to 20 carbon atoms (i.e., C 1~20 alkyl), 1 to 12 carbon atoms (i.e., C 1~12 alkyl), 1 to 8 carbon atoms (i.e., C 1~8 alkyl), 1 to 6 carbon atoms (i.e., C 1~6 alkyl), or 1 to 4 carbon atoms (i.e., C 1~4 alkyl). Examples of alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. When an alkyl residue having a specific number of carbon atoms is named by a chemical name or specified by a molecular formula, all positional isomers having that number of carbon atoms may be encompassed. Thus, for example, "butyl" includes n-butyl (i.e., -(CH2)3CH3), sec-butyl (i.e., -CH(CH3)CH2CH3), isobutyl (i.e., -CH2CH(CH3)2), and tert-butyl (i.e., -C(CH3)3), and "propyl" includes n-propyl (i.e., -(CH2)2CH3) and isopropyl (i.e., -CH(CH3)2). Certain commonly used alternative chemical names may be used. For example, a divalent group such as a divalent "alkyl" group may also be referred to as "alkylene." Additionally, for purposes of this specification, replacement of an atom with a corresponding isotope of that atom is intended to be within the scope of the present invention. For example, whenever hydrogen (H) is listed, the hydrogen can be replaced with deuterium (D), unless specifically stated otherwise.

[0068] Pharmaceutically acceptable salts, isotopically enriched analogs, deuterated analogs, stereoisomers, mixtures of stereoisomers, and prodrugs of the compounds described herein are also provided. "Pharmaceutically acceptable" or "physiologically acceptable" refers to compounds, salts, compositions, dosage forms, and other materials useful in preparing pharmaceutical compositions suitable for veterinary or human medical use.

[0069] The term "pharmaceutically acceptable salt" of a given compound refers to a salt that retains the biological effectiveness and properties of the given compound and is not biologically or otherwise undesirable. "Pharmaceutically acceptable salts" or "physiologically acceptable salts" include, for example, salts with inorganic acids and salts with organic acids. Furthermore, if a compound described herein is obtained as an acid addition salt, the free base can be obtained by basifying a solution of the acid salt. Conversely, if the product is a free base, an addition salt, particularly a pharmaceutically acceptable addition salt, can be produced by dissolving the free base in a suitable organic solvent and treating the solution with an acid, according to conventional procedures for preparing acid addition salts from base compounds. Those skilled in the art will recognize various synthetic methods that can be used to prepare non-toxic pharmaceutically acceptable addition salts. Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids. Salts derived from inorganic acids include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include, for example, acetic acid, propionic acid, glucuronic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Similarly, pharmaceutically acceptable base addition salts can be prepared from inorganic or organic bases. Salts derived from inorganic bases include, by way of example only, sodium, potassium, lithium, aluminum, ammonium, calcium, and magnesium salts.Salts derived from organic bases include alkylamines (i.e., NH2(alkyl)), dialkylamines (i.e., HN(alkyl)2), trialkylamines (i.e., N(alkyl)3), substituted alkylamines (i.e., NH2(substituted alkyl)), di(substituted alkyl)amines (i.e., HN(substituted alkyl)2), tri(substituted alkyl)amines (i.e., N(substituted alkyl)3), alkenylamines (i.e., NH2(alkenyl)), dialkenylamines (i.e., HN(alkenyl)2), trialkenylamines (i.e., N(alkenyl)3), substituted alkenylamines (i.e., NH2(substituted alkenyl)), di(substituted alkenyl)amines (i.e., HN(substituted alkenyl)2), tri(substituted alkenyl)amines The salts of suitable amines include, but are not limited to, salts of primary, secondary, and tertiary amines such as (substituted alkenyl)amines (i.e., N(substituted alkenyl)3, mono-, di-, or tricycloalkylamines (i.e., NH2(cycloalkyl), HN(cycloalkyl)2, N(cycloalkyl)3), mono-, di-, or triarylamines (i.e., NH2(aryl), HN(aryl)2, N(aryl)3), or mixed amines. Specific examples of suitable amines include, by way of example only, isopropylamine, trimethylamine, diethylamine, tri(isopropyl)amine, tri(n-propyl)amine, ethanolamine, 2-dimethylaminoethanol, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0070] Some compounds exist as tautomers. Tautomers are in equilibrium with each other. For example, an amide-containing compound may be in equilibrium with an imidic acid tautomer. Regardless of which tautomer is shown and the nature of the equilibrium between the tautomers, one skilled in the art will understand that the compound includes both the amide and imidic acid tautomers. Thus, amide-containing compounds are understood to include their imidic acid tautomers. Similarly, imidic acid-containing compounds are understood to encompass their amide tautomers. Other examples of tautomeric structures are well known in the art.

[0071] The compounds, or pharmaceutically acceptable salts thereof, may contain asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or (D)- or (L)- for amino acids. The present disclosure is intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) and (S), or (D) and (L) isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques, such as chromatography and / or fractional crystallization. Conventional techniques for preparing / isolating individual enantiomers include chiral synthesis from suitable optically pure precursors or separation of the racemate (or racemate of a salt or derivative) using, for example, chiral high-pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers.

[0072] "Stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds but with different, incompatible three-dimensional structures. The present disclosure contemplates various stereoisomers, or mixtures thereof, and includes "enantiomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another.

[0073] "Diastereomers" are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other.

[0074] "Subject" refers to a mammal. A mammal can be a human or non-human mammalian organism. "Patient" refers to a human subject.

[0075] "Treating" a disease or disorder in a subject or "treating" a disease or disorder refers to 1) preventing the onset of the disease or disorder in a subject prone to or not yet showing symptoms of the disease or disorder, 2) inhibiting or halting the progression of the disease or disorder, or 3) ameliorating or regressing the disease or disorder. The disease or disorder being treated is "inflammatory bowel disease." "Inflammatory bowel disease," "inflammatory bowel disorder," and "IBD," which are used interchangeably herein, are used in the broadest sense to encompass all diseases and pathological conditions whose etiology involves recurrent inflammation of the intestine, including the small intestine and colon. Inflammatory bowel disease includes ulcerative colitis and Crohn's disease, as well as animal models used to mimic ulcerative colitis and Crohn's disease. Ulcerative colitis may include moderate or severe ulcerative colitis, as well as moderate and severe forms of Crohn's disease.

[0076] An "effective amount" refers to an amount of a protein-drug conjugate described herein sufficient to treat an IBD afflicting a subject, or to prevent recurrence of an IBD that has developed in said subject or patient, reduce the incidence of IBD, or reduce the severity of IBD. An effective amount of a compound (protein-drug conjugate) can improve one or more of the following conditions in a subject administered the protein-drug conjugate: i) inhibiting de-epithelialization of the intestinal epithelial barrier; ii) inhibiting intestinal microbial infections; iii) protecting intestinal goblet cells during infection; iv) enhancing epithelial cell integrity; v) increasing epithelial cell proliferation; vi) enhancing epithelial cell differentiation; and vii) Initiating re-epithelialization in damaged areas of the intestinal epithelial barrier.

[0077] The term "improvement" means a clinically significant change in one or more characteristics compared to the patient's pre-treatment state.

[0078] "Administration" refers to any art-recognized form of administration to a subject, including oral (including oral gavage), pulmonary, transdermal, sublingual, parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, oral, colonic, topical), transmucosal (e.g., colonic, nasal, etc.), etc. Oral administration contemplates that the conjugate is enteric coated to allow release in the intestine and not in other parts of the gastrointestinal tract. The route of administration is selected by the attending clinician and is based on factors such as the age, weight, and general health of the patient, as well as the severity of the condition. In one embodiment, the compounds and pharmaceutical compositions described herein are administered orally. Here, the compound for administration is a protein-drug conjugate described herein, comprising a thioredoxin molecule (or a thioredoxin variant), IL22 (or a functional IL22 variant), and laquinimod or a laquinimod derivative conjugated to a fusion protein. The protein-drug conjugate (PDC) can be glycosylated, non-glycosylated (aglycosylated), or has reduced glycosylation compared to that normally expressed in the human thioredoxin protein or human IL22 protein. When synthesized in bacteria such as E. coli, the fusion protein is not glycosylated.

[0079] The term "linker" refers to a group that connects a first group to a second group. In the case of Formula I above, the linker is used to connect one or more laquinimod molecules (or derivatives thereof) to the thioredoxin portion of the fusion protein to form a protein-drug conjugate. In one embodiment, the linker contains at least one and up to about 40 non-hydrogen atoms, including carbon, nitrogen, oxygen, sulfur, and phosphorus. Where appropriate, these atoms include hydrogen (including all isotopes) or halo to satisfy valency. The linker is a cleavable linker and can be either monovalent or polyvalent.

[0080] The term "cleavable linker" means that the linker comprises a cleavable functional group, i.e., the functional group or covalent bond is readily cleaved, for example by an intracellular enzyme, into the first and second components.

[0081] The term "free cysteine" refers to a cysteine ​​amino acid in a polypeptide in which the thiol group (-SH) is retained and which is not part of a disulfide group (-SS-).

[0082] The term "laquinimod derivative" refers to a laquinimod compound of the formula

[0083] [ka] (In the formula, R, R 1 , and R 2 are defined as above, except that R, R 1 , and R 2 cannot all be hydrogen).

[0084] The term "intestinal epithelial barrier" or "IEB" is well known as one of the largest interfaces in the body's environment and the internal environment.

