Soluble tagged molecules and related methods

Abstract

The present invention relates to molecules having a soluble tag and methods for increasing the solubility of molecules. Additionally, the present invention relates to antibody-drug conjugates having a soluble tag, methods and compounds for preparing such antibody-drug conjugates, methods for increasing the solubility of antibody-drug conjugates, antibody-drug conjugates prepared by such methods, and the use of such antibody-drug conjugates in medicine.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority from European Patent Application EP20196705.6, filed September 17, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing which has been provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 16, 2021, is entitled P20-059_WO-PCT_SL.txt, and is 6,527 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to molecules having solubility tags and methods for improving the solubility of molecules. Furthermore, the present invention relates to antibody-drug conjugates provided with solubility tags, methods and compounds for preparing such antibody-drug conjugates, methods for improving the solubility of antibody-drug conjugates, antibody-drug conjugates prepared by such methods, and the use of such antibody-drug conjugates in medical treatments. [Background technology]

[0004] Background of the Invention Highly specialized chemical molecules are essential elements in many important applications of modern life, for example, in clinical diagnosis, in medical treatment or as research tools.However, often the choice of which molecules can be used for these applications is severely limited because poor solubility prevents the use of molecules that would otherwise be perfectly suited for a specific purpose.Therefore, frequently and in many technical fields, there arises a need to create molecules with improved solubility or to improve the solubility of specific molecules.

[0005] A classic example of this problem is antibody-drug conjugates (ADCs) in medical diagnostics and treatment. In recent years, ADCs have emerged as a new class of biologics that allow combining the specificity of antibodies with the efficacy of small molecule therapeutics.

[0006] Structurally, ADCs are cell-targeting conjugates that typically contain three covalently linked main components: (i) an antibody component, (ii) a linker, and (iii) a drug ("payload"). Upon administration to a patient, the antibody component guides the ADC to its target antigen, target cell, or desired site in the body via specific binding of the antibody component. Typically, the ADC is then internalized into the cell, for example, by receptor-mediated endocytosis. The drug is then released, for example, by protease- or pH-dependent linker cleavage or antibody degradation. The released drug then performs its therapeutic function within the cell.

[0007] While non-targeted drugs typically reach their site of action through systemic distribution and passive diffusion, ADCs are not uniformly distributed throughout the body. Rather, ADCs are primarily concentrated at their site of action through the interaction of the antibody component with its target antigen. As a result, ADCs require smaller dosages while still allowing the drug to reach therapeutically effective levels within target cells, thus improving the therapeutic window. Targeting through the formation of ADCs is therefore a powerful method for enhancing specificity, reducing the systemic toxicity of drugs, and enabling the therapeutic use of drugs that are less suitable or inappropriate as systemic drugs.

[0008] The suitability of an ADC for clinical use depends on multiple factors, including the selection of appropriate target antigen and antibody components, linker design and stability, and the use of a drug payload with appropriate characteristics and efficacy.

[0009] Another important factor is the overall solubility of the ADC. Common drug payloads, such as camptothecin and duocarmycin, are often hydrophobic, significantly reducing the solubility of the ADC molecule. Therefore, newly developed ADCs often have solubility issues and are prone to aggregation and particle formation. This, in turn, means that only ADCs with low drug-to-antibody ratios (DARs) can be synthesized, while the synthesis of ADCs with large or even more hydrophobic payloads is completely impossible. Even when synthesis is possible, reduced solubility and aggregation tendency still complicate analytical characterization of the ADC, hinder efficient production with high reproducibility, and reduce long-term stability. In clinical applications, low solubility of ADCs often leads to reduced efficacy, significant side effects, and a narrow therapeutic window that are unacceptable for patient safety reasons.

[0010] In light of these issues, lack of solubility is a significant risk in the development of new ADCs, raising costs and often leading to project termination.

[0011] The solubility of ADCs can be improved, to some extent, by selecting a more hydrophilic linker or payload. However, such compromises limit ADC design options and do not allow for the adjustment and fine-tuning of the drug-to-antibody ratio necessary for ADC optimization. Therefore, this approach often results in ADCs with inferior therapeutic characteristics.

[0012] Alternatively, incorporating PEG chains of various lengths into the linker is a common approach to addressing solubility issues in the ADC landscape (e.g., Simmons et al., Toxicology and Applied Pharmacology (2020), vol. 392, pp. 114932; Tiberghien, Organic Process Research & Development (2018), vol. 22(9), pp. 1241-1256). However, administration of ADCs with PEG chains often results in the development of anti-PEG antibodies, which counteract the positive effect of PEG chains on solubility and often leads to an immune response against the ADC (Thi et al., Polymers (2020), vol. 12(2), pp. 298; Hong, Journal of Pharmacological and Toxicological Methods (2020), vol. 102, pp. 106678).

[0013] Thus, there is a need in the art for improved methods of improving the solubility of ADCs. Furthermore, there is a need in the art for improved methods of reducing / preventing aggregation of ADCs. Furthermore, there is a need in the art for improved methods of increasing the drug-to-antibody ratio (DAR) of ADCs. Furthermore, there is a need in the art for improved ADCs, particularly those with longer serum half-lives, improved pharmacokinetics, greater efficacy, fewer side effects, reduced toxicity, reduced immunogenicity, a larger therapeutic window, and / or improved patient safety. Furthermore, there is a need in the art for improved methods of achieving optimization of the clinical characteristics of ADCs (e.g., pharmacokinetics, efficacy, side effects, therapeutic window, patient safety). Furthermore, there is a need in the art for approaches that allow for the formation of ADCs from payloads that are too hydrophobic to otherwise produce ADCs. Furthermore, there is a need in the art for approaches that allow for the formation of ADCs with multiple hydrophobic payloads that cannot otherwise be produced due to solubility and / or aggregation issues. Furthermore, there is a need in the art for methods that address the above needs with a "standardized" approach that can be broadly used for a variety of ADCs. Furthermore, there is a need in the art for methods that address these needs with a modular approach that is flexible enough to accommodate a variety of ADCs and hydrophobicities of a particular payload, allowing for adjustment and fine-tuning of the drug-to-antibody ratio, and that address these needs with an approach that allows rapid synthetic access.

[0014] Similar needs to those outlined above exist for other types of molecules. Summary of the Invention [Problem to be solved by the invention]

[0015] The present invention solves the above problems and addresses the above needs. [Means for solving the problem]

[0016] Summary of the Invention The present invention addresses the needs set forth above in the "Background of the Invention" section through the following various aspects and embodiments.

[0017] The present invention is based, in part, on the surprising observation that covalent attachment of the oligosaccharide-based solubility tags described herein to molecules, such as antibody-drug conjugates, can confer a variety of beneficial effects on the molecule. For example, as shown with ADCs, the beneficial effects can include, but are not limited to, improved solubility, reduced aggregation, access to molecules that would otherwise be unavailable due to solubility issues, access to high drug-to-antibody ratios, and improved clinical characteristics. Furthermore, it has been found that this approach is not limited to a particular structure, but rather can be used broadly and as a modular approach, allowing for flexible adaptation to, for example, specific ADCs.

[0018] In certain embodiments, the present invention relates to molecules comprising a targeting moiety and at least one soluble tag.

[0019] In another embodiment, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety and at least one soluble tag.

[0020] In another aspect, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety, one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, and at least one solubility tag.

[0021] In certain embodiments, the at least one functional moiety is a payload that is a therapeutic agent or a detectable label. In certain embodiments, the soluble tag comprises at least 3 and up to 12 monosaccharide units.

[0022] In certain embodiments, the one or more soluble tags are covalently attached to the at least one payload and / or the linker.

[0023] In some embodiments, the monosaccharide units are independently selected from the group consisting of aldoses, ketoses, and chemically modified forms of the aldoses or ketoses. In some embodiments, the monosaccharide units are independently selected from the group consisting of tetroses, pentoses, hexoses, and chemically modified forms of the tetroses, pentoses, and hexoses, wherein the tetroses are independently selected from the group consisting of erythrose and threose, the pentoses are independently selected from the group consisting of ribose, arabinose, xylose, and lyxose, and the hexoses are independently selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose, and talose.

[0024] In some embodiments, the soluble tag comprises or consists of a chitooligosaccharide, hi some embodiments, the chitooligosaccharide is a chitooligosaccharide having 3 to 7 monosaccharide units selected from Table 1 below.

[0025] In certain embodiments, the soluble tag comprises or is a chemical group having a structural formula selected from the group consisting of structural formulas (I)-(IV) defined below.

[0026] In certain embodiments, the targeting moiety is selected from the group consisting of a protein, a peptide, a peptidomimetic, a nucleic acid, an oligonucleotide, and a small molecule.

[0027] In certain embodiments, the antibody is an antibody to an antigen present on the surface of a target cell, or an antigen-binding fragment of such an antibody.

[0028] In certain embodiments, the targeting moiety specifically binds to a tumor antigen.

[0029] In certain embodiments, the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent, or an immunosuppressant.

[0030] In certain embodiments, the / each of the linkers has a molecular weight of up to 1,500 Da.

[0031] In another embodiment, the present invention relates to a molecule consisting of a targeting moiety and at least one soluble tag.

[0032] In another embodiment, the present invention relates to a molecule consisting of a targeting moiety, at least one functional moiety and at least one soluble tag.

[0033] In another aspect, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety, one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, and at least one soluble tag.

[0034] In another aspect, the invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0035] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety and at least one functional moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0036] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of the functional moieties to the targeting moiety, comprising covalently linking at least one solubility tag to the molecule.

[0037] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0038] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety and at least one functional moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0039] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, comprising covalently linking at least one solubility tag to said molecule.

[0040] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0041] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety and at least one functional moiety, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0042] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound, wherein the chemical compound comprises a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of the functional moieties to the targeting moiety, wherein the method comprises producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0043] In certain embodiments, the at least one functional moiety is a payload that is a therapeutic agent or a detectable label. In certain embodiments, the soluble tag comprises at least 3 and up to 12 monosaccharide units.

[0044] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0045] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety and at least one functional moiety, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0046] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, comprising producing a molecule in which said chemical compound is covalently linked to at least one solubility tag.

[0047] In another aspect, the invention relates to the use of soluble tags to enhance the solubility of molecules that include targeting moieties.

[0048] In another embodiment, the invention relates to the use of a soluble tag to enhance the solubility of a molecule comprising a targeting moiety and at least one functional moiety.

[0049] In another aspect, the invention relates to the use of a soluble tag to enhance the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of the functional moieties to the targeting moiety.

[0050] In another aspect, the invention relates to the use of soluble tags to enhance the solubility of molecules comprising a targeting moiety.

[0051] In another embodiment, the present invention relates to the use of a soluble tag to enhance the solubility of a molecule comprising a targeting moiety and at least one functional moiety.

[0052] In another aspect, the invention relates to the use of soluble tags to enhance the solubility of molecules comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of the functional moieties to the targeting moiety.

[0053] In certain embodiments, the present invention relates to antibody-drug conjugates comprising: (i) an antibody component; (ii) at least one payload that is a therapeutic agent or a detectable label; (iii) one or more linkers that covalently link the one or more payloads to the antibody component; and (iv) at least one solubility tag.

[0054] In another aspect, the present invention relates to an antibody-drug conjugate comprising: (i) an antibody component; (ii) at least one payload that is a therapeutic agent or a detectable label; (iii) one or more linkers that covalently link the one or more payloads to the antibody component; and (iv) at least one solubility tag.

[0055] In another aspect, the present invention relates to a method for improving the solubility of an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, the method comprising covalently attaching at least one solubility tag to the antibody-drug conjugate.

[0056] In another aspect, the present invention relates to a method for improving the solubility of an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, the method comprising covalently attaching at least one solubility tag to the antibody-drug conjugate.

[0057] In another aspect, the present invention relates to a method for improving the solubility of an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, the method comprising preparing the antibody-drug conjugate in a form in which the antibody-drug conjugate is covalently linked to at least one solubility tag.

[0058] In another aspect, the present invention relates to a method for improving the solubility of an antibody-drug conjugate comprising an antibody component, (ii) at least one payload which is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, the method comprising preparing the antibody-drug conjugate in a form in which the antibody-drug conjugate is covalently linked to at least one solubility tag.

[0059] In another aspect, the present invention relates to a method for improving the solubility of a chemical compound comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag, wherein the molecule is an antibody-drug conjugate.

[0060] In another aspect, the present invention relates to a method for improving the solubility of a chemical compound comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, the method comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag, wherein the molecule is an antibody-drug conjugate.

[0061] In another aspect, the present invention relates to a method for improving the solubility of a molecule comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, comprising preparing the molecule in a form in which the molecule is covalently linked to at least one solubility tag, thus resulting in an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, (iii) one or more linkers covalently linking the one or more payloads to the antibody component, and (iv) at least one solubility tag.

[0062] In another aspect, the present invention relates to a method for improving the solubility of a molecule comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the one or more payloads to the antibody component, wherein the method comprises preparing the molecule in a form in which the molecule is covalently linked to at least one solubility tag, thus resulting in an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, (iii) one or more linkers covalently linking the one or more payloads to the antibody component, and (iv) at least one solubility tag.

[0063] In another aspect, the present invention relates to the use of a soluble tag to enhance the solubility of an antibody-drug conjugate. In certain embodiments, the use comprises covalently binding the soluble tag to the antibody-drug conjugate.

[0064] In certain embodiments of the use, the antibody-drug conjugate comprises (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers that covalently link the one or more payloads to the antibody component.

[0065] In certain embodiments of the use, the antibody-drug conjugate comprises (i) an antibody component, (ii) at least one payload that is a therapeutic agent or a detectable label, and (iii) one or more linkers that covalently link the one or more payloads to the antibody component.

[0066] In another aspect, the invention relates to a method of making an antibody-drug conjugate as defined herein, comprising the steps of: performing a reaction that results in the formation of a covalent bond between (a) a molecule comprising an antibody component as defined herein, a payload as defined herein and a linker as defined herein, and (b) a solubility tag as defined herein; or performing a reaction that results in the formation of a covalent bond between (a) an antibody component as defined herein and a molecule comprising (b) a payload as defined herein, a linker as defined herein and a solubility tag as defined herein; or performing a reaction that results in the formation of a covalent bond between (a) an antibody component as defined herein, a linker as defined herein and a solubility tag as defined herein, and (b) a payload as defined herein; or performing a reaction that results in the formation of a covalent bond between (a) a molecule comprising an antibody component as defined herein and a linker as defined herein and a solubility tag as defined herein, and (b) a payload; or performing a reaction that results in the formation of a covalent bond between (a) an antibody component as defined herein and a linker as defined herein, and a molecule comprising (b) a solubility tag as defined herein and a payload as defined herein, to produce an antibody-drug conjugate having a covalently attached solubility tag.

[0067] In another aspect, the present invention relates to a compound for use in the preparation of an antibody-drug conjugate according to the invention, the compound comprising a soluble tag as defined herein attached to an activator group.

[0068] In another aspect, the invention relates to an antibody-drug conjugate prepared by the method of the invention.

[0069] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody-drug conjugate of the present invention or an antibody-drug conjugate prepared by a method of the present invention.

[0070] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent and / or excipient.

[0071] In another embodiment, the present invention relates to an antibody-drug conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament or for use in the treatment of a disease as defined below.

[0072] In another aspect, the present invention relates to a method for treating a disease in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention.

[0073] In another aspect, the present invention relates to the use of an antibody-drug conjugate of the present invention or of a pharmaceutical composition of the present invention for the manufacture of a medicament, preferably for the treatment of a disease or disorder as defined below. [Brief explanation of the drawings]

[0074] Reference is made below to the figures: All methods referred to in the figure descriptions below were carried out as detailed in the Examples.

[0075] [Figure 1] Figure 1A shows the mean body weight curves of subcutaneous SK-OV-3 tumor-bearing BALB / c nude mice obtained in Example 8. Each point represents the mean body weight recorded per group. Animals were randomized and treated on D31. D97 was the last day of the study. Values ​​are shown for all time points at which at least 80% of the animals in each group were still present. "Q1Dx1" indicates that the treatment was administered in one single dose (administered on D31). Figure 1A shows the mean body weight curves of Group 1 (control) and Groups 2, 3, and 4. Figure 1B shows the mean body weight curves of Group 1 (control) and Groups 5, 6, and 7. Figure 1C shows the mean body weight curves of Group 1 (control) and Groups 8, 9, and 10.

[0076] [Figure 2]Figure 2 shows median body weight curves for BALB / c nude mice bearing subcutaneous SK-OV-3 tumors obtained in Example 8. Each point represents the median body weight recorded per group. Values ​​are shown for all time points at which at least 80% of the animals in each group were still present. Figure 2A shows median body weight curves for Group 1 (control) and Groups 2, 3, and 4. Figure 2B shows median body weight curves for Group 1 (control) and Groups 5, 6, and 7. Figure 2C shows median body weight curves for Group 1 (control) and Groups 8, 9, and 10.

[0077] [Figure 3] Figure 3 shows the mean tumor volume curves of BALB / c nude mice bearing subcutaneous SK-OV-3 tumors obtained in Example 8. Each point represents the mean tumor volume recorded per group. Values ​​are shown for all time points when at least 80% of the animals in each group were still present. Figure 3A shows the mean tumor volume curves for Group 1 (control) and Groups 2, 3, and 4. Figure 3B shows the mean tumor volume curves for Group 1 (control) and Groups 5, 6, and 7. Figure 3C shows the mean tumor volume curves for Group 1 (control) and Groups 8, 9, and 10.

[0078] [Figure 4] Figures 4A and 4B show median tumor volume curves for BALB / c nude mice bearing subcutaneous SK-OV-3 tumors obtained in Example 8. Values ​​are shown for all time points at which at least 80% of the animals in each group were still present. Figure 4A shows median tumor volume curves for Group 1 (control) and Groups 2, 3, and 4. Figure 4B shows median tumor volume curves for Group 1 (control) and Groups 5, 6, and 7. Figure 4C shows median tumor volume curves for Group 1 (control) and Groups 8, 9, and 10.

[0079] [Figure 5] 1 shows a summary of tumor volumes in BALB / c nude mice bearing subcutaneous SK-OV-3 tumors at D59 in Example 8.

[0080] [Figure 6]Figure 6 shows tumor growth inhibition (T / C%) in treated BALB / c nude mice bearing subcutaneous SK-OV-3 tumors in Example 8. Group 1 was used as a control. After D83, further calculations were not possible because fewer than four mice remained in the control group. Figure 6A shows tumor growth inhibition (T / C%) in Group 1 (control) and Groups 2, 3, and 4. Figure 6B shows tumor growth inhibition (T / C%) in Group 1 (control) and Groups 5, 6, and 7. Figure 6C shows tumor growth inhibition (T / C%) in Group 1 (control) and Groups 8, 9, and 10. DETAILED DESCRIPTION OF THE INVENTION

[0081] Detailed Description of Certain Embodiments Although the present invention is described in detail above and below, it is to be understood that the invention is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0082] Below, certain elements of the present invention are described in more detail, including descriptions of specific embodiments. However, the various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. The description should be construed as supporting and embracing the explicitly described embodiments and embodiments combining the disclosed and / or preferred elements in any number and in any manner. Furthermore, any permutation and combination of all described elements herein should be construed as being disclosed by the description herein, unless a logical contradiction arises or the context indicates otherwise.

[0083] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques referred to herein, such as those of organic chemistry, chemical synthesis, biology, medicinal and pharmaceutical chemistry, medicine, pharmacology, or toxicology, are well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the cited references and throughout the specification, unless otherwise specified.

[0084] According to one embodiment, the present invention relates to a molecule comprising a targeting moiety and at least one soluble tag.

[0085] In another embodiment, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety and at least one soluble tag.

[0086] In another aspect, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety, one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, and at least one solubility tag.

[0087] In another embodiment, the present invention relates to a molecule consisting of a targeting moiety and at least one soluble tag.

[0088] In another embodiment, the present invention relates to a molecule consisting of a targeting moiety, at least one functional moiety and at least one soluble tag.

[0089] In another aspect, the present invention relates to a molecule comprising a targeting moiety, at least one functional moiety, one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, and at least one soluble tag.

[0090] In another aspect, the invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0091] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety and at least one functional moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0092] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of the functional moieties to the targeting moiety, comprising covalently linking at least one solubility tag to the molecule.

[0093] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0094] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety and at least one functional moiety, comprising covalently attaching at least one solubility tag to the molecule.

[0095] In another aspect, the present invention relates to a method for increasing the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and one or more linkers covalently linking one or more of said functional moieties to said targeting moiety, comprising covalently linking at least one solubility tag to said molecule.

[0096] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising a targeting moiety, comprising producing a molecule in which the chemical compound is covalently linked to at least one solubility tag.

[0097] As will be understood by those skilled in the art, this "molecule in which the compound is covalently linked to at least one soluble tag" is a molecule as referred to in the first six embodiments described in the "Antibody-Drug Conjugates" section, and can be characterized by all of the other features disclosed in this application with respect to any of these embodiments.

[0098] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Targeting The present invention relates to a method for increasing the solubility of a compound comprising a moiety and at least one functional moiety, the method comprising preparing a molecule in which the compound is covalently linked to at least one solubility tag.

[0099] In another aspect, the present invention relates to a method for increasing the solubility of a compound, the compound comprising: Targeting a portion, at least one functional portion, and a combination of the functional portion and the Targeting The method comprises preparing a molecule in which the compound is covalently linked to at least one soluble tag.

[0100] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Targeting The present invention relates to a method for increasing the solubility of a compound comprising at least one targeting moiety, the compound comprising preparing a molecule wherein the compound comprises at least one targeting moiety, the compound being covalently linked to at least one solubility tag.

[0101] In another aspect, the present invention provides a method for increasing the solubility of a compound, the method comprising: Targeting The compound comprises a moiety and at least one functional moiety, and the method comprises covalently linking the compound to at least one soluble tag.

[0102] In another aspect, the present invention relates to a method for increasing the solubility of a compound, the compound comprising: Targeting The present invention relates to a method for preparing a molecule comprising the steps of: preparing a molecule comprising a moiety, at least one functional moiety, and one or more linkers, wherein the functional moiety is covalently attached to at least one soluble tag.

[0103] In certain embodiments of any of the above methods, all components of the compound are covalently bonded.

[0104] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Targeting The present invention relates to the use of soluble tags to increase the solubility of molecules that contain the moiety.

[0105] In another aspect, the invention relates to the use of a soluble tag to increase the solubility of a molecule, the molecule being Targeting The protein comprises a portion and at least one functional portion.

[0106] In another aspect, the invention relates to the use of a soluble tag to increase the solubility of a molecule comprising a targeting moiety, at least one functional moiety, and a linker that covalently links the functional moiety and the targeting moiety to the functional moiety.

[0107] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: Targeting The present invention relates to the use of soluble tags to increase the solubility of molecules comprising the moiety.

[0108] In another aspect, the present invention relates to the use of soluble tags to increase the solubility of molecules comprising a targeting moiety and at least one functional moiety.

