Water-soluble prodrugs, conjugates and uses thereof

Abstract

The present invention provides prodrugs and conjugates thereof, their uses, kits and pharmaceutical compositions.

Description

[Technical field]

[0001] The present invention relates to prodrugs and conjugates thereof, their uses, kits and pharmaceutical compositions. [Background technology]

[0002] Targeted therapy using small molecules or biopharmaceuticals has attracted more attention in the past decade. One special class of targeted therapy is ADC (antibody drug conjugates). Antibody drug conjugates (ADCs) present an unparalleled opportunity to improve the safety of highly toxic drugs by exploiting the specificity of antibodies to obtain targeted delivery of potent drugs to specific tissues. Much of the success of ADCs is due to technological advances in the design of the linkage between the antibody and the therapeutic payload.

[0003] To be selective and potent ADCs, the linker technology employed must have three important properties: (1)High stability in circulation (2) High water solubility, which aids in bioconjugation and avoids the formation of inactive ADC aggregates (3) Efficient release of highly cytotoxic payload-linker metabolites We must aim for this.

[0004] Cleavable linkers are generally preferred over non-cleavable linkers due to their scope of application, but are much more likely to become unstable in circulation. Thus, the success of a cleavable linker depends on its ability to effectively distinguish between circulating and target cell conditions. The poor tumor penetration of large IgG antibodies, coupled with the low efficiency of internalization, intracellular trafficking and drug release, necessitates a robust cleavable linker approach that maximizes the delivery of potent cytotoxins to cancer cells.

[0005] The stability of the ADC linker is not only defined by the cleavable linker technology, but also by the attachment site on the antibody: conjugation to a large antibody makes the linker less accessible to chemical and enzymatic triggers, thereby increasing plasma stability and slowing the rate of target cell release.

[0006] This effect can be further amplified when site-selective bioconjugation techniques are used to attach the linker-payload to sites that are poorly solvent accessible.

[0007] Although much research has been done on conjugation techniques and cleavage mechanisms, the addition of multiple drugs per cleavage site has not been fully explored. Considering that drugs with low potency require higher DAR (drug-to-antibody ratio), the identification of such modules would be beneficial as it would allow the release of more drug per cleavage step. Summary of the Invention

[0008] In one embodiment, the present invention provides a compound of formula (I): [ka] (In the formula, R 1 is a reactive moiety; R 2 and R 3 are the same or different active pharmaceutical ingredient moieties; R 4 is a hydrophilic moiety; and X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo or polyethylene glycol moiety, an oligo or polyvinyl alcohol moiety, or an oligo or polyglycerol moiety. The present invention provides a compound represented by the formula:

[0009] In a further embodiment, the present invention provides a compound of formula (I(a)): [ka] (In the formula, R 2 and R 3 are the same or different active pharmaceutical ingredient moieties; R 4 is a hydrophilic moiety; R 5 is an antibody or antigen moiety; X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo or polyethylene glycol moiety, or an oligo or polyvinyl alcohol moiety, or an oligo or polyglycerol moiety; X 2 is the linker; and (n is a number between 0.01 and 10) The present invention provides a complex represented by the formula:

[0010] In a further embodiment, the present invention provides a compound or conjugate as described herein above for use in the treatment of cancer.

[0011] In a further embodiment, the present invention relates to a method for producing a pharmaceutical composition comprising the steps of: i) a compound as described herein above; and ii) Antibody or antigen A kit comprising: [Brief description of the drawings]

[0012] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with its objects, features and advantages, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

[0013] [Figure 1A]Cytotoxicity effect of the complex of the present invention on various cell viability assays (exposure time: 5 days). Cell viability of HCC1954 after 120 hours (5 days) treatment with serial dilutions of the complex. Data show mean ± SD; representative of two separate experiments. [Figure 1B] Cytotoxic effect of the conjugates of the invention on various cell viability assays (exposure time: 5 days). MDA-MB-231 cell viability after 120 hours (5 days) of treatment with serial dilutions of the conjugates. Data are shown as mean ± SD; representative of two separate experiments. [Figure 1C] The cytotoxic effect of the complex of the present invention on various cell viability assays (exposure time: 5 days). The table lists the IC50 of various treatments in HCC1954 cells. Data show the mean ± SD; representative of two separate experiments. [Figure 1D] The cytotoxic effect of the complexes of the present invention on various cell viability assays (exposure time: 5 days). The table lists the IC50 of various treatments in MDA-MB-231 cells. Data are shown as mean ± SD; representative of two separate experiments. [Figure 2A] Cytotoxic effect of the conjugate of the present invention on various cell viability assays (exposure time: 6 days). Viability of HCC1954 cells after 144 hours (6 days) of treatment with serial dilutions of trastuzumab, exatecan, or Trans-Exa-1. Data show mean ± SD; representative of three separate experiments. [Figure 2B] Cytotoxicity effect of the conjugate of the present invention on various cell viability assays (exposure time: 6 days). Viability of JIMT-1 cells after 144 hours (6 days) of treatment with serial dilutions of trastuzumab, exatecan, or Trans-Exa-1. Data show mean ± SD; representative of three separate experiments. [Figure 2C] Cytotoxic effect of the conjugates of the invention on various cell viability assays (exposure time: 6 days). Viability of MDA-MB-468 cells after 144 hours (6 days) of treatment with serial dilutions of trastuzumab, exatecan, or Trans-Exa-1. Data are shown as mean ± SD; representative of three separate experiments. [Figure 2D] The cytotoxic effect of the complexes of the invention on various cell viability assays (exposure time: 6 days) is shown. The table lists the IC50 of the various treatments, where the first two rows are the IC50 values, "Tras-Exa / Exa fold change" is the Tras-Exa divided by the exa value, and "-", "+" and "++" indicate the her2 levels found in the literature, indicating undetectable, low and high levels, respectively (e.g., relative levels as described in Yang, L.; Li, Y.; Bhattacharya, A.; Zhang, YS A recombinant human protein targeting HER2 overcomes drug resistance in HER2-positive breast cancer. Sci. Transl. Med. 2019, 11, 11). Data are shown as mean ± SD; representative of three individual experiments. [Figure 3A] Figure 2 shows that trastuzumab-based ADC induces dose-dependent inhibition of tumor growth. Growth curves of HCC1954 tumors after a single intravenous injection of trastuzumab-based ADC at a concentration of 10 mg / kg (n=9 mice / group). Data are presented as mean ± sem. [Figure 3B] Figure 2 shows that trastuzumab-based ADC induces dose-dependent inhibition of tumor growth. Growth curves of HCC1954 tumors after a single intravenous injection of trastuzumab-based ADC at a concentration of 3 mg / kg (n=9 mice / group). Data are presented as mean ± sem. [Figure 3C] Figure 2 shows that trastuzumab-based ADC induces dose-dependent inhibition of tumor growth. Growth curves of HCC1954 tumors after a single intravenous injection of trastuzumab-based ADC at a concentration of 1 mg / kg (n=9 mice / group). Data are presented as mean±sem. [Figure 3D] Figure 2 shows that trastuzumab-based ADC induces dose-dependent inhibition of tumor growth. Body weight change is expressed as percentage change from the day of tumor cell inoculation. Data are shown as mean ± sem. [Figure 4A]1 shows that trastuzumab-based ADCs extend mouse survival in a dose-dependent manner. Kaplan-Meier overall survival curves of HCC1954 tumor-bearing SCID mice treated with a single intravenous injection of trastuzumab-based ADCs at a concentration of 10 mg / kg (n=9 mice / group). [Figure 4B] Figure 2 shows that trastuzumab-based ADCs extend mouse survival in a dose-dependent manner. Kaplan-Meier overall survival curves of HCC1954 tumor-bearing SCID mice treated with a single intravenous injection of trastuzumab-based ADCs at a concentration of 3 mg / kg (n=9 mice / group). [Figure 4C] 1 shows that trastuzumab-based ADCs extend mouse survival in a dose-dependent manner. Kaplan-Meier overall survival curves of HCC1954 tumor-bearing SCID mice treated with a single intravenous injection of trastuzumab-based ADCs at a concentration of 1 mg / kg (n=9 mice / group). [Figure 4D] Figure 1 shows that trastuzumab-based ADCs extend mouse survival in a dose-dependent manner. Median survival time to death and individual time points. The dashed line indicates the predefined endpoint of the study (152 days).