[0085] The term "thioredoxin polypeptide" refers to a thioredoxin protein or functional portion thereof having redox activity. Exemplary thioredoxin polypeptides include mammalian and primate thioredoxin polypeptides. Human thioredoxin is another exemplary polypeptide. Thioredoxin may be glycosylated or non-glycosylated. The C-terminus of thioredoxin may be attached to the N-terminus of IL22. Less preferably, the N-terminus of thioredoxin may be attached to the C-terminus of IL22. In some embodiments, one or more thioredoxins may be linked, such as Trx-Trx-IL22 or Trx-Trx-Trx-IL22. Another variant includes placing IL22 in the middle of the thioredoxin sequence, as exemplified by the APT sequence discussed in the Examples.

[0086] The terms "interleukin-22 polypeptide," "IL22 polypeptide," and "IL-22 polypeptide" each refer to an IL22 protein or functional portion thereof that can bind to the IL22 receptor (i.e., a heterodimer consisting of IL10R2 and IL22R1 subunits) and induce the IL22 receptor signaling pathway. IL22 polypeptides may be full-length polypeptides or mature proteins lacking amino-terminal signaling proteins. Exemplary IL22 polypeptides include mammalian and primate IL22 polypeptides, such as monkey IL22 (XP_001117159), as discussed in Neto et al., "Interleukin-22 Forms Dimers that are Recognized by Two Interleukin-22R1 Receptor Chains," Biophysical J. 94:1754-1765, 2008. Human IL22 polypeptides are another exemplary polypeptide. IL22 polypeptides may be glycosylated or non-glycosylated. The term "IL22 receptor" or "IL22R" refers to a heterodimer consisting of IL22R1 and IL10R2 or naturally occurring variants thereof. See, e.g., Ouyang et al., 2011 Ann. Rev. Immunol. 29:159-63. Naturally occurring variants of IL22R and IL22 can include alternatively spliced ​​forms and allelic variants of the polypeptides. Mutants of IL22 are also being considered for use in protein-drug conjugates, including high-affinity IL22 mutants with improved binding to their receptors. Exemplary IL22 mutants of human and mouse sequences are described, for example, in Saxton et al., "The tissue protective functions of interleukin-22 can be decoupled from pro-inflammatory actions through structure-based design," Immunity 54:660-672, 2021.

[0087] The term "pharmaceutically acceptable carrier" refers to a compound that serves to aid in drug delivery. Drug carriers can improve the selectivity, efficacy, and / or safety of administering drugs, such as drugs conjugated with fusion proteins. As used herein, the term "pharmaceutically acceptable" generally means that it is useful for preparing safe and non-toxic pharmaceutical compositions.

[0088] A "pharmaceutically acceptable excipient" is a pharmacologically inactive compound, such as a diluent, disintegrant, lubricant, glidant, or binder, found in an active pharmaceutical agent. A pharmaceutically acceptable excipient is an excipient useful in preparing a pharmaceutical composition containing an active pharmaceutical ingredient, e.g., a drug conjugated with a thioredoxin-IL22 fusion protein. Pharmaceutically acceptable excipients are generally safe, non-toxic, and acceptable for animal use, such as veterinary or human use. Reference to an "excipient" includes both one and more than one such excipient.

[0089] The term "X is a reactive functional group capable of forming a covalent sulfide bond with a thiol (SH) group" refers to those functional groups that react with the thiol (SH) group of cysteine ​​to form a covalent sulfide bond. The following table shows some of the reactive functional groups known in the art:

[0090] [Table 1]

[0091] General synthesis method The protein-drug conjugates described herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, it will be understood that other process conditions can also be used unless otherwise specified. Optimal reaction conditions may vary with the particular reagents or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0092] The protein-drug conjugates described herein can be lyophilized after synthesis for stable storage. After lyophilization, the conjugates can be placed in glass vials, preferably restricting light from entering the vial. The lyophilized form can later be solubilized using, for example, DMSO (dimethyl sulfoxide) and then placed in saline for administration. Lyophilization involves freezing, followed by sublimation, and optionally desorption under a sterile lyophilization environment (ISO5 environment). The conjugate salt form can include glycine.

[0093] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting specific functional groups, are well known in the art. For example, many protecting groups are described in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and the references cited therein.

[0094] The starting materials for the following reactions are known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Sigma-Aldrich (St. Louis, Missouri, USA), Bachem (Torrance, California, USA), and Emka-Chemce (St. Louis, Missouri, USA). Others can be prepared by procedures, or obvious modifications thereof, described in standard references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 2016), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 2001), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 2019), March's Advanced Organic Chemistry, (John Wiley and Sons, 8th edition, 2019), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0095] A. Laquinimod-Linker (L 2 ) synthesis In the illustrated synthetic schemes below, the specific linkers used are for illustrative purposes only, and other linkers known in the art can be used instead.

[0096] [ka]

[0097] In one embodiment illustrated in Scheme 1, laquinimod is converted to its corresponding oxide salt (Compound 1) as the first step in the Williamson reaction. The reaction proceeds by adding a stoichiometric amount of a base, such as sodium carbonate, potassium carbonate, sodium methoxide, sodium hydroxide, potassium hydroxide, or lithium diisopropylamide, to laquinimod (Compound 1) in a suitable polar aprotic solvent, such as acetonitrile, methyl t-butyl ether, methyl ethyl ketone (MEK), or N,N-dimethylformamide (DMF). The salt used is preferably, but not necessarily, a pharmaceutically acceptable salt. The reaction is maintained under conventional conditions defined by the known Williamson reaction to form the corresponding 4-oxide (not shown). Alternatively, if the linker contains a hydroxyl group, the group can be converted to the corresponding mesylate salt by a conventional mesylation reaction and then conjugated to the hydroxyl group of laquinimod.

[0098] The laquinimod oxide (compound 1) can be recovered and purified, or used as is, by combining it with at least a stoichiometric equivalent of the dipeptide (compound 2) and maintaining the reaction at a suitable temperature, typically between 0°C and 100°C. The reaction is maintained until the reaction is substantially complete, typically occurring within approximately 30 minutes to 48 hours. The resulting product (compound 3) can be recovered by conventional procedures, including concentration, precipitation, crystallization, chromatography, etc., or used directly in the next step of the overall reaction. Alternatively, if the benzyl chloride group of the linker is replaced with benzyl alcohol, the hydroxyl group can be converted to the corresponding benzyl mesylate by a conventional mesylation reaction. The mesylate can be conjugated with the laquinimod oxide (compound 1) to form an ether bond.

[0099] Scheme 2 then illustrates the formation of N-maleimide (compound 5) from compound 3.

[0100] [ka]

[0101] Specifically, the t-Boc group of compound 3 is removed under conventional conditions known in the art to give compound 4. For example, the t-Boc group of compound 3 can be removed by contacting the compound with about 10% to about 30% trifluoroacetic acid in dichloromethane (v / v). The solution is maintained under gentle stirring for about 1 hour to about 4 hours. Compound 4 is then recovered by conventional procedures, including concentration, precipitation, crystallization, chromatography, etc., but is preferably used in the next step without isolation or purification.

[0102] In one embodiment, the free amine of compound 4 is then contacted with at least a stoichiometric amount of maleic anhydride under conditions well known in the art to form the maleimide (compound 5). Purification of compound 5 is accomplished by well-recognized procedures including chromatography, precipitation, crystallization, and the like.

[0103] Alternatively, maleimide synthesis utilizes at least a stoichiometric amount (e.g., a slight excess) of N-methoxycarbonylmaleimide. The reaction proceeds in a suitable solvent, such as tetrahydrofuran / saturated sodium bicarbonate, initially maintained at approximately 0°C and allowing the solution to slowly warm to room temperature over approximately 1-5 hours. The desired product (compound 5) contains laquinimod attached to a linker, which in turn contains a reactive maleimide for conjugation to free thiol groups on the fusion protein.

[0104] In another embodiment, an enzymatically cleavable linker, laquinimod, and a glucuronic acid-based linker containing a reactive group that can react with a thiol functional group to form a sulfide bond (compound 6) can be used, and a representative example of such a linker is shown below.

[0105] [ka]

[0106] A key feature of Compound 6 is its ability to enzymatically degrade in the intracellular environment, releasing free laquinimod from the remainder of the linker. Without being limited to any particular theory, degradation is initiated in the presence of glucuronidase during endocytosis of the conjugate in epithelial cells, including epithelial stem cells. Once the glucuronyl group is removed (Compound 6A), degradation proceeds via the resonance structure of the compound's phenolic group in the acidic medium of the endosome, resulting in the removal of the oxy group. Such removal is disclosed in Greenwald, et al., J. Med. Chem., 42:3657-3667 (1999), the entire contents of which are incorporated herein by reference. A schematic diagram of the degradation of this linker is shown in Scheme 3 below to provide Compound 6B (laquinimod) and Compound 6C.

[0107] [ka]

[0108] Specifically, enzymatic removal of the glucuronic acid group is achieved by glucuronidases found in the endosomes of epithelial cells. After removal, the resulting compound (compound 6A) contains a 4-hydroxybenzyloxy group. The acidic medium found in endosomes releases the oxy moiety of the benzyloxy group, resulting in the intracellular release of free laquinimod (compound 6B) and the remainder of the linker conjugated to the maleimide group, as seen in figure 6C. Note, however, that in practice, the maleimide group is converted to a succinimide group when conjugated to a fusion protein, as previously described.

[0109] Regarding the linker shown in Scheme 3 above, one synthetic route to compound 6 begins with the commercially available compound 11 (Boc Sciences, 45-16 Ramsey Road, Shirley, NY 11967, USA), which in turn begins with compound 10 as an intermediate reagent, as shown in Scheme 3A below.