[0109] In another aspect, the invention relates to the use of a soluble tag to increase the solubility of a molecule, the molecule comprising: Targeting The moiety comprises at least one functional moiety and one or more linkers covalently linking the functional moiety and the targeting moiety.

[0110] In some embodiments, the use comprises covalently attaching at least one soluble tag to the molecule.

[0111] The following embodiments relate to any of the molecules, methods or uses defined above.

[0112] In some embodiments, all components of the molecule are covalently linked.

[0113] In one embodiment, the Targeting The moiety is a molecular group that specifically binds to a target molecule or a fragment thereof. In some embodiments, the target molecule is a biomolecule. In some embodiments, the target molecule is a cell surface receptor. In some embodiments, the target molecule is an antigen present on the surface of a target cell.

[0114] In one embodiment, the Targeting The moiety is capable of specifically binding to an antigen present on the surface of a target cell. Targeting The moiety comprises a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule. Targeting The moiety is selected from the group consisting of a protein, a peptide, a peptidomimetic, a nucleic acid, an oligonucleotide, and a small molecule.

[0115] In one embodiment, the Targeting In some embodiments, the moiety comprises a protein. Targeting In some embodiments, the moiety is a protein. Targeting The moiety comprises or is a protein that is a protein ligand that specifically binds to a cell surface receptor. Targeting The moiety comprises or is a protein that is an antibody or an antigen-binding fragment thereof. Targeting The moiety comprises or is a protein that is an antibody component. Targeting The portion comprises or includes a protein containing at least 30 amino acids. Targeting The moiety is a peptide comprising or consisting of a peptide of 2 to 30 amino acids.

[0116] In one embodiment, the Targeting In some embodiments, the moiety comprises a peptide. Targeting The moiety is a peptide.

[0117] In one embodiment, the TargetingIn some embodiments, the moiety comprises a peptidomimetic. Targeting The moiety is a peptidomimetic.

[0118] In one embodiment, the Targeting In some embodiments, the moiety comprises a nucleic acid. Targeting In some embodiments, the moiety is a nucleic acid. Targeting The moiety comprises or is a nucleic acid, which may be DNA or RNA.

[0119] In one embodiment, the Targeting In some embodiments, the moiety comprises an oligonucleotide. Targeting The moiety is an oligonucleotide.

[0120] In one embodiment, the Targeting The moiety comprises or is a small molecule with a molecular weight <1000 Da. Targeting In some embodiments, the moiety comprises a small molecule. Targeting The moiety is a small molecule.

[0121] In one embodiment, the Targeting In some embodiments, the moiety is not a sugar. Targeting The portion contains no sugar.

[0122] In one embodiment, the Targeting The moiety has a molecular weight of at least 100 Da. Targeting The moiety has a molecular weight of at least 500 Da. Targeting The moiety has a molecular weight of at least 1000 Da. Targeting The moiety has a molecular weight of at least 2000 Da. Targeting In one embodiment, the moiety has a molecular weight of at least 10 kDa. Targeting In one embodiment, the moiety has a molecular weight of at least 50 kDa. Targeting In one embodiment, the moiety has a molecular weight of at least 100 kDa. Targeting The moiety has a molecular weight of up to 1000 Da. Targeting The moiety has a molecular weight of up to 2000 Da. Targeting The moiety has a molecular weight of up to 10 kDa. Targeting The moiety has a molecular weight of up to 50 kDa. Targeting The moiety has a molecular weight of up to 200 kDa. Targeting The moiety has a molecular weight of up to 1 MDa. Targeting The moiety has a molecular weight of up to 5 MDa. Targeting The moieties have a molecular weight of up to 10 MDa.

[0123] In certain embodiments, the at least one functional moiety is a chemical entity capable of performing a biological, chemical, therapeutic and / or diagnostic function in the human body.

[0124] As will be understood by those skilled in the art, the term "chemical entity" includes any kind of chemical group or molecule of any substance class, and is limited only by the recited requirement that it must be capable of fulfilling a biological, chemical, therapeutic and / or diagnostic function in the human body (in the context of the molecules according to the present invention).

[0125] In some embodiments, the at least one functional moiety is a therapeutic agent or a detectable label. In some embodiments, the at least one functional moiety is a therapeutic agent. In some embodiments, the at least one functional moiety is a detectable label.

[0126] In some embodiments, the at least one functional moiety is a payload that is a therapeutic agent or a detectable label. In some embodiments, the payload is a therapeutic agent. In some embodiments, the payload is a detectable label.

[0127] In certain embodiments, the at least one functional moiety comprises a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule. In certain embodiments, the at least one functional moiety is a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule.

[0128] In some embodiments, the at least one functional moiety comprises a protein. In some embodiments, the at least one functional moiety is a protein. In some embodiments, the at least one functional moiety comprises or comprises a protein comprising at least 30 amino acids. In some embodiments, the at least one functional moiety is a peptide comprising or consisting of a peptide of 2 to 30 amino acids.

[0129] In certain embodiments, the at least one functional moiety comprises a peptide. In certain embodiments, the at least one functional moiety is a peptide.

[0130] In certain embodiments, the at least one functional moiety comprises a peptidomimetic. In certain embodiments, the at least one functional moiety is a peptidomimetic.

[0131] In some embodiments, the at least one functional moiety comprises a nucleic acid. In some embodiments, the at least one functional moiety is a nucleic acid. In some embodiments, the at least one functional moiety comprises or is a nucleic acid that is DNA or RNA.

[0132] In some embodiments, the at least one functional moiety comprises an oligonucleotide. In some embodiments, the at least one functional moiety is an oligonucleotide.

[0133] In some embodiments, the at least one functional moiety comprises a small molecule. In some embodiments, the at least one functional moiety is a small molecule. In some embodiments, the at least one functional moiety is a small molecule.

[0134] In certain embodiments, the at least one functional moiety comprises or is a small molecule with a molecular weight <1000 Da. In certain embodiments, the at least one functional moiety comprises a small molecule.

[0135] In some embodiments, the at least one functional moiety is not a sugar. In some embodiments, the at least one functional moiety does not comprise a sugar.

[0136] In some embodiments, the at least one functional moiety has a molecular weight of at least 100 Da. In some embodiments, the at least one functional moiety has a molecular weight of at least 500 Da. In some embodiments, the at least one functional moiety has a molecular weight of at least 1000 Da. In some embodiments, the at least one functional moiety has a molecular weight of at least 2000 Da. In some embodiments, the at least one functional moiety has a molecular weight of at least 10 kDa. In some embodiments, the at least one functional moiety has a molecular weight of at least 50 kDa. In some embodiments, the at least one functional moiety has a molecular weight of at least 100 kDa. In some embodiments, the at least one functional moiety has a molecular weight of at most 1000 Da. In some embodiments, the at least one functional moiety has a molecular weight of at most 2000 Da. In some embodiments, the at least one functional moiety has a molecular weight of at most 10 kDa. In some embodiments, the at least one functional moiety has a molecular weight of at most 50 kDa. In some embodiments, the at least one functional moiety has a molecular weight of up to 200 kDa. In some embodiments, the at least one functional moiety has a molecular weight of up to 1 MDa. In some embodiments, the at least one functional moiety has a molecular weight of up to 5 MDa.

[0137] In one embodiment, a comparison molecule having the same structure as the molecule but lacking the solubility tag has an isoelectric point (pI) of 5-9.

[0138] In one embodiment, a comparison molecule having the same structure as the molecule but lacking the solubility tag has a solubility (expressed as grams of compound per ml of PBS; solubility is measured in PBS (phosphate buffer) at 25°C) within ±50%, preferably ±30%, of the solubility in saline (physiological saline: 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO, 1.8 mM KHPO, pH 7.4). Conditions for a compound consisting of the antibody trastuzumab and two copies of auristatin covalently linked to its Fc region.

[0139] In some embodiments, the number of functional moieties per molecule ranges from 1 to 15. In some embodiments, the number of functional moieties per molecule ranges from 1 to 10. In some embodiments, the number of functional moieties per molecule ranges from 1 to 8. In some embodiments, the number of functional moieties per molecule ranges from 1 to 4.

[0140] In some embodiments, a molecule contains 1, but not more than 1, functional moiety. In some embodiments, the number of functional moieties per molecule is in the range of 2 to 8. In some embodiments, the number of functional moieties per molecule is in the range of 4 to 8.

[0141] In certain embodiments, the number of targeting moieties per molecule ranges from 1 to 15. In certain embodiments, the number of targeting moieties per molecule ranges from 1 to 10. In certain embodiments, the number of targeting moieties per molecule ranges from 1 to 10. Targeting The number of moieties ranges from 1 to 8. In one embodiment, Targeting The number of parts ranges from 1 to 4.

[0142] In some embodiments, the molecule comprises one but not more than one Targeting In some embodiments, the moiety is Targeting The number of moieties ranges from 2 to 8. In one embodiment, Targeting The number of parts is in the range of 4 to 8.

[0143] In certain embodiments, the molecule is an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload (wherein the at least one payload is a therapeutic agent or a detectable label), (iii) a covalent linker connecting the payload and the antibody component, and (iv) at least one solubility tag.

[0144] In certain embodiments, the molecule is an antibody-drug conjugate consisting of (i) an antibody component, (ii) at least one payload (wherein the at least one payload is a therapeutic agent or a detectable label), (iii) a linker covalently attaching the payload to the antibody component, and (iv) at least one solubility tag.

[0145] Antibody-drug conjugates

[0146] In another aspect, the present invention relates to an antibody-drug conjugate comprising: (i) an antibody component; (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker covalently attaching the payload to the antibody component; and (iv) at least one solubility tag.

[0147] As used herein, an "antibody-drug conjugate" (abbreviated "ADC") is a molecule comprising an antibody (the "antibody component of the ADC, see below) attached to a payload (the "drug") via a linker. In the ADCs of the invention, the payload is a therapeutic agent or a detectable label. The various components of the antibody-drug conjugate are covalently linked.

[0148] The antibody component of an ADC functions as a targeting component that can guide the ADC to its target site by binding to its antigen. For example, if the antigen of the antibody component is a tumor antigen, the ADC is directed, for example, to tumor cells that express this tumor antigen on their cell surface. Once the ADC is mobilized to the target site, the payload can mediate a therapeutic effect (e.g., killing cancer cells, locally reducing inflammation, locally stimulating or suppressing the immune system), or if the payload is a detectable label, the payload can mediate a therapeutic effect. The target site can be identified by detecting the detectable label.

[0149] Non-targeted drugs typically reach their site of action through systemic distribution and passive diffusion. In contrast, ADCs are targeted compounds that are not uniformly distributed throughout the body. Due to the interaction between the antibody component and its target antigen, the ADC preferentially concentrates at the target site. Therefore, ADCs with a therapeutic agent as a payload require a lower dose to exert a therapeutic effect, improving the therapeutic range.

[0150] In many cases, upon binding to a target cell, an ADC is internalized into the cell, for example, by receptor-mediated endocytosis. If the linker is cleavable, the linker can be cleaved (e.g., by enzymatic or chemical cleavage) after cell degradation. Alternatively, the antibody may be degraded within the cell. In either case, the payload is released into the cell. If the payload is a drug, it can perform a therapeutic function within the cell. If the payload is a detectable label, it may be detected within the cell.

[0151] Antibody-drug conjugates, their structure, preparation and use are described, for example, in Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer, 1st edition (2016), editors Olivier and Hurvitz, publisher John Wiley & Sons, Inc. (US); Toader, Topics in Medicinal Chemistry (2018), vol. 28 (Cancer II), pp. 289-332; Chau, Lancet (2019), vol. 394 (10200), pp. 793-804; Nimoy, Pharmaceuticals (2018), vol. 11 (2), pp. 32 / 1-32 / 22; Gorka et al., Accounts of Chemical Research (2018), vol. 51(12), pp. 3226-3235; Tiberghien et al., Journal of Organic Chemistry (2018), vol. 51(12), pp. 3226-3235. This is described in detail in Chemistry (2019), vol. 84(8), pp. 4830-4836; Rohrer, in: Process Scale Purification of Antibodies, 2nd edition (2017), editor: Gottschalk, John Wiley & Sons, Inc., pp. 595-614; Vaklavas and Forero, Methods in Molecular Biology (2012), vol. 899, pp. 489-497.

[0152] As will be appreciated by those skilled in the art, in practice, ADCs are often populations of molecules that vary slightly in terms of their properties. For example, a population of ADC molecules may contain mostly ADC molecules with four payloads per ADC molecule, but may also contain a small percentage of ADC molecules with three payloads per ADC molecule and a small percentage of ADC molecules with five payloads per ADC molecule. When there is slight variation in the ADC population in terms of properties, the values ​​shown below typically relate to rounded average values ​​for the entire population.

[0153] For the purposes of the present invention, high homogeneity among ADCs within a population of interest is generally desirable. Higher homogeneity can usually be achieved by additional purification / separation steps, such as HIC (hydrophobic interaction chromatography), SEC (size exclusion chromatography), and HPLC / reverse-phase HPLC. The homogeneity of an ADC population can be determined, for example, by HIC, HPLC / reverse-phase HPLC, SDS-PAGE analysis, and MS (mass spectrometry) analysis. For analysis that distinguishes between antibody heavy and light chains, SEC or SDS-PAGE under reducing conditions, followed by MS analysis, can be performed.

[0154] antibody component

[0155] As used herein, the term "antibody component" refers to an immunoglobulin molecule that is or can be used as part of an antibody-drug conjugate. The term "antibody component" can encompass intact antibodies and antigen-binding fragments of intact antibodies (i.e., fragments of intact antibodies that can still bind to the same antigen as the corresponding intact antibody). In certain embodiments, the term also includes molecules in which an intact antibody or antigen-binding fragment of an intact antibody is covalently linked to one or more additional intact antibodies and / or antigen-binding fragments of one or more additional antibodies and / or another antibody. Molecular structure. Thus, an antibody component is an immunoglobulin molecule that recognizes and specifically binds to a target (antigen, see below) via at least one antigen-binding site within the variable region of the immunoglobulin molecule.

[0156] As used herein, an "intact" antibody refers to an antibody that contains the complete full-length sequence of an antibody of each antibody class. Thus, an intact antibody includes, depending on the antibody class, an antigen-binding region (i.e., complete VL and VH domains), as well as complete light and heavy chain constant domains, and the antibody domains remain bound together through at least one non-covalent interaction. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof.

[0157] As used herein, an antibody "fragment" is a portion of an intact antibody. An "antigen-binding fragment" of an (intact) antibody is a portion of the antibody that binds to the same antigen as the intact antibody. Usually, this means that the fragment contains the same antigen-binding region as the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, as well as single-chain Fv (scFv) antibodies. In certain embodiments, the term "fragment" of an antibody also encompasses bivalent or multivalent antibody constructs generated by linking together two or more of the aforementioned antibody fragments.

[0158] As used herein, "antigen" refers to a substance that can specifically bind to the variable region of an antibody. Antigens can be, for example, proteins, polypeptides, peptides, carbohydrates, polynucleotides, lipids, or combinations thereof.

[0159] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antibody's antigen-binding site. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variation (Sequences of Proteins of Immunological Interest, 5th ed. (1991), editors Kabat et al., National Institutes of Health (Bethesda, USA)); (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol. (1997), vol. 273, pp. 927-948)). Moreover, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0160] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to the portion of an antigen that is recognized and specifically bound by a particular antibody. When an antigen is a polypeptide, epitopes are formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in a unique spatial structure.

[0161] Each ADC molecule according to the invention contains one antibody component (but may contain multiple payloads and multiple linkers).

[0162] With regard to the type and source of antibody, antibody component is not particularly limited, as long as it comprises at least one antigen binding site and shows binding to its target antigen.Standard techniques for designing and preparing antibody are known to those skilled in the art (see, for example, Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (US); Antibody Engineering - Methods and Protocols, 2nd edition (2010), editors Nevoltris and Chames, publisher Springer (Germany); Handbook of Therapeutic Antibodies (2014), editors Duebel and Reichert, publisher Wiley-VCH Verlag GmbH & Co. KGaA (Germany); Harper, Methods in Molecular Biology (2013), vol. 1045, p. 41-49).

[0163] The following embodiments relate to any of the molecules, antibody-drug conjugates, methods or uses defined above.

[0164] In some embodiments, the antibody component is an intact antibody or an antigen-binding fragment thereof. In some embodiments, the antibody component is an intact antibody. In some embodiments, the antibody component is an antigen-binding fragment of an intact antibody.

[0165] In one embodiment, antibody components (each of which is a "molecule" as defined above) TargetingThe antibody component (which may be included as a "molecule" as defined above) is a monoclonal or polyclonal antibody. Targeting The antibody included as a moiety is a monoclonal antibody.

[0166] As used herein, a "monoclonal" antibody refers to an antibody that arises from a nearly homogeneous antibody population. More specifically, the individual antibodies in the population are identical except for a few possible naturally occurring mutations that may be found in minimal proportions. In other words, a monoclonal antibody is composed of homogeneous antibodies resulting from the propagation of a single cell clone and is generally characterized by a heavy chain of only one class and subclass and a light chain of only one type. Furthermore, in contrast to polyclonal antibody preparations, which typically contain various antibodies against different epitopes, each monoclonal antibody is directed against a single epitope of an antigen. Monoclonal antibodies are typically produced by a single clone of B lymphocytes ("B cells"). Monoclonal antibodies can be obtained using a variety of techniques known to those skilled in the art, including standard hybridoma technology (see, for example, Koehler and Milstein, Eur. J. Immunol. (1976), vol. 5, pp. 511-519; Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (USA); Immunobiology, 5th ed. (2001), editors Janeway et al., Garland Publishing (USA)) and by expression from eukaryotic host cells transfected with DNA molecules encoding the homogeneous antibody or from prokaryotic host cells transfected with DNA molecules encoding the homogeneous antibody.

[0167] As used herein, "polyclonal" antibodies refer to heterogeneous antibody populations that are typically obtained by purification from the serum of immunized animals by standard techniques known to those skilled in the art (see, e.g., Antibodies: A Laboratory Manual, 2nd edition (2014), editor Greenfield, Cold Spring Harbor Laboratory Press (USA)).

[0168] In one embodiment, antibody components (each of which is a "molecule" as defined above) Targeting The antibody included as a moiety may be a monospecific antibody or a bispecific antibody.

[0169] As used herein, a "monospecific antibody" is an antibody that can bind to only one antigen.

[0170] The term "bispecific antibody" as used herein refers to an antibody that can specifically bind to two different epitopes simultaneously. The epitopes may be derived from the same antigen or from two different antigens. Preferably, the epitopes are derived from two different antigens. Typically, a bispecific antibody has two antigen-binding sites, e.g., two pairs of heavy and light chains (HC / LC), each specifically binding to a different antigen. That is, the first heavy chain and the first light chain specifically bind to a first antigen, and the second heavy chain and the second light chain specifically bind to a second antigen. Methods for producing bispecific antibodies are known in the art. For example, bispecific antibodies can be recombinantly produced using coexpression of two immunoglobulin heavy / light chain pairs (see, e.g., Milstein et al., Nature (1983), vol. 305, pp. 537-539). Alternatively, bispecific antibodies can be prepared using chemical conjugation (see, for example, Brennan et al., Science (1985), vol. 229, p. 81). Bispecific antibodies can also be prepared, for example, by SEED technology (an approach for generating bispecific antibodies in which structurally related sequences within the conserved CH3 domains of human IgA and IgG are exchanged to form two asymmetric but complementary domains; see WO 2016 / 087650).

[0171] In one embodiment, the antibody components (each of which is a "molecule" as defined above) Targeting The bispecific antibody (the antibody included as a moiety) is a bispecific antibody or an antigen-binding fragment thereof that can bind to both antigens for which the bispecific antibody is specific. Thus, the antigen-binding fragment of the bispecific antibody binds to the same two antigens as the bispecific antibody.

[0172] In one embodiment, within the above-defined "molecule" Targeting The antibody included as a moiety is a bispecific antibody.

[0173] The antibody components of the present invention (each within the "molecule" defined above) TargetingThe antibody (including the antibody moiety) may be monovalent, bivalent, or multivalent. A "monovalent" antibody / antibody component has one antigen-binding site. A "bivalent" antibody / antibody component has two antigen-binding sites. These two antigen-binding sites may bind to the same antigen or different antigens. A "multivalent" antibody / antibody component has two or more antigen-binding sites. These three or more antigen-binding sites may bind to the same antigen or different antigens.

[0174] In one embodiment, the antibody components (each of which is a "molecule" as defined above) Targeting The antibody included as a moiety is an antibody selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody.

[0175] As used herein, a "chimeric" antibody is an antibody in which a portion of the heavy and / or light chain is identical to or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (US Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci USA (1984), vol. 81, pp. 6851-6855). As used herein, "humanized antibody" is used as a subset of "chimeric antibody."

[0176] As used herein, a "humanized antibody" is a "humanized" form of a non-human (e.g., murine) antibody. A "humanized antibody" is a chimeric antibody that contains minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR (defined below) of the recipient are replaced by residues from an HVR (donor antibody) of a non-human species, such as mouse, rat, or rabbit, having the desired specificity, affinity, and / or capacity. In some instances, framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of the non-human immunoglobulin sequence and all or substantially all of the hypervariable loops corresponding to those of the non-human immunoglobulin sequence. The FR regions are those of a human immunoglobulin sequence, but may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions within the FR is as follows: The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.For further details, see, for example, Jones et al., Nature (1986), vol. 321, pp. 522-525; Riechmann et al., Nature (1988), vol. 332, pp. 323-329; and Presta, Curr. Op. Struct. Biol. (1992), vol. 2, pp. 593-596. See also, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. (1998), vol. 1, pp. 105-115; Harris, Biochem. Soc. Transactions (1995), vol. 23, pp. 1035-1038; Hurle and Gross, Curr. Op. Biotech. (1994), vol. 5, pp. 428-433; U.S. Patent 6,982,321; US ​​Patent 7,087,409.

[0177] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody made using any of the techniques for making human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced by various techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. (1991), vol. 227, p. 381; Marks et al., J. Mol. Biol. (1991), vol. 222, p. 581). For the preparation of human monoclonal antibodies, the methods described in Cole et al., in: Monoclonal Antibodies and Cancer Therapy (1985), editors Reisfeld and Sell, publisher Alan R. Liss Inc. (New York), pp. 77-96; Boerner et al., J. Immunol. (1991), vol. 147(1), pp. 86-95; van Dijk and van de Winkel, Curr. Opin. Pharmacol. (2001), vol. 5, pp. 368-374, can also be used. Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenogeneic animals, which have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (e.g., XENOMOUSE). TM (See U.S. Patent Nos. 6,075,181 and 6,150,584 for technology.) See also Li et al., Proc. Natl. Acad. Sci. USA (2006), vol. 103, pp. 3557-3562, for example, regarding human antibodies produced via human B-cell hybridoma technology.