[0014] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, dimensions of some elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to obscure the present invention.

[0016] Prodrug In one embodiment, the present invention provides a compound of formula (I): [ka] (In the formula, R 1 is a reactive moiety; R 2 and R 3 are the same or different active pharmaceutical ingredient moieties; R 4 is a hydrophilic moiety; and X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo or polyethylene glycol moiety, an oligo or polyvinyl alcohol moiety, or an oligo or polyglycerol moiety. The present invention provides a compound represented by the formula:

[0017] In one embodiment, R 1 is a reactive moiety. In another embodiment, a "reactive moiety" in the context of the present invention is defined as a chemical moiety (e.g., an alkene group) that may contain one functional group or multiple functional groups (same or different), where at least one of such functional groups is capable of reacting or interacting with an antibody or antigen to conjugate the moiety to the antibody or antigen. In some other embodiments, a thiol of Cys or an amine of Lys from an antibody or antigen reacts with the moiety in a 1,4 (Michael) addition. In another embodiment, R 1 comprises a dihydropyridazinemaleimide, bromoacetamine, tetrazine, alkyne or amine moiety, or a combination thereof. 1 In another embodiment, R 1 contains a constrained alkyne moiety. In another embodiment, R 1comprises a cycloalkyne moiety. In another embodiment, non-limiting examples of cyclooctyne moieties include dibenzocyclooctyne (DBCO), bicyclo[6.1.0]nonyne (BCN), and biarylazacyclooctynone (BARAC) moieties. 1 teeth, [ka] It is expressed by:

[0018] In one embodiment, R of formula (I) 2 is an anti-cancer agent, an immunostimulant (immuno-oncology), or an immunosuppressant (e.g., immunological steroids). 2 is an agent used in the treatment of immunology, immune-oncology, cancer-related diseases and / or disorders, or immune system deficiencies / dysfunctions. In other embodiments, the cancer-related / associated pathologies / disorders and / or immune system deficiencies / dysfunctions include at least one of the following non-limiting examples: autoimmune diseases, arthritis, ectopic dermatitis, Crohn's disease, colitis, IBD, fibrosis, psoriasis, lupus erythematosus, multiple sclerosis, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, ALS, myasthenia gravis, Graves' disease, ankylosing fibrosis), and respiratory diseases (COPD, asthma). In another embodiment, R 2 is an exatecan, belotecan, camptothecin, auristatin, monomethylauristatin E (MMAE), or doxorubicin moiety. 2 is exatecan, auristatin or belotecan.

[0019] In one embodiment, R of formula (I) 3 is an anti-cancer agent, an immunostimulant (immuno-oncology), or an immunosuppressant (e.g., immunological steroids). 3is an agent used in the treatment of immunology, immune-oncology, cancer-related diseases and / or disorders, or immune system deficiencies / dysfunctions. In other embodiments, the cancer-related / associated pathologies / disorders and / or immune system deficiencies / dysfunctions include at least one of the following non-limiting examples: autoimmune diseases, arthritis, ectopic dermatitis, Crohn's disease, colitis, IBD, fibrosis, psoriasis, lupus erythematosus, multiple sclerosis, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, ALS, myasthenia gravis, Graves' disease, ankylosing fibrosis), and respiratory diseases (COPD, asthma). In another embodiment, R 3 is an exatecan, belotecan, camptothecin, auristatin, monomethylauristatin E (MMAE), or doxorubicin moiety. 3 are exatecan, auristatin, monomethylauristatin E (MMAE) or belotecan.

[0020] In one embodiment, R of formula (I) 2 and R 3 is an anti-cancer agent, an immunostimulant (for immuno-oncology), or an immunosuppressant (e.g., steroid), or an agent used in the treatment of immunological, immuno-oncological, cancer-related diseases and / or disorders, or in the treatment of immune system deficiencies / dysfunctions, wherein the cancer-associated / related pathology / disorder and / or immune system deficiencies / dysfunctions are as described herein above (R 2 and / or R 3 In another embodiment, R 2 and R 3 are each independently an exatecan, belotecan, auristatin, monomethylauristatin E (MMAE), camptothecin, or doxorubicin moiety. 2 and R 3 are each independently an exatecan, auristatin, monomethylauristatin E (MMAE), or belotecan moiety. 2 and R 3are each independently an exatecan moiety. 2 and R 3 are each independently a belotecan moiety.

[0021] In one embodiment, R of formula (I) 4 comprises an oligo- or poly-carboxylic acid moiety, or an oligo- or poly-ethylene glycol moiety, or an oligo- or poly-alcohol moiety, or an oligo- or poly-vinyl alcohol moiety, or an oligo- or poly-glycerol moiety. 4 teeth, [ka] It is expressed by:

[0022] In one embodiment, X in formula (I) 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, or an oligo- or poly-ethylene glycol moiety, or an oligo- or poly-vinyl alcohol moiety, or an oligo- or poly-glycerol moiety. 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide containing any amino acid: natural, non-natural (artificial), saturated or unsaturated, or combinations thereof, in any order / sequence. 1 In another embodiment, X is CGKRK, -Val-Cit-, -Ala-Ala-, AAN, GGFG, Val-Arg- or Val-Ala, or any combination thereof. 1 In another embodiment, X is -Val-Cit-, -Ala-Ala-, AAN, GGFG, -Val-Arg- or -Val-Ala-, or any combination thereof. 1 includes sequences (eg, Ala-Ala) cleavable by specific proteases, including, by way of non-limiting examples, cathepsin B and legumain. Each possibility represents a separate embodiment of the present invention.

[0023] In one embodiment, the compound of formula (I) has the formula 1, 17, 18, 19 or 20: [ka] JPEG2024532677000008.jpg197147 JPEG2024532677000009.jpg76139 (wherein TFA is trifluoroacetic acid) It is represented by the structure:

[0024] In some embodiments, the compound of formula (I) comprises an R that reacts and / or interacts with an antibody or antigen and a relevant moiety / portions / functional group / amino acid of the antibody or antigen. 1 and the product of this reaction is a conjugate represented below in the sections that follow herein, e.g., by formula (I(a)), 1(a), 2(a) and / or compound 21. In one specific embodiment, a lysine or cysteine ​​of an antibody or antigen is coupled to a maleimide moiety, R 1 In a further embodiment, R 1 reacts with an artificial amino acid such as azido-tyrosine or with an alkyne to produce the above conjugate. In another embodiment, an antibody / antigen reacts with a compound of formula (I), which then interacts with some portion of the compound (e.g., some peptide recognition motif / trigger), and the interaction may trigger the compound to release the covalently loaded drug. In one embodiment, the antibody is trastuzumab or rituximab.