[0110] [ka]

[0111] This reaction uses the commercially available reagent (Sigma-Aldrich, St. Louis, MO, USA) β-alanine N-Fmoc acid chloride under conventional amidation conditions to give compound 11. In one embodiment, compound 10 is obtained from the corresponding nitro derivative (not shown) by treatment with hydrogen / palladium.

[0112] The hydroxy group of compound 11 is then mesylated using mesyl anhydride (MsO) under conventional conditions. Preferably, mesylation of the benzyl alcohol group is accomplished using mesylic anhydride to give compound 12, as shown in Scheme 3B below.

[0113] [ka]

[0114] The resulting product, compound 12 (hereinafter referred to as compound 13), can be isolated and optionally purified by conventional methods such as isolation, precipitation, chromatography, crystallization, etc., or can be used in the next step of the reaction without isolation and / or purification.

[0115] The alkali or alkaline salt of compound 14 is then combined with compound 13 to attach laquinimod as shown in Scheme 3C below.

[0116] [ka]

[0117] Scheme 3C illustrates a method for attaching laquinimod oxide (compound 14) to compound 13, thereby obtaining compound 15. Compound 14 is formed using the Williamson reaction as in Scheme 1 above, with the oxide existing as a salt, such as the sodium salt. Alternatively, the sodium salt of compound 14 is available from Simson Pharma Ltd., India.

[0118] According to Scheme 3C, approximately equimolar amounts of compounds 13 and 14 are combined in a suitable solvent, such as acetone, methyl ethyl ketone (MEK), etc. The reaction is carried out at a temperature of about 15° C. to about 70° C. for approximately 12 to 72 hours. The product (compound 15) can be isolated and optionally purified by methods known in the art, such as chromatography, crystallization, precipitation, etc., or can be used directly in subsequent reactions.

[0119] Compound 15 was first deprotected by the addition of base, which deacetylated the protected hydroxy group, deesterified the methyl carboxyl ester, and removed the Fmoc group, as shown in Scheme 3D below.

[0120] [ka]

[0121] The reaction proceeds by adding an excess of a base, such as lithium hydroxide, cyclohexylamine, or ethanolamine, to compound 15 in a suitable solvent, such as an aqueous combination of C1-C3 alkanols (methanol, ethanol, isopropanol), or an aqueous combination of water and tetrahydrofuran (e.g., 10:2 MeOH / THF and LiOH in 2 volumes of water). The reaction is maintained at about 0°C to about 10°C for about 1-5 hours, and then allowed to return to room temperature.

[0122] The resulting free amine (compound 16) can be isolated and optionally purified by conventional methods such as isolation, precipitation, chromatography, crystallization, etc., or can be used in the next step of the reaction without isolation and / or purification.

[0123] In the final step of Scheme 3D, compound 6 is prepared by amidating the free amino group of compound 16 with at least a stoichiometric amount of commercially available pentafluorophenyl ester 3-[oxyethyloxyethyloxyethyl-(ethyl-2-maleimido)]propanoic acid (obtained from BroadPharm, 6625 Top Gun Dr., #103, San Diego, CA, USA 92121) under conventional conditions known in the art to yield compound 6. The reaction proceeds in a suitable solvent, such as N,N-dimethylformamide or DMSO. The reaction is maintained at about -20°C to about 10°C for about 0.3 to 3 hours. The product (compound 6) is preferably isolated and purified by direct injection onto preparative HPLC prior to use in conjugation to fusion proteins.

[0124] Additionally, cleavable ester-based peptide linkers containing laquinimod can be prepared as shown in Scheme 4 below.

[0125] [ka]

[0126] Specifically, the free amino group of a valine-citrulline dipeptide (compound 17) is contacted with maleic anhydride under conventional conditions known in the art to yield compound 18. Next, at least a stoichiometric equivalent of laquinimod (compound 19) is contacted with compound 18 under conventional esterification conditions to yield the desired product (compound 20). Each step is preferably carried out using an inert solvent, generally at a temperature of about 0°C to about 60°C, with gentle stirring. The reaction is maintained under these conditions until the reaction is substantially complete. The reaction mixture is then quenched, and the desired product is isolated and optionally purified, or can be used without isolation or purification.

[0127] In addition to the above, other esters can be formed using well-known aminocarboxylic acids such as the amino-protected di-β-alanine or its free carboxylmaleimide derivative shown below:

[0128] [ka] (In the formula, Pg 1 is a carboxyl-protecting group. Polyvalent linkers with three laquinimod units attached can be achieved using either aspartic acid-aspartic acid dimers or glutamic acid-glutamic acid dimers. Polyvalent linkers with four laquinimod units attached can be achieved using aspartic acid-aspartic acid-aspartic acid trimers or glutamic acid-glutamic acid-glutamic acid trimers. Mixtures of aspartic acid and glutamic acid dimers and trimers can also be used.

[0129] B. Inclusion of an Additional Releasable Laquinimod Compound in the Drug Conjugate In a further embodiment, the number of laquinimod compounds that can be releasably delivered via the drug conjugates described herein is determined by the linker (L 2 ) can be doubled, tripled, quadrupled, etc. by including additional carboxyl groups. An example is shown in Figure 5 below:

[0130] [ka]

[0131] Specifically, in Scheme 5, compound 30 can be prepared in a similar manner to the preceding reaction scheme. For example, as described above, the amino functionality of compound 11 can be obtained by reduction of the nitro group of the precursor compound. In such cases, the nitro group is retained during the conjugation of laquinimod, followed by reduction of the nitro group to an amino group using hydrogen / palladium to obtain compound 30. The introduction of the known N-Fmoc aspartic anhydride allows the free amino group of compound 30 to open the anhydride ring, which occurs under conventional conditions to yield an additional carboxyl group as well as an Fmoc-protected amino group, as exemplified in compound 31.

[0132] The additional carboxyl group can be reacted with compound 30 to give a second laquinimod compound (compound 32) attached to a cleavable linker. The Fmoc group is removed with LiOH as described above, as well as the acetyl group and methyl ester found in the glucuronic acid portion of the compound, which is then reacted with POOC(CH2CHO)3CH2CH2-N-maleimide (P = PFP or pentafluorophenyl) as described above to give compound 33, suitable for reaction with a free cysteine ​​residue of a fusion protein.

[0133] Using this approach, up to six laquinimod groups can be releasably attached to the fusion protein. Similarly, by using a tricarboxylic acid group instead of a tetracarboxylic acid group, up to nine or twelve laquinimod groups, respectively, can be releasably attached to the fusion protein.

[0134] In one embodiment, other multimeric, non-peptide linkers can be used to conjugate the drug (e.g., laquinimod) to the fusion protein. Examples of such linkers include, by way of example only:

[0135] [ka] Both of the above are commercially available.

[0136] C. IL22 thioredoxin fusion protein The preparation of fusion proteins containing IL22 has been disclosed in the art. See, for example, International Publication No. 2019 / 148,026, the entire contents of which are incorporated herein by reference. Furthermore, Example 7 below provides the synthesis of an IL22-thioredoxin fusion protein. Interestingly, the IL22 portion of the fusion protein does not contain a free cysteine ​​residue, while the thioredoxin portion of the fusion protein contains three free cysteine ​​residues. This means that the linkers described herein covalently bind only to the thioredoxin portion of the fusion protein, thereby retaining the IL22 functional group.

[0137] D. Conjugation of Laquinimod Linkers to Fusion Proteins Formation of the laquinimod-linker-fusion protein conjugate proceeds as described below in Scheme 6. In this scheme, the linker is any of the laquinimod-linkers described above, but may also be any suitable linker known in the art.

[0138] [ka]

[0139] In Scheme 6, a linker compound (compound 21) containing laquinimod is contacted with a fusion protein (compound 22). As described above, the fusion protein (compound 22) is a fusion protein having a linker compound (compound 21) containing laquinimod as defined above. 1The fusion protein comprises a thioredoxin moiety and an IL22 moiety connected by a -SH group. The thioredoxin portion of the fusion protein contains three free cysteine ​​(Cys) residues; for illustrative purposes, these residues are identified by their -SH groups. Each of these groups can react with the maleimide group of compound 21 in a classical Michael addition manner to form up to three sulfide bonds, as shown in compound 23, converting the maleimide group to a succinimide group. The extent of sulfide bond formation depends on the stoichiometry used, the reaction conditions (time and temperature), and the location of the thiol group in three-dimensional space.

[0140] The reaction is carried out under conventional conditions well known in the art, and Example 6 below further illustrates one known method for conjugation.

[0141] E. Biology Upon administration to a patient, the conjugate is targeted by the IL22 portion of the fusion protein to epithelial cells expressing the IL22 heterodimeric receptor, composed of the IL22R1 and IL10R2 pair. Because intestinal epithelial cells, including stem cells, constantly express these receptors, the IL2 portion of the fusion protein provides directional guidance for targeting the conjugate to these epithelial cells. The conjugate is then absorbed into these cells by endosomes (endocytosis). These endosomes contain one or more enzymes that initiate degradation of the conjugate, thereby providing free laquinimod and the fusion protein.

[0142] Without being limited to any theory, the following Scheme 7 illustrates this pathway. For illustrative purposes, Scheme 7 uses a single linker in the fusion protein. It is understood that if additional linkers are used, the degradation mechanism will occur in the same manner.

[0143] [ka]

[0144] In Scheme 7, the IL22 portion of the drug-fusion protein conjugate targets epithelial cells, including epithelial stem cells, that express the IL22 heterodimeric receptor, composed of the IL22R1 and IL10R2 pair. Ligand-receptor binding initiates intracellular absorption through endocytosis through endocytic sites. The endocytic site contains the enzyme glucuronidase, which cleaves the glucuronic acid moiety from the conjugate. The acidic environment of the endocytic site promotes degradation of the laquinimod molecule from the remainder of the conjugate, thereby enabling target-specific intracellular delivery of both laquinimod and IL22 to intestinal epithelial cells.