[0178] Antibody components according to the invention can be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1). Different classes of immunoglobulins have different, well-known subunit structures and three-dimensional configurations (Immunobiology, 5th ed. (2001), editors Janeway et al., Garland Publishing (USA)).

[0179] In one embodiment, the antibody components (each of which is a "molecule" as defined above) Targeting The antibody included as a moiety is selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgA antibody, an IgM antibody, and hybrids thereof.

[0180] An antibody consisting of a "hybrid" of two antibodies of different classes / subclasses refers to an antibody that contains sequences from these two antibodies of different classes / subclasses. For example, bispecific antibodies prepared by SEED technology (WO 2016 / 087650) typically contain both IgG and IgA sequences and are therefore considered "hybrids" of IgG and IgA antibodies.

[0181] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, scFv, diabody and VHH.

[0182] "Fab" fragments are obtained by papain digestion of antibodies, producing two identical antigen-binding fragments called "Fab" fragments and a remaining fragment called the "Fc" fragment, reflecting its ability to crystallize readily. Fab fragments consist of an entire L chain, the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding; that is, it has a single antigen-binding site.

[0183] "F(ab')2" fragments can be obtained by pepsin treatment of an antibody to yield a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities.

[0184] "Fab" fragments differ from Fab fragments in that they have a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0185] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by the sequences in the Fc region. This region is also recognized by Fc receptors (FcRs) found on certain types of cells.

[0186] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This fragment consists of a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops from the H chain and three from the L chain), which contribute antigen-binding amino acid residues and confer antigen-binding specificity to the antibody. However, a single variable domain (or half of an Fv containing only the three antigen-specific HVRs) is capable of recognizing and binding antigen, albeit with lower affinity than the entire binding site.

[0187] "Single-chain Fv," also abbreviated as "scFv," is an antibody fragment containing the VH and VL antibody domains linked in a single polypeptide chain. Preferably, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun, in: The Pharmacology of Monoclonal Antibodies, vol. 113 (1994), editors Rosenburg and Moore, Springer-Verlag (New York), pp. 269-315.

[0188] The term "diabody" refers to small antibody fragments prepared by constructing scFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the VH and VL domains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the VH and VL domains of the two antibodies are present on different polypeptide chains. Diabodies are described in detail, for example, in EP 0404097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA (1993), vol. 90, pp. 6444-6448.

[0189] As used herein, the terms "VHH" and "nanobody" have the same meaning. They refer to single-domain antibodies, which are antibody fragments consisting of a single monomeric variable region of an antibody heavy chain. Like intact antibodies, VHHs can selectively bind to specific antigens. VHHs have a molecular weight of only 12-15 kDa, much smaller than typical antibodies (150-160 kDa). The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids (Gibbs and Wayt, "Nanobodies," Scientific American Magazine (2005)). Generally, antibodies that naturally lack the light and heavy chain constant region 1 (CH1) are first isolated. Therefore, the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0190] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, and Fv. In some embodiments, the antigen-binding fragment is selected from the group consisting of scFv, diabody, and VHH. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of a monoclonal antibody or a polyclonal antibody. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of a monoclonal antibody. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of a monospecific antibody or a bispecific antibody. In some embodiments, the antigen-binding fragment is an antigen-binding fragment of a bispecific antibody that can bind to both antigens for which the bispecific antibody is specific.

[0191] In certain embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody.

[0192] In certain embodiments, the antigen-binding fragment is an antigen-binding fragment of an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgA antibody, an IgM antibody, and hybrids thereof.

[0193] In one embodiment, the Targeting The antibody moiety / component (each of which is a "molecule" as defined above) Targeting The antibody included as a moiety is capable of specifically binding to an antigen present on the surface of a target cell.

[0194] As used herein, "antigen present on the surface of target cell" refers to the antigen present on the surface of target cell that can be accessed from the extracellular environment (i.e., antibody can bind from the extracellular environment).For example, CD8 is a transmembrane protein of cytotoxic T cells, and its extracellular domain is accessible to the antibody against the extracellular domain of CD8 from the extracellular environment.Therefore, in the sense of the present invention, CD8 is an antigen present on the surface of cytotoxic T cells.In one embodiment, the "antigen present on the surface of target cell" is a protein present on the surface of target cell.

[0195] An antibody / antibody component that "binds" an antigen of interest is an antibody / antibody component that can bind to that antigen with sufficient affinity so that the antibody / antibody component is useful for targeting cells that express the antigen.

[0196] When the present invention refers to a first molecule / molecular group (e.g., an antibody / antibody component) that "specifically binds" / "specifically binds" to a second molecule / molecular group (e.g., an antigen of interest), this means that the first molecule / molecular group (e.g., an antibody / antibody component) binds to the second molecule / molecular group (in this example, the antigen of interest) with an affinity that is at least 10 times greater than the affinity of the molecule / molecular group (in this example, an antibody) to other molecules / molecular groups, or specific other molecules / molecular groups in the human body (in this example, at least 10 times greater than the binding affinity to non-specific antigens other than the antigen of interest (or closely related antigens), e.g., BSA, casein). In a preferred embodiment, a first molecule / molecular group (e.g., an antibody / antibody component) that "specifically binds" to a second molecule / molecular group (e.g., an antigen of interest) binds to the antigen with an affinity of at least 100. Its affinity for other molecules / molecular groups, particularly other molecules / molecular groups in the human body, is many times greater (in this example, at least 100 times greater than its affinity for binding to nonspecific antigens other than the antigen of interest (or closely related antigens)). Typically, the binding is determined under physiological conditions. A first molecule / molecular group that "specifically binds" to a second molecule / molecular group has an affinity of at least about 1 x 10 7 M -1 An antibody / antibody component that "specifically binds" to an antigen of interest can bind to that second molecule / molecular group with an affinity of at least about 1 x 10 7 M -1 can bind to its antigen with an affinity of

[0197] In one embodiment, the antibody components (each a "molecule" as defined above) Targeting The antibody included as a moiety is an antibody directed against an antigen present on the surface of a target cell or an antigen-binding fragment of such an antibody.

[0198] An antibody / antibody component "directed against" a particular antigen is an antibody / antibody component that has an antigen-binding site that binds to that antigen. Whether an antibody binds to an antigen can be determined, for example, by testing whether the antibody binds to cells that express the antigen on their cell surface in an immunofluorescence experiment using cultured cells.

[0199] In some embodiments, the antigen present on the surface of the target cell is more abundant on the surface of the target cell than on the surface of other cell types.

[0200] The abundance of a surface antigen on a cell type can be determined by standard methods known to those of skill in the art, such as flow cytometry (e.g., by exposing cells of the cell type to an antibody of interest, followed by staining with a fluorescently labeled secondary antibody against the antibody of interest, and detecting the fluorescent label by flow cytometry).

[0201] In certain embodiments, the antigen present on the surface of the target cell is "present on the surface of the target cell but substantially absent from the surface of other cell types."

[0202] As used herein, an antigen "present on the surface of the target cell but substantially absent on the surface of other cell types" is sufficiently abundant on the surface of the target cell to allow the antibody component to recruit an ADC to act on the antigen under physiological conditions. In contrast, the abundance of the antigen on the surface of other cell types is so low that the recruitment of the ADC under physiological conditions is only slightly above background binding.

[0203] The antigen present on the surface of the target cell is present on the surface of the target cell but not on the surface of other cell types.

[0204] As used herein, an antigen "present on the surface of the target cell but not on the surface of other cell types" refers to an antigen that is sufficiently abundant on the surface of the target cell to allow recruitment of an ADC with an antibody component directed against the antigen under physiological conditions. In contrast, the abundance of the antigen on the surface of other cell types is so low that recruitment of the ADC under physiological conditions does not exceed background binding.

[0205] In one embodiment, the antibody component (each of which is a "molecule" as defined above) is directed to the antigen present on the surface of the target cell. TargetingThe binding of the antibody (which is included as a moiety) allows for the specific recruitment of the antibody-drug conjugate to the target cell.

[0206] The term "enabling the antibody-drug conjugate to be specifically recruited to the target cells" means that the ADC is recruited to the target cells under physiological conditions with at least 10-fold, and preferably at least 100-fold, greater efficiency than to other cell types (i.e., other cell types to which the ADC may be exposed in the body during administration of the ADC).

[0207] In some embodiments, the antigen present on the surface of the target cell is selected from the group consisting of a tumor antigen and an immune cell antigen, hi some embodiments, the antigen present on the surface of the target cell is a tumor antigen.

[0208] In one embodiment, the Targeting Moieties / components of the antibody (each within the "molecule" defined above) Targeting The antibody (included as a moiety) is capable of specifically binding to an antigen selected from the group consisting of a tumor antigen and an immune cell antigen.

[0209] In one embodiment, the Targeting Moieties / components of the antibody (each within the "molecule" defined above) Targeting The antibody (included as a moiety) is capable of specifically binding to a tumor antigen.

[0210] As used herein, the term "tumor" refers to an abnormal mass of cells formed by the proliferation of neoplastic cells. Tumors can be benign or malignant. Preferably, in the present invention, the term "tumor" refers to a malignant tumor. The tumor may be a cancer such as, but not limited to, myeloma, blood cancers such as leukemia and lymphoma (e.g., B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), hematopoietic neoplasms, thymoma, head and neck cancer, sarcoma, lung cancer, liver cancer, genitourinary cancer (e.g., ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, bladder cancer, testicular cancer, prostate cancer, penile cancer), adenocarcinoma, breast cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, neural tumors including brain tumors such as astrocytoma and glioblastoma, angiosarcoma, hemangiosarcoma, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, vascular cancer, gastrointestinal cancer (e.g., gastric cancer, stomach cancer, or colon cancer) (Rosenberg, Ann. Rev. Med. (1996), vol. 47, p. See 481-491).

[0211] The term "cancer" as used herein refers to a malignant neoplasm. Cancer includes blood cancer or solid tumor. For example, cancer can be leukemia (e.g., acute myeloid leukemia (AML), acute monocytic leukemia, promyelocytic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia (ALL), such as acute B-lymphoblastic leukemia (B-ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)) or lymphoma (e.g., non-Hodgkin's lymphoma), myelodysplastic syndrome (MDS), melanoma, lung cancer (e.g., non-small cell lung cancer; NSCLC), ovarian cancer, endometrial cancer, peritoneal cancer, pancreatic cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, or cervical cancer. Preferably, in the present invention, the term "cancer" refers to a solid malignant tumor.

[0212] As used herein, a "tumor antigen," in its broadest sense, is an antigen that enables recruitment of an ADC to a tumor site so that a therapeutic effect or diagnosis (e.g., labeling of the tumor site) can be achieved. Tumor antigens can be either antigens present on the surface of tumor cells or antigens associated with the tumor microenvironment.

[0213] Sources of information on cell surface expression and methods for identifying and validating tumor antigens are known to those skilled in the art and are described in the literature (e.g., Bornstein, AAPS J. (2015), vol. 17(3), pp. 525-534; Bander, Methods Mol Biol (2013), vol. 1045, pp. 29-40; "Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer", 1st edition (2016), editors Olivier and Hurvitz, publisher John Wiley & Sons, Inc. (US); Vigneron et al., Cancer Immun. (2013), vol. 13, p. 15; Hong et al., BMC Syst Biol. (2018), vol. 12 (Suppl 2), p. 17; de Souza et al., Cancer Immun. (2012), vol. 12, p. 15; Immune Epitope Database and Analysis Resource (https: / / www.iedb.org); Cancer Cell Line Encyclopedia (https: / / portals.broadinstitute.org / ccle); OASIS Database (http: / / oasis-genomics.org / )).

[0214] In preferred embodiments, the tumor antigen is an antigen present on the surface of tumor cells. In these embodiments, the term "tumor antigen" refers to an antigen present on the cell surface of tumor cells, allowing tumor cells to be distinguished from other cell types. A tumor antigen can be a part of a molecule (e.g., a protein) expressed by tumor cells and accessible from the extracellular environment. A tumor antigen can be different (i.e., qualitatively different) from its counterpart on corresponding non-tumor cells (e.g., when the molecule is a protein by one or more amino acid residues). Alternatively, a tumor antigen can be identical to its counterpart on corresponding non-tumor cells but present at a higher level on the surface of tumor cells than on the surface of corresponding non-tumor cells. For example, a tumor antigen can be present only on the surface of tumor cells and not on the surface of non-tumor cells, or the tumor antigen can be present at a higher level on the surface of tumor cells (e.g., at least 5-fold, preferably at least 100-fold higher than on the surface of non-tumor cells). In one embodiment, a tumor antigen is present on the surface of tumor cells at a level at least 1000-fold higher than on the surface of non-tumor cells.

[0215] Preferably, the tumor with which the tumor antigen is associated is a cancer (ie, the tumor antigen is present on the surface of tumor cells and is present on cancer cells).

[0216] In one embodiment, the tumor antigen is CD11a, CD4, CD19, CD20, CD21, CD22, CD23, CD25, CD52, CD30, CD33, CD37, CD40L, CD52, CD56, CD70, CD72, CD74, CD79a, CD79b, CD138, CD163, Her2, Her3, EGFR, Muc18, integrin, PSMA, CEA, BLys, ROR1, NaPi2b, NaPi3b, CEACAM5, Muc1, integrin avb6, Met, Trop2, BCMA, disialoganglioside GD2, B-PR1B, E16, STEAP1, 0772P, Sema 5b, ETBR, MSG783, STRAP2, Trp4, CRIPTO, FcRH1, FcRH2, NCA, IL20R-alpha, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CXCR5, HLA-DOB, P2X5, LY 64, IRTA2, TENB2, PSMA, FOLH1, STR5, SSTR1, SSTR2, SSTR3, SSTR4, TGAV, ITGB6, CA9, EGFRvlll, IL2RA, AXL, CD3Q, TNFRSF8, TNFRSF17, CTAG, CTA; CD174 / fucosyltransferase 3 (Lewis blood group), CLEC14A, GRP78, HSPA5, ASG-5, ENPP3, PRR4, GCC, GUCY2C, Liv-1, SLC39A8, 5T4, NCMA1, CanAg, FOLR1, GPN B, TIM-1, HAVCR1, Mindin / RG-1, B7-H4, VTCN1, PTK7, SDC1, claudins (preferably claudin 18.2), RON, MST1R, EPHA2, MS4A1, TNC (tenascin C), FAP, DKK-1, CS1 / SLAMF7, ENG (endoglin), ANXA1 (annexin A1), VCAM-1 (CD106) and folate receptor alpha.

[0217] In one embodiment, the tumor antigen is xCT, gpNMB, carbonic anhydrase IX (CAIX), cKIT, c-MET, tumor-associated glycoprotein 72 (TAG-72), TROP-2, TRA-1-60, TRA, TNF-α, TM4SF1, TIM-1, TAA, TA-MUC1 (tumor-specific epitope of mucin-1), sortilin (SORT1), STn, STING, STEAP-1, SSTR2, SSEA-4, SLITRK6, SLC44A4, SLAMF7, SAIL, receptor tyrosine kinase (RTK), ROR2, ROR1, RNF43, prolactin receptor (PRLR), polymorphic epithelial mucin (PEM), phosphatidylserine (PS), phosphatidylserine, PTK7, PSMA, PD-L1, P -Cadherin, OX001L, OAcGD2, Nectin-4, NaPi2b, NOTCH3, Mesothelin (MSLN), MUC16, MTX5, MTX3, MT1-MMP, MRC2, MET, MAGE, Ly6E, Lewis Y antigen, LRRC15, LRP-1, LIV- 1, LHRH, LGR5, LGALS3BP, LAMP-1, KLK2, KAAG-1, IL4R, IL7R, IL1RAP, IL-4, IL-3, IL-2, IL-13R, IGF-1R, HSP90, HLA-DR, HER-3, HER-2, Globo H, GPR20, GPC3, GPC-1, GD3, GD2, GCC, FSH, FOLR-alpha, FOLR, FLT3, FGFR3, FGFR2, FCRH5, EphA3, EphA2, EpCAM, ETBR, ENPP3, EGFRviii, EGFR, EFNA4, dysadherin, DR5 (death receptor 5), DPEP3, DLL3, DLK-1, DCLK1, Cripto, cathepsin D, CanAg, CXCR5, CSP-1, CLL-1, CLDN6, CLDN18. 2, CEACAM6, CEACAM5, CEA, CDH6, CD79b, CD74, CD71, CD70, CD56, CD51, CD48, CD46, CD45, CD44v6, CD40L, CD38, CD37, CD352, CD33, CD 317, CD30, CD300f, CD3, CD25, CD248, CD228, CD22, CD205, CD20, CD19, CD184, CD166, CD147, CD142, CD138, CD123, CCR7, CA9, CA6, C4.4a, BCMA, B7-H4, B7, -H3, Axl, ASCT2, AMHRII, ALK, AG-7, ADAM-9, 5T4, and 4-1BB.

[0218] Insofar as the names of antigens referred to in the present invention are names of genes, these names refer to the proteins encoded by said genes.

[0219] In one embodiment, the antibody components (each of which is a "molecule" as defined above) Targeting The antibody (including as a moiety) has a first and a second antigen-binding site. Preferably, the first and second antigen-binding sites are capable of binding to different antigens. In one embodiment, the first antigen-binding site is capable of specifically binding to a tumor antigen, and the second antigen-binding site is capable of specifically binding to a tumor antigen.

[0220] In one embodiment, the Targeting Moieties / components of the antibody (each within the "molecule" defined above) Targeting The antibody (included as a moiety) is capable of specifically binding to an immune cell antigen.

[0221] Preferably, the immune cell antigen is an antigen present on the surface of an immune cell, an antigen that is a molecule secreted by an immune cell, or an antigen that is a molecule that interacts with a receptor on an immune cell, and more preferably, the immune cell antigen is an antigen present on the surface of an immune cell.

[0222] In one embodiment, the antigen present on the surface of the target cell is an immune cell antigen present on the surface of an immune cell. In one embodiment, the immune cell is a B cell, a T cell, or a dendritic cell. Preferably, the immune cell is a T cell.

[0223] In one embodiment, the immune cell antigen is selected from the group consisting of CD80, CD86, B7H3, TNF-α, TGF-β, TGF-β2, TGF-1, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-22, IL-23, interferon receptor, PD-1, PD-L1, CTLA4, MSR1, and folate receptor beta.

[0224] Binding of the antibody components (each of which is comprised as a targeting portion of a "molecule" as defined above) to the immune cell antigen can have an immunostimulatory or immunosuppressive effect.

[0225] payload

[0226] In some embodiments, a "molecule" as defined above comprises only one type of functional moiety. As noted above, the ADCs of the invention comprise a payload.

[0227] The term "payload" as used herein refers to a chemical moiety attached to an antibody component as part of an antibody-drug conjugate. In the antibody-drug conjugates of the present invention, the payload is covalently attached to the antibody component via a linker. As described above, the payload (respectively a functional moiety) in the ADCs of the present invention is a therapeutic agent or a detectable label. When the ADC is recruited to its target site by binding of the antibody component to its target antigen, the payload can perform its function at the target site. For example, if the antibody component is specific for a tumor antigen, the payload may be a cytotoxic agent that kills tumor cells, such as a maytansinoid or duocarmycin. Alternatively, if the antibody component is specific for an antigen that indicates inflammation, the payload may be an anti-inflammatory agent, such as a glucocorticoid receptor antagonist, such as cortisol or prednisolone. Alternatively, the payload may be a detectable agent that enables detection of the presence of the target antigen or identification of the target site.

[0228] The payload can be introduced into the ADC at various preparatory stages. In one approach, a linker-payload construct (i.e., a construct in which the payload is covalently attached to the linker) is synthesized by standard methods of organic chemistry (as shown in the Examples), and then this linker-payload construct is attached to the antibody component. However, the antibody component, linker, and payload can also be prepared and attached in a different order (e.g., the linker is attached to the antibody component, followed by the payload being attached to the linker).

[0229] Various payloads, their preparation, conjugation and use in antibody-drug conjugates are described, for example, in Nicolaou et al., Accounts of Chemical Research (2019), vol. 52(1), pp. 127-139; Maderna et al., Molecular Pharmaceutics (2015), vol. 12(6), pp. 1798-1812; Gromek et al., Current Topics in Medicinal Chemistry (2014), vol. 14(24), pp. 2822-2834.

[0230] ADCs according to the invention may comprise only one type of payload (i.e., one ADC molecule is conjugated to only one type of payload, e.g., auristatin E, and one or more copies of the payload (in this example, auristatin E may be conjugated to the ADC molecule)) or several types of payloads (i.e., one ADC molecule is conjugated to two or more types of payloads, e.g., auristatin E and DM4, and may be linked to one or more copies of each payload (in this example, one or more copies of auristatin E and one or more copies of DM4)). Preferably, antibody-drug conjugates according to the invention comprise only one type of payload.

[0231] The copy number payload linked to one ADC molecule (i.e., in the first example above, the number of auristatin E molecules linked to one ADC molecule; in the second example above, the number of auristatin E molecules linked to one ADC molecule plus the number of DM4 molecules linked to one ADC molecule) is reflected in the drug-to-antibody ratio.

[0232] As used herein, the "drug-antibody ratio" of an ADC (abbreviated as "DAR") is the (average) number of payloads per ADC molecule divided by the number of antibody components per ADC molecule. The DAR of an ADC can be determined, for example, by identifying the molecular components of the ADC molecule by mass spectrometry, followed by dividing the number of payload molecules ("drug" molecules that also include the detectable label if the payload is a detectable label). The DAR is equal to the number of antibody components in the ADC molecule (an ADC according to the present invention contains one antibody component per ADC molecule). The DAR values ​​of the embodiments defined below are preferably determined by this approach, i.e., calculated from structural information obtained by mass spectrometry.

[0233] ADCs with different DARs can be prepared by linking different numbers of payloads to ADC molecules. For example, a linker-payload construct containing one payload copy per linker can be prepared, and then multiple copies of this linker-payload construct are attached to each antibody component. As known to those skilled in the art, the number of linker-payload constructs attached to each antibody component can be influenced by reaction conditions (e.g., component concentration, degree of activation of the component, duration of the attachment reaction, etc.). This is described in Example 3 below. See also the section on conjugation below.

[0234] Typically, the drug-antibody ratio (DAR) of an antibody-drug conjugate according to the present invention is in the range of 1 to 15, preferably in the range of 1 to 10, and more preferably in the range of 1 to 10. In another embodiment, the drug-antibody ratio (DAR) of an antibody-drug conjugate according to the present invention is in the range of 4 to 8, particularly preferably in the range of 1 to 4. In one embodiment, the drug-antibody ratio (DAR) of an antibody-drug conjugate according to the present invention is in the range of 2 to 8.

[0235] As noted above, the payload (respectively the functional moiety) of the ADC of the invention can be a therapeutic agent.

[0236] As used herein, a "therapeutic agent" is an agent that, when administered to a patient, exerts an effect associated with a therapeutic benefit (e.g., by killing tumor cells, reducing unwanted inflammation, stimulating immune system activity, suppressing the immune response to infection or in the case of autoimmune disease). Therapeutic agents useful according to the present invention include, but are not limited to, cytotoxic agents, anti-inflammatory agents, immunostimulatory agents, and immunosuppressants.