[0025] Conjugates of prodrugs with antibodies or antigens In one embodiment, the present invention provides a compound represented by formula (I(a)): [ka] (In the formula, R 5 is an antibody or antigen moiety; X 2 is a linker; n is a number between 0.01 and 10; and R 2 ~R 4 and X 1 is described herein above) The present invention provides a complex represented by the formula:

[0026] In one embodiment, the present invention provides a conjugate represented by formula (I(a)) prepared by reacting a prodrug or a compound of formula (I) with an antibody or antigen, all definitions of formula and structures are as hereinbefore or hereinafter defined.

[0027] In some embodiments, the antigen / antibody moiety (R 5 The bond between R and the other moieties of formula (I(a)) may be a covalent bond or a non-covalent bond. The term "non-covalent bond" within the meaning of the present invention includes, as known in the art, van der Waals / interactions / London dispersion forces, hydrogen bonds, halogen bonds, pi-pi (π-π) interactions, ionic bonds (e.g., R 5 antibody or antigen and X 1 In some embodiments, R refers to any non-covalent interactions / bonds, such as salt bridges between the peptide moiety of R 5 is connected at more than one position to other moieties of formula (I(a)) with the same or different interactions / bonds at each position. 5 is covalently linked to one or more compounds that are other moieties of formula (I(a)) and also non-covalently linked / interacted with such one or more compounds at a different location compared to the point of covalent attachment. In one embodiment, "other moieties of formula (I(a))" are [ka] (wherein the curly bond is R 5 (not shown) showing a bond to (not shown). Each possibility represents a separate embodiment of the present invention.

[0028] In one embodiment, X of formula (I(a)) 2 In another embodiment, X comprises a succinimide, acetamide, dihydropyridazine, alkene or amine moiety, or any combination thereof. 2 In another embodiment, X 2 In another embodiment, X 2 teeth [ka] Represented by JPEG2024532677000013.jpg90132.

[0029] In some embodiments, n is between 0.01 and 10. In one embodiment, n is between 0.01 and 1. In another embodiment, n is between 0.01 and 2. In another embodiment, n is between 0.01 and 5. In another embodiment, n is between 0.05 and 1. In another embodiment, n is between 0.05 and 2. In another embodiment, n is between 0.1 and 1. In another embodiment, n is between 0.1 and 2. In another embodiment, n is between 0.5 and 1. In another embodiment, n is between 0.01 and 0.5. In another embodiment, n is between 0.1 and 0.5. In another embodiment, n is between 0.5 and 2. Each possibility represents a separate embodiment of the present invention.

[0030] In one embodiment, R of formula (I(a)) 5 is an antibody or an antigen. In another embodiment, R 5 is a tumor-targeting antibody. 5 is an immune cell specific antigen. 5is an anti-EGFR or anti-CD33 antibody. 5 The antibodies of are model antibodies or "stimulating" parts / fragments of antibodies, such models / parts / fragments being represented inter alia by the CGKRK peptide. 5 is any one of CEACAM family, folate receptor, ER, PR, notch receptor, notch ligand, PSMA, glypican-3, mesothelin, MET, EGFR, Erb2, EpCAM, NCAM, ephrin A4, IGF-1R, FAP, Ly6E, cadherin family, VEGFR-2, RNF43, MUC family, PD-1, PD-L1, FAK, CCR2, CCR4, CXCR1, CXCR2, nectin-4, transferrin, TIM-1, LAG-3, Axl, CTLA-4, 4-1BB, MART-1, TIGIT, SLAMF6, CX40, trastuzumab, or rituximab. 5 is a trastuzumab or rituximab or CGKRK moiety. 5 is a trastuzumab moiety. In another embodiment, R 5 is a rituximab moiety. In another embodiment, R 5 is a CGKRK moiety. Each possibility represents a separate embodiment of the present invention.

[0031] In one embodiment, the compound of formula (I(a)) has formula 1(a) or 2(a): [ka] JPEG2024532677000015.jpg111147 (in the formula, R 5 is as described above in this specification) It is represented by the structure:

[0032] In one embodiment, the compound of formula (I(a)) is compound 21: [ka] It is represented by the structure:

[0033] In some embodiments, for example, a compound represented by formula (I) or structure 1, 17, 18, 19, or 20, also referred to as a "prodrug," can bind to an antibody or an antigen to form a conjugate. The term "conjugate," in the context of the present invention, can be interchangeably referred to as an "antibody drug conjugate" (ADC).

[0034] Proposed mechanism of release In some embodiments, without being bound by any mechanism or theory, the drug is released from the covalently linked complex via reaction with proteases and / or hydrases and / or other enzymes that subsequently lead to a cascade of additional reactions. In one embodiment, an example of this mechanism / cascade is shown below (cathepsin B is the proposed protease). Scheme 1: Examples of drug release mechanisms [ka]

[0035] Thereby, the peptide or R in the above scheme 5 Any other viable antibody / antigen can be used for targeted delivery of the complex to the desired (biological) site and is activated by some trigger, such as reaction with hydrolases / proteases as exemplified above.

[0036] Pharmaceutical Compositions Comprising Prodrugs, Antibodies or Antigens and / or Complexes Thereof In one embodiment, the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a prodrug or its conjugate as described hereinabove, and optionally at least one pharma- ceutically acceptable carrier, diluent, vehicle or excipient. In another embodiment, the composition comprises a combination of a prodrug and an antibody or antigen, and at least one pharma- ceutically acceptable carrier, diluent, vehicle or excipient. In another embodiment, the pharmaceutical composition is in a form selected from the group consisting of tablets (including, for example, film-coated tablets), powders, granules, capsules (including soft capsules), orally disintegrating tablets, pills, pellets, lozenges, sachets, cachets, patches, elixirs, suspensions, dispersions, emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, suppositories, sterile injection solutions, sterile packaged powders, and sustained release formulations, as known in the art. In another embodiment, the composition is a solid-state composition (e.g., tablets, pills, capsules, pellets, granules, powders, etc.). Each possibility represents a separate embodiment of the present invention.

[0037] In some embodiments, pharma- ceutically acceptable carriers, diluents, vehicles or excipients that may be used in the context of the present invention include, but are not limited to, surfactants, lubricants, binders, fillers, compression aids, disintegrants, water-soluble polymers, inorganic salts, preservatives, antioxidants, colorants, sweeteners, acidulants, effervescent agents and flavorings. Each possibility represents a separate embodiment of the present invention.

[0038] In some embodiments, non-limiting examples of suitable carriers, diluents, vehicles or excipients within the scope of the present invention include, for example, lactose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, and titanium oxide. Each possibility represents a separate embodiment of the present invention. Suitable surfactants include, for example, lecithin and phosphatidylcholine. Each possibility represents a separate embodiment of the present invention. Suitable Suitable lubricants include, for example, magnesium stearate, sucrose fatty acid esters, polyethylene glycol, talc, and stearic acid. Each possibility represents a separate embodiment of the present invention. Suitable binders include, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, α-starch, polyvinylpyrrolidone, acacia powder, gelatin, pullulan, and low-substituted hydroxypropylcellulose. Each possibility represents a separate embodiment of the present invention. Suitable disintegrants include, for example, cross-linked povidone (any cross-linked 1-ethenyl-2-pyrrolidone homopolymer such as polyvinylpyrrolidone (PVPP) and 1-vinyl-2-pyrrolidone homopolymer), cross-linked carmellose sodium, carmellose calcium, sodium carboxymethyl starch, low-substituted hydroxypropylcellulose, corn starch, and the like. Each possibility represents a separate embodiment of the present invention. Suitable water-soluble polymers include, for example, cellulose derivatives such as hydroxypropylcellulose, polyvinylpyrrolidone, hydroxypropylmethylcellulose, methylcellulose and sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, and the like. Each possibility represents a separate embodiment of the present invention. Suitable inorganic salts include, for example, basic inorganic salts of sodium, potassium, magnesium and / or calcium, and the like. Each possibility represents a separate embodiment of the present invention. Particular embodiments include basic inorganic salts of magnesium and / or calcium. Basic inorganic salts of sodium include, for example, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, and the like. Each possibility represents a separate embodiment of the present invention. Basic inorganic salts of potassium include, for example, potassium carbonate, potassium bicarbonate, and the like. Each possibility represents a separate embodiment of the present invention. Basic inorganic salts of magnesium include, for example, magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium aluminometasilicate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite, magnesium alumina hydroxide, and the like. Each possibility represents a separate embodiment of the present invention.Basic inorganic salts of calcium include, for example, precipitated calcium carbonate, calcium hydroxide, etc. Each possibility represents a separate embodiment of the present invention.