[0145] Upon degradation, the liberated IL22 portion of the fusion protein stimulates the proliferation of intestinal epithelial stem cells. Similarly, the liberated laquinimod or its derivative initiates the differentiation and maturation of stem cells into epithelial cells. Thus, the IL22 portion of the fusion protein and laquinimod or its derivative act synergistically and cooperatively to induce the proliferation of stem cells associated with the differentiation and maturation of epithelial cells, leading to the repair of the intestinal barrier.

[0146] F. Biological considerations Without being limited to any theory, AhR (aryl hydrocarbon receptor)-mediated responses have two important aspects in maintaining intestinal homeostasis. First, AhR-mediated responses are involved in intestinal epithelial stem cell homeostasis and barrier integrity. Second, AhR-mediated responses are primarily involved in anti-inflammatory and pro-regenerative immune responses in epithelial cells of the gastrointestinal tract. Under inflammatory conditions, epithelial cells in the liver, lung, and skin respond to IL-22 and, therefore, to its associated AhR agonists. LGR5 plays a key role in epithelial cell homeostasis / barrier integrity. + It has been demonstrated that the absence of AhR in the epithelial stem cell compartment impairs the differentiation of stem cells into functional intestinal epithelial cells (IECs). The intestinal epithelium is renewed throughout an individual's adult life, with active proliferation occurring in the crypt compartment. This process is facilitated by the stem cell (SC) compartment, which is known to reside near the bottom of the crypt compartment. LGR5 +has been found to be an epithelial stem cell marker.

[0147] The failure to produce functional intestinal epithelial cells (IECs) leads to the eventual loss of barrier integrity, resulting in increased inflammation and ultimately tumorigenesis. All of this demonstrates the importance of AhR-mediated pathways in intestinal biology. In addition to their effects on epithelial stem cells, dietary AhR ligands directly activate AhR function in IECs, controlling the expression of genes that enhance cell-cell adhesion and antimicrobial activity, thus strengthening barrier integrity.

[0148] Again, without being limited by theory, in addition to IL22's role in regulating various functional aspects underlying intestinal barrier integrity, IL22 aids in mucosal healing by promoting epithelial cell proliferation and regeneration after injury. Both epithelial barrier homeostasis and repair depend on the asymmetric division of intestinal stem cells, which produce intestinal stem cells (ISCs) and transit-amplifying cells (TAs). TA cells proliferate, migrate from the crypt compartment, and then differentiate into mature intestinal epithelial cells (IECs). IL22 functions by promoting TA cell proliferation and simultaneously inhibiting ISC proliferation. The presence of IL22 also accelerates apoptosis of older intestinal epithelial cells, thereby promoting intestinal epithelial cell renewal.

[0149] Binding of IL22 to its heterodimeric receptor IL22-IL22R also activates the pro-proliferative MAPK (mitogen-activated protein kinase) pathway and the pro-survival AKT pathway. IL22 also induces JAK-mediated phosphorylation of STAT1 under some inflammatory conditions, such as in combination with type I interferon (IFN) signaling.

[0150] As with any growth-promoting signaling pathway, a negative feedback loop exists to ensure that cells do not overproliferate, avoiding tissue hyperplasia and tumorigenesis. In the case of tyrosine kinase-related cytokine receptors, this negative feedback loop is anchored by members of the suppressor of cytokine signaling (SOCS) family. SOCS protein members are induced as immediate early genes following cytokine signaling. SOCS proteins contain a Scr homology 2 domain (SH2), which binds to tyrosine-phosphorylated residues of cytokine receptors and JAK kinases, inhibiting their kinase activity and inducing their proteasomal degradation as part of the Cullin-related ligase (CRL) E3 ubiquitin ligase complex. SOCS functions as the substrate-binding domain of the CRL / E3 ubiquitin ligase complex. Under conditions of high basal inflammation, pleiotropic pro-inflammatory cytokines, such as interleukin-6 (IL6), signal by binding to the IL6 receptor and increasing the expression of SOCS family proteins. Increased expression of the SOCS family of proteins cross-inhibits multiple unrelated cytokine receptors, leading to the development of a phenomenon known as cytokine resistance.

[0151] Cytokine resistance may explain the previously reported inconsistent effects of IL22 therapy in mediating experimental colitis. Therefore, to establish an effective treatment that can help promote healing, reestablish intestinal barrier function, and reduce inflammation, we must be able to provide all the specific elements necessary to reestablish homeostasis in a tissue-specific manner.

[0152] Although not limited to this mode of action, (i) AhR agonists that can induce IL22-independent activities such as maintaining the epithelial stem cell niche, promoting functional differentiation of Treg T cells, and inhibiting differentiation of TH17 cells (CD4 T cells capable of producing IL17), and (ii) their combination with IL22 (involved in epithelial cell proliferation, induction of antimicrobial proteins, tight junction proteins, and mucins) should be able to promote therapeutic reconstruction of the normal structure and function of the intestine.

[0153] G. Pharmaceutical Compositions and Dosages The protein-drug conjugates described herein (also referred to herein as conjugates) are best administered to patients as pharmaceutical compositions. Such compositions are not limited to a particular formulation or pharmaceutical carrier, which may vary. Generally, the conjugates described herein are administered as liquid pharmaceutical compositions by any of a number of known routes of administration, including topical administration, mucosal delivery, e.g., oral administration via enteric-coated capsules, or intracolonic administration, as well as intravenous, intramuscular, subcutaneous, and intraperitoneal administration. However, compositions suitable for intravenous delivery are preferred.

[0154] The liquid pharmaceutical compositions described herein have a concentration of the conjugate ranging from about 0.2 milligrams / mL to about 50 milligrams / mL. In some embodiments, the liquid pharmaceutical composition contains from about 0.5 mg / mL to about 20 mg / mL. These compositions are preferably aqueous pharmaceutical compositions, and in one embodiment, are sterile aqueous pharmaceutical compositions formulated for intravenous delivery, such as saline.

[0155] The protein-drug conjugates and pharmaceutical compositions comprising said conjugates are administered to a patient in an amount effective to treat IBD, including any one or more of the following conditions associated with IBD: -Inhibiting de-epithelialization of the intestinal epithelial barrier, - inhibiting intestinal microbial infections, Protecting intestinal goblet cells during infection -Enhancing epithelial cell integrity, -Increasing epithelial cell proliferation, Enhancement of epithelial cell differentiation, and · Initiating re-epithelialization of damaged areas of the intestinal epithelial barrier.

[0156] The dosage of the protein-drug conjugate of the present invention is based on the patient's age, sex, weight, severity of the condition, general health, and other factors familiar to the treating clinician. In one embodiment, the patient is treated with an effective amount of the drug conjugate based in part on the amount of laquinimod released into the intestinal epithelial cells, which depends on the amount of laquinimod per drug conjugate. Generally, the amount of drug conjugate administered ranges from about 50 micrograms to 1 gram, preferably about 100 micrograms to 100 milligrams, per day.

[0157] H. Combination The protein-drug conjugates described herein can be used in combination with TNF blockers, steroids, and 5-aminosalicylic acid, as well as other conventional therapies currently used to treat IBD. [Example]

[0158] The present disclosure will be further understood by reference to the following examples, which are intended to be merely exemplary of the present disclosure. The present disclosure is not limited in scope by the exemplified embodiments, which are intended only to illustrate single aspects of the disclosure. Any functionally equivalent methods are within the scope of the present disclosure. Various modifications of the present disclosure, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and any accompanying drawings. Such modifications are within the scope of the appended claims.

[0159] In this specification and in the examples that follow, all temperatures are in degrees Celsius unless otherwise specified. Additionally, the following abbreviations have the following meanings: If not defined, these abbreviations have their art-recognized meanings. Abbreviation Meaning Δ Chemical shift (ppm) AcOH acetic acid Boc or t-Boc tert-butoxycarbonyl Cbz benzyloxycarbonyl DCM dichloromethane DIEA Diisopropylethylamine DMF N,N-dimethylformamide eq. equivalent amount ESI electrospray ionization EtOAc ethyl acetate EtOH ethanol FICZ 6-Formylindolo[3,2-b]carbazole Fmoc Fluorenylmethyloxycarbonyl g grams GI gastrointestinal 1 H NMR Proton Nuclear Magnetic Resonance Spectroscopy H time HPLC High Performance Liquid Chromatography IEB intestinal epithelial barrier IFN Interferon IL Interleukins (including IL6 and IL22) IL10R2 Interleukin 10 receptor 2 IPA Isopropyl Alcohol Laq Lakinimod L liters LDA Lithium diisopropylamide M mole MEK Methyl ethyl ketone MeOH Methanol μg and μL are micrograms and microliters, respectively mg milligram mmol millimole mL milliliter m / z mass-to-charge ratio MsOH methanesulfonic acid Min N Normal Pd / C Palladium Carbon t-Bu tert-butyl THF tetrahydrofuran TR thioredoxin (also called Trx) UV ultraviolet light v / v capacity / capacity ratio wt% weight percent

[0160] Preparation Example A: Synthesis of (9H-fluoren-9-yl)methyl (3-chloro-3-oxopropyl)carbamate (Reagent A)

[0161] [ka]

[0162] Commercially available Fmoc-β-alanine (3.5 g, 11.2 mmol) was converted to reagent A according to well-known literature procedures.