[0237] In some embodiments, the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent, or an immunosuppressant agent. In some preferred embodiments, the therapeutic agent is a cytotoxic agent.

[0238] As used herein, a "cytotoxic agent" is a substance that is toxic to cells (i.e., causes cell death or destruction). Cytotoxic agents according to the present invention are typically small molecule compounds, peptides, or nucleic acid molecules. Various cytotoxic agents that can be used in ADCs are known to those skilled in the art (Nicolaou et al., Accounts of Chemical Research (2019), vol. 52(1), pp. 127-139; Maderna et al., Molecular Pharmaceutics (2015), vol. 12(6), pp. 1798-1812; Gromek et al., Current Topics in Medicinal Chemistry (2014), vol. 14(24), pp. 2822-2834; Garcia-Echeverria, Journal of Medicinal Chemistry (2014), vol. 57(19), pp. 7888-7889). Examples of cytotoxic agents include, but are not limited to, auristatins (e.g., auristatin E, MMAE (monomethylauristatin E), MMAF (monomethylauristatin F), dolastatin 10, dolastatin 15), maytansinoids (e.g., maytansine, DM1, DM2, DM3, DM4; maytansinoids are derived from maytansine and therefore may also be referred to herein as "maytansines"), tubulysin, exatecan, camptothecin, SN38, Dxd, exatecan, duocarmycin, CBI dimers (cyclopropanebenz[e]indoline dimers, also sometimes referred to herein as "CBI"), doxorubicin, or diazepines (e.g., pyrrolobenzodiazepines or indolinobenzodiazepines).

[0239] In one embodiment, the cytotoxic agent according to the present invention is a chemotherapeutic agent or a radioisotope. Preferably, the cytotoxic agent is a chemotherapeutic agent.

[0240] In some embodiments, the therapeutic agent is an Eg5 inhibitor, a V-ATPase inhibitor, an HSP90 inhibitor, an IAP inhibitor, an mTor inhibitor, a microtubule stabilizer, a microtubule destabilizer, an auristatin, a dolastatin, a MetAP (methionine aminopeptidase), an inhibitor of protein nuclear export CRM1, a DPPIV inhibitor, an inhibitor of mitochondrial phosphotransferase, a protein synthesis inhibitor, a kinase inhibitor, a CDK2 inhibitor, a CDK9 inhibitor, a proteasome inhibitor, a kinesin inhibitor, an HDAC inhibitor, a DNA damaging agent, a DNA alkylating agent, a DNA intercalator, a DNA minor groove binder, an RNA polymerase inhibitor, a topoisomerase inhibitor, or a DHFR inhibitor. Methods for linking each of these to an antibody-compatible linker and the methods of the present invention are known in the art (see, e.g., Singh et al., Therapeutic Antibodies: Methods and Protocols (2009), vol. 525, pp. 445-457).

[0241] In a preferred embodiment, the chemotherapeutic agent is, for example, a maytansinoid (such as DM1, DM2, DM3, or DM4), an antimetabolite (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), an ablative agent (e.g., mechlorethamine, thiotepa, chlorambucil, meifalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan), or a chemotherapeutic agent (e.g., cyclophosphamide, cyclosulfan). , dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC)), and antimitotics (e.g., vincristine, vinblastine) (e.g., Seattle Genetics US 2009 / 0304721), benzodiazepine compounds (e.g., pyrrolobenzodiazepines or indolinobenzodiazepines), taxoids, CC-1065, CC-1065 analogs, duocarmycins, duocarmycin analogs, enediynes (such as calicheamicin), dolastatins or dolastatin analogs (e.g., auristatins), tomaymycin derivatives, leptomycin derivatives, adriamycin, cisplatin, carboplatin, etoposide, melphalan, chlorambucil, calicheamicin, taxanes (WO 01 / 038318 and WO 03 / 097625), DNA alkylating agents (e.g., CC-1065 or CC-1065 analogs), anthracyclines, tubulysin analogs, cytochalasin B, gramicidin D, ethidium bromide, emetine (including derivatives thereof). Further details regarding cytotoxic payloads for ADCs can be found, for example, in Cytotoxic Payloads for Antibody-Drug Conjugates (Drug Discovery, Band 71), 1st edition (2019), editors Thurston and Jackson, Royal Society of Chemistry (UK).

[0242] An example of a cytotoxic agent that is a radioisotope is At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , P 212 , Zr 89 and a radioactive isotope of Lu.

[0243] Cytotoxic agents can achieve cell death through a variety of mechanisms and are therefore classified into various classes depending on their mechanism of action (Nicolaou et al., Accounts of Chemical Research (2019), vol. 52(1), pp. 127-139). In one embodiment, the cytotoxic agent comprised in the ADC of the invention is selected from the group consisting of a microtubule formation inhibitor, an EG5 inhibitor, and a DNA damaging agent (e.g., Anderl et al., Methods in Molecular Biology (2013), vol. 1045, pp. 51-70).

[0244] As used herein, an "inhibitor of microtubule formation" is an inhibitor that acts by inhibiting tubulin polymerization or microtubule assembly and therefore has an anti-proliferative / toxic effect on cells. In a preferred embodiment, the microtubule formation inhibitor is selected from the group consisting of an auristatin (preferably auristatin E, MMAE, or MMAF), a maytansinoid (preferably maytansine, DM1, DM2, DM3, or DM4), and a tubulysin.

[0245] As used herein, "EG5 inhibitor" refers to an inhibitor that inhibits the protein EG5 and is therefore toxic to cells. EG5 refers to human kinesin family member 11, also known as KIF11, HKSP, KNSL1, or TRIP5. EG5 inhibitors are, for example, those described in Macroni et al., Molecules (2019), vol. 24(21), p. 3948 or Karpov et al., ACS Medicinal Chemistry Letters (2019), vol. 10(12), pp. 1674-1679. In a preferred embodiment, the EG5 inhibitor is selected from the group consisting of ispenisib, filanesib, ritronesib, and K858 (Chen et al., ACS Chem Biol. (2017), vol. 12(4), pp. 1038-1046). 1046).

[0246] As used herein, a "DNA damaging agent" refers to an agent that damages cellular DNA, for example, by inducing double-strand breaks, cross-linking specific sites in DNA, or intercalating between DNA base pairs. In a preferred embodiment, the DNA damaging agent is selected from the group consisting of a topoisomerase I inhibitor, a topoisomerase II inhibitor, and a DNA alkylating agent. In some embodiments, the cytotoxic agent is a topoisomerase I inhibitor. In some embodiments, the cytotoxic agent is a topoisomerase II inhibitor. In some embodiments, the cytotoxic agent is a DNA alkylating agent. Preferably, the topoisomerase I inhibitor is selected from the group consisting of exatecan, camptothecin, SN38, Dxd, and variants thereof, and preferably, the topoisomerase I inhibitor is exatecan, SN38, or Dxd. Preferably, the topoisomerase II inhibitor is doxorubicin or a variant thereof, preferably doxorubicin. Preferably, the DNA alkylating agent is selected from the group consisting of duocarmycins, CBI dimers, pyrrolobenzodiazepines and variants thereof, and preferably, the DNA alkylating agent is selected from the group consisting of duocarmycins, CBI dimers and diazepines (preferably pyrrolobenzodiazepines or indolinobenzodiazepines).

[0247] In certain embodiments, the cytotoxic agent is exatecan, duocarmycin, or a CBI dimer.

[0248] In some embodiments, the therapeutic agent is selected from the group consisting of auristatins, MMAE (monomethylauristatin E), duocarmycins, CBI (cyclopropanebenzo[e]indoline) dimers, maytansines, pyrrolobenzodiazepines, and indolinobenzodiazepines. In some embodiments, the therapeutic agent is selected from the group consisting of auristatins, duocarmycins, CBI (cyclopropanebenz[e]indoline) dimers, and maytansinoids. In some embodiments, the therapeutic agent is selected from the group consisting of MMAE (monomethylauristatin E), duocarmycins, CBI (cyclopropanebenz[e]indoline) dimers, and the maytansinoid DM4.

[0249] In some embodiments, the therapeutic agent is selected from the group consisting of dolastatin, auristatin, MMAE, MMAF, amberstatin 269, auristatin 101, auristatin f, auristatin w, CEN-106, CM1, DGN462, DGN549, DM1, DM2, DM4, doxorubicin, duocarmycin, exatecan, OX-4235, PNU-159682, rapamycin, SG3199, SG1882, SN-38, tublysin, amanitin, aminopterin, anthracycline, calicheamicin, camptothecin, fujimycin, hemiasterlin, maytansinoid, PBD, rapamycin, and vinblastine.

[0250] In one embodiment, the therapeutic agent is an anti-inflammatory agent. As used herein, an "anti-inflammatory agent" is a substance that reduces inflammation. This means that the anti-inflammatory agent reduces unwanted inflammation compared to administration of a control molecule that does not contain the anti-inflammatory agent. By recruiting specific immune cells as target cells, the anti-inflammatory effect can be focused on the site of inflammation (where these immune cells may be concentrated) or on specific types of immune cells. Preferably, the anti-inflammatory agent can be a glucocorticoid receptor agonist.

[0251] In certain embodiments, the anti-inflammatory agent is a steroid, preferably selected from the group consisting of cortisol, cortisone acetate, beclomethasone, prednisone, prednisolone, methylprednisolone, betamethasone, tricinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, and mometasone.

[0252] In some embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent, such as a Cox2 inhibitor.

[0253] In one embodiment, the therapeutic agent is an immunostimulatory agent. As used herein, an "immunostimulatory agent" is a substance that enhances the generation or maintenance of an immune response. An immunostimulatory agent may be an agonist of an immunostimulatory molecule or an antagonist of a molecule that inhibits an immune response. In one embodiment, the immunostimulatory agent comprises an agonist of an immunostimulatory molecule, such as an agonist of a costimulatory molecule found on immune cells (e.g., T cells) or an agonist of a costimulatory molecule found on cells involved in innate immunity (e.g., T cells or NK cells). In one embodiment, the immunostimulatory agent comprises an antagonist of an immunosuppressive molecule, e.g., an antagonist of a costimulatory molecule found on cells involved in innate immunity (e.g., NK cells). Preferably, administration of an ADC comprising an immunostimulatory agent as a payload results in an improvement in the desired immune response. In one embodiment, administration of an ADC comprising an immunostimulatory agent as a payload results in an improved anti-tumor response in an animal cancer model, such as a xenograft model, compared to administration of a control molecule that does not contain the immunostimulatory agent.

[0254] In some embodiments, the immunostimulatory agent is or comprises an antagonist of an inhibitor of T cell activation. In some embodiments, the immunostimulatory agent is or comprises an agonist of a stimulator of T cell activation. In some embodiments, the immunostimulatory agent is or comprises an agent that antagonizes or blocks cytokines that inhibit T cell activation, such as IL-6, IL-10, TGFβ, or VEGF. In some embodiments, at least one immunostimulatory agent comprises an antagonist of a chemokine, such as CXCR2, CXCR4, CCR2, or CCR4. In some embodiments, the immunostimulatory agent is or comprises an agonist of a cytokine that stimulates T cell activation, such as IL-2, IL-7, IL-12, IL-15, IL-21, and IFNα.

[0255] In a preferred embodiment, the immunostimulant is selected from the group consisting of a TLR7 agonist, a TLR8 agonist, a TLR7 antagonist, a TLR8 antagonist, a Sting inhibitor, a TGFbeta inhibitor, an alpha2A inhibitor and an alpha2B inhibitor.

[0256] In some embodiments, the therapeutic agent is an immunosuppressant.

[0257] As used herein, an "immunosuppressant" is an agent that inhibits the development or maintenance of an immune response. Such inhibition by an immunosuppressant can include, for example, elimination of immune cells (e.g., T or B lymphocytes); induction or generation of immune cells that can modulate (e.g., downregulate) the functional capabilities of other cells; induction of an unresponsive state in immune cells (e.g., anergy); or an increase, decrease, or alteration of immune cell activity or function, including, for example, alteration of the pattern of proteins expressed by these cells (e.g., altered production and / or secretion of specific classes of molecules, such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors). In typical embodiments, an immunosuppressant has a cytotoxic or cytostatic effect on immune cells that promote an immune response. In some embodiments, the immunosuppressant results in a reduction of an undesired immune response compared to administration of a control molecule that does not contain the immunosuppressant.

[0258] As used herein, "immune cell" refers to a T cell (T lymphocyte), a B cell (B lymphocyte), or any cell of the hematopoietic system involved in regulating the immune response to an antigen (e.g., an autoantigen). Dendritic cells. Preferably, the immune cell according to the present invention is a T cell or a B cell.

[0259] In a preferred embodiment, the immunosuppressant according to the present invention is selected from the group consisting of IMDH (inosine monophosphate dehydrogenase) inhibitors, mTor (mechanistic target of rapamycin) inhibitors, SYK (spleen tyrosine kinase) inhibitors, JAK (Janus kinase) inhibitors and calcineurin inhibitors.

[0260] In certain embodiments of the antibody-drug conjugates according to the invention, the payload is a detectable label. As used herein, a "detectable label" refers to a molecule that is detectable (i.e., detectable using detection methods known in the art, such as radiography, fluorescence, chemiluminescence, enzyme activity, or absorbance-based detection methods).

[0261] ADCs with a detectable label as a payload can be useful for monitoring or prognosing the onset, development, progression, and / or severity of a disease or disorder as part of a clinical trial procedure, such as diagnosing a disease, identifying the site of a disease, or determining the effectiveness of a particular therapy. There are even situations in which a detectable label can be used in combination with a therapeutic agent (e.g., Rondon and Degoul, Bioconjugate Chemistry (2020), vol. 31(2), pp. 159-173).

[0262] Detectable labels as payloads include, for example, enzymes (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase), prosthetic groups (such as streptavidin / biotin and avidin / biotin), fluorophores (Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 70 ... Fluor® 750, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin), luminescent substances (e.g., luminol), bioluminescent substances (e.g., luciferase, luciferin, or aequorin), radioactive substances (iodine (131I, 125I, 123I) または 121 I) ,carbon など )( 14 C), sulfur ( 35S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In or 111 In), technetium ( 99 Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F). 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 64 Cu, 113 Sn and 117 Sn), positron-emitting metals (for positron emission tomography) or non-radioactive paramagnetic metal ions, etc. Alternatively, the detectable label can be, for example, a fluorophore, spin label, infrared probe, affinity probe, spectroscopic probe, radioactive probe or quantum dot.

[0263] In some embodiments, the detectable label is a radioisotope, a fluorophore, a chromophore, an enzyme, a dye, a metal ion, a ligand (such as biotin, avidin, streptavidin, or a hapten), or a quantum dot. In some embodiments, the detectable label is a radioisotope, a fluorescent compound, or an enzyme.

[0264] In certain embodiments, the detectable label is a cyanine dye, a sulfocyanine dye, an Alexa Fluor® dye (Molecular Probes / Thermo Fisher Scientific), a DyLight® Fluor dye (Dyomics / Thermo Fisher Scientific), a FluoProbes® dye (Interchim), a Seta® dye (SETA BioMedicals), and an IRIS. TM dyes (Cyanine Technologies). Preferably, the detectable label is a cyanine dye or a sulfocyanine dye.

[0265] In one embodiment, the detectable label is a cyanine dye selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7.

[0266] In certain embodiments, the detectable label is a sulfo-cyanine dye selected from the group consisting of sulfo-Cy2, sulfo-Cy3, sulfo-Cy3B, sulfo-Cy3.5, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7.

[0267] Linker

[0268] As noted above, the antibody-drug conjugates according to the invention comprise a linker, which is a molecular group that covalently connects the payload and the antibody component of the ADC.

[0269] Various linkers that can be used in the ADCs and related methods of the invention are described in WO 2004 / 010957 entitled "Drug conjugates and uses thereof for the treatment of cancer, autoimmune or infectious diseases."

[0270] Typically, there is one linker per payload (i.e., one linker molecule for each occurrence of the payload in the ADC; this means that if two copies of the payload are present in the ADC, then two linkers will be present: the first linker covalently attaches the first payload to the antibody component, and the second linker covalently attaches the second payload to the antibody component). However, it is also possible for a single linker to attach multiple payload moieties to the antibody component of an ADC.

[0271] Depending on the number of payloads linked to the antibody component and the number of payloads per linker, there may be one or more linkers in the ADC.

[0272] Covalent attachment of the antibody component to the payload via a linker can be achieved, for example, by a linker having two reactive functional groups (i.e., a linker that is bivalent in the sense of reactivity). Bivalent linker agents useful for linking two or more functional or biologically active components are known to those skilled in the art (see, for example, Hermanson, Bioconjugate Techniques (1996), Academic Press (New York), pp. 234-242).

[0273] Alternatively, linker-payload constructs comprising a payload covalently attached to a linker can be prepared by methods of organic synthesis. One or more copies of this linker-payload construct can then be conjugated to an antibody component by methods known to those skilled in the art (e.g., Behrens et al., Molecular Pharmaceutics (2015), vol. 12(11), pp. 3986-3998; Stefano, Methods in Molecular Biology (2013), vol. 1045, pp. 145-171; Dickgiesser et al., in: Methods in Molecular Biology: Enzyme-Mediated Ligation Methods (2019), editors Nuijens and Schmidt, vol. 2012, pp. 135-149; Dickgiesser et al., Bioconjugate Chem. (2020), vol. 31(4), pp. 1070-1076) and described in Example 3 below.

[0274] The linker in the antibody-drug conjugate of the present invention is preferably stable extracellularly (i.e., outside of cells, for example, in plasma). Thus, before transport or delivery to cells, the ADC preferably remains stable and intact, i.e., the antibody remains bound to the payload. An effective linker (i) maintains the specific binding properties of the antibody; (ii) enables intracellular delivery of the payload; (iii) remains stable and intact, i.e., is not cleaved, until the conjugate is delivered or transported to its target site; and (iv) maintains the therapeutic effect of the payload (e.g., the cytotoxicity, cell-killing effect of the payload).

[0275] Whether a linker is stable in the extracellular environment can be determined, for example, by independently incubating with plasma both (a) the ADC ("ADC sample") and (b) an equimolar amount of unbound antibody or therapeutic agent ("ADC sample"). The "control sample") is allowed to stand for a predetermined period of time (e.g., 8 hours), after which the amount of unbound antibody or therapeutic agent present in the ADC sample is compared to the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0276] A linker that is stable outside a target cell may be cleaved at a certain effective rate inside the target cell. In some embodiments, a linker that is cleavable under intracellular conditions can be cleaved by a cleaving agent present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal protease or an endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs within target cells (e.g., Dubowchik and Walker, Pharm. Therapeutics (1999), vol. 83, pp. 67-123). For example, a peptidyl linker that can be cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissues, can be used (e.g., a Phe-Leu linker or a Gly-Phe-Leu-Gly linker (SEQ ID NO: 3)). Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In certain embodiments, the peptidyl linker that can be cleaved by intracellular proteases is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a Val-Cit linker). One advantage of using intracellular proteolytic release of therapeutic agents is that the therapeutic agent is usually weakened upon binding, and the serum stability of the conjugate is usually high.

[0277] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes can be used (see, for example, U.S. Patent No. 5,122,368; U.S. Patent No. 5,122,368; U.S. Patent No. 5,824,805; U.S. Patent No. 5,622,929; Dubowchik and Walker, Pharm. Therapeutics (1999), vol. 83, pp. 67-123; Neville et al., Biol. Chem. (1989), vol. 264, pp. 14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but become unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to a therapeutic agent via an acylhydrazone bond; see, e.g., U.S. Pat. No. 5,622,929).

[0278] In yet other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT (e.g., Thorpe et al., Cancer Res. (1987), vol. 47, pp. 5924-5931; Wawrzynczak et al., in: Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (1987), editor Vogel, Oxford U. Press; U.S. Patent 4,880,935).

[0279] In other embodiments, the linker is not cleavable within the target cell, but the payload is released, for example, by antibody degradation.

[0280] In yet another specific embodiment, the linker is a malonic acid linker (Johnson et al., Anticancer Res. (1995), vol. 15, pp. 1387-1393), a maleimidobenzoyl linker (Lau et al., Bioorg-Med-Chem. (1995), vol. 3(10), pp. 1299-1304), or a 3'-N-amide analog (Lau et al., Bioorg-Med-Chem. (1995), vol. 3(10), pp. 1305-1312).

[0281] The linker may be cleavable under intracellular conditions (as described above) or cleavable under intracellular conditions. In some embodiments, the linker is not cleavable under intracellular conditions. In other embodiments, the linker is cleavable under intracellular conditions. Such linkers are particularly preferred when the payload is a therapeutic agent. Preferably, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the payload from the antibody component in the intracellular environment.

[0282] Whether the linker of an ADC is stable or cleaved can be determined by exposing the ADC to the conditions to be tested and then verifying the integrity of the linker in treated and untreated control samples by standard analytical techniques such as mass spectrometry, HPLC, or the separation / analysis technique LC / MS.

[0283] In one embodiment, the / each of the linkers has a molecular weight of at most 1,500 Da, preferably at most 1,000 Da, more preferably at most 500 Da.

[0284] In some embodiments, the linker is stable in the extracellular environment.

[0285] The designation that a linker is "stable in an extracellular environment" preferably means that the linker is stable in human serum. In an assay in which an ADC molecule containing the linker is exposed to human serum, a linker is "stable in human serum" if at least 50%, preferably at least 75%, of the linker is stable in human serum after 48 hours of incubation at 37°C. The ADC is not cleaved or degraded. In one embodiment, the linker is stable in an intracellular environment.

[0286] Preferably, a linker that is "stable in an intracellular environment" is one in which, when an ADC molecule containing the linker is taken up by a cell (i.e., enters the intracellular environment of the cell), at least 50%, preferably at least 75%, of the linker in the ADC molecule is not cleaved or degraded even when left at 37°C for 24 hours.

[0287] In one embodiment, the linker is cleaved upon exposure to the intracellular environment. Preferably, a linker that is "cleaved upon exposure to the intracellular environment" is one that has a structure that allows the linker to be degraded when an ADC molecule containing the linker is taken up by a cell (i.e., enters the intracellular environment of a cell). The linker in the ADC molecule is efficiently cleaved (preferably, at least 90% of the linker is cleaved within 24 hours, more preferably within 12 hours). As will be understood by those skilled in the art, this allows the release of the payload into the target cell.

[0288] In one embodiment, the linker is stable in an extracellular environment but cleaved when exposed to an intracellular environment. Preferably, a linker that is "stable in an extracellular environment but cleaved when exposed to an intracellular environment" refers to a linker that is stable in human serum, but has a structure that allows the linker in the ADC molecule to be efficiently cleaved when the ADC molecule containing the linker is taken up by a cell (i.e., when it enters the intracellular environment of the cell) (preferably, at least 90% of the linker is cleaved within 24 hours, more preferably within 12 hours). As will be understood by those skilled in the art, this allows the release of the payload into the target cell.