[0039] Suitable preservatives include, for example, sodium benzoate, benzoic acid, and sorbic acid. Each possibility represents a separate embodiment of the present invention. Suitable antioxidants include, for example, sulfites, ascorbic acid, and alpha-tocopherol. Each possibility represents a separate embodiment of the present invention. Suitable coloring agents include, for example, food dyes such as Food Color Yellow No. 5, Food Color Red No. 2, and Food Color Blue No. 2. Each possibility represents a separate embodiment of the present invention. Suitable sweetening agents include, for example, dipotassium glycyrrhetinate, aspartame, stevia, and thaumatin. Each possibility represents a separate embodiment of the present invention. Suitable acidulants include, for example, citric acid (citric acid anhydrous), tartaric acid, and malic acid. Each possibility represents a separate embodiment of the present invention. Suitable foaming agents include, for example, sodium bicarbonate. Suitable flavoring agents include, for example, synthetic or natural, including lemon, lime, orange, menthol, and strawberry. Each possibility represents a separate embodiment of the present invention.

[0040] In one embodiment, the compounds of the present invention (prodrugs and / or conjugates thereof) are useful as pharmaceuticals for medical treatment. The present invention thus provides pharmaceutical compositions comprising a prodrug or conjugate thereof, or a combination of a prodrug and an antibody or antigen, as disclosed herein, and at least one pharma- ceutically acceptable carrier, diluent, vehicle or excipient. The compounds of the present invention can be safely administered orally or parenterally. Routes of administration include, but are not limited to, oral, topical, subcutaneous, intraperitoneal, rectal, intravenous, intraarterial, transdermal, intramuscular, topical, and nasal. Each possibility represents a separate embodiment of the present invention. Further routes of administration include, but are not limited to, mucosal, nasal, parenteral, gastrointestinal, intrathecal, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intraventricular, intracerebral, ophthalmic, oral, epidural, and sublingual. Each possibility represents a separate embodiment of the present invention.

[0041] In another embodiment, tablets and other solid dosage forms of the pharmaceutical compositions described herein can be optionally stored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.They can also be formulated to provide slow or controlled release of the active ingredient contained therein, for example, using hydroxypropylmethylcellulose, other polymer matrices, etc., in various proportions to provide the desired release profile.The active ingredient can also be in microencapsulated form, where appropriate, with one or more of the above excipients.Each possibility represents a separate embodiment of the present invention.

[0042] Use of Prodrugs and Conjugates In one embodiment, the present invention provides a method of treating cancer or a related disease / condition comprising administering to a subject in need thereof a pharmaceutical composition comprising a prodrug (e.g., a compound of formula (I)), a conjugate thereof (e.g., a compound of formula (I(a))), or a combination of a prodrug with an antibody or antigen, or a prodrug / conjugate / combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. In another embodiment, the present invention provides a method of treating cancer or a related disease / condition comprising administering to a subject in need thereof a pharmaceutical composition comprising a conjugate (e.g., a compound of formula (I(a))), or a combination of a prodrug (e.g., a compound of formula (I)) with an antibody or antigen, or a conjugate / combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. In one embodiment, the method is an immunological method. In another embodiment, the method is an immuno-oncological method. Each possibility represents a separate embodiment of the present invention.

[0043] In one embodiment, the present invention provides a method of treating a condition and / or disease associated with immune system deficiency / dysfunction, comprising administering to a subject in need thereof a pharmaceutical composition comprising a prodrug (e.g., a compound of Formula (I)), a conjugate thereof (e.g., a compound of Formula (I(a))), or a combination of a prodrug with an antibody or antigen, or a prodrug / conjugate / combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. In another embodiment, the present invention provides a method of treating cancer or a related disease / condition, comprising administering to a subject in need thereof a pharmaceutical composition comprising a conjugate (e.g., a compound of Formula (I(a))), or a combination of a prodrug (e.g., a compound of Formula (I)) with an antibody or antigen, or a conjugate / combination and at least one pharmaceutically acceptable carrier, diluent, vehicle or excipient. In another embodiment, the method is an immunological method. In another embodiment, the method is an immuno-oncological method. Each possibility represents a separate embodiment of the present invention.

[0044] In other embodiments, the pathology and / or disease associated / related to cancer and / or immune system deficiency / dysfunction includes at least one of the following non-limiting examples: autoimmune disease, arthritis, ectopic dermatitis, Crohn's disease, colitis, IBD, fibrosis, psoriasis, lupus erythematosus, multiple sclerosis, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease, ALS, myasthenia gravis, Graves' disease, ankylosing fibrosis), and respiratory diseases (COPD, asthma). In one embodiment, the present invention provides a method for administering to a subject in need thereof: a) a prodrug (e.g., a compound of formula (I)); and b) Antibody or antigen or Administering a conjugate of a prodrug and an antibody or antigen The present invention provides a method for treating cancer, comprising:

[0045] In some embodiments, the cancer is breast cancer, kidney cancer, brain cancer, skin cancer, ovarian cancer, lung cancer, pancreatic cancer, colon cancer, head and neck cancer, prostate cancer, endometrial cancer, liver cancer, blood cancer, gastric cancer, fibrosarcoma, bone tumor / osteosarcoma or duodenal cancer. In one embodiment, the cancer is breast cancer. In another embodiment, the administration of the prodrug (compound of formula (I)) and the antibody or antigen is performed subsequently or simultaneously. Each possibility represents a separate embodiment of the present invention.

[0046] In some embodiments, the subject is a mammal, preferably a human.

[0047] In some embodiments, the present invention provides a pharmaceutical composition comprising a prodrug (e.g., a compound of Formula (I)), a conjugate thereof (e.g., a compound of Formula (I(a))), or a combination of a prodrug with an antibody or an antigen, or the prodrug / conjugate / combination and at least one pharma- ceutically acceptable carrier, diluent, vehicle or excipient, for use in the treatment of cancer, or conditions and / or diseases related / associated with cancer, and / or the treatment of immune system deficiencies / dysfunctions, as described in the method embodiments above.

[0048] In one embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: i) a prodrug as described herein above (e.g., a compound of formula (I)); and ii) Antibody or antigen A kit comprising:

[0049] In other embodiments, the kit comprises instructions for use.

[0050] definition As used herein, the term "therapeutically effective amount" is defined to refer to an amount of a drug that, upon administration to a subject in a single dose or multiple doses, is effective to provide a therapeutic effect to the subject. In additional embodiments, the crystalline forms of the present invention are used for the manufacture of a medicament for treating the aforementioned diseases or disorders.

[0051] As used herein, with respect to medical treatment, the term "treating" is defined to refer to medical care or the alleviation, or the inhibition or prevention of a condition, disease or disorder in a subject following its appearance / identification.