[0163] Preparation Example B - Laquinimod sodium salt (Compound 14)

[0164] [ka]

[0165] 5-Chloro-N-ethyl-1,2-dihydro-4-hydroxy-1-methyl-2-oxo-N-phenyl-3-quinolinecarboxamide sodium salt (laquinimod sodium salt) is a commercially available reagent, see, for example, Finetech Industry Limited (Wuhan, Hubei, China).

[0166] [Example 1] Synthesis of compound 13

[0167] [ka] Approximately equimolar amounts of compound 11 and reagent A were combined in a suitable inert solvent in the presence of sufficient DIEA (diisopropylethylamine) to scavenge the acid generated during the reaction. The reaction was maintained at about 20° C. for about 0.5 hours, which is sufficient time for amidation to occur to give compound 12.

[0168] Alternatively, methyl-(4-triacetyl-β-D-glucopyranuronate)-3-Fmoc (compound 12) is commercially available—see above.

[0169] A solution of compound 12 (1.33 g, 1.8 mmol) in DCM (20 mL) was cooled in an ice bath under argon. DIPEA (320 μL, 1.8 mmol) was added, followed by MsO (313 mg, 1.8 mmol) in one portion. The reaction was stirred for 30 min, and additional DIPEA (320 μL, 1.8 mmol) and MsO (313 mg, 1.8 mmol) were added. The reaction was stirred for 1 h, quenched with saturated sodium bicarbonate solution, and diluted with DCM. The layers were separated, and the DCM layer was washed with brine and dried over sodium sulfate. The solution was concentrated on a Roto Vap at 25 °C water bath temperature. The residue was purified by silica gel chromatography using a gradient of 50–100% ethyl acetate / hexanes.

[0170] [Example 2] Synthesis of compound 15

[0171] [ka] Approximately equimolar amounts of compound 13 and compound 14 were dissolved in MEK. The resulting solution was heated to approximately 55° C. and maintained at that temperature for 30 hours. The reaction was then terminated and the solvent removed to give a solid, which was used in the following examples without purification or isolation.

[0172] [Example 3] Synthesis of compound 16 (formate salt)

[0173] [ka] Compound 15 (147 mg, 0.135 mmol) was dissolved in MeOH (10 mL) and THF (2 mL) and cooled in an ice bath. LiOH solution (16 mg in 1 mL of water) was added in 200 μL increments over 1 h. The ice bath was removed, and the reaction was stirred for an additional 3 h. The reaction solution was concentrated on a Roto Vap in a 25 °C water bath to remove THF and MeOH. The residue was treated with methyl t-butyl ether (10 mL) and water (2 mL) and vortexed for 1 min. The aqueous layer was directly purified by preparative HPLC (XBridge C18 10 μm OBD, 19 × 250 mm column, 245 nm, flow rate 30 mL / min, 0.1% formic acid in water / ACN, 10% ACN in water to 100% ACN, 10 min ramp). The fraction containing the desired mass at 4.59 min was lyophilized to give the product as the formate salt. MS(ESI+)m / z:725.15(M+)

[0174] [Example 4] Synthesis of compound 6

[0175] [ka]

[0176] Compound 16 was combined with an approximately equimolar amount of commercially available 3-[oxyethyloxyethyloxyethyl-(ethyl-2-maleimido)]propanoic acid pentafluorophenyl ester, Reagent C (PFP ​​is pentafluorophenyl), in DMF, as described above. The amidation reaction was allowed to proceed to completion by conventional techniques. The reaction mixture (DMF) was then analyzed by preparative HPLC (XBridge C). 18 Purification was performed directly on a 10 μm OBD, 19x250 mm column, 245 nm, flow rate 30 mL / min, 0.1% formic acid in water / ACN, 10% ACN in water to 100% ACN, 10 min ramp. Fractions containing the desired mass were collected and lyophilized to give the product. MS (ESI+) m / z: 1008.47, 1030.34 [M+Na + ] + .

[0177] [Example 5] Method for producing IL22 and thioredoxin (IL22 / Trx fusion protein) The following materials and methods were used to prepare the thioredoxin-IL22 conjugate. pET-27b plasmid vector and BL21(DE3) competent cells were obtained from Sigma-Millipore. Terrific Broth, antibiotics, restriction enzymes, and isopropyl β-D-thiogalactopyranoside (IPTG) were obtained from ThermoFisher. All gene synthesis constructs were performed by GeneArt (Thermo Scientific). Cloning and expression were performed according to standard molecular biology protocols (Sambrook et al.). Briefly, the coding sequence for a His-tagged human thioredoxin-IL22 fusion protein (with a GSAM peptide linker between thioredoxin and IL22) was cloned into pET-27b and transformed into Escherichia coli (E. coli) cells (BL21(DE3)). The fusion protein also contains a 6His tag at the amino terminus of the thioredoxin protein. Instead of the His tag, alternative tags such as glutathione-S-transferase (GST) affinity tag, HaloTag® protein, Nano-Glo® HiBiT, NanoLuc® luciferase, etc. may also be used.

[0178] BL21(DE3) Escherichia coli (E. coli) cells were grown in Terrific Broth / kanamycin medium and induced with IPTG. The culture was harvested by centrifugation after 20 hours at 20°C. The cell pellet was lysed in 20 mM phosphate buffer, pH 8, 5% sorbitol, 5% glycerol, 100 mM KCl, 1% n-dodecyl-β-D-maltoside, EDTA-free protease inhibitor cocktail, 300 μg / ml lysozyme, and 1 IU / ml universal DNA nuclease. The lysate was incubated in lysis buffer with gentle agitation for 30 minutes and then sonicated in ice for 10 cycles (e.g., 1 cycle = 15 seconds on, 1 minute off, 60% amplitude). Insoluble material was pelleted by centrifugation at 21,000 x g for 30 minutes at 4°C. The supernatant was applied to an HP Histrap Nickel-Sepharose column (Cytiva Life Sciences) and chromatographed according to the manufacturer's instructions. The eluted peak was analyzed for the presence of IL22 by ELISA. Biological activity was assessed using an IL22 reporter cell line.

[0179] The thioredoxin-IL22 fusion protein was further purified by size-exclusion chromatography using a Superdex 75PG column (Sigma) and reanalyzed as described above. The purified thioredoxin-IL22 fusion protein was stored at -80°C in storage buffer (i.e., 20 mM phosphate pH 7.5, 100 mM KCl, and 300 mM L-arginine).

[0180] The exemplified His-tagged thioredoxin-IL22 fusion protein was made using the following sequence: The human thioredoxin protein ("Trx") used in the examples has the following sequence:

[0181] [ka]

[0182] The double underlined cysteine ​​("C") in Trx indicates an unpaired cysteine. The single underlined cysteine ​​represents a disulfide-paired cysteine. Trx is attached to the IL22 protein (at the amino terminus of the IL22 protein) at the carboxy terminus of Trx. The Trx methionine (M) is included at the amino terminus of a 6-histidine tag (SEQ ID NO: 5). A different tag can be used instead of the 6-His tag (SEQ ID NO: 5); therefore, the Trx protein used in the fusion protein is not limited to one containing a 6-His tag (SEQ ID NO: 5).

[0183] The human IL22 protein used lacks the 33 amino acid signal protein (i.e., MAALQKSVSS FLMGTLATSC LLLLALLVQG GAA, SEQ ID NO: 6) present at the amino terminus of immature IL22. One of the IL22 sequences used is as follows:

[0184] [ka]

[0185] In the IL22 protein, cysteines are bold and underlined; double underlined cysteines represent cysteines that form disulfide bonds, and single underlined cysteines do not form disulfide bonds.

[0186] The Trx (thioredoxin) protein is linked to the IL22 protein via a peptide linker using a four-amino acid linker, i.e., GSAM (glycine-serine-alanine-methionine) (SEQ ID NO: 4). The peptide linker is indicated by brackets and underline. A peptide linker is not required to link the two proteins. Alternative peptide linkers can be used, ranging in length from 1 amino acid to 100 amino acids. Therefore, embodiments should not be limited to four amino acids or peptide linkers, but can include any peptide linker having a sequence of 1 to 100 amino acids and any integer number therebetween. Alternatively, a chemical linker can be used in place of a peptide linker. For example, tyrosine can be used via click chemistry by reacting it with PTAD (4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione). Alternatively, lysine (K) can be used via its ε-amino group, but this is undesirable given its abundance and the associated lack of reproducibility.

[0187] An exemplary complete fusion protein discussed in this example is shown below for human thioredoxin (C32SC35S)-IL22, with cysteine ​​to serine mutations at positions 32 and 35, as indicated by the cysteines with asterisks:

[0188] [ka]

[0189] The underlined cysteine ​​shown in the fusion protein of SEQ ID NO: 6 is the cysteine ​​that forms the disulfide bond. The peptide linker is boxed. The sequence order is therefore N-terminus-His tag-Trx-peptide linker-IL22-C-terminus. Interestingly, when thioredoxin is expressed at the carboxy terminus of IL22, the biological activity in the form of inducing STAT3 activation is reduced by a full log. Thus, it has been observed that the protein order of IL22 and thioredoxin can be reversed and still retain activity, but with reduced activity.

[0190] The nucleic acid encoding the His-tagged thioredoxin-IL22 fusion protein was inserted into the pET27b (Novagen-Millipore-Sigma) vector. The designations "m" and "h" indicate "mouse" and "human," respectively. The variants of the nucleic acid sequence are as follows:

[0191] mThio-mIL22 (protein)

[0192] [ka]

[0193] mThio-mIL22 (nucleic acid)

[0194] [ka]

[0195] mThioC32S35S-mIL22 (protein)

[0196] [ka]

[0197] mThioC32S35S-mIL22 (nucleic acid)

[0198] [ka]

[0199] HThio-HIL22-APT (protein)

[0200] [ka]

[0201] The HThio-HIL22-APT variant has the human thioredoxin VKQIESKTAFQEALDAAGDKLVVVDFSATWC (SEQ ID NO: 12) sequence, followed by 1L22, followed by the remainder of thioredoxin PCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATlNELV (SEQ ID NO: 13).