[0289] In certain embodiments, the linker is cleavable by enzymatic or chemical cleavage.

[0290] As used herein, an "enzymatically cleavable" linker is a linker that is cleaved in the presence of a specific enzyme but is stable in the absence of this enzyme. For the purpose of the ADC of the present invention, this enzyme is typically not exposed to the ADC in the extracellular environment, but is exposed upon uptake of the ADC into target cells, resulting in an extracellular linker. Stable but cleaved upon entry into target cells.

[0291] As used herein, a "chemically cleavable" linker is a linker that is cleaved by a non-enzymatic reaction that results in cleavage of a covalent chemical bond. Examples include linkers that are pH sensitive or cleavable under reducing conditions (see above).

[0292] In some embodiments, the linker is cleavable by enzymatic cleavage, hi some embodiments, the enzymatic cleavage is by exposure to a glycosidase, protease, or esterase.

[0293] Glycosidases are enzymes in EC 3.2.1 that catalyze the hydrolysis of glycosidic bonds in complex sugars. Proteases are enzymes in EC 3.4 that catalyze the cleavage of peptide bonds. Esterases are enzymes in EC 3.1 that catalyze the cleavage of ester bonds. Preferably, the glycosidase is a glucuronidase. Glucuronidases are enzymes in EC 3.2.1.31 that catalyze the cleavage of β-glucuronides.

[0294] Preferably, the protease is a cathepsin (most preferably cathepsin B). Cathepsins are a group of proteases in EC 3.4 that catalyze the proteolytic cleavage of peptide bonds. For the purposes of the present invention, the use of endolysosomal proteolytic cathepsins is particularly advantageous because they are activated at low pH (as in lysosomes) and cleave within peptide sequences. Cathepsin B is a cathepsin classified as EC 3.4.22.1.

[0295] In one embodiment, the enzymatic cleavage is by exposure to a tumor-specific enzyme, preferably a tumor-specific protease or esterase. A "tumor-specific" enzyme is an enzyme that is present in a particular tumor (i.e., there is enzymatic activity of the enzyme within the tumor), but is substantially absent in other cells and tissues (i.e., outside the tumor). There is substantially no, preferably no, enzymatic activity of the enzyme.

[0296] In one embodiment, the linker comprises a protease cleavage site, preferably a cathepsin B cleavage site.

[0297] In some embodiments, the linker comprises / the linker comprises a glucuronide (a molecular group that can be cleaved by glucuronidase).

[0298] In some embodiments, the linker is cleavable by chemical cleavage. In some embodiments, the linker is cleavable by chemical cleavage is a pH-sensitive linker / the linker is cleavable by chemical cleavage is a pH-sensitive linker. Preferably, the linker comprises a hydrazone / the linker comprises a hydrazone.

[0299] In one embodiment, the linker cleavable by chemical cleavage is cleavable under reducing conditions, and the linker cleavable by chemical cleavage is cleavable under reducing conditions. Preferably, the linker comprises a disulfide bond / the linker comprises a disulfide bond.

[0300] In certain embodiments, the linker comprises a cathepsin B cleavage site, a glucuronide bond, or a disulfide bond.

[0301] Soluble Tags

[0302] The antibody-drug conjugates (each a "molecule" as defined above) according to the present invention comprise a soluble tag.

[0303] As will be understood by those skilled in the art, a "solubility tag" is a molecular group attached to a molecule of interest that has the purpose of increasing the solubility of the molecule of interest in an aqueous environment compared to the same molecule of interest without the solubility tag. Thus, it is intended that a molecule to which a solubility tag is attached will have a higher solubility in an aqueous environment than the same molecule without the solubility tag attached.

[0304] The soluble tags of the present invention are based on oligosaccharides, and as shown in the examples of the present invention, the inclusion of such soluble tags in ADCs provides a variety of advantageous effects.

[0305] ADCs according to the invention may contain one or more solubility tags per ADC molecule. Typically, the solubility tags are covalently attached to the antibody-drug conjugate.

[0306] Typically, in an ADC according to the invention, the soluble tag is attached to the ADC of the invention by a covalent bond between the soluble tag and the linker. However, the soluble tag can also be attached to the ADC by a covalent bond between the soluble tag and a component of the ADC other than the linker.

[0307] When the present invention states that one molecule or moiety A is "covalently" attached / bonded to another molecule or moiety B, this indicates that there are no additional atoms or molecular groups between molecule / moiety A and molecule / moiety B, and that molecule / moiety A is directly bonded to molecule / moiety B by a chemical bond.

[0308] In some embodiments, the soluble tag is covalently attached to Targeting In some embodiments, the soluble tag is covalently attached to the targeting moiety, functional moiety, or linker. In some embodiments, the soluble tag is covalently attached to the targeting moiety. In some embodiments, the soluble tag is covalently attached to the functional moiety. In some embodiments, the soluble tag is covalently attached to the linker.

[0309] In some embodiments, the soluble tag is covalently bound to the antibody component, at least one payload, or linker. In some embodiments, the soluble tag is covalently bound to the antibody component. In some embodiments, the soluble tag is covalently bound to the at least one payload. In some embodiments, the soluble tag is covalently bound to the linker.

[0310] In certain embodiments, the soluble tag is (i) covalently attached to the antibody component but not to at least one payload or linker, or (ii) covalently attached to at least one payload or linker, but included in the payload but not included in the antibody component or linker, or (iii) included in the linker but not included in the antibody component or at least one payload.

[0311] In some embodiments, the soluble tag is covalently attached only to the antibody component (and not to at least one payload or linker). In some embodiments, the soluble tag is covalently attached only to at least one payload (and not to an antibody component or linker). In some embodiments, the soluble tag is covalently attached only to the linker (and not to an antibody component or at least one payload).

[0312] In one embodiment, the soluble tag is covalently attached to at least one of: (i) an antibody component, (ii) at least one payload, and (iii) a linker covalently linking the payload and the antibody component of an ADC according to the invention.

[0313] In some embodiments, the antibody component (i), the payload (ii), and the linker (iii) are covalently linked. In some embodiments, the antibody component (i), the payload (ii), the linker (iii), and the solubility tag (iv) are covalently linked.

[0314] The antibody-drug conjugate according to the present invention can comprise one or more solubility tags. In some embodiments, the molecule comprises only one solubility tag. In some embodiments, the molecule comprises at least one, preferably at least two, more preferably at least three, more preferably at least four solubility tags. In some embodiments, the molecule comprises up to 10, preferably up to 8, more preferably up to 6, more preferably up to 4, more preferably up to two solubility tags, more preferably only one solubility tag. In some embodiments, the molecule comprises at least one and up to four solubility tags. In some embodiments, the molecule comprises at least three and up to 10 solubility tags.

[0315] In some embodiments, at least one soluble tag is covalently bound to the molecule. In other embodiments, at least two, preferably at least three, more preferably at least four soluble tags are covalently bound to the molecule. In some embodiments, up to 10, preferably up to 6, more preferably up to 4, more preferably up to 2 soluble tags are covalently bound to the molecule, more preferably only one soluble tag is covalently bound to the molecule. In some embodiments, at least one and up to four soluble tags are covalently bound to the molecule. In some embodiments, at least three and up to 10 soluble tags are covalently bound to the molecule. As will be understood by those skilled in the art, the number of soluble tags covalently bound to a molecule is an average number (which is determined for a population of the molecule). Preferably, the population is a homogeneous population.

[0316] In certain embodiments, the antibody-drug conjugate comprises at least one solubility tag. In certain embodiments, the antibody-drug conjugate comprises at least two, preferably at least three, and more preferably at least four solubility tags. In certain embodiments, the antibody-drug conjugate comprises up to 10, preferably up to 8, more preferably up to 6, more preferably up to 4, and more preferably up to 2 solubility tags. In certain embodiments, the antibody-drug conjugate comprises at least one and up to four solubility tags. In certain embodiments, the antibody-drug conjugate comprises at least three and up to 10 solubility tags. In certain embodiments, the antibody-drug conjugate comprises only one solubility tag.

[0317] In some embodiments, at least one solubility tag is covalently attached to the antibody-drug conjugate. In other embodiments, at least two, preferably at least three, and more preferably at least four solubility tags are covalently attached to the antibody-drug conjugate. In some embodiments, only one, preferably up to two, more preferably up to four, more preferably up to six, more preferably up to eight, and more preferably up to ten solubility tags are covalently attached to the antibody-drug conjugate. In some embodiments, at least one and up to four solubility tags are covalently attached to the antibody-drug conjugate. In some embodiments, at least three and up to ten solubility tags are covalently attached to the antibody-drug conjugate. As will be understood by those skilled in the art, the number of solubility tags covalently attached to an antibody-drug conjugate is an average number (determined across a population of molecules of an ADC). Preferably, the population is a homogeneous population.

[0318] In some embodiments, no more than three soluble tags are covalently attached per linker. In preferred embodiments, no more than two soluble tags are covalently attached per linker. In more preferred embodiments, no more than one soluble tag is covalently attached per linker.

[0319] In some embodiments, no more than three soluble tags are covalently attached per payload. In preferred embodiments, no more than two soluble tags are covalently attached per payload. In more preferred embodiments, no more than one soluble tag is covalently attached per payload.

[0320] In one embodiment, no more than three solubility tags are covalently attached per antibody component. In a preferred embodiment, no more than two solubility tags are covalently attached per antibody component. In a more preferred embodiment, no more than one solubility tag is covalently attached per antibody component.

[0321] In a preferred embodiment, only one type of solubility tag is covalently attached to an antibody-drug conjugate, meaning that all solubility tags covalently attached to the antibody-drug conjugate are identical (they are of the same type in terms of molecular structure).

[0322] In certain embodiments, more than one type of solubility tag is covalently attached to the antibody-drug conjugate, meaning that there are at least two different types of solubility tags with different structures covalently attached to the antibody-drug conjugate (i.e., there are more than one type of solubility tag covalently attached to the ADC, in terms of molecular structure).

[0323] In some embodiments, up to two soluble tags are covalently attached to the antibody-drug conjugate.

[0324] In a preferred embodiment, the antibody-drug conjugate comprises only one type of solubility tag, meaning that all solubility tags comprised in the antibody-drug conjugate are identical (they are of the same type in terms of molecular structure).

[0325] In certain embodiments, the antibody-drug conjugate comprises two or more types of solubility tags, meaning that the antibody-drug conjugate comprises at least two different types of solubility tags having different structures (i.e., the solubility tags covalently attached to the ADC are of multiple types with respect to their molecular structures).

[0326] In certain embodiments, the antibody-drug conjugate comprises up to two different types of soluble tags.

[0327] Typically, the soluble tag of the ADC of the invention will comprise a covalently attached monosaccharide unit.

[0328] In some embodiments, the soluble tag comprises / consists of a monosaccharide unit. In some embodiments, the soluble tag consists / consists of a monosaccharide unit.

[0329] As used herein, a "monosaccharide" is a sugar that cannot be hydrolyzed to simpler sugars, is classified as either an aldose or a ketose, and contains one or more hydroxyl groups (-OH) per molecule. Examples of monosaccharides include glucose (dextrose), fructose (levulose), and galactose. Monosaccharides are the building blocks of disaccharides (such as sucrose and lactose), oligosaccharides, and polysaccharides (such as cellulose and starch). In the present invention, the terms "monosaccharide" or "monosaccharide unit" are used to refer to a single monosaccharide residue in an oligosaccharide. Within the context of an oligosaccharide, a monosaccharide unit is a monosaccharide that is linked to another monosaccharide via a covalent bond (e.g., a glycosidic bond) formed by the hydroxyl group of the monosaccharide.

[0330] As used herein, "oligosaccharide" refers to a compound containing two or more monosaccharide units. Preferably, the term "oligosaccharide" refers to a compound containing 2 to 12 monosaccharide units joined by glycosidic bonds. In accordance with accepted nomenclature, oligosaccharides are depicted herein with the non-reducing end on the left and the reducing end on the right.

[0331] Monosaccharides and oligosaccharides can be chemically synthesized by standard methods of carbohydrate chemistry (see, for example, Preparative Carbohydrate Chemistry (1997), editor Hanessian, publisher Marcel Dekker, Inc. (New York); Carbohydrate Chemistry: Proven Synthetic Methods (2015), editors Roy and Vidal, CRC Press; Carbohydrate Chemistry: State of the Art and Challenges for Drug Development (2016), editor Cipolla, Imperial College Press (London); CRC Handbook of Oligosaccharides (1990), Vol. I-III, (published 2019), editors: Liptak et al., CHR Press, Inc.; Liaqat and Eltem, Carbohydrate Polymers (2018), Vol. 184, pp. 243-259).

[0332] Alternatively, oligosaccharides can be prepared by biotechnological methods (see, for example, Meyer et al., Biotechnological Production of Oligosaccharides - Applications in the Food Industry, Food Production and Industry (2015), Ayman Hafiz Amer Eissa, IntechOpen, DOI: 10.5772 / 60934; Liaqat and Eltem, Carbohydrate Polymers (2018), vol. 184, pp. 243-259; Samain et al., Carbohydrate Research (1997), vol. 302, pp. 35-42; Samain et al., Biotechnol. (1999), vol. 72, pp. 33-47).

[0333] To further increase the purity of the oligosaccharides prepared by the above approaches, the oligosaccharides may be purified by, for example, precipitation, recrystallization, ultrafiltration, nanofiltration, gel permeation chromatography, ion exchange chromatography, capillary electrophoresis, HPLC purification, UPLC purification, or other methods as described, for example, in Pinelo et al., Separation and Purification Technology (2009), vol. 70(1), pp. 1-11.

[0334] Monosaccharides and oligosaccharides can be characterized by standard methods known to those skilled in the art (see, for example, Carbohydrate Chemistry (1988), editor El Khadem, Academic Press (San Diego)), including, for example, LC-MS / ESI MS, 1D and 2D NMR, gel permeation chromatography, or ion mobility mass spectrometry (Seeberger et al., Nature (2015), vol. 526 (7572), pp. 241-244).

[0335] In some embodiments, the soluble tag comprises an oligosaccharide consisting of monosaccharide units. In some embodiments, the soluble tag comprises an oligosaccharide consisting of monosaccharide units.

[0336] In one embodiment, the soluble tag comprises up to 25, preferably up to 20, more preferably up to 15, more preferably up to 12, more preferably up to 10, more preferably up to 9, up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5 monosaccharide units.

[0337] In one embodiment, the soluble tag comprises at least 2, preferably at least 3, more preferably at least 4, more preferably at least 5 monosaccharide units.

[0338] In one embodiment, the soluble tag comprises / each comprises 5 monosaccharide units.

[0339] In one embodiment the / each soluble tag consists of 3 to 8, preferably 4 to 8, more preferably 4 to 7, more preferably 4 to 6, more preferably 4 or 5 monosaccharide units.

[0340] In one embodiment the / each soluble tag consists of 3 to 8, preferably 4 to 8, more preferably 4 to 7 monosaccharide units.

[0341] In a particularly preferred embodiment, the / each soluble tag consists of 4 to 6 monosaccharide units.

[0342] In some embodiments, the / each soluble tag consists of 4 or 5 monosaccharide units. In some embodiments, the / each soluble tag consists of 5 or 6 monosaccharide units. In some embodiments, the / each soluble tag consists of 5 monosaccharide units.

[0343] In one embodiment, the monosaccharide units comprising the / each soluble tag are covalently linked to form an oligosaccharide.

[0344] In a particularly preferred embodiment, the soluble tag of the antibody-drug conjugate according to the invention may comprise or consist of a chitooligosaccharide.

[0345] As used herein, the term "chitooligosaccharides" (abbreviated herein as "CO") refers to oligosaccharides obtained by hydrolysis of chitin (non-deacetylated, partially deacetylated, or fully deacetylated) with dilute aqueous mineral acid. This results in a mixture of various chitooligosaccharides that can be further separated into different chitooligosaccharide species, for example, by ultrafiltration, gel permeation chromatography, cation exchange chromatography, and capillary electrophoresis. Preparation of chitooligosaccharides by this approach is described, for example, in Schmitz et al., Marine Drugs (2019), vol. 17(8), p. 452.

[0346] Alternatively, chitooligosaccharides can be obtained by chemical synthesis (Bohe and Crich, in: Comprehensive Organic Synthesis, 2nd edition (2014), vol. 6, editors Knochel and Molander, Elsevier Ltd.; Solid Support Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries (2001), editor Seeberger, John Wiley & Sons, Inc.). As a further alternative, chitooligosaccharides can be obtained by biotechnological approaches (see, for example, Samain et al., Carbohydrate Research (1997), vol. 302, pp. 35-42; Samain et al., Biotechnol. (1999), vol. 72, pp. 33-47). The chitooligosaccharides can then be further purified and characterized as described above for general oligosaccharides.

[0347] Chitooligosaccharides are typically composed of D-glucosamine (GlcN) and / or N-acetyl-D-glucosamine (GlcNAc) and have the general formula (GlcNAc) m (GlcN) n where m and n are integers. The monosaccharide units of chitooligosaccharides are usually linked by β-(1,4)-glycosidic bonds.

[0348] As used herein, "N-acetyl-D-glucosamine," also known as "N-acetylglucosamine" and abbreviated as "GlcNAc," is 2-acetylamino-2-deoxy-D-glucose (also called 2-acetamido-2-deoxy-D-glucose). Glucose is abbreviated herein as "Glc."

[0349] As used herein, D-glucosamine, also known as "glucosamine" and abbreviated as "GlcN," is 2-amino-2-deoxy-D-glucose.

[0350] Examples of chitooligosaccharides are summarized below in Table 1. CAS® Registry Numbers for the exact definitions of the compounds are provided. [Table 1-1] [Table 1-2]

[0351] In one embodiment, the / each soluble tag comprises a chitooligosaccharide.

[0352] In a preferred embodiment, the / each soluble tag is a chitooligosaccharide (ie the / each soluble tag consists of a chitooligosaccharide).

[0353] In certain embodiments, the chitooligosaccharide is selected from the chitooligosaccharides shown in Table 1. Preferably, the chitooligosaccharide is a chitooligosaccharide having 3 to 7 monosaccharide units shown in Table 1. More preferably, the chitooligosaccharide is a chitooligosaccharide having 4 to 6 monosaccharide units shown in Table 1.

[0354] In one embodiment, the monosaccharide units of the soluble tag according to the present invention (i.e., the monosaccharide units contained in the soluble tag according to the present invention, each of which constitutes a soluble tag according to the present invention) are independently selected from the group consisting of aldoses, ketoses, and chemically modified forms of the aldoses or ketoses.

[0355] n (H2O) n Aldoses refer to monosaccharides that are polyhydroxyaldehydes having n (n is an integer) and a single aldehyde group (-CH=O) per molecule (during ring formation, the aldehyde group reacts with a hydroxyl group to form a hemiacetal). Non-limiting examples of aldoses include aldohexoses (e.g., all six-carbon, aldehyde-containing sugars, including glucose, mannose, and galactose), aldopentoses (e.g., all five-carbon, aldehyde-containing sugars, including xylose and arabinose), and aldotetroses (all four-carbon, aldehyde-containing sugars, including erythrose).

[0356] As used herein, the term "ketose" refers to a compound of the general chemical formula C n (H2O) n (n is an integer) refers to a monosaccharide that is a polyhydroxyketone with a single ketone group (=O) per molecule. (During ring formation, the ketone group reacts with a hydroxyl group to form a hemiketal.) Non-limiting examples of ketoses include ketohexoses (e.g., all 6-carbon ketone-containing sugars, including fructose), ketopentoses (e.g., all 5-carbon ketone-containing sugars, including xylulose and ribulose), and ketotetroses (all 4-carbon ketone-containing sugars). Ketose-containing sugars include, for example, erythrulose.

[0357] For purposes of the present invention, aldoses and ketoses as defined above are considered to be "unmodified" monosaccharides.

[0358] A "chemically modified" form of a monosaccharide refers to a monosaccharide that is not an unmodified monosaccharide as defined above, but that differs from the unmodified monosaccharide as defined above by the addition, removal, or substitution of at least one atom or molecular group.

[0359] The oligosaccharide used in the Examples, CO-V (see structural formula (I)), is lightly modified compared to unmodified oligosaccharides: one GlcN to one Glc (i.e., one substitution of a glucose hydroxyl group with an amino group) and four GlcNAc to one Glc (i.e., four substitutions of a glucose hydroxyl group with an N-acetylamino group). As one skilled in the art will recognize, more intensive chemical modifications compared to the soluble tags of the Examples may result in more pronounced changes in the properties of the soluble tag compared to the soluble tags of the Examples. Thus, while the present invention encompasses soluble tags with additional chemical modifications compared to the Examples, it is understood that a degree of chemical modification that results in less deviation from the soluble tags of the Examples is preferred over a more extensive degree of chemical modification.

[0360] In some embodiments, the monosaccharide units are independently selected from the group consisting of aldoses and chemically modified forms of the aldoses.

[0361] In certain embodiments, the monosaccharide units are individually selected from the group consisting of tetrose, pentose, hexose, heptose, octose, and chemically modified forms of tetrose, pentose, hexose, heptose, and octose. Preferably, the monosaccharide units are individually selected from the group consisting of tetrose, pentose, hexose, and chemically modified forms of tetrose, pentose, and hexose. More preferably, the monosaccharide units are individually selected from the group consisting of pentose, hexose, and chemically modified forms of pentose and hexose. More preferably, the monosaccharide units are selected from the group consisting of hexose and chemically modified forms of hexose.

[0362] The term "individually selected" indicates that each individual monosaccharide unit can be independently selected from the listed options. For example, if the monosaccharide units of a three-monosaccharide oligosaccharide are "individually selected" from the group consisting of pentose (P) and hexose (H), then the first, second, and third monosaccharide units of the oligosaccharide can each be independently selected from the group consisting of pentose (P) and hexose (H). Thus, in this example, combinations include PPP, PPH, PHP, PHH, HPP, HPH, HHP, and HHH.

[0363] As those skilled in the art are aware, tetroses, pentoses, hexoses, heptoses and octose have the chemical formula C n (H2O) n where n=4 for tetroses, n=5 for pentoses, n=6 for hexoses, n=7 for heptoses, and n=8 for octose. Tetroses, pentoses, hexoses, heptoses, and octose can exist as aldoses or ketoses.

[0364] In some embodiments, the monosaccharide units are individually selected from the group consisting of tetrose, pentose, hexose, heptose, and octose. Preferably, the monosaccharide units are individually selected from the group consisting of tetrose, pentose, and hexose. More preferably, the monosaccharide units are individually selected from the group consisting of pentose and hexose. More preferably, the monosaccharide unit is a hexose.

[0365] In some embodiments, the monosaccharide units of the soluble tag are not modified by chemical modification.

[0366] In one embodiment, the tetrose, pentose, hexose, heptose, and octose have the formula C n (H2O) n where n=4 for tetrose, n=5 for pentose, n=6 for hexose, n=7 for heptose, and n=8 for octose.