[0052] The term "DAR" or "Drug-Antibody Ratio" is defined herein as equal to the number of drug equivalents per antibody or antigen equivalent. In one embodiment, the DAR is between 0.5 and 200. In another embodiment, the DAR is between 0.5 and 50. In another embodiment, the DAR is between 0.5 and 20. In another embodiment, the DAR is between 0.5 and 10. In another embodiment, the DAR is between 0.5 and 5. In another embodiment, the DAR is between 1 and 10. In another embodiment, the DAR is between 1 and 20. In another embodiment, the DAR is between 1 and 50. In another embodiment, the DAR is between 1 and 100. In another embodiment, the DAR is between 1 and 200. In another embodiment, the DAR is between 5 and 10. In another embodiment, the DAR is between 5 and 20. In another embodiment, the DAR is between 5 and 50. In another embodiment, the DAR is between 10 and 20. In another embodiment, the DAR is between 20 and 50. In another embodiment, the DAR is between 50 and 100. In another embodiment, the DAR is between 50 and 200. In another embodiment, the DAR is between 100 and 200. In some embodiments, the DAR is explicitly mentioned (e.g., Table 1 in Example 4). In other embodiments, the DAR can be calculated via "n" within the meaning of formula (I(a)). A few examples of such calculations are given below. Here, for the compounds / complexes of the present invention, i.e., formula (I(a)), R 5 For each compound bound to 2 and R 3 is known to exist. - n=1, R 5 Two drug moieties are attached to one compound, the DAR is 2 / 1 or 2. - n=0.5, R 5 Two drug moieties are bound to one compound, with a DAR of 2 / 0.5 or 4. - n=0.33, R 5For two drug moieties bound to one compound, the DAR is 2 / 0.33 or 6. EXAMPLES

[0053] Example 1: Synthesis of Compound 1 Scheme 2: Total synthesis of compound 1 [ka] JPEG2024532677000019.jpg238150

[0054] Experimental procedure [ka] Compound 4: Compound 2 (see E. Danieli, D. Shabat, Bioorg. Med. Chem. 2007, 15, 7318-7324; RJ Amir, E. Danieli, D. Shabat, Chem. - A Eur. J. 2007, 13, 812-821; and A. Gopin, S. Ebner, B. Attali, D. Shabat, Bioconjug. Chem. 2006, 17, 1432-1440) (1.07 g, 2.32 mmol) was dissolved in dry DMF (5 mL) under argon atmosphere and cooled to 0° C. Sodium hydride (97 mg, 2.44 mmol) was added and the reaction was allowed to warm to room temperature. After stirring at room temperature for 15 min, compound 3 (ME Roth-Konforti, CR Bauer, D. Shabat, Angew. Chemie Int. Ed. 2017, 56, 15633-15638) (1.65 g, 2.32 mmol) was added and the reaction was monitored by TLC (MeOH:EtOAc 5:95). Upon completion, the reaction was diluted with EtOAc (20 mL) and NH4Cl (10 mL). The biphasic mixture was then extracted with EtOAc (3×20 mL), washed with brine (20 mL), dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel to give the title compound 4 (1.61 g, 67%) as an off-white solid. MS (ES+) m / z C 57 H 78 Calculated for N6O9Si2: 1046.5, [M+2H] + Actual measured value: 1048.9.

[0055] [ka] Compound 6: Compound 4 (1.2 g, 1.15 mmol) was dissolved in DMF (6 mL) and diethylamine (2 mL) was added. The reaction was monitored by TLC. Once the starting material disappeared, the solvent and diethylamine were removed under reduced pressure. The product was dried under vacuum and used directly for the next reaction.

[0056] The crude material was dissolved in DMF (5 mL) and compound 5 (327 mg, 1.15 mmol) was added. The mixture was stirred at room temperature for 3 h and the reaction was monitored by TLC. Upon completion, the solvent was removed under reduced pressure. The crude product was purified by column chromatography on silica gel to give the title compound 6 (630 mg, 55% for two steps) as an off-white solid. MS (ES+) m / z C 50 H 79 N7O 10 Calculated for Si2: 993.5, [M+2H] + Actual measured value: 995.0.

[0057] [ka] Compound 7: Compound 6 (625 mg, 0.63 mmol) was dissolved in MeOH (4 mL) and 4-toluenesulfonic acid monohydrate (PTSA, 12 mg, 0.063 mmol) was added. The reaction was monitored by TLC. Upon completion, the reaction mixture was diluted with EtOAc (20 mL) and saturated NaHCO3 (5 mL) was added. The biphasic mixture was then extracted with EtOAc (3 x 20 mL), washed with brine (20 mL), dried over Na2SO4, and evaporated under reduced pressure. The crude product was dissolved in dry THF / DMSO solvent system (8 mL, THF:DMSO 10:1) and then DIPEA (0.45 mL, 2.52 mmol) and 4-DMAP (4 mg, 0.03 mmol) were added. The mixture was cooled to 0 °C. 4-Nitrophenyl chloroformate (PNP-Cl, 317 mg, 1.58 mmol) was then added in portions and the reaction was stirred at RT for 2 h. The progress of the reaction was monitored by TLC. Upon completion, the solution was loaded directly onto silica gel and the product was purified by column chromatography to give compound 7 (289 mg, 42% for two steps) as an off-white solid. MS (ES+) m / z C 52 H 57 N9O 18 Calculated value for: 1095.4, [M+2H] +Found value for compound 7: 1096.9. Analytical RP-HPLC: Column C18 5μ, 250×4.6 mm. Eluent: ACN / H2O (H2O with 0.1% TFA). Method: 30-100% ACN gradient. R :15.1 minutes.

[0058] [ka] Compound 8: Compound 7 (80 mg, 0.07 mmol) was dissolved in dry DMF (2 mL) at RT. Exatecan mesylate (77 mg, 0.14 mmol) was added followed by Et3N (60 μL, 0.44 mmol). The reaction mixture was stirred for 2 days and the reaction progress was monitored by RP-HPLC. Once the starting material had disappeared, the product was precipitated by adding MeOH (5 mL). The precipitate was filtered, washed repeatedly with EtOAc and dried under vacuum to give compound 8 (76 mg, 62%) as a yellow solid. MS (ES+) m / z C 88 H 91 F2N 13 O 20 Calculated value for: 1687.6, [M+2H] + Found value for t: 845.5. Analytical RP-HPLC: Column C18 5μ, 250×4.6 mm. Eluent: ACN / H2O (H2O with 0.1% TFA). Method: 30-100% ACN gradient. t for compound 8 R :11.2 minutes.

[0059] [ka] Compound 10: Compound 8 (65 mg, 0.04 mmol) and azide 9 (86 mg, 0.2 mmol) were dissolved in the solvent system DMSO / HO (10:1, 2 mL) followed by the addition of CuSO 5HO (5 mg, 0.02 mmol) and sodium ascorbate (3.2 mg, 0.03 mmol). The solution was degassed with argon for 30 min and then stirred at room temperature. After stirring for 30 min, the progress of the reaction was monitored by RP-HPLC. Upon completion, the product was isolated by reverse phase preparative HPLC (30-100% ACN in water with 0.1% TFA, 20 min) to give compound 10 (64 mg, 79%) as a yellow solid. MS (ES+) m / z C 103 H 112 F2N 18 O 30 Calculated value for: 2119.9, [M / 2+H] + Found value for compound 10: 1061.4. Analytical RP-HPLC: Column C18 5μ, 250×4.6 mm. Eluent: ACN / H2O (H2O with 0.1% TFA). Method: 30-100% ACN gradient. R :10.7 minutes.