[0202] HThio-HIL22-APT (nucleic acid)

[0203] [ka]

[0204] HThio-HIL22 (protein)

[0205] [ka]

[0206] HThio-HIL22 (nucleic acid)

[0207] [ka]

[0208] HThioC3235S-HIL22 (protein)

[0209] [ka]

[0210] HThioC3235S-HIL22 (nucleic acid)

[0211] [ka]

[0212] A complete list of each identified sequence number follows: SEQ ID NO: 1 mature human IL22 SEQ ID NO: 2 Gly-Ser-Ala-Met SEQ ID NO: 3 Human thioredoxin C32C35-IL22 fusion protein SEQ ID NO: 4 Gly-Ser-Ala-Met SEQ ID NO: 5 6His tag SEQ ID NO: 6 IL22 signal peptide of human IL22, cleaved during maturation SEQ ID NO: 7 mThio-mIL22 (protein) SEQ ID NO: 8 mThio-mIL22 (nucleic acid) SEQ ID NO: 9 mThioC32S35S-mIL22 (protein) SEQ ID NO: 10 mThioC32S35S-mIL22 (nucleic acid) SEQ ID NO: 11 HThio-HIL22-APT (protein) SEQ ID NO: 12: The amino acid sequence of Trx upstream of this sequence is the same as that of human thio-HIL22-APT. SEQ ID NO: 13: The IL22 protein has a carboxyl group of Trx upstream of this sequence compared to human thio-HIL22-APT. SEQ ID NO: 14 HThio-HIL22-APT (nucleic acid) SEQ ID NO: 15 HThio-HIL22 (protein) SEQ ID NO: 16 Hthio-HIL22 (nucleic acid) SEQ ID NO: 17 HThioC3235S-HIL22 (protein) SEQ ID NO: 18 HThioC3235S-HIL22 (nucleic acid)

[0213] The vectors were expressed in E. coli BL21(DE3) after nucleic acid optimization for expression in E. coli. Genes were synthesized by ThermoFisher's GeneArt service. GeneArt has a proprietary nucleic acid optimization tool that optimizes sequences based on codon usage for any given expression platform.

[0214] [Example 6] Conjugation of laquinimod to Trx-IL22 fusion protein Glucoronide-maleimide-laquinimod is a drug payload conjugable moiety and is represented by compound 6.

[0215] [ka] Compound 6 consists of a thiol-reactive maleimide group, a glucuronidase-cleavable glucuronide linker, and a releasable drug payload (laquinimod).

[0216] A. In the first example, lyophilized Compound 6 was dissolved in DMSO (dimethyl sulfoxide) to prepare a 10 mM stock solution. Purified thioredoxin-IL22 fusion protein from Example 7 above was dissolved in 20 mM phosphate buffer, 100 mM KCl, maintained at pH 7.5, and a sufficient amount of stock solution was added to provide a 10-fold molar excess of Compound 6 relative to the number of free cysteine ​​residues in the fusion protein. The solution was stirred at room temperature for 2 hours, then incubated overnight at 4°C with gentle agitation.

[0217] B. Separately, 2 mg / ml of the fusion thioredoxin-IL22 fusion protein (50 μM) from Example 7 was reacted with a 3-fold molar excess of the glucuronide-maleimide-laquinimod conjugate at room temperature for 1 hour with gentle stirring. The reaction was quenched with excess L-cysteine, and the mixture was desalted using a 26 / 10 HiPrep™ desalting column (Sigma-Aldrich) according to the manufacturer's instructions. The desalted protein was placed in storage buffer (i.e., 20 mM phosphate pH 7.5, 100 mM KCl, and 300 mM L-arginine).

[0218] The conjugated fusion protein was then exposed to a 10-fold molar excess (over the disulfide cysteine ​​content) of TCEP to reduce disulfides and incubated at room temperature (RT) for 1 h (with gentle agitation), followed by the addition of a 2-fold molar excess (over TCEP; TCEP is the reducing agent, tris(2-carboxyethyl)phosphine) of dibromo-PEG-maleimide-amino (recrosslinker). The mixture was incubated at room temperature for 2 h with gentle agitation, desalted using a HiTrap desalting column (Cytiva) as described above, and stored at -80 °C until use.

[0219] The degree of conjugation was measured by comparing the number of free thiols in protein samples before and after conjugation using a commercially available thiol measurement kit, Measure-IT™ (Thermo Scientific), according to the manufacturer's instructions. The drug-to-protein ratio was calculated by optical density to be approximately 2.97 drugs per protein molecule. Because the calculation assumed that a lack of thiol reactivity equates to the presence of drug, measuring thiols before and after conjugation allowed for an estimation of the number of drugs, i.e., the number of drug / moles of protein. It was observed that the HThio-HIL22-APT protein variant (SEQ ID NO: 11) was able to conjugate more laquinimod molecules than the other constructs. The APT variant was able to deliver five laquinimod molecules to cells, compared to only three molecules for the other variants.

[0220] [Example 7] Characterization of drug-conjugated Trx-IL22 fusion protein The fusion protein was quantified by ELISA. The biological activity of the fusion protein was determined using an IL22-dependent reporter bioassay using the HEK Blue IL22 cell line from InvivoGen according to the manufacturer's instructions. The fusion protein was calibrated with commercially available recombinant IL22 (R&D Systems, Cat. No. 782-IL-010 / CF).

[0221] The biological activity bioassay uses HEK293, an epithelial cell line engineered to constitutively express the murine IL-22 receptor heterodimer. HEK293 cells have been engineered to contain the IL-22-responsive transcription factor murine STAT3 and harbor an integrated copy of a plasmid containing a STAT3-sensitive promoter driving expression of a secreted alkaline phosphatase reporter protein. The cells are the InvivoGen IL-22 reporter cell line.

[0222] The results in Figure 1 show that both thioredoxin-IL22 fusion proteins analyzed (i.e., Thio-IL22 and Thio-C3235S-IL22) are expressed at high levels in the E. coli BL21(DE3) host. Thio-C3235S-IL22 (mouse SEQ ID NO: 9, human SEQ ID NO: 17) is a mutant in which the cysteines at positions 32 and 35 are changed to serine. The IL22 and thioredoxin sequences are otherwise identical to the Thio-IL22 construct. Expression of the constructs is as described above using the listed sequences. The expression method is the same for both.

[0223] ELISA was performed using the R&D Systems Mouse and Human IL22 Duo-Set ELISA package according to the manufacturer's instructions. Samples were diluted in 10-fold (log) serial dilutions.

[0224] Figure 2 shows the results of reporter bioassays of samples analyzed using the human Thio-IL22 and human Thio-C3235S-IL22 constructs. Similar mouse constructs showed similar results (data not shown). C32 and C35 are the active cysteines in the thioredoxin catalytic domain. When mutated to serine, thioredoxin no longer has redox activity. However, a C32S / C35S thioredoxin double mutant maintains chaperone-like activity but does not have redox activity. In the example of the IL22 / thioredoxin fusion protein, we observed that mutating thioredoxin to avoid its redox activity improved the biological activity of the IL22 / thioredoxin fusion protein. While eliminating the redox activity of thioredoxin does not necessarily improve the biological performance of the fusion partner in all thioredoxin fusion proteins, it does in the case of IL22.

[0225] "recHIL22" is recombinant human IL22 protein obtained from R&D (same as above).

[0226] The total Trx-IL22 fusion protein content of the samples was measured using the ThermoFisher Bradford assay. ThermoFisher Bradford reagent and protein stock solution were adjusted to 1 mg / ml according to the manufacturer's instructions. Protein samples were added to IL22 reporter cells (InvivoGen IL22 reporter cell line), and the cells were cultured according to the manufacturer's instructions. Protein samples were assayed at the concentrations shown in Figure 3. Supernatants from cultured IL22 reporter cells were collected after 24 hours of culture. Secreted alkaline phosphatase activity was measured using the colorimetric substrate provided with the InvivoGen IL22 cell line (i.e., secreted embryonic alkaline phosphatase (SEAP) substrate) according to InvivoGen's instructions.

[0227] Figure 3 illustrates the mechanism underlying the design of an IL22 reporter cell line that can independently measure the biological activity of IL22 and the activity of the drug payload released intracellularly after IL22 receptor-mediated endocytosis of protein-drug conjugates (PDCs). Both Thio-IL22 and ThioC3235S-IL22 share similar biological activity to recombinant human IL22. Data are shown for the human Thio-IL22 and ThioC3235S-IL22 constructs. Tests performed with murine analogs of the human constructs also yielded similar results (data not shown).

[0228] The glucuronide linker is cleaved by endosomal glucuronidase in the target cell, releasing the laquinimod payload. The red star in Figure 3 represents laquinimod. The IL22-AHR reporter cell line was constructed using the IL22 reporter HEK cell line described above as a starting point. The HEK cell line was then transfected with a plasmid carrying the AHR-GFP gene (Sino Biologicals, Cat. No. HG10456-ACG) and a drug-responsive element (DRE) promoter (Promega Corp. pGL4.43 [luc2P XRE Hygro]). This promoter binds to the active drug-loaded AHR and drives the expression of the firefly luciferase gene contained in the Promega plasmid.