[0367] In one embodiment, the aldose has the formula C n (H2O) n wherein preferably n is an integer and 4≦n≦12, more preferably n is an integer and 4≦n≦8, more preferably n is an integer and 5≦n≦7, more preferably n is 5 or 6, and even more preferably n is 6.

[0368] In one embodiment, the ketose has the formula C n (H2O) n wherein preferably n is an integer and 4≦n≦12, more preferably n is an integer and 4≦n≦8, more preferably n is an integer and 5≦n≦7, more preferably n is 5 or 6, and even more preferably n is 6.

[0369] Preferably, the tetroses are individually selected from the group consisting of erythrose and threose.

[0370] Preferably, the pentoses are individually selected from the group consisting of ribose, arabinose, xylose and lyxose.

[0371] Preferably, the hexoses are individually selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose and talose.

[0372] In some embodiments, the monosaccharide unit is a D-sugar. As used herein, the term "D-sugar" refers to the Fischer projection known to those skilled in the art.

[0373] In some embodiments, at least one monosaccharide unit of the soluble tag is modified by at least one chemical modification. In some embodiments, all monosaccharide units of the soluble tag are modified by at least one chemical modification. In some embodiments, no monosaccharide unit of the soluble tag is modified by more than three, preferably more than two, more preferably more than one chemical modification.

[0374] In one embodiment, the average number of chemical modifications per monosaccharide unit of the soluble tag is ≦3. Preferably, the average number of chemical modifications per monosaccharide unit of the soluble tag is ≦2. More preferably, the average number of chemical modifications per monosaccharide unit of the soluble tag is ≦2. The soluble tag has ≦1.5 monosaccharide units. More preferably, the average number of chemical modifications per monosaccharide unit of the soluble tag is ≦1.

[0375] As one of skill in the art will understand, the average number of chemical modifications per monosaccharide unit of the soluble tag is calculated by dividing the total number of chemical modifications for all monosaccharide units of the soluble tag by the number of monosaccharide units of the soluble tag.

[0376] In some embodiments, the chemically modified form of the monosaccharide unit is the form of the monosaccharide unit with at least one chemical modification.In some embodiments, the chemically modified form of the monosaccharide unit is the form of the monosaccharide unit with up to three chemical modifications, preferably up to two chemical modifications, more preferably one chemical modification.

[0377] In certain embodiments, the chemical modification is independently selected from the following: (i) hydrogen, alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, halogen, amino, N-acylamino, azide, sulfate, selenyl, and azide; (ii) substitution of a hydroxyl group with a sulfur-containing moiety selected from the group consisting of sulfoxide, sulfone, sulfate, sulfate ester, thiosulfate, thioester, thioether, and sulfoximine; (iii) substitution of a hydroxyl group with a phosphorus-containing moiety selected from the group consisting of phosphate, phosphonate, phosphine, phosphoric acid, and phosphate ester; (iv) substitution of a hydroxyl group with a silyl group or covalent attachment of a silyl group to the hydroxyl group by formation of a silyl ether; and (v) substitution of a hydroxyl group with a branched polyhydric alcohol. (vi) substitution of a hydroxyl group with an amino acid or a peptide of up to three amino acids or covalent attachment of an amino acid or a peptide of up to three amino acids to the monosaccharide unit; (vi) acetal formation with the hydroxyl group of the monosaccharide unit; (vii) covalent attachment of a PEG (polyethylene glycol) group to the monosaccharide unit; (viii) covalent attachment to an aromatic or heteroaromatic substituent; (ix) formation of an intracyclic double bond within the sugar ring of the monosaccharide unit.

[0378] Chemical modifications of carbohydrates are known to those skilled in the art and are described, for example, in The Organic Chemistry of Sugars, editors Levy and Fuegedi (2005), CRC Press / Taylor & Francis (USA); Tamburrini et al., Medicinal Research Reviews (2020), vol. 40(2), pp. 495-531; Koviach-Cote and Pirinelli, Chemical Reviews (2018), vol. 118(17), pp. 7986-8004; Saloranta and Leino, Synlett (2015), vol. 26(4), pp. 421-425; Corsaro et al., in: Microwaves in Organic Synthesis, 2nd edition (2006), publisher Wiley-VCH Verlag, pp. 579-614; Lichtenthaler, in: Methods and Reagents for Green Chemistry (2007), editors Tundo et al., publisher John Wiley & Sons, pp. 23-63.

[0379] In certain embodiments, the chemical modifications are individually selected from the following: replacement of a hydroxyl group with a substituent selected from the group consisting of hydrogen, alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, halogen, amino, N-acylamino, azido, sulfate, selenyl, and azido.

[0380] In certain embodiments, the alkyl is substituted or unsubstituted C1-C5 alkyl, the acyl is substituted or unsubstituted C1-C5 acyl, the acyloxy is substituted or unsubstituted C1-C5 acyloxy, the alkenyl is substituted or unsubstituted C1-C5 alkenyl, the alkynyl is substituted or unsubstituted C1-C5 alkynyl, the O-alkyl is substituted or unsubstituted C1-C5 O-alkyl, the S-alkyl is substituted or unsubstituted C1-C5 S-alkyl, the carboxyalkyl is substituted or unsubstituted C1-C5 carboxyalkyl, and the N-acylamino is substituted or unsubstituted C1-C5 N-acylamino. Preferably, the alkyl is unsubstituted C1-C5 alkyl, the acyl is unsubstituted C1-C5 acyl, the acyloxy is unsubstituted C1-C5 acyloxy, the alkenyl is unsubstituted C1-C5 alkenyl, the alkynyl is unsubstituted C1-C5 alkynyl, the O-alkyl is unsubstituted C1-C5 O-alkyl, the S-alkyl is unsubstituted C1-C5 S-alkyl, the carboxyalkyl is unsubstituted C1-C5 carboxyalkyl, and the N-acylamino is unsubstituted C1-C5 N-acylamino.

[0381] In some preferred embodiments, the alkyl is substituted or unsubstituted C1-C2 alkyl, the acyl is substituted or unsubstituted C1-C2 acyl, the acyloxy is substituted or unsubstituted C1-C2 acyloxy, the alkenyl is substituted or unsubstituted C1-C2 alkenyl, the alkynyl is substituted or unsubstituted C1-C2 alkynyl, the O-alkyl is substituted or unsubstituted C1-C2O-alkyl, the S-alkyl is substituted or unsubstituted C1-C2S-alkyl, the carboxyalkyl is substituted or unsubstituted C1-C2 carboxyalkyl, and the N-acylamino is substituted or unsubstituted C1-C2N-acylamino. Preferably, the alkyl is unsubstituted C1-C2 alkyl, the acyl is unsubstituted C1-C2 acyl, the acyloxy is unsubstituted C1-C2 acyloxy, the alkenyl is unsubstituted C1-C2 alkenyl, the alkynyl is unsubstituted C1-C2 alkynyl, the O-alkyl is unsubstituted C1-C2O-alkyl, the S-alkyl is unsubstituted C1-C2S-alkyl, the carboxyalkyl is unsubstituted C1-C2 carboxyalkyl and the N-acylamino is unsubstituted C1-C2N-acylamino.

[0382] In some embodiments, the alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, or N-acylamino is linear or branched.Preferably, the alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, or N-acylamino is linear.

[0383] In certain embodiments, the substituted alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, or N-acylamino is substituted with a group selected from halogen, CN, OH, amino, methyl, ethyl, methoxy, or ethoxy.

[0384] In certain embodiments, the substituted alkyl, acyl, acyloxy, alkenyl, alkynyl, O-alkyl, S-alkyl, carboxyalkyl, or N-acylamino is substituted with an atom or group having a molecular weight of ≦100 Da, preferably ≦100 Da, ≦80 Da, more preferably ≦50 Da.

[0385] In certain embodiments, the chemical modification is independently selected from the following: replacement of a hydroxyl group with a sulfur-containing moiety selected from the group consisting of sulfoxide, sulfone, sulfate, sulfate ester, thiosulfate, thioester, thioether, and sulfoximine. In some preferred embodiments, the sulfur-containing moiety has a molecular weight of up to 100 Da, more preferably up to 50 Da.

[0386] In certain embodiments, the chemical modification is independently selected from the following: replacement of a hydroxyl group with a phosphorus-containing moiety selected from the group consisting of phosphate, phosphonate, phosphine, phosphoric acid, and phosphate ester. In some preferred embodiments, the fluorophore-containing moiety has a molecular weight of up to 100 Da, more preferably up to 50 Da.

[0387] In certain embodiments, the chemical modification is independently selected from the following: substitution of a hydroxyl group with a silyl group or covalent attachment of a silyl group to the hydroxyl group by formation of a silyl ether. In some preferred embodiments, the silyl group has a molecular weight of up to 150 Da, more preferably up to 100 Da.

[0388] In some embodiments, the chemical modification is individually selected from: substitution of a hydroxyl group with an amino acid or a peptide of up to three amino acids, or addition of covalently bound amino acids of up to three amino acids or peptides to the monosaccharide unit. In some embodiments, the chemical modification is individually selected from: acetal formation with a hydroxyl group of the monosaccharide unit. In some preferred embodiments, each acetal substituent that is not covalently attached to a monosaccharide unit has a molecular weight of up to 100 Da, more preferably up to 50 Da.

[0389] In certain embodiments, the chemical modification is independently selected from the following: replacement of hydroxyl groups with a branched polyalcohol, hi some preferred embodiments, the branched polyalcohol is a branched polyalcohol having up to 8 hydroxyl groups, more preferably up to 5 hydroxyl groups.

[0390] In certain embodiments, the chemical modification is independently selected from the following: covalent attachment of a PEG (polyethylene glycol) group to the monosaccharide unit, hi some preferred embodiments, the PEG group has a molecular weight of up to 1000 Da, more preferably up to 500 Da, more preferably up to 120 Da.

[0391] In certain embodiments, the chemical modifications are independently selected from covalent attachment to an aromatic or heteroaromatic substituent, hi some preferred embodiments, the aromatic or heteroaromatic substituent has a molecular weight of up to 200 Da, more preferably up to 100 Da.

[0392] Formation of an intracyclic double bond within the sugar ring of said monosaccharide unit.

[0393] In certain embodiments, the chemical modification of the monosaccharide is a replacement of a hydroxyl group of the monosaccharide with a substituent selected from the group consisting of hydrogen, amino, N-acetylamino, methyl, methoxy, and sulfate.

[0394] In certain embodiments, the chemical modification of the monosaccharide is a replacement of a hydroxyl group of the monosaccharide with a substituent selected from the group consisting of hydrogen, amino, N-acetylamino, methyl, and methoxy.

[0395] In certain embodiments, the chemical modification of the monosaccharide is a replacement of a hydroxyl group of the monosaccharide with a substituent selected from the group consisting of hydrogen, amino, and N-acetylamino.

[0396] In one embodiment, the chemically modified form of the monosaccharide unit is a form of the monosaccharide unit having 1, 2 or 3 chemical modifications, preferably 1 or 2 chemical modifications, more preferably 1 chemical modification.

[0397] In some embodiments, the chemically modified form of the monosaccharide unit is the form of the monosaccharide unit in which 1, 2 or 3 hydroxyl groups are independently replaced.In some preferred embodiments, the chemically modified form of the monosaccharide unit is the form of the monosaccharide unit in which 1 or 2 hydroxyl groups are independently replaced.In some more preferred embodiments, the chemically modified form of the monosaccharide unit is the form of the monosaccharide unit in which one hydroxyl group is independently replaced.

[0398] In one embodiment, the chemically modified form of the monosaccharide unit is a form of the monosaccharide unit in which one or two hydroxyl groups have been independently replaced with hydrogen, an amino group, an N-acetylamino group, a methyl group, a methoxy group, or a sulfate group.

[0399] In one embodiment, the chemically modified form of the monosaccharide unit is a form of the monosaccharide unit in which one or two hydroxyl groups have been independently replaced with hydrogen, an amino group, an N-acetylamino group, a methyl group, or a methoxy group.

[0400] In certain embodiments, the chemically modified form of the monosaccharide unit is a form of the monosaccharide unit in which one or two hydroxyl groups have been independently replaced with hydrogen, an amino group, or an N-acetylamino group.

[0401] In one embodiment, the substituents on the carbon backbone of the monosaccharide units resulting from the chemical modification have a molecular weight of 800 daltons or less, preferably 400 daltons or less, more preferably 200 daltons or less, more preferably 100 daltons or less.

[0402] In one embodiment, the soluble tag comprises / does not comprise a monosaccharide unit other than a monosaccharide unit selected from the group consisting of glucose, chemically modified forms of glucose, galactose, and chemically modified forms of galactose.

[0403] The soluble tag does not contain any monosaccharide units other than those selected from glucosamine (GlcN), N-acetyl-glucosamine (GlcNAc), fucose (Fuc), and 6-methyl-fucose. In some preferred embodiments, the soluble tag contains only monosaccharide units selected from glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc). In some more preferred embodiments, the soluble tag contains / contains only N-acetyl-glucosamine (GlcNAc) as a monosaccharide unit.

[0404] As used herein, "fucose" (abbreviated as "Fuc") is 6-deoxy-L-galactose.

[0405] In certain embodiments, the soluble tag consists solely of covalently linked monosaccharide units selected from the group consisting of glucosamine (GlcN), N-acetyl-glucosamine (GlcNAc), and 6-methyl-fucose. In some preferred embodiments, the soluble tag consists solely of covalently linked monosaccharide units selected from the group consisting of glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc). In some more preferred embodiments, the soluble tag consists solely of covalently linked N-acetyl-glucosamine (GlcNAc) units.

[0406] In one embodiment, the soluble tag comprises at least three N-acetyl-glucosamine (GlcNAc) units, preferably at least four N-acetyl-glucosamine (GlcNAc) units.

[0407] In some embodiments, the soluble tag comprises one glucosamine (GlcN) unit and four N-acetyl-glucosamine (GlcNAc) units. In some preferred embodiments, the soluble tag consists of one glucosamine (GlcN) unit and four N-acetyl-glucosamine (GlcNAc) units.

[0408] In some embodiments, the monosaccharide units of the soluble tag are linked in an unbranched linear fashion. In some embodiments, the monosaccharide units of the soluble tag are linked in a branched fashion.

[0409] In certain embodiments, the monosaccharide units are covalently linked by a bond independently selected from the group consisting of a glycosidic bond, an ether bond, an ester bond, and exchange of a hydroxy group at the anomeric center of a monosaccharide unit for a bond to another monosaccharide. In certain embodiments, the monosaccharide units are covalently linked by a glycosidic bond. As will be appreciated by those skilled in the art, this means that the monosaccharide units are linked by a covalent bond formed between a hemiacetal or hemiketal group of a monosaccharide unit and a hydroxyl group of another monosaccharide unit.

[0410] In some embodiments, the monosaccharide units of the soluble tag are cyclic. As those skilled in the art will appreciate, monosaccharides can exist in either a cyclic ("cyclic") or open-chain form. It is understood that in the case of terminal monosaccharides of oligosaccharides, the cyclic form may be in equilibrium with the open-chain form. According to the present invention, such terminal monosaccharides are still considered to be cyclic.

[0411] In certain embodiments, the soluble tag is attached to the antibody-drug conjugate (respectively the molecule) via a covalent bond between a GlcN monosaccharide unit of the soluble tag and the linker.

[0412] In certain embodiments, the soluble tag is attached to the antibody-drug conjugate (respectively the molecule) via a beta-alanine group that covalently links the GlcN monosaccharide unit of the soluble tag to the linker.

[0413] In certain embodiments, the soluble tag is attached to the antibody-drug conjugate (respectively the molecule) by formation of a covalent bond between a GlcN monosaccharide unit of the soluble tag and the linker.

[0414] In some embodiments, the soluble tag is attached to the antibody-drug conjugate (respectively, the molecule) via a covalent bond between the linker and an amino group attached to carbon 2 of the monosaccharide unit of the soluble tag. The "carbon 2" number refers to the standard numbering of the carbon atoms in the carbon backbone of a monosaccharide (i.e., carbon atoms are numbered from 1 to x along the backbone, starting from the end closest to the C=O group). The C=O group of an oligosaccharide can form an acetal or hemiacetal.

[0415] Preferably, the amino group is attached to carbon C-2 of the terminal monosaccharide unit at the non-reducing end of the oligosaccharide (ie, the oligosaccharide contained in / making up the soluble tag).

[0416] The number of soluble tags per ADC molecule can be conveniently controlled if the soluble tags are attached to the linker or linker-payload construct by a chemical reaction prior to conjugation with the antibody component. To increase the number of soluble tags in the final ADC, more soluble tags can be included per linker / linker-payload construct or the number of soluble-tagged neighbor- / linker-payload constructs in the ADC can be increased.

[0417] As those skilled in the art are aware, the attachment of more soluble tags per linker / linker-payload construct can be facilitated, for example, by the use of appropriate reactive groups in the linker motif. By appropriately combining two reactive groups, one on the linker / linker-payload construct and the other on the tag (e.g., combinations such as carboxylic acid or azide / alkyne or halogen / thiol for amide bond formation with the amino group of the glucosamine unit of the oligosaccharide tag), the tag can be added at an early or late stage of the synthesis of the linker or linker-payload construct, respectively.

[0418] Alternatively or additionally, the number of linker / linker-payload constructs per antibody component can be controlled by modifying the conjugation method. Site-specific conjugation, such as with transglutaminase, limits the number of linker / linker-payload constructs per antibody component to the number of recognition sequences (typically, full-length antibodies have two transglutaminase recognition sequences, although this can be accommodated by altering the antibody sequence through mutation). The ratio can be adjusted by using nonspecific lysine linkages or more specific interchain cysteines. These approaches also allow for control of the number of soluble tags per antibody component if they are attached directly to the antibody component (i.e., without a linker or payload).

[0419] In one embodiment, the soluble tag comprises / each comprises the oligosaccharide GlcN-GlcNAc-GlcNAc-GlcNAc-GlcNAc.

[0420] In one embodiment, the soluble tags consist of / each consist of an oligosaccharide GlcN-GlcNAc-GlcNAc-GlcNAc-GlcNAc.

[0421] Preferably, the oligosaccharide GlcN-GlcNAc-GlcNAc-GlcNAc-GlcNAc is attached to another component of the antibody-drug conjugate by a covalent bond to a GlcN monosaccharide unit of the oligosaccharide.

[0422] In one embodiment, the solubility tag is or comprises O-(2-desoxy-2-amino-β-D-glucopyranosyl)-(14)-O-(→2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranose.

[0423] In one embodiment, the solubility tag is or comprises O-(2-desoxy-2-amino-β-D-glucopyranosyl)-(14)-O-(→2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-{(6-desoxy-2-O-methyl-α-L-galactopyranosyl)-(16→)-O}-(2-acetamido-2-desoxy-D-glucopyranose).

[0424] In one embodiment, the solubility tag is or comprises O-(2-desoxy-2-amino-β-D-glucopyranosyl)-(14)-O-(→2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranose).

[0425] In one embodiment, the solubility tag is or comprises the sodium salt of O-(2-desoxy-2-amino-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranosyl)-(1→4)-O-(2-acetamido-2-desoxy-β-D-glucopyranosyl)-(14→)-O-(2-acetamido-2-desoxy-6-O-sulfo-D-glucopyranose).

[0426] In one embodiment, the solubility tag is or comprises O-(2-deoxy-2-amino-β-D-glucopyranosyl)-(14)-O-→(2-acetamido-2-desoxy-D-glucopyranose).

[0427] In certain embodiments, the soluble tag is or comprises N-[(3R,4R,6R)-5-[(2S,3R,4R,6R)-3-acetamido-5-[(2S,3R,4R,6R)]-3-acetamido-5-[(2S,3R,4R,6R)-3-acetamido-5-[(2S,3R,4R,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-2,4-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide.

[0428] In one embodiment, the solubility tag is N-[(3R,4R,5S,6R)-5-[(2S,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-6-[[(2R,3S,4R,5S,6S)-4,5-dihydroxy-3-methoxy-6-methyl-tetrahydropyran[-2-yl]oxymethyl]-2,4-dihydroxy-tetrahydropyran-3-yl]acetamide.

[0429] In certain embodiments, the soluble tag is or comprises N-[(3R,4R,5S,6R)-5-[(2S,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-acetamido-5-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-4-hydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-2,4-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide.

[0430] In certain embodiments, the soluble tag is or comprises ((2R,3S,4R,5R)-5-acetamido-3-(((2S,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4R,5S,6R)-3-acetamido-5-(((2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-4,6-dihydroxytetrahydro-2H-pyran-2-yl)methyl sodium sulfate (I).

[0431] In some embodiments, the soluble tag is or comprises N-[(3R,4R,5S,6R)-5-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxy-2,4-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide.

[0432] In certain embodiments, the soluble tag comprises a chemical group having the structural formula (I): [ka]

[0433] In certain embodiments, the soluble tag comprises a chemical group having the structural formula (II): [ka]

[0434] In some embodiments, the soluble tag comprises a chemical group having the structural formula (III): [ka]

[0435] In some embodiments, the soluble tag comprises a chemical group having the structural formula (IV): [ka]

[0436] In certain embodiments, the soluble tag is a chemical group having the structural formula (I): [ka]

[0437] In certain embodiments, the soluble tag is a chemical group having the structural formula (II): [ka]

[0438] In certain embodiments, the soluble tag is a chemical group having the structural formula (III): [ka]

[0439] In certain embodiments, the soluble tag is a chemical group having the structural formula (IV): [ka]

[0440] As will be understood by those skilled in the art, chemical groups having structural formulas (I)-(IV) can be attached to a molecule / antibody-drug conjugate / component thereof by, for example, any of the bonds described above or in the Examples (e.g., a covalent bond between the molecule / antibody-drug conjugate / component thereof and an amino group attached to carbon 2 of a monosaccharide unit within the chemical group). When a chemical group having structural formulas (I)-(IV) is covalently attached to a molecule / antibody-drug conjugate, respectively, as a component thereof, it is understood that, for example, a hydrogen atom in structural formulas (I)-(IV) may be replaced by the molecule / antibody-drug conjugate (i.e., the covalent bond shown in structural formulas (I)-(IV) as being instead attached to a hydrogen atom forms the covalent bond by which the chemical group is attached to the molecule / antibody-drug conjugate / component thereof).

[0441] As will be appreciated by those skilled in the art, the glycosylation of native antibodies is not a soluble tag according to the present invention.

[0442] In certain embodiments, the soluble tag is not an N-linked glycan. In certain embodiments, the soluble tag is not an N-linked glycan, does not comprise an N-linked glycan, or is not a molecular group within an N-linked glycan.

[0443] As used herein, the term "N-linked glycan" refers to a monosaccharide, oligosaccharide, or polysaccharide that is covalently attached to the nitrogen of an asparagine side chain in a polypeptide as part of a glycoprotein.

[0444] As used herein, a molecular group "within an N-linked glycan" is a molecular group that is part of the chemical structure of the N-linked glycan.