[0060] [ka] Compound 11: Compound 10 (52 mg, 0.025 mmol) was dissolved in DMF (2 mL) under argon, then Pd(PPh3)4 (15 mg, 0.012) and 1,3-dimethylbarbituric acid (8.0 mg, 0.05 mmol) were added. The mixture was stirred under argon at 45 °C, and the progress of the reaction was monitored by RP-HPLC. After 30 min, the HPLC chromatogram shows that about 50% conversion had occurred. Therefore, another batch of Pd(PPh3)4 and 1,3-dimethylbarbituric acid was added. After 2 h reaction was complete, the product was isolated by reverse phase preparative HPLC (10-90% ACN in water with 0.1% TFA, 20 min) to give compound 11 (30 mg, 61%) as a yellow solid. MS (ES+) m / z C 99 H 108 F2N 18 O 28 Calculated value for: 2035.7, [M / 2+H]+ Found value for t: 1019.2. Analytical RP-HPLC: Column C18 5μ, 250×4.6 mm. Eluent: ACN / H2O (H2O with 0.1% TFA). Method: 10-90% ACN gradient. t for compound 11 R :12.3 minutes.

[0061] [ka] Compound 1: Compound 11 (25 mg, 0.012 mmol) and compound 12 (4 mg, 0.014 mmol) were dissolved in dry DMF (1.5 mL). Et3N was added and the reaction mixture was stirred at room temperature for 1 h. Upon completion as monitored by RP-HPLC, the product was purified by reverse-phase preparative HPLC (10-90% ACN in water with 0.1% TFA, 20 min) to give the final prodrug 1 (19 mg, 71%) as a yellow solid. MS (ES+) m / z C 106 H 113 F2N 19 O 31 Calculated value for: 2186.79, [M / 2+H] + Found value for t: 1094.8. Analytical RP-HPLC: Column C18 5μ, 250×4.6 mm. Eluent: ACN / H2O (H2O with 0.1% TFA). Method: 10-90% ACN gradient. t for prodrug 1 R :13.2 minutes.

[0062] Scheme 3: Synthesis of compound 4 [ka] Compound 4: Compound 13 (PD Jeffrey, SW McCombie, J. Org. Chem. 1982, 47, 587-590) (750 mg, 4.0 mmol) was dissolved in DCM (8 mL) and cooled to 0° C. N-hydroxysuccinimide (692 mg, 6.0 mmol) was added followed by N,N′-dicyclohexylcarbodiimide (908 mg, 4.4 mmol). The reaction was allowed to warm to room temperature and stirring was continued for 4 h. Upon completion, the cloudy mixture was filtered and washed with DCM. The combined organic solution was concentrated and the product was purified by silica gel column chromatography to give compound 4 (1.08 g, 95%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 5.95-5.85 (m, 1H), 5.28 (dd, J = 17.2, 1.1 Hz, 1H), 5.19 (dd, J = 10.4, 1.1 Hz, 1H), 4.54 (d, J = 5.2 Hz, 2H), 3.28 (q, J = 6.5 Hz, 2H), 2.82 (s, 4H), 2.66 (t, J = 7.3 Hz, 2H), 1.99-1.92(m, 2H). MS(ES+)m / z C 12 H 16 Calculated for N2O6: 284.1, [M+H] + Actual measured value: 285.3.

[0063] Scheme 4: Synthesis of compound 9 [ka] Compound 16: Compound 16 was prepared according to literature procedures (E. Roussakis, Z. Li, NH Nowell, AJ Nichols, CL Evans, Angew. Chemie Int. Ed. 2015, 54, 14728-14731). L-azidoglutamic acid 14 (J. Bachl, J. Mayr, FJ Sayago, C. Cativiela, D. Diaz Diaz, Chem. Commun. 2015, 51, 5294-5297) (222 mg, 1.3 mmol) was dissolved in dry DMF (10 mL) under argon atmosphere. HBTU (1.23 g, 3.25 mmol) was added to the solution and the mixture was stirred at room temperature for 5 min. DIPEA (2.30 mL, 13.0 mmol) was added to the solution in one portion, followed immediately by L-glutamic acid di-tert-butyl ester 15 (760 mg, 2.6 mmol). The reaction mixture was stirred overnight, after which the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography to give compound 16 (597 mg, 71% relative to 14) as a gummy liquid. MS (ES+) m / z C 31 H 53 N5O 10 Calculated value for: 655.4, [M+H] + Actual measured value: 656.7.

[0064] [ka] Compound 9: Compound 16 (550 mg, mmol) was dissolved in DCM (3 mL) and TFA (3 mL) was added. The reaction was stirred overnight. Upon completion as monitored by TLC, the solvent and TFA were removed under reduced pressure. Product formation was confirmed by MS and the crude material was used directly in the click reaction. MS (ES+) m / z C 15 H 21 N5O 10 Calculated value for: 431.1, [M+H] + Actual measured value: 432.3.

[0065] Example 2: Synthesis of Compound 20 Scheme 5: Total synthesis of compound 20 [ka] JPEG2024532677000031.jpg108164 Compound 20 was synthesized according to the above scheme.

[0066] Example 3: Synthesis of Compound 21 Scheme 6: Total synthesis of compound 21 [ka] JPEG2024532677000033.jpg203164 JPEG2024532677000034.jpg150164 Compound 21 was synthesized according to the above scheme and confirmed by mass spectrometry.

[0067] Example 4: Trastuzumab-exatecan / belotecan conjugate inhibits proliferation of HER2-positive HCC1954 cells material and method cell line HCC1954 and MDA-MB-468 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC1954 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 U / mL nystatin, 2 mM L-glutamine, and 100 μg / mL sodium pyruvate. MDA-MB-468 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 U / mL nystatin, and 2 mM L-glutamine. Cells were grown at 37°C in 5% CO2.

[0068] Cell viability assay Human breast cancer HCC1954 and MDA-MB231 cells were seeded onto 24-well culture plates (5,000 cells / well and 10,000 cells / well, respectively) and incubated for 24 h. Cells were then exposed to serial dilutions of exatecan or belotecan, either free or combined with trastuzumab. Cell viability was assessed using MTT (3 μg / mL, Sigma) over the following 5-6 days of incubation. MTT absorbance was measured at 570 nm using a SpectraMax M5e multidetector reader.

[0069] result Trastuzumab-exatecan / belotecan conjugate inhibits proliferation of HER2-positive HCC1954 cells Several batches of trastuzumab conjugated to exatecan or belotecan were obtained from ITL (Table 1). The different conjugates were evaluated for their inhibitory effect compared to the free drug on the proliferation of two human breast cancer cell lines, HCC1954 (HER2-positive) and MDA-MB-468 (HER2-negative) cells. Belotecan and exatecan showed similar IC 50 The conjugates showed potent cytotoxicity values ​​of 7 and 10 nM in HCC1954; 5 and 2.5 nM in MDA-MB-468. However, whereas the cytotoxicity of the conjugates was similar to that of the free drugs in HCC1954 cells (except for Trans-Bel PPB-4435), in MDA-MB-468 cells the conjugates were slightly less active. Trastuzumab had no inhibitory effect on the proliferation of both cell lines (Figures 1A-1D).

[0070] [Table 1]

[0071] Trans-Exa-1 was selected for further evaluation due to its enhanced cytotoxicity. To evaluate whether increasing exposure time of cells to the conjugate would result in enhanced activity compared to free exatecan, cells were exposed to the drug for an additional 24 hours (6 days total). Also, to further demonstrate the benefit of the conjugate in HER2-positive cells, another HER2+ breast cancer cell line, JIMT-1, was evaluated. In correlation with the previous experiment, HCC1954 cells were significantly more sensitive to Tras-Exa-1 compared to MDA-MB-468. Tras-Exa-1 did not show any advantage compared to free exatecan in JIMT-1 cells (Figures 2A-2D).