[0229] Figures 4A and 4B show the ability of IL22-fused PDC to activate both pathways as expected when IL22 is internalized and releases laquinimod intracellularly. All constructs used and shown in Figures 4A and 4B use human sequences. Mouse analogs of the human constructs yielded similar results (data not shown).

[0230] The tryptophan metabolite FICZ, a highly affinity AHR agonist, was used as a positive control, along with the pesticide TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a non-metabolizable AHR agonist. In Figure 4A, the reporter assay measures STAT3 activity at various concentrations of IL22 PDC (µg / ml). PDC was diluted in complete medium (DMEM, 5% FCS, 2 mM L-glutamine) and then administered to cells. IL22 PDC was added directly to the cell culture at various concentrations diluted in complete medium. Cells were plated at approximately 50,000 cells / well in a 96-well plate. Once IL22 PDC was administered to the cells, they were cultured in the presence of PDC at 37°C and 7% CO2 for 21–24 hours before assay. The results in Figure 4A reflect measurements of the thioredoxin-IL22 fusion without drug (ThioIL22-no laquinimod), with drug (ThioIL22-Liq, "Liq" = laquinimod), a thioredoxin-IL22 fusion without the laquinimod payload called ThioC3235SIL22, with drug (ThioC3235SIL22-Liq), human recombinant IL22 (recIL22, recombinant IL22 obtained from R&D Systems), the tryptophan metabolite (FICZ), and the pesticide TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, or dioxin). TCDD is used as a positive control. IL22 (recIL22, human recombinant IL22) is the negative control. FICZ and TCDD were administered to cells at a concentration of 10 nM diluted in complete DMEM medium as described above. FICZ and TCDD were from Sigma Chemical Co.

[0231] In Figure 4B, fusion proteins with IL22-Liq (i.e., ThioIL22-Liq and ThioC3235SIL22-Liq) were measured at 30 nM (i.e., 90 nM equivalent of laquinimod). Given that there are three cysteines (Cys or C) available for conjugation, a 30 μM protein solution contains 90 μM cysteines, each conjugated with one drug, for 3 nM of the IL22-Liq form (i.e., 9 nM equivalent of laquinimod). In Figure 4B, 30 nM activity reflects 1 μg / mL of IL22 PDC, and 3 nM reflects 0.1 μg / mL of IL22 PDC.

[0232] The activity at 30 nM and 3 nM was measured using an AHR luciferase activity assay using recombinant HEK293 cells engineered to express the IL-22-responsive transcription factor mouse STAT3, as described above. HEK cells were cultured at approximately 50,000 cells / well in 96-well plates. Cells were treated with various PDC reagents and controls, then cultured at 37°C and 7% CO2 for 21–24 hours before assay. For luciferase assays, Promega's ONE-Glo™ Homogeneous Luciferase Cell Assay was used according to the manufacturer's instructions. The amounts of FICS AhR agonist and TCDD AhR agonist used in Figure 4B were again 10 nM diluted in complete medium. "Laq" refers to laquinimod. This experiment assumes a drug-to-protein ratio of 3, as described above.

[0233] One advantage of conjugated drug fusion proteins is that drugs such as laquinimod may be less toxic when administered in this form because the dose is restricted to receptor-bearing cells. Delivery of laquinimod is related to both stoichiometry and timing, resulting in intracellular localization and not systemic administration of the drug.

[0234] [Example 8] Cleavage of laquinimod by β-glucuronidase In this example, the following was prepared: a) 0.25 mL of a 30 mM solution of compound 16 was prepared as the formate salt in distilled water; b) 0.10 mL β-glucuronidase solution (400–800 units / mL); c) 0.50 mL of 75 mM potassium phosphate buffer, containing 1% (w / v) bovine serum albumin, adjusted to pH 6.8 at 37°C; and d) 0.65 mL of distilled water.

[0235] Each of a, c, and d was combined into the first solution and mixed by inversion. The enzyme solution was added to the first solution to form a second solution, which was then incubated at 37°C.

[0236] The purpose of this experiment was to determine whether the enzyme could release free laquinimod from the conjugate. Therefore, samples were taken at different time points, starting at T = 0 min and T = 30 min. The molecular weight of compound 16 is 724.2147, while the molecular weight of laquinimod is 356.0928.

[0237] LC showed a new peak corresponding to laquinimod at T = 30. Similarly, mass spectrometry also demonstrated the release of laquinimod at T = 30, confirming that enzymatic cleavage of laquinimod was achieved as described herein.

[0238] Embodiment Also described herein are one or more of the following embodiments.

[0239] Embodiment 12. a) a fusion protein comprising an IL22 moiety and a thioredoxin moiety linked by a covalent bond or a first linker; and b) each comprising one or more laquinimod groups or derivatives thereof and one to about three second linkers attached to sulfur groups of cysteine ​​side chains in the fusion protein; A conjugate wherein the first linker and the second linker, if present, contain from 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that the remaining valences of the non-hydrogen atoms are filled with hydrogen or deuterium atoms.

[0240] Embodiment 13. The conjugate of embodiment 1, wherein the one to about three second linkers are each cleavable linkers that cleave in the presence of an intracellular enzyme found in intestinal epithelial cells or intestinal epithelial stem cells.

[0241] Embodiment 14. The conjugate of embodiment 1, wherein each of the 1 to about 3 second linkers is attached to laquinimod or a derivative thereof via a covalent bond to a free cysteine ​​residue in the thioredoxin portion of the fusion protein.

[0242] Embodiment 15. The conjugate of embodiment 1, wherein each of the 1 to about 3 second linkers connecting the laquinimod or laquinimod derivative to the fusion protein is a cleavable linker that is selectively cleaved upon intracellular absorption of the conjugate into intestinal epithelial cells.

[0243] Embodiment 16. A conjugate of Formula A, or a pharmaceutically acceptable salt thereof: [(LAQ) n -L 2 -S-] b -FP-Y A (wherein n is 1 to 4, Y is hydrogen or L 1 -TR, b is 1 to 3 when Y is hydrogen, and 1 -TR is 1 to 6, L 2 is an enzymatically cleavable linker, FP is the formula TR-L 1 -IL22, TR is thioredoxin or a biologically active fragment thereof, and L 1 is a linker or covalent bond, and IL22 is 1 or L1 is a covalent bond, interleukin-22 or a biologically active fragment thereof bound to a TR; LAQ is laquinimod or its derivatives, S is the sulfur atom of the thiol group of a free cysteine ​​in the thioredoxin portion of the fusion protein).

[0244] Embodiment 17. The conjugate of embodiment 5, represented by formula IA

[0245] [ka] (In the formula, L 2 is a monovalent linker).

[0246] Embodiment 18. The conjugate of embodiment 5, represented by formula IB

[0247] [ka] (wherein q is 1 to 10).

[0248] Embodiment 19. The conjugate of embodiment 5, having formula IC.

[0249] [ka]

[0250] Embodiment 20. The conjugate of embodiment 5, represented by formula ID

[0251] [ka] where b is 1, 2, or 3.

[0252] Embodiment 21. The conjugate of embodiment 5, represented by formula IE

[0253] [ka] wherein the maleimide linker group is represented by the following formula:

[0254] [ka] q is between 1 and 10).

[0255] Embodiment 22. The conjugate of embodiment 5, represented by formula IF

[0256] [ka] wherein the succinimide linker attached to FP is represented by the formula:

[0257] [ka] q is between 1 and 10).

[0258] Embodiment 23. A protein-drug conjugate comprising a thioredoxin polypeptide linked directly, via a chemical linker, or via a peptide linker to an interleukin-22 (IL22) polypeptide, wherein one or more laquinimod molecules are conjugated to one or more cysteine ​​residues present in the thioredoxin polypeptide.

[0259] Embodiment 24 The conjugate of embodiment 12, wherein the thioredoxin polypeptide is linked to the amino terminus of the IL22 polypeptide.

[0260] Embodiment 25 The conjugate of embodiment 12, wherein the thioredoxin polypeptide comprises two or three thioredoxin polypeptides, optionally linked by peptide linkers.

[0261] Embodiment 26 The conjugate of embodiment 12, which is not glycosylated.

[0262] Embodiment 27. The conjugate of embodiment 12, wherein one or more laquinimod molecules are joined by linkers, each of said linkers comprising one or more laquinimod molecules, and wherein the linker comprises 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that the valences are satisfied with hydrogen or deuterium atoms.

[0263] Embodiment 28 The conjugate of embodiment 12, wherein the chemical or peptide linker is a cleavable linker that is cleaved in the presence of an intracellular enzyme located in intestinal epithelial cells and intestinal epithelial stem cells.

[0264] Embodiment 29 The conjugate of embodiment 12, wherein the thioredoxin polypeptide is linked to the IL22 polypeptide by a peptide linker or via chemical conjugation.

[0265] Embodiment 30. The conjugate of embodiment 13, wherein the peptide linker comprises 1 to 100 amino acids.

[0266] Embodiment 31 The conjugate of embodiment 15, wherein the peptide linker comprises 10 or fewer amino acids.

[0267] Embodiment 32 The conjugate of embodiment 16, wherein the peptide linker is Gly-Ser-Ala-Met (SEQ ID NO: 4).

[0268] Embodiment 33 The conjugate of any one of embodiments 12 to 22, wherein the thioredoxin polypeptide is human thioredoxin.

[0269] Embodiment 34. The conjugate of any one of embodiments 12 to 22, wherein the IL22 polypeptide is human IL22.

[0270] Embodiment 35 The conjugate of any one of embodiments 12 to 23, wherein the thioredoxin polypeptide comprises SEQ ID NO: 1.

[0271] Embodiment 36. The conjugate of any one of embodiments 12 to 24, wherein IL22 is SEQ ID NO: 2, or SEQ ID NO: 2 in which Cys32 and Cys35 are mutated to serine.