[0445] In certain embodiments, the soluble tag is not an N-linked or O-linked glycan. In certain embodiments, the soluble tag is not an N-linked or O-linked glycan, does not comprise an N-linked or O-linked glycan, or is not a molecular group within an N-linked or O-linked glycan.

[0446] As used herein, an "O-linked glycan" is a monosaccharide (typically N-acetylgalactosamine, galactose, or xylose) covalently attached to the oxygen of a serine or threonine side chain in a polypeptide as part of a glycoprotein.

[0447] In certain embodiments, the antibody component is either unglycosylated or has glycosylation only at position Asn297 (EU numbering) of the IgG1 heavy chain. Preferably, the glycosylation at Asn297 is the glycosylation of a native antibody.

[0448] The term "EU numbering" refers to the numbering of antibody heavy chains as taught in Sequences of Proteins of Immunological Interest, 5th ed. (1991), editors Kabat et al., National Institutes of Health (Bethesda, USA). The EU index is based on the residue numbering of the human IgG1 EU antibody (Edelman et al., Proc Natl Acad Sci USA (1969), vol. 63, pp. 78-85).

[0449] In some embodiments, the antibody component (respectively, the antibody or antigen-binding fragment of item

[38] ) has no monosaccharides or no monosaccharides other than those in the glycosylation of the antibody. In some embodiments, the antibody component (respectively, the antibody or antigen-binding fragment of item

[38] ) does not have any antibody glycosylation.

[0450] In some embodiments, the antibody component (respectively, the antibody or antigen-binding fragment of item

[38] ) does not comprise any chitooligosaccharides. In some embodiments, the antibody component does not comprise any monosaccharide units.

[0451] In some embodiments, the antibody-drug conjugate (each of the molecules) does not contain any chitooligosaccharides beyond the chitooligosaccharide of the soluble tag. In some embodiments, the antibody-drug conjugate does not contain any other monosaccharide units beyond the monosaccharide units of the soluble tag and, optionally, the monosaccharide units that are part of the glycosylation of the antibody component (the antibody or antigen-binding fragment, respectively) (see item

[38] ). In some embodiments, the antibody-drug conjugate (each of the molecules) does not contain any other monosaccharide units beyond the monosaccharide units of the soluble tag.

[0452] In some embodiments, the molecule with the solubility tag has a higher solubility than a molecule of the same structure but without the solubility tag.

[0453] In certain embodiments, the ADC with a solubility tag has a higher solubility than an ADC of the same structure but without the solubility tag.

[0454] The solubility of an ADC can be assessed during formulation development by measuring aggregate formation at various ADC concentrations in an appropriate buffer (Kalonia et al., J. Phys. Chem. B (2016), vol. 120, pp. 7062-7075; Duerr and Friess, European Journal of Pharmaceutics and Biopharmaceutics (2019), vol. 139, pp. 168-176), preferably by the method of Duerr and Friess. This approach allows for comparison of the solubility of an ADC with and without a specific solubility tag. A suitable initial assessment of whether a tag increases the solubility of an ADC can also be performed by HIC (hydrophobic interaction chromatography) experiments, as described in the experimental section below.

[0455] In one embodiment, the corresponding molecule without the payload (i.e., a molecule consisting only of the antibody component of the antibody drug conjugate, a linker, and a soluble tag) is non-toxic to mice at a dose of 6 mg / kg body weight of the mouse when administered intravenously.

[0456] In certain embodiments, the antibody drug conjugate is such that the corresponding molecule without the payload (i.e., a molecule consisting only of the antibody component, linker, and solubility tag of the antibody drug conjugate) does not elicit signs of hepatotoxicity in animal studies using mice.

[0457] Preferably, the animal experiment using mice involves administering a single intravenous dose of the molecule without the payload to adult female BALB / c nude mice at a dose of 6 mg of ADC per kg of mouse body weight, preparing formalin-fixed liver tissue stained with hematoxylin and eosin, and performing pathological analysis under a light microscope, where the absence of visible lesions under a light microscope indicates the absence of liver toxicity. Further details on how this experiment can be performed can be found in Example 8 below.

[0458] In another aspect, the invention relates to an antibody-drug conjugate comprising: (i) an antibody component; (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker covalently attaching the payload to the antibody component; and (iv) at least one solubility tag.

[0459] Preferably, the antibody-drug conjugate, the antibody component, the at least one payload, the therapeutic agent, the detectable label, the linker / linkers, and the at least one soluble tag are as defined in any of the above embodiments or as defined by any combination of the above embodiments (unless this would lead to a logical contradiction).

[0460] How to Increase Solubility

[0461] As described above and in the Examples section below, the covalent attachment of a solubility tag according to the present invention can increase the solubility of an antibody-drug conjugate. Thus, in a further aspect, the present invention provides a method for increasing the solubility of an antibody-drug conjugate.

[0462] In another aspect, the invention relates to a method for increasing the solubility of an antibody-drug conjugate, the antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the payload and the antibody component, the method comprising covalently attaching at least one solubility tag to the antibody-drug conjugate.

[0463] In another aspect, the invention relates to a method for increasing the solubility of an antibody-drug conjugate, the antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the payload and the antibody component, the method comprising covalently attaching at least one solubility tag to the antibody-drug conjugate.

[0464] Preferably, the antibody-drug conjugate, the antibody component, the at least one payload, the therapeutic agent, the detectable label, the linker / linkers, and the at least one soluble tag are as defined in any of the above embodiments, or as defined by any combination of the above embodiments (unless this would lead to a logical contradiction).

[0465] General methods for preparing ADCs using various approaches are described, for example, in Matsuda et al., Organic Process Research & Development (2019), vol. 23(12), pp. 2647-2654; Walker et al., Bioconjugate Chemistry (2019), vol. 30(9), pp. 2452-2457; and Barfield and Rabuka, Methods in Molecular Biology (2018), vol. 1728 (Noncanonical Amino Acids), pp. 3-16.

[0466] The antibody component, payload and linker can be prepared and covalently attached by standard biotechnology and synthetic organic chemistry methods, as described above in the "Antibody-Drug Conjugates" section and detailed in the Examples section.

[0467] As described above in the section "Antibody-Drug Conjugates," the oligosaccharide-based soluble tags according to the present application can be chemically synthesized by standard methods of carbohydrate chemistry (see, for example, Preparative Carbohydrate Chemistry (1997), editor Hanessian, publisher Marcel Dekker, Inc. (New York); Carbohydrate Chemistry: Proven Synthetic Methods (2015), editors Roy and Vidal, CRC Press; Carbohydrate Chemistry: State of the Art and Challenges for Drug Development (2016), editor Cipolla, Imperial College Press (London); CRC Handbook of Oligosaccharides (1990), Vol. I-III, (published 2019), editors: Liptak et al., CHR Press, Inc.; Liaqat and Eltem, Carbohydrate Polymers (2018), Vol. 184, pp. 243-259).

[0468] Alternatively, oligosaccharide tags can be prepared by biotechnological methods (see, for example, Meyer et al., Biotechnological Production of Oligosaccharides - Applications in the Food Industry, Food Production and Industry (2015), Ayman Hafiz Amer Eissa, IntechOpen, DOI: 10.5772 / 60934; Liaqat and Eltem, Carbohydrate Polymers (2018), vol. 184, pp. 243-259; Samain et al., Carbohydrate Research (1997), vol. 302, pp. 35-42; Samain et al., Biotechnol. (1999), vol. 72, pp. 33-47).

[0469] Further purification can be carried out by standard methods of organic chemistry, including, for example, recrystallization or precipitation, nanofiltration, ultrafiltration, gel permeation chromatography, ion exchange chromatography, capillary electrophoresis, HPLC purification, UPLC purification or the membrane and support approaches described, for example, in Pinelo et al., Separation and Purification Technology (2009), vol. 70(1), pp. 1-11.

[0470] The intermediate monosaccharides and oligosaccharides, as well as the final soluble tag before conjugation, can be characterized by standard methods known to those skilled in the art, such as LC-MS / ESI MS, 1D and 2D NMR, GPC (gel permeation chromatography), or ion mobility mass spectrometry (see, for example, Carbohydrate Chemistry (1988), editor El Khadem, Academic Press (San Diego); Seeberger et al., Nature (2015), vol. 526(7572), pp. 241-244).

[0471] A high level of control can be achieved by using orthogonal conjugation methods (e.g., a combination of transglutaminase and cysteine ​​linkages) to attach the different components. Attaching a soluble tag to a linker has the advantage that it is usually straightforward to include an appropriate reactive group on the linker during synthesis. On the other hand, specific attachment of a soluble tag to an antibody component can usually be achieved by an enzymatic conjugation approach.

[0472] Although the reaction environment during enzymatic attachment of soluble tags is generally not harmful to the protein component of an ADC, the chemical attachment conditions must be appropriately selected to avoid damage to the protein component. Suitable conditions can be inferred from the conditions for chemical linker attachment to antibody components. Thus, tags can be attached via cycloaddition reactions such as click chemistry or Diels-Alder modifications (Rossin et al., Bioconjugate Chemistry (2016), vol. 27(7), pp. 1697-1706) or using aldehydes (Barfield and Rabuka, Methods in Molecular Biology (2018), vol. 1728, pp. 3-16).

[0473] In some cases, the covalent attachment of a soluble group to an antibody-drug conjugate can be achieved using an intermediate compound, as described below. To obtain this intermediate compound, the soluble tag is activated by introducing an activating group using standard organic synthesis methods. The activating group is a reactive functional group, such as maleimide, halogen acetamide, alkyl halogen, Michael acceptor (such as vinylpyridine), or a group suitable for cycloaddition (such as ketone, hydrazone, semicarbazone, carboxylic acid, acid, alkene, or alkyne suitable for cycloaddition). The soluble tag is then covalently attached to the antibody-drug conjugate by reaction of the activating group with an appropriate molecular group within the antibody-drug conjugate. As will be understood by those skilled in the art, the covalent attachment of a soluble tag to an antibody-drug conjugate can also be achieved using an activating group on the antibody-drug conjugate rather than the soluble tag.

[0474] Successful covalent attachment of the soluble tag to the antibody-drug conjugate can then be confirmed by standard methods known to those skilled in the art, such as LC-MS.

[0475] When the present invention indicates that a modification or effect "increases the solubility of an antibody-drug conjugate," this refers to a situation in which the solubility of the antibody-drug conjugate after performing the method is higher than that before performing the method.

[0476] To verify whether an increase in the solubility of an antibody-drug conjugate has actually been achieved, the solubility of the antibody-drug conjugate before and after covalent attachment of a solubility tag can be evaluated by measuring aggregate formation, for example, under the following conditions: different ADC concentrations in an appropriate buffer during formulation development (Kalonia et al., J. Phys. Chem. B (2016), vol. 120, pp. 7062-7075; Duerr and Friess, European Journal of Pharmaceutics and Biopharmaceutics (2019), vol. 139, pp. 168-176), preferably according to the Duerr and Friess method. This approach allows for comparison of the solubility of ADCs with and without specific solubility tags.

[0477] Alternatively, HIC (hydrophobic interaction chromatography) experiments can be performed as described in Example 4 below. Comparability between samples can be ensured by using an internal standard. A shorter retention time in hydrophobic interaction chromatography indicates increased solubility.

[0478] In another aspect, the invention relates to the use of a soluble tag to increase the solubility of an antibody-drug conjugate.

[0479] In some embodiments, the use comprises covalently attaching the soluble tag to the antibody-drug conjugate.

[0480] In one embodiment of the use, the antibody-drug conjugate comprises (i) an antibody component, (ii) at least one payload (wherein the at least one payload is a therapeutic agent or a detectable label), and (iii) a linker, and covalently attaching the payload or payloads to the antibody component.

[0481] In one embodiment of the use, the antibody-drug conjugate consists of (i) an antibody component, (ii) at least one payload (wherein the at least one payload is a therapeutic agent or a detectable label), and (iii) a linker that covalently links the payload or the antibody component.

[0482] Preferably, in such a use, the antibody-drug conjugate, the antibody component, the at least one payload, the therapeutic agent, the detectable label, the linker and the at least one solubility tag are (unless this leads to a logical contradiction) as defined in any of the above embodiments or any combination of the above embodiments (see the "Antibody-Drug Conjugates" section above).

[0483] Preparation and covalent attachment of antibody components, payloads and linkers, preparation, purification and characterization of soluble tags, covalent attachment of soluble tags, confirmation of successful attachment of the soluble tags and verification that the soluble tags did in fact provide an enhancement, and tuning of the solubility of antibody-drug conjugates can all be performed as described above for methods of increasing the solubility of antibody-drug conjugates.

[0484] In another aspect, the invention relates to a method for increasing the solubility of an antibody-drug conjugate, the antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload, the at least one payload comprising a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the payload and the antibody component, the method comprising preparing the antibody-drug conjugate in a form in which the antibody-drug conjugate is covalently linked to at least one solubility tag.

[0485] In another aspect, the invention relates to a method for increasing the solubility of an antibody-drug conjugate, the antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) one or more linkers covalently linking the payload to the antibody component, wherein the method comprises covalently linking the antibody-drug conjugate to at least one solubility tag.

[0486] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) a linker covalently linking the payload and the antibody component, the method comprising preparing a molecule in which the chemical compound is covalently linked to at least one solubility tag, the molecule being an antibody-drug conjugate.

[0487] In another aspect, the present invention relates to a method for increasing the solubility of a chemical compound comprising an antibody component, (i) the antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) a linker covalently linking the payload and the antibody component, the method comprising preparing a molecule in which the chemical compound is covalently linked to at least one solubility tag, the molecule being an antibody-drug conjugate.

[0488] In another aspect, the invention relates to a method for increasing the solubility of a molecule comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) the payload / linker / linker covalently linking the payload and the antibody component, the method comprising preparing the molecule in a form in which the molecule is covalently attached to at least one solubility tag, resulting in an antibody-drug conjugate comprising (i) the antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, (iii) a linker covalently linking the payload and the antibody component, and (iv) at least one solubility tag.

[0489] In another aspect, the invention relates to a method for increasing the solubility of a molecule comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, and (iii) a linker / linker covalently linking the payload and the antibody component, the method comprising preparing the molecule in a form in which the molecule is covalently attached to at least one solubility tag, resulting in an antibody-drug conjugate comprising (i) an antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label, (iii) a linker / linker covalently connecting the payload and the antibody component, and (iv) at least one solubility tag.

[0490] Preferably, in such methods, the antibody-drug conjugate, the antibody component, the at least one payload, the therapeutic agent, the detectable label, the linker / linkers and the at least one solubility tag (unless this leads to logical problems) (a contradiction) are defined in any of the above embodiments or by any combination of the above embodiments (see the "Antibody-Drug Conjugates" section above).

[0491] Preparation of the antibody component, payload and linker, as well as preparation, purification and characterization of the solubility tag, can be performed as described above for methods of increasing the solubility of antibody-drug conjugates.

[0492] Preparing an antibody-drug conjugate / molecule "in a form in which the antibody-drug conjugate / molecule is covalently linked to at least one solubility tag" means producing a compound in which the antibody-drug conjugate / molecule is covalently linked to at least one solubility tag. This can be achieved by preparing and characterizing the individual components (i.e., antibody component, payload, linker, and solubility tag) and then covalently linking them, as described above for methods of increasing the solubility of antibody-drug conjugates. The order in which the individual components are linked is not limited. Thus, for example, it is possible to prepare a linker-payload construct covalently linked to a soluble tag by synthetic organic chemistry methods, and then in the final step conjugate this construct to the antibody component by standard conjugation methods as described in the literature (e.g., Behrens et al., Molecular Pharmaceutics (2015), vol. 12(11), pp. 3986-3998; Stefano, Methods in Molecular Biology (2013), vol. 1045, pp. 145-171; Dickgiesser et al., in: Methods in Molecular Biology: Enzyme-Mediated Ligation Methods (2019), editors Nuijens and Schmidt, vol. 2012, pp. 135-149; Dickgiesser et al., Bioconjugate Chem. (2020), vol. 31(4), p. 1070-1076) and in Example 3 below.

[0493] Confirmation that the desired compound has been obtained and verification that the inclusion of the solubility tag does in fact increase solubility can be performed as described above.

[0494] Methods and Tools for Preparing ADCs According to the Invention

[0495] Antibody-drug conjugates with oligosaccharide-based solubility tags according to the present invention can be prepared as described above in the sections "Antibody-drug conjugates" and "Methods for increasing solubility." Further methods and tools for preparing antibody-drug conjugates according to the present invention are disclosed below.

[0496] In another aspect, the present invention relates to a method for preparing an antibody-drug conjugate as defined in the present invention, the method comprising: (i) performing a reaction that results in the formation of a covalent bond between a molecule comprising (a) an antibody component as defined in the present disclosure, a payload as defined in the present disclosure, and a linker as defined in the present disclosure, and (b) a soluble tag as defined in the present disclosure; or (ii) a reaction that results in the formation of a covalent bond between (a) an antibody component as defined in the present disclosure, and a molecule comprising (b) a payload as defined in the present disclosure, a linker as defined in the present disclosure, and a soluble tag as defined in the present disclosure; or (iii) a reaction that results in the formation of a covalent bond between (a) an antibody component as defined in the present disclosure, a linker as defined in the present disclosure, and a soluble tag as defined in the present disclosure, and (b) a payload as defined in the present disclosure; or (iv) performing a reaction that results in the formation of a covalent bond between (a) an antibody component as defined in the present disclosure and a linker as defined in the present disclosure, and (b) a molecule comprising a soluble tag as defined in the present disclosure, and a payload as defined in the present disclosure, to generate an antibody-drug conjugate with a covalently attached soluble tag. Preferably, the payload is a therapeutic agent or a detectable label. Preferably, the payload is a therapeutic agent or a detectable label.

[0497] Preferably, in such methods, the antibody-drug conjugate, the antibody component, the payload, the therapeutic agent, the detectable label, the linker and the soluble tag are (unless this causes a logical contradiction) as defined in any of the above embodiments or any combination of the above embodiments (see section "Antibody-Drug Conjugates" above).

[0498] Preparation, purification and characterization of antibody components, payloads, linkers and solubility tags can be performed as described.

[0499] Alternatively, intermediates in ADC synthesis (e.g., constructs containing a linker, payload, and solubility tag) can be constructed not by covalently linking pre-prepared components, but by gradually synthesizing the entire construct in one molecule (e.g., as shown in Example 1 below). The synthetic sequence and its flexibility can be driven to achieve the desired structure, and various approaches have been described (see, for example, Quiles et al., Journal of Medicinal Chemistry (2010), vol. 53(2), pp. 586-594; Feuillatre et al., ACS Omega (2020), vol. 5(3), pp. 1557-1565; Sonzini, Bioconjugate Chemistry (2020), vol. 31(1), pp. 123-129; Watkinson, BioProcess International (2017), vol. 15(10), pp. 22-33).

[0500] Covalent attachment of the individual components can be accomplished using standard methods of synthetic organic chemistry (see above). Confirmation of successful preparation of ADCs containing soluble tags can be performed as described above and in the Examples.

[0501] Covalent attachment of a soluble tag to another component of the antibody-drug conjugate can be achieved by "activating" the soluble tag (i.e., by forming an intermediate having a reactive chemical group), followed by carrying out a reaction in which the activated intermediate covalently binds to the other component of the antibody-drug conjugate.

[0502] Thus, in another aspect, the present invention relates to a compound for use in the preparation of an antibody-drug conjugate according to the present invention, said compound comprising a soluble tag as defined in the present invention attached to an activator group.

[0503] In one embodiment, the compound comprises a soluble tag as defined herein attached to an activating group.

[0504] Preferably, the antibody-drug conjugate and the soluble tag are as defined in any of the above embodiments or as defined by any combination of the above embodiments (unless this creates a logical contradiction) (see the "Antibody-Drug Conjugates" section above).

[0505] As used herein, an "activating group" is a reactive chemical group useful for covalently linking a soluble tag to another compound or to an antibody-drug conjugate, such as an antibody component, linker, or payload as defined herein. As will be understood by those skilled in the art, the reactive group must be selected based on compatibility and selectivity, as described above for conjugation reactions.

[0506] In some embodiments, the activator group is selected from the group consisting of maleimides, halogen acetamides, alkyl halogens, Michael acceptors (wherein the Michael acceptor is preferably vinylpyridine), and groups suitable for cycloaddition (wherein the Michael acceptor is preferably vinylpyridine). The groups suitable for cycloaddition are preferably ketones, hydrazones, semicarbazones, carboxylic acids, alkenes, or alkynes suitable for cycloaddition.

[0507] In another aspect, the present invention relates to an antibody-drug conjugate prepared by a method according to the present invention, which may be any method for increasing the solubility of an antibody-drug conjugate / molecule according to the present invention, such as the use of a soluble tag to increase the solubility of an antibody-drug conjugate according to the present invention. The present invention also relates to a method for preparing an antibody-drug conjugate according to the present invention.

[0508] Preferably, the antibody-drug conjugate is as defined in any of the above embodiments or as defined by any combination of the above embodiments (unless this creates a logical contradiction) (see the "Antibody-Drug Conjugates" section above).

[0509] Pharmaceutical Compositions and Medical Uses

[0510] In another aspect, the present invention relates to a pharmaceutical composition comprising an antibody-drug conjugate of the present invention or an antibody-drug conjugate prepared by a method according to the present invention.

[0511] Preferably, the antibody-drug conjugate and the method are as defined in any of the above embodiments or as defined by any combination of the above embodiments (see above, section "Antibody-Drug Conjugates").

[0512] Methods for preparing pharmaceutical compositions are known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press).

[0513] In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent and / or excipient.

[0514] The term "pharmaceutically acceptable" indicates that the carrier, diluent, or excipient is a non-toxic, inert material that is compatible with the other ingredients of the pharmaceutical composition and is not harmful to a patient receiving the pharmaceutical composition. It can be used in pharmaceutical preparations. Substances suitable as carriers, diluents, or excipients in pharmaceutical compositions are known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press). The pharmaceutical composition may further contain, for example, additional adjuvants, antioxidants, buffers, bulking agents, colorants, emulsifiers, fillers, flavoring agents, preservatives, stabilizers, suspending agents, and / or other conventional pharmaceutical auxiliary substances.

[0515] In certain embodiments, the pharmaceutical composition further comprises at least one additional adjuvant, antioxidant, buffer, bulking agent, colorant, emulsifier, filler, flavoring agent, preservative, stabilizer, suspending agent and / or other conventional pharmaceutical auxiliary.

[0516] In another aspect, the present invention relates to an antibody-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0517] In another aspect, the present invention relates to an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of cancer. In another aspect, the present invention relates to an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of malignant tumors. In another aspect, the present invention relates to an antibody-drug conjugate according to the present invention or a pharmaceutical composition according to the present invention for use in the treatment of inflammatory diseases.

[0518] In certain embodiments, the antibody-drug conjugates and the pharmaceutical compositions are for use in treating humans.

[0519] Preferably, the antibody-drug conjugate and the pharmaceutical composition are as defined in any of the above embodiments or as defined by any combination of the above embodiments (see in particular the "Antibody-Drug Conjugates" section above).