[0072] Example 5: Trastuzumab-Exatecan / Belotecan Conjugate Tumor Growth Compared to Commercially Available Enhertu® material and method cell line HCC1954 human breast cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, 12.5 U / mL nystatin, 2 mM L-glutamine, and 100 μg / mL sodium pyruvate. Cells were grown at 37°C in 5% CO2.

[0073] In vivo testing All animals were housed in the Tel Aviv University specific pathogen-free (SPF) animal facility. Experiments were approved by the animal care and use committee (IACUC) of Tel Aviv University (approval number 01-19-088) and performed in accordance with NIH guidelines.

[0074] HCC1954 cells (1 x 10 6) was injected into the mammary fat pad of 6-week-old female severe combined immunodeficiency (SCID) mice. Tumor growth was monitored by calvarial and the volume was calculated as 0.52 × width × length. 2 Tumor volume was defined as approximately 95 mm 3 Once tumor size reached 1000 mm, mice were randomized into groups (n=9) and injected intravenously with a single dose of trastuzumab-deruxtecan (Enhertu®; DAR=8), trastuzumab-exatecan (Tras-Exa; DAR=4) or trastuzumab-belotecan (Tras-Bel; DAR=4) at three different concentrations (1, 3 or 10 mg / kg). As controls, mice were treated with either rituximab-deruxtecan (Ritux-DXd; DAR=8) at 10 mg / kg or vehicle control (10 mM histidine, 3% trehalose, 100 mM NaCl, pH 5.5). All treatments were administered in a volume of 200 μl / 20 g mouse. Tumor volumes of 1000 mm were established. 3 Mice were euthanized when the tumor reached 0.1 mg / kg / day, when the tumor became necrotic and ulcerated, or when the mice lost 15% of their body weight.

[0075] statistical analysis Data are expressed as mean ± standard deviation (SEM) unless otherwise stated. Statistical significance in overall survival was determined by the log-rank (Mantel-Cox) test using Graphpad Prism 9 software. Statistical significance was defined as p<0.05.

[0076] result Trastuzumab-based antibody-drug conjugates (ADCs) conjugated to exatecan ("Tras-Exa"; DAR=4) or belotecan ("Tras-Bel"; DAR=4) were evaluated for their in vivo antitumor efficacy. To this end, HCC1954 (HER2+) human breast cancer xenograft-bearing mice were treated with a single dose of Tras-Exa or Tras-Bel at three concentrations (1, 3 and 10 mg / kg) and tumor growth and survival were monitored. The therapeutic efficacy of our ADCs was compared with the commercially available Enhertu® (Tras-deruxtecan; DAR=8), also administered at three concentrations (1, 3 and 10 mg / kg), and with the isotopic control of rituximab-deruxtecan ("Ritux-DXd"; DAR=8), administered only at the highest dose of 10 mg / kg. An additional control group was treated with vehicle. Dose-dependent inhibition of tumor growth was observed, with the most potent antitumor effect observed at 10 mg / kg of the three trastuzumab-based ADCs (Figures 3A-3C). Interestingly, a single dose of Tras-Exa or Enhertu® at the highest concentration (10 mg / kg) resulted in complete tumor regression. Conversely, 4 out of 9 (44%) Tras-Bel-treated mice (10 mg / kg) relapsed 60 days after treatment (Figure 3A). Furthermore, moderate antitumor activity was observed at 3 mg / kg for all ADCs, with Enhertu® showing more potent tumor growth inhibition among the various ADCs (Figure 3B). Overall, Tras-Exa and Enhertu® showed superior antitumor activity at 3 and 10 mg / kg compared to the Tras-Bel ADC, but the effects between these two ADCs were not significantly different at both concentrations. In contrast, treatment with the lowest dose (1 mg / kg) had no effect on tumor growth compared to controls for either ADC (Figure 3C).

[0077] Of note, after treatment, mice in all experimental groups, including vehicle and isotope control, showed a transient weight loss that recovered after 5 days (Figure 3D). Overall, our trastuzumab-based ADC as well as Enhertu®, similar to vehicle or isotope control, were well tolerated at all concentrations evaluated, with final positive weight gain over the course of the study (Figure 3D).

[0078] Consistent with the data presented above, Kaplan-Meier analysis showed a dose-dependent survival benefit of the various ADCs evaluated. At the end of the study (day 152), 100% of mice treated with 10 mg / kg Tras-Exa or Enhertu® and 89% of mice treated with the same concentration of Tras-Bel were alive; whereas no control-treated mice survived beyond 109 days (vehicle) and 116 days (Ritux-DXd) (Figures 4A and 4D). Furthermore, treatment with 3 mg / kg significantly extended the median survival time of mice treated with Tras-Exa (116 days) or Enhertu® (137 days) compared to vehicle control (60 days) and Ritux-DXd (71 days) (Figures 4B, 4D and Table 2). Tras-Bel (3 mg / kg) had no significant effect on mouse survival compared to controls, with a median survival of 84 days (Figures 4B and 4D and Table 2). At the lowest dose of 1 mg / kg, neither ADC showed any therapeutic effect compared to vehicle and isotopic controls (Figures 4C and 4D and Table 2).

[0079] [Table 2] JPEG2024532677000037.jpg65123

[0080] Example 6: Antibody conjugation techniques Conjugation by transglutaminase was performed to the light chain of transtuzumab by standard methods, following the procedure also described in Dickgiesser et al., Bioconjugate Chem. (2020), vol. 31(4), p. 1070-1076, using wild-type microbial transglutaminase (WT transglutaminase purchased from Zedira, Germany). For this purpose, one equivalent of antibody was mixed with a solution of the respective payload (10- or 20-fold excess over the concentration of the antibody, depending on the number of binding sites) and 6 U / ml of transglutaminase in a buffer with 150 mM NaCl, 25 mM Tris (pH 8.0). The mixture was incubated for 16 h in a thermomixer at 37 °C and 450 rpm.

[0081] For conjugation via maleimide chemistry, a 5 mg / mL solution of each antibody component in PBS (pH 7.4) was prepared (~33 μM antibody). The antibody was reduced with TCEP (tris(2-carboxyethyl)phosphine); 1:2 to 1:6 ratio of antibody component to TCEP depending on the desired DAR; TCEP was used at 2 mM stock solution, pH 7.0) or in some cases with DTT (dithiothreitol; 20 mM). After incubation in a 37°C water bath for 0.5 to 2 hours (depending on the number of cysteines to be activated), the reaction was allowed to cool to room temperature. The solution was desalted on a Sephadex G25 column into conjugation buffer (10 mM sodium phosphate, pH 6.0, 2 Mm EDTA, N2 degassed) and adjusted to an antibody concentration of 0.2 mg / mL in conjugation buffer. Conjugation was initiated by adding reduced antibody to a solution of the appropriate maleimide-activated linker payload construct at the appropriate ratio (e.g., antibody to linker payload construct ratio 1:4 to 1:8 for DAR=4; e.g., antibody to linker payload construct ratio 1:20 for DAR=8). The reaction was incubated for 1 hour with slow rocking at 22°C, after which conjugation was confirmed by LCMS. If necessary, the reaction was continued until the desired DAR was reached.