[0272] Embodiment 37. A conjugate wherein the thioredoxin polypeptide is SEQ ID NO: 1, the IL22 polypeptide is SEQ ID NO: 2, and the peptide linker is Gly-Ser-Ala-Met (SEQ ID NO: 4).

[0273] Embodiment 38. The conjugate of any one of embodiments 12 to 26, wherein the one or more laquinimod molecules or derivatives thereof are not conjugated to a cysteine ​​of the IL22 polypeptide.

[0274] Embodiment 39. A pharmaceutical composition comprising the conjugate of any one of embodiments 1 to 27 and a pharmaceutically acceptable carrier or excipient.

[0275] Embodiment 40. A method of treating a subject having inflammatory bowel disease (IBD), comprising administering to the subject the conjugate of any one of embodiments 1 to 27 or the pharmaceutical composition of embodiment 28.

[0276] Embodiment 41. A method for inhibiting des-epithelialization of the intestinal barrier in a patient at risk for an inflammatory bowel disease episode, or inhibiting further des-epithelialization of the intestinal barrier during an ongoing inflammatory bowel disease episode, comprising administering to the patient an effective amount of a conjugate as described herein and / or the conjugate of any one of embodiments 1 to 27, or the pharmaceutical composition of embodiment 28, to inhibit des-epithelialization or further des-epithelialization of the intestinal barrier.

[0277] Embodiment 42. A method for initiating re-epithelialization of the intestinal barrier in a patient suffering from an inflammatory bowel disease episode, comprising administering to the patient an effective amount of a conjugate as described herein and / or the conjugate of any one of embodiments 1 to 27, or the pharmaceutical composition of embodiment 28, to initiate re-epithelialization of the intestinal barrier.

[0278] Embodiment 43 The method of any one of embodiments 29-31, wherein the conjugate is administered to the subject in an amount sufficient to clinically improve one or more of the following conditions: i) inhibiting de-epithelialization of the intestinal epithelial barrier; ii) inhibiting intestinal microbial infections; iii) protecting intestinal goblet cells during infection; iv) enhancing epithelial cell integrity; v) increasing epithelial cell proliferation; vi) enhancing epithelial cell differentiation; and vii) Initiating re-epithelialization in damaged areas of the intestinal epithelial barrier.

[0279] Embodiment 44. The method of any one of embodiments 29-32, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0280] Embodiment 45. The conjugate of any one of embodiments 1 to 27 for use in treating inflammatory bowel disease.

[0281] Embodiment 46. Use of the conjugate of any one of embodiments 1 to 27 in the manufacture of a medicament for the treatment of inflammatory bowel disease.

Claims

1. a) a fusion protein comprising an IL22 moiety and a thioredoxin moiety linked by a covalent bond or a first linker; and b) conjugates each comprising one or more laquinimod groups or derivatives thereof and one to about three second linkers attached to sulfur groups of cysteine ​​side chains in said fusion protein; A conjugate wherein said first linker and said second linker, if present, contain from 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that the remaining valences of said non-hydrogen atoms are filled with hydrogen or deuterium atoms.

2. 2. The conjugate of claim 1, wherein each of the one to about three second linkers is a cleavable linker that is cleaved in the presence of an intracellular enzyme found in intestinal epithelial cells or intestinal epithelial stem cells.

3. 2. The conjugate of claim 1, wherein each of the one to about three second linkers is attached to laquinimod or a derivative thereof via a covalent bond to a free cysteine ​​residue of the thioredoxin portion of the fusion protein.

4. 10. The conjugate of claim 1, wherein each of the one to about three second linkers connecting laquinimod or a laquinimod derivative to the fusion protein is a cleavable linker that is selectively cleaved upon intracellular absorption of the conjugate into the intestinal epithelial cells.

5. A conjugate of formula A, or a pharmaceutically acceptable salt thereof: [(LAQ) ｎ -L ２ -S-] ｂ -FP-Y A (wherein n is 1 to 4, Y is hydrogen or L １ -TR, b is 1 to 3 when Y is hydrogen, and １ -TR is 1 to 6, L ２ is an enzymatically cleavable linker, FP is a compound of the formula TR-L １ -IL22, TR is thioredoxin or a biologically active fragment thereof, and L １ is a linker or covalent bond, and IL22 is １ or L １ is a covalent bond, interleukin-22 or a biologically active fragment thereof bound to a TR; LAQ is laquinimod or a derivative thereof, S is the sulfur atom of the thiol group of a free cysteine ​​in the thioredoxin portion of the fusion protein).

6. The conjugate of claim 5 represented by formula IA 【Chemical 1】 (In the formula, L ２ is a monovalent linker).

7. The conjugate of claim 5 having the formula IB 【Chemistry 2】 (wherein q is 1 to 10).

8. The conjugate of claim 5 having formula IC. 【Chemistry 3】

9. The conjugate of claim 5, represented by formula ID 【Chemistry 4】 (wherein b is 1, 2, or 3).

10. The conjugate of claim 5 represented by formula IE 【Chemistry 5】 wherein the maleimide linker group is represented by the following formula: 【Chemistry 6】 and q is 1 to 10.

11. The conjugate of claim 5 represented by formula IF 【Chemistry 7】 wherein the succinimide linker attached to the FP is represented by the formula: 【Chemistry 8】 and q is 1 to 10.

12. A protein-drug conjugate comprising a thioredoxin polypeptide linked directly, via a chemical linker, or via a peptide linker to an interleukin-22 (IL22) polypeptide, wherein one or more laquinimod molecules are conjugated to one or more cysteine ​​residues present in the thioredoxin polypeptide.

13. The conjugate of claim 12 , wherein the thioredoxin polypeptide is linked to the amino terminus of the IL22 polypeptide.

14. 13. The conjugate of claim 12, wherein the thioredoxin polypeptide comprises two or three thioredoxin polypeptides, optionally linked by peptide linkers.

15. The conjugate of claim 12, which is non-glycosylated.

16. 13. The conjugate of claim 12, wherein the one or more laquinimod molecules are joined by linkers, each of the linkers comprising one or more laquinimod molecules, and the linkers comprising 1 to about 40 non-hydrogen atoms selected from carbon, nitrogen, oxygen, sulfur, and phosphorus, with the proviso that valences are satisfied with hydrogen or deuterium atoms.

17. The conjugate of claim 12, wherein the chemical or peptide linker is a cleavable linker that cleaves in the presence of intracellular enzymes located in intestinal epithelial cells and intestinal epithelial stem cells.

18. The conjugate of claim 12, wherein the thioredoxin polypeptide is linked to the IL22 polypeptide by a peptide linker or via chemical conjugation.

19. The conjugate of claim 13, wherein the peptide linker comprises 1 to 100 amino acids.

20. 16. The conjugate of claim 15, wherein the peptide linker comprises 10 or fewer amino acids.

21. 17. The conjugate of claim 16, wherein the peptide linker is Gly-Ser-Ala-Met (SEQ ID NO: 4).

22. The conjugate of any one of claims 12 to 22, wherein the thioredoxin polypeptide is human thioredoxin.

23. The conjugate of any one of claims 12 to 22, wherein the IL22 polypeptide is human IL22.

24. The conjugate of any one of claims 12 to 23, wherein the thioredoxin polypeptide comprises SEQ ID NO:

1.

25. The conjugate according to any one of claims 12 to 24, wherein the IL22 is SEQ ID NO: 2 or SEQ ID NO: 2 in which Cys32 and Cys35 have been mutated to serine.

26. 25. The conjugate of claim 24, wherein the thioredoxin polypeptide is SEQ ID NO: 1, the IL22 polypeptide is SEQ ID NO: 2, and the peptide linker is Gly-Ser-Ala-Met (SEQ ID NO: 4).

27. The conjugate of any one of claims 12 to 26, wherein the one or more laquinimod molecules or derivatives thereof are not conjugated to a cysteine ​​of the IL22 polypeptide.

28. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 27 and a pharmaceutically acceptable carrier or excipient.

29. 29. A method of treating a subject with inflammatory bowel disease (IBD), comprising administering to the subject a conjugate according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 28.

30. 29. A method of inhibiting de-epithelialization of the intestinal barrier in a patient at risk for an inflammatory bowel disease episode, or inhibiting further de-epithelialization of the intestinal barrier during an ongoing inflammatory bowel disease episode, comprising administering to the patient an effective amount of a conjugate according to any one of claims 1 to 27, or a pharmaceutical composition according to claim 28, to inhibit de-epithelialization or further de-epithelialization of the intestinal barrier.

31. 29. A method for initiating re-epithelialization of the intestinal barrier in a patient suffering from an episode of inflammatory bowel disease, comprising administering to the patient an effective amount of a conjugate according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 28 to initiate re-epithelialization of the intestinal barrier.

32. 32. The method of any one of claims 29-31, wherein the conjugate is administered to the subject in an amount sufficient to clinically improve one or more of the following conditions: viii) inhibiting de-epithelialization of the intestinal epithelial barrier; ix) inhibiting microbial infection of the intestine; x) protecting goblet cells in the intestine during infection; xi) enhancing epithelial cell integrity; xii) increasing epithelial cell proliferation; xiii) enhancing epithelial cell differentiation; and xiv) initiating re-epithelialization of damaged areas of the intestinal epithelial barrier.

33. 33. The method of any one of claims 29 to 32, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

34. A conjugate according to any one of claims 1 to 27 for use in the treatment of inflammatory bowel disease.

35. Use of a conjugate according to any one of claims 1 to 27 in the manufacture of a medicament for the treatment of inflammatory bowel disease.

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