[0520] The manufacture of medicaments containing the antibody-drug conjugates according to the present invention or the pharmaceutical compositions according to the present invention can be carried out according to well-known pharmaceutical methods. Further details regarding formulation and administration techniques can be found, for example, in Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), Pharmaceutical Press.

[0521] As used herein, "treatment" of a disease and "treating" a disease refer to a process in which a subject is provided with pharmaceutical treatment, e.g., the administration of a drug, such that the disease is alleviated, alleviated, minimized, stopped, or halted, and may even be cured and / or the likelihood of recurrence of the disease is reduced or recurrence of the disease is prevented.

[0522] The use of antibody-drug conjugates in the treatment of diseases is known to those skilled in the art (see, for example, Coats et al., Clinical Cancer Research (2019), vol. 25(18), pp. 5441-5448; Rudra, Bioconjugate Chemistry (2020), vol. 31(3), pp. 462-473). Therefore, those skilled in the art will recognize that for successful treatment, the components of the antibody-drug conjugate, particularly the antibody component and the payload, must be appropriately selected. For example, when treating a specific cancer, the antibody component of the ADC needs to be selected so that binding of the antibody component to its target antigen directs the ADC to the cancer (e.g., by using an antibody component against a tumor antigen that is specifically found on the surface of cancer cells). Similarly, the payload needs to be selected so that the desired therapeutic effect is achieved. For example, in the treatment of cancer, a cytotoxic drug may be selected as the payload.

[0523] In another aspect, the present disclosure relates to a method for treating a disease in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an antibody-drug conjugate of the present disclosure or a pharmaceutical composition thereof.

[0524] Preferably, the antibody-drug conjugate and the pharmaceutical composition are as defined in any of the above embodiments or as defined by any combination of the above embodiments (see in particular the "Antibody-Drug Conjugates" section above).

[0525] A "therapeutically effective amount" refers to the amount of drug required to ameliorate the symptoms of a disease. The effective amount of an active agent (e.g., an antibody-drug conjugate (ADC)) used to treat a disease according to the present invention will vary depending on the method of administration, age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as a "therapeutically effective" amount.

[0526] As used herein, the term "patient" refers to a mammal (such as a human, rat, mouse, monkey, pig, goat, cow, horse, dog, or cat). Preferably, the patient is a human.

[0527] In some embodiments, the disease is cancer. In some embodiments, the disease is a malignant tumor. In some embodiments, the disease is an inflammatory disease.

[0528] In another aspect, the present invention relates to the use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for the manufacture of a medicament.

[0529] In another aspect, the present invention relates to the use of an antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of cancer. In another aspect, the present invention relates to the use of an antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of malignant tumors. In another aspect, the present invention relates to the use of an antibody-drug conjugate of the present invention or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment of inflammatory diseases.

[0530] Preferably, the antibody-drug conjugate and the pharmaceutical composition are as defined in any of the above embodiments or as defined by any combination of the above embodiments (see in particular the "Antibody-Drug Conjugates" section above).

[0531] In some embodiments, the medicament is prepared for administration to a human.

[0532] In certain embodiments of the different medical uses disclosed above, the inflammatory disease is an autoimmune disease.

[0533] In certain embodiments of the different medical uses disclosed above, the inflammatory disease is selected from the group consisting of inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome, and hidradenitis suppurativa (HS).

[0534] In certain embodiments of the different medical applications disclosed above, the cancer, malignancy or inflammatory disease is a human disease. In relation to the above subject matter, the following is also disclosed:

[0535] [1] -Targeting section and - at least one soluble tag A molecule containing

[0536] [2] - Targeting portion, - at least one functional part and - at least one soluble tag A molecule containing

[0537] [3] - Targeting portion, - at least one functional part, - the functional part and the Targeting a linker covalently linking the moiety to - at least one soluble tag A molecule containing

[0538] [4] -Targeting section and - at least one soluble tag A molecule consisting of

[0539] [5] - Targeting portion, - at least one functional part and - at least one soluble tag A molecule consisting of

[0540] [6] - Targeting portion, - at least one functional part, - the functional part and the Targeting a linker covalently linking the moiety to - at least one soluble tag A molecule consisting of

[0541] [7] A method for increasing the solubility of a molecule that contains a targeting moiety, the method comprising covalently attaching at least one soluble tag to the molecule.

[0542] [8] A method for increasing the solubility of a molecule, comprising: -Targeting section and - at least one functional part, wherein said method comprises covalently attaching at least one soluble tag to said molecule.

[0543] [9] A method for increasing the solubility of a molecule, comprising: - Targeting portion, - at least one functional part and - the functional part and the Targeting a linker that covalently bonds the moiety to wherein said method comprises covalently attaching at least one soluble tag to said molecule.

[0544]

[10] Targeting A method for increasing the solubility of a molecule comprising a moiety, said method comprising covalently attaching at least one solubility tag to said molecule.

[0545]

[11] A method for increasing the solubility of a molecule, comprising: -Targeting section and - at least one functional part, wherein said method comprises covalently attaching at least one soluble tag to said molecule.

[0546]

[12] A method for increasing the solubility of a molecule, comprising: - Targeting portion, - at least one functional part and - the functional part and the Targeting a linker that covalently bonds the moiety to wherein said method comprises covalently attaching at least one soluble tag to said molecule.

[0547]

[13] A method for increasing the solubility of a compound, wherein the compound comprises a targeting moiety, the method comprising preparing a molecule in which the compound is covalently linked to at least one solubility tag.

[0548]

[14] A method for increasing the solubility of a compound, comprising: -Targeting section and - at least one functional part, wherein the compound is covalently attached to at least one soluble tag.

[0549]

[15] A method for increasing the solubility of a compound, comprising: - Targeting portion, - at least one functional part and - the functional part and the Targeting a linker that covalently bonds the moiety to wherein the compound is covalently attached to at least one soluble tag.

[0550]

[16] A method for increasing the solubility of a compound, comprising: Targeting A method comprising preparing a molecule comprising a compound covalently linked to at least one soluble tag.

[0551]

[17] A method for increasing the solubility of a compound, comprising: -Targeting section and - at least one functional part, wherein said compound is covalently attached to at least one soluble tag.

[0552]

[18] A method for increasing the solubility of a compound, comprising: - Targeting portion, - at least one functional part and - the functional part and the Targeting a linker that covalently bonds the moiety to wherein said compound is covalently attached to at least one soluble tag.

[0553]

[19] Any of the methods

[13] to

[18] , wherein all components of the compound are covalently bonded.

[0554]

[20] The use of a soluble tag to enhance the solubility of a molecule, wherein the molecule is Targeting Use including parts.

[0555]

[21] The use of a soluble tag to increase the solubility of a molecule, wherein the molecule is - Targeting section and - at least one functional part Including, use.

[0556]

[22] The use of a soluble tag to increase the solubility of a molecule, wherein the molecule is - Targeting portion, - at least one functional part and - the functional part(s) and the Targeting Linkers that covalently link moieties Including, use.

[0557]

[23] The use of a soluble tag to enhance the solubility of a molecule, wherein the molecule is Targeting Use consisting of parts.

[0558]

[24] The use of a soluble tag to increase the solubility of a molecule, wherein the molecule is -Targeting section and - at least one functional part Consists of, use.

[0559]

[25] The use of a soluble tag to increase the solubility of a molecule, wherein the molecule is - Targeting portion, - at least one functional part and - the functional part(s) and the Targeting Linkers that covalently link moieties Consists of, use.

[0560]

[26] The use of any of items

[20] to

[25] , wherein the use comprises a step of covalently attaching at least one soluble tag to the molecule.

[0561]

[27] , any of the molecules of items [1] to [6], any of the methods of items [7] to

[19] , or any of the uses of items

[20] to

[26] , wherein all components of the molecule are covalently bonded.

[0562]

[28] The molecule of any of items [1] to [6] or

[27] , the method of any of items [7] to

[19] or

[27] , or the use of any of items

[20] to

[27] , wherein the targeting moiety is a molecular group that specifically binds to a target molecule or a fragment thereof.

[0563]

[29] The molecule, method or use of item

[28] , wherein the target molecule is a biomolecule.

[0564]

[30] The molecule, method or use of any of items

[28] to

[29] , wherein the target molecule is a cell surface receptor.

[0565]

[31] The molecule, method, or use of any of items

[28] to

[30] , wherein the target molecule is an antigen present on the surface of a target cell.

[0566]

[32] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[31] , a method of any of items [7] to

[19] or

[27] to

[31] , or a use of any of items

[20] to

[31] , wherein the moiety is capable of specifically binding to an antigen present on the surface of a target cell.

[0567]

[33] The molecule of any of items [1] to [6] or

[27] to

[32] , the method of any of items [7] to

[19] or

[27] to

[32] , or the use of any of items

[20] to

[32] , wherein the targeting moiety comprises a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide, or small molecule.

[0568]

[34] Applicable TargetingThe molecule of any of items [1] to [6] or

[27] to

[33] , the method of any of items [7] to

[19] or

[27] to

[33] or the use of any of items

[20] to

[33] , wherein the moiety is selected from the group consisting of proteins, peptides, peptidomimetics, nucleic acids, oligonucleotides and small molecules.

[0569]

[35] The molecule of any of items [1] to [6] or

[27] to

[34] , the method of any of items [7] to

[19] or

[27] to

[34] , or the use of any of items

[20] to

[34] , wherein the targeting moiety comprises a protein.

[0570]

[36] The molecule of any of items [1] to [6] or

[27] to

[35] , the method of any of items [7] to

[19] or

[27] to

[35] , or the use of any of items

[20] to

[35] , wherein the targeting moiety is a protein.

[0571]

[37] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[36] , a method of any of items [7] to

[19] or

[27] to

[36] or a use of any of items

[20] to

[36] , wherein the moiety comprises or is a protein that is a protein ligand that specifically binds to a cell surface receptor.

[0572]

[38] The molecule of any of items [1] to [6] or

[27] to

[37] , the method of any of items [7] to

[19] or

[27] to

[37] , or the use of any of items

[20] to

[37] , wherein the targeting moiety comprises or is a protein that is an antibody or an antigen-binding fragment thereof.

[0573]

[39] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[38] , a method of any of items [7] to

[19] or

[27] to

[38] , or a use of any of items

[20] to

[38] , wherein the moiety comprises or is a protein that is an antibody component.

[0574]

[40] The molecule of any of items [1] to [6] or

[27] to

[39] , the method of any of items [7] to

[19] or

[27] to

[39] , or the use of any of items

[20] to

[39] , wherein the target moiety comprises or is a protein comprising at least 30 amino acids.

[0575]

[41] The molecule of any of items [1] to [6] or

[27] to

[40] , or the method of any of items [7] to

[19] or

[27] to

[40] . The use of any of items

[20] to

[40] , wherein the targeting moiety comprises or is a peptide consisting of 2 to 30 amino acids.

[0576]

[42] The molecule of any of items [1] to [6] or

[27] to

[41] , the method of any of items [7] to

[19] or

[27] to

[41] , or the use of any of items

[20] to

[41] , wherein the targeting moiety comprises a peptide.

[0577]

[43] The molecule of any of items [1] to [6],

[27] to

[34] or

[41] to

[42] , the method of any of items [7] to

[19] ,

[27] to

[34] or

[41] to

[42] or the use of any of items

[20] to

[34] or

[41] to

[42] , wherein the targeting moiety is a peptide.

[0578]

[44] The molecule of any of items [1] to [6],

[27] to

[35] or

[37] to

[42] , the method of any of items [7] to

[19] ,

[27] to

[35] or

[37] to

[42] or the use of any of items

[20] to

[35] or

[37] to

[42] , wherein the targeting moiety comprises a peptidomimetic.

[0579]

[45] The molecule of any of items [1] to [6] or

[27] to

[34] or

[44] , the method of any of items [7] to

[19] or

[27] or the use of any of items

[20] to

[34] or

[44] , wherein the targeting moiety is a peptidomimetic.

[0580]

[46] Applicable TargetingA molecule of any of items [1] to [6] or

[27] to

[35] or

[37] to

[42] or

[44] , a method of any of item [7],

[19] or

[27] to

[35] or

[37] to

[42] or

[44] or the use of any of items

[20] to

[35] or

[37] to

[42] or

[44] , wherein the molecule comprises or is a nucleic acid, the nucleic acid being DNA or RNA.

[0581]

[47] The molecule of any of items [1] to [6] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] , wherein the targeting moiety comprises a nucleic acid, any of the following methods or uses of items [7] to

[19] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] or any of items

[20] to

[35] or

[37] to

[42] or

[44] or

[46] .

[0582]

[48] ​​The molecule of any of items [1] to [6] or

[27] to

[34] or

[46] to

[47] , the method of any of items [7] to

[19] or the use of any of items

[27] to

[34] or

[46] to

[47] or the use of any of items

[20] to

[34] or

[46] to

[47] , wherein the targeting moiety is a nucleic acid.

[0583]

[49] The molecule of any one of items [1] to [6] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] to

[48] , the method of any of items [7] to

[19] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] to

[48] or the use of items

[20] to

[35] or

[37] to

[42] or

[44] or

[46] to

[48] , wherein the targeting moiety comprises an oligonucleotide.

[0584]

[50] The molecule of any of items [1] to [6],

[27] to

[34] , or

[46] to

[49] , the method of any of items [7] to

[19] ,

[27] to

[34] , or

[46] to

[49] , or the use of any of items

[20] to

[34] , or

[46] to

[49] , wherein the targeting moiety is an oligonucleotide.

[0585]

[51] A molecule of any of items [1] to [6] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] to

[47] or

[49] , a method of any of items [7] to

[19] or

[27] to

[35] or

[37] to

[42] or

[44] or

[46] to

[47] or

[49] or a use of any of items

[20] to

[35] or

[37] to

[42] or

[44] or

[46] to

[47] or

[49] , comprising or being a small molecule of molecular weight < 1000 Da.

[0586]

[52] Applicable Targeting The moiety comprises a small molecule, a molecule of any of items [1] to [6],

[27] to

[35] ,

[37] to

[42] ,

[44] ,

[46] to

[47] ,

[49] or

[51] , a method of any of items [7] to

[19] ,

[27] to

[35] ,

[37] to

[42] ,

[44] ,

[46] to

[47] ,

[49] or

[51] or a use of any of items

[20] to

[35] ,

[37] to

[42] ,

[44] ,

[46] to

[47] ,

[49] or

[51] .

[0587]

[53] The molecule of any of items [1] to [6],

[27] to

[34] or

[51] to

[52] , the method of any of items [7] to

[19] ,

[27] to

[34] or

[51] to

[52] or the use of any of items

[20] to

[34] or

[51] to

[52] , wherein the targeting moiety is a small molecule.

[0588]

[54] The molecule of any of items [1] to [6] or

[27] to

[53] , the method of any of items [7] to

[19] or

[27] to

[53] , or the use of any of items

[20] to

[53] , wherein the targeting moiety is not a sugar.

[0589]

[55] The molecule of any of items [1] to [6] or

[27] to

[54] , the method of any of items [7] to

[19] or

[27] to

[54] , or the use of any of items

[20] to

[54] , wherein the targeting moiety does not comprise a sugar.

[0590]

[56] Applicable Targeting A molecule according to any of items [1] to [6] or

[27] to

[55] , a method according to any of items [7] to

[19] or

[27] to

[55] , or a use according to any of items

[20] to

[55] , wherein the moiety has a molecular weight of at least 100 Da.

[0591]

[57] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[56] , a method of any of items [7] to

[19] or

[27] to

[56] , or a use of any of items

[20] to

[56] , wherein the moiety has a molecular weight of at least 500 Da.

[0592]

[58] The molecule of any of items [1] to [6] or

[27] to

[50] to

[57] , the method of any of items [7] to

[19] or

[27] to

[50] to

[57] , or the use of any of items

[20] to

[50] to

[57] , wherein the targeting moiety has a molecular weight of at least 1000 Da.

[0593]

[59] The molecule of any of items [1] to [6],

[27] to

[52] , or

[54] to

[58] , the method of any of items [7] to

[19] ,

[27] to

[52] , or

[54] to

[58] , or the use of any of items

[20] to

[52] , or

[54] to

[58] , wherein the targeting moiety has a molecular weight of at least 2000 Da.

[0594]

[60] The molecule of any of items [1] to [6] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[59] , the method of any of items [7] to

[19] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[59] or the use of any of items

[20] to

[42] or

[44] to

[49] or

[52] or

[54] to

[59] , wherein the targeting moiety has a molecular weight of at least 10 kDa.

[0595]

[61] The molecule of any of items [1] to [6] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[60] , the method of any of items [7] to

[19] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[60] or the use of any of items

[20] to

[42] or

[44] to

[49] or

[52] or

[54] to

[60] , wherein the targeting moiety has a molecular weight of at least 50 kDa.

[0596]

[62] The molecule of any of items [1] to [6] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[61] , the method of any of items [7] to

[19] or

[27] to

[42] or

[44] to

[49] or

[52] or

[54] to

[61] or the use of any of items

[20] to

[42] or

[44] to

[49] or

[52] or

[54] to

[61] , wherein the targeting moiety has a molecular weight of at least 100 kDa.

[0597]

[63] The molecule of any of items [1] to [6] or

[27] to

[62] , the method of any of items [7] to

[19] or

[27] to

[62] , or the use of any of items

[20] to

[62] , wherein the targeting moiety has a molecular weight of up to 10 MDa.

[0598]

[64] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[63] , a method of any of items [7] to

[19] or

[27] to

[63] , or a use of any of items

[20] to

[63] , wherein the moiety has a molecular weight of up to 5 MDa.

[0599]

[65] Applicable Targeting A molecule of any of items [1] to [6] or

[27] to

[64] , a method of any of items [7] to

[19] or

[27] to

[64] , or a use of any of items

[20] to

[64] , wherein the moiety has a molecular weight of up to 1 MDa.

[0600]

[66] Applicable TargetingA molecule according to any of items [1] to [6] or

[27] to

[65] , a method according to any of items [7] to

[19] or

[27] to

[65] , or a use according to any of items

[20] to

[65] , wherein the moiety has a molecular weight of up to 200 kDa.

[0601]

[67] The molecule of any of items [1] to [6],

[27] to

[61] , or

[63] to

[66] , the method of any of items [7] to

[19] ,

[27] to

[61] , or

[63] to

[66] , or the use of any of items

[20] to

[61] , or

[63] to

[66] , wherein the targeting moiety has a molecular weight of up to 50 kDa.

[0602]

[68] The molecule of any of items [1] to [6],

[27] to

[60] or

[63] to

[67] , the method of any of items [7] to

[19] ,

[27] to

[60] or

[63] to

[67] or the use of any of items

[20] to

[60] or

[63] to

[67] , wherein the targeting moiety has a molecular weight of at most 10 kDa.

[0603]

[69] The molecule of any of items [1] to [6],

[27] to

[59] , or

[63] to

[68] , the method of any of items [7] to

[19] ,

[27] to

[59] , or

[63] to

[68] , or the use of any of items

[20] to

[59] , or

[63] to

[68] , wherein the targeting moiety has a molecular weight of up to 2,000 Da.

[0604]

[70] The molecule of any of items [1] to [6],

[27] to

[58] or

[63] to

[69] , the method of any of items [7] to

[19] ,

[27] to

[58] or

[63] to

[69] or the use of any of items

[20] to

[58] or

[63] to

[69] , wherein the targeting moiety has a molecular weight of up to 1000 Da.

[0605]

[71] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[70] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[70] or a use of any of items

[21] -

[22] or

[24] -

[70] , wherein the at least one functional moiety is a chemical entity capable of performing a biological, chemical, therapeutic and / or diagnostic function in the human body.

[0606]

[72] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[71] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[71] or the use of any of items

[21] -

[22] or

[24] -

[71] , wherein the at least one functional moiety is a therapeutic agent or a detectable label.

[0607]

[73] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[72] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[72] or the use of any of items

[21] -

[22] or

[24] -

[72] , wherein the at least one functional moiety is a therapeutic agent.

[0608]

[74] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[72] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[72] or the use of any of items

[21] -

[22] or

[24] -

[72] , wherein the at least one functional moiety is a detectable label.

[0609]

[75] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[74] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[74] or the use of any of items

[21] -

[22] or

[24] -

[74] , wherein the at least one functional moiety is a payload that is a therapeutic agent or a detectable label.

[0610]

[76] The molecule, method or use of item

[75] , wherein the payload is a therapeutic agent.

[0611]

[77] The molecule, method or use of item

[75] , wherein the payload is a detectable label.

[0612]

[78] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[77] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[77] or the use of any of items

[21] -

[22] or

[24] -

[77] , wherein the at least one functional moiety comprises a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide or small molecule.

[0613]

[79] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[78] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[78] or the use of any of items

[21] -

[22] or

[24] -

[78] , wherein the at least one functional moiety is a protein, peptide, peptidomimetic, nucleic acid, oligonucleotide or small molecule.

[0614]

[80] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or a use of any of items

[21] -

[22] or

[24] -

[79] , wherein at least one functional moiety comprises a protein.

[0615]

[81] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[80] or the use of any of items

[21] -

[22] or

[24] -

[80] , wherein the at least one functional moiety is a protein.

[0616]

[82] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[81] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[81] or the use of any of items

[21] -

[22] or

[24] -

[81] , wherein the at least one functional portion comprises or is a protein comprising at least 30 amino acids.

[0617]

[83] The molecule of any of items [2]-[3], [5]-[6], or

[27] -

[82] , the method of any of items [8]-[9],

[11] -

[12] ,

[14] -

[15] ,

[17] -

[19] , or

[27] -

[82] , or the use of any of items

[21] -

[22] , or

[24] -

[82] , wherein the at least one functional moiety comprises or is a peptide consisting of 2 to 30 amino acids.

[0618]

[84] The molecule of any of items [2]-[3], [5]-[6], or

[27] -

[83] , the method of any of items [8]-[9], or the use of any of items

[11] -

[12] ,

[14] -

[15] ,

[17] -

[19] ,

[27] -

[83] , or items

[21] -

[22] , or

[24] -

[83] , wherein at least one functional moiety comprises a peptide.

[0619]

[85] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[83] -

[84] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[83] -

[84] or the use of any of items

[21] -

[22] or

[24] -

[79] or

[83] -

[84] , wherein the at least one functional moiety is a peptide.

[0620]

[86] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or the use of any of items

[21] -

[22] or

[24] -

[79] , wherein the at least one functional moiety comprises a peptidomimetic.

[0621]

[87] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[86] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[86] or the use of any of items

[21] -

[22] or

[24] -

[79] or

[86] , wherein the at least one functional moiety is a peptidomimetic.

[0622]

[88] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] or

[82] -

[84] or

[86] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[80] or

[82] -

[84] or

[86] or the use of any of items

[21] -

[22] or

[24] -

[80] or

[82] -

[84] or

[86] , wherein the at least one functional moiety comprises or is a nucleic acid that is DNA or RNA.

[0623]

[89] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] , or the