[0082] The combined samples were purified by hydrophobic interaction chromatography (HIC) on a 15PHE (phenyl) column (GE Healthcare) or a HiTrap HP or FF Butyl Sepharose column (GE Healthcare). The eluted fractions were concentrated and buffer exchanged into PBS pH 7.4 or 10 mM potassium phosphate, 200 mM NaCl, 10 mM histidine, 50 mM trehalose, pH 7.0. The identity and purity of each prepared ADC were confirmed by LC-MS and SDS-PAGE. The DAR of each prepared ADC was calculated and confirmed from HIC (hydrophobic interaction chromatography) data and mass spectrometry data.

[0083] The stability of each prepared ADC was examined by freeze-thaw experiments. Specifically, ADCs in histidine buffer were shock-frozen in liquid nitrogen to -80°C and stored at this temperature for several weeks and up to several months. After the samples were placed at room temperature until they had completely thawed, the materials were subjected to SE-HPLC (size-exclusion high performance liquid chromatography) analysis to check the degradation of the compounds and to examine the activity of the thawed ADCs. Specifically, target antigen binding was examined by an octet binding assay, and payload-mediated cytotoxicity was tested by a cell titer glow assay on positive and negative cell lines. The results for the freeze-thawed ADCs were compared to the corresponding non-freeze-thawed ADCs. In each case, the ADCs with solubility tags were found to be stable during this freeze-thaw procedure.

[0084] Endotoxin was measured by the PTS (Portable Test System) cartridge method (Nexgen) under standard conditions according to the manufacturer's instructions. For each prepared ADC, endotoxin levels were found to be <5.0 endotoxin units (EU) / mg.

[0085] While particular embodiments of the present invention have been illustrated and described, it will be apparent that the invention is not limited to the embodiments described herein. Numerous modifications, changes, variations, substitutions and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. Formula (I): 【Chemical 1】 (wherein R 1 is a reactive moiety comprising a maleimide, bromoacetamide, tetrazine, alkyne, amine moiety, or any combination thereof; R 2 and R 3 are the same or different active pharmaceutical ingredient moieties; R 4 comprises an oligo- or poly-carboxylic acid moiety or an oligo- or poly-ethylene glycol or an oligo- or poly-alcohol or an oligo- or poly-vinyl alcohol or an oligo- or poly-glycerol moiety; and X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, an oligo- or polyethylene glycol or an oligo- or polyvinyl alcohol or an oligo- or polyglycerol moiety) is a compound represented by

2. R 1 is 【Chemical 2】 【Chem.】 The compound according to claim 1, represented by

3. R 2 The compound according to claim 1, wherein R is an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

4. R 2 The compound according to claim 3, wherein R is exatecan, verotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or doxorubicin moiety.

5. R 2 The compound according to claim 4, wherein R is exatecan, auristatin, monomethyl auristatin E (MMAE) or verotecan moiety.

6. R 3 The compound according to claim 1, wherein R is an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

7. R 3 The compound according to claim 6, wherein R is exatecan, verotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or doxorubicin moiety.

8. R 3 The compound according to claim 7, wherein R is exatecan, auristatin, monomethyl auristatin E (MMAE) or verotecan moiety.

9. R 2 and R 3 The compound according to claim 1, wherein R and R are an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

10. R 2 and R 3 The compound according to claim 9, wherein each of them is independently exatecan, velotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or a doxorubicin moiety.

11. R 2 and R 3 The compound according to claim 10, wherein R and R are an exatecan, an auristatin or a velotecan moiety.

12. R 4 is [Chemical Formula 3] The compound according to claim 1, represented by

13. X 1 The compound according to claim 1, wherein X is -Val-Cit-, -Ala-Ala-, AAN, GGFF, -Val-Ala- or -Val-Arg-.

14. Formula 1, 17, 18, 19 or 20: [Chemical Formula 4] 【Chem.】 【Chem.】 (wherein TFA is trifluoroacetic acid) The compound according to claim 1, represented by the structure of

15. Formula (I(a)): [Chemical Formula 5] (wherein R 2 and R 3 are the same or different active pharmaceutical ingredient moieties; R 4 comprises an oligo- or poly-carboxylic acid moiety or an oligo- or poly-ethylene glycol or an oligo- or poly-alcohol or an oligo- or poly-vinyl alcohol or an oligo- or poly-glycerol moiety; and R 5 is an antibody or antigenic portion; X 1 is a mono-, di-, tri-, tetra-, oligo- or polypeptide moiety, oligo- or polyethylene glycol, or oligo- or polyvinyl alcohol, or oligo- or polyglycerol moiety; X 2 is a linker comprising a succinimide, acetamide, dihydropyridazine, alkene or amine moiety or any combination thereof; and n is a number between 0.01 and 10) is a complex represented by

16. R 2 The conjugate according to claim 15, wherein R is an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

17. R 2 The conjugate according to claim 16, wherein R is exatecan, verotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or a doxorubicin moiety.

18. R 2 The conjugate according to claim 17, wherein R is exatecan, auristatin, monomethyl auristatin E (MMAE) or verotecan moiety.

19. R 3 The conjugate according to claim 15, wherein R is an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

20. R 3 The conjugate according to claim 19, wherein R is exatecan, velotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or a doxorubicin moiety.

21. R 3 The conjugate according to claim 20, wherein R is exatecan, auristatin, monomethyl auristatin E (MMAE) or verotecan moiety.

22. R 2 and R 3 The conjugate according to claim 15, wherein R and R are an anticancer agent moiety, an immunostimulant moiety or an immunosuppressant moiety.

23. R 2 and R 3 The conjugate according to claim 22, wherein each of R and R is independently exatecan, verotecan, camptothecin, auristatin, monomethyl auristatin E (MMAE) or a doxorubicin moiety.

24. R 2 and R 3 The conjugate according to claim 23, wherein R and R are an exatecan, an auristatin, a monomethyl auristatin E (MMAE) or a verotecan moiety.

25. R 4 is [Chemical Formula 6] The complex according to claim 15, represented by

26. X 1 The complex according to claim 15, wherein X is -Val-Cit-, -Ala-Ala-, AAN, GGFFG, -Val-Ala- or -Val-Arg-.

27. X 2 is 【Chemical Formula 7】 The complex according to claim 15, represented by

28. R 5 The conjugate according to claim 15, wherein R is an anti-EGF or anti-CD33 antibody.

29. R 5 The conjugate according to claim 15, wherein R is trastuzumab or rituximab or the CGKRK moiety.

30. R 5 The conjugate according to claim 29, wherein R is the trastuzumab moiety.

31. 1(a) or 2(a): [Chemical Formula 8] The complex according to claim 15, represented by the structure of

32. R 5 The conjugate according to claim 31, wherein R is a trastuzumab moiety or a rituximab moiety.

33. R 5 The conjugate according to claim 32, wherein R is the trastuzumab moiety.

34. Compound 21: 【Chemical Formula 9】 The complex according to claim 15, represented by the structure of

35. The complex according to claim 15, wherein the drug-to-antibody ratio (DAR) is between 0.5 and 200

36. The complex according to claim 15, wherein n is between 0.1 and 1

37. A medicament for the treatment of cancer, comprising the compound according to any one of claims 1 to 14, or the complex according to any one of claims 15 to 36

38. The medicament according to claim 37, wherein the cancer is breast cancer, kidney cancer, brain tumor, skin cancer, ovarian cancer, lung cancer, pancreatic cancer, colon cancer, head and neck cancer, prostate cancer, endometrial cancer, liver cancer, blood cancer, stomach cancer, fibrosarcoma, bone tumor, osteosarcoma or duodenal cancer

39. i) a compound according to any one of claims 1 to 14; and ii) an antibody or antigen A kit comprising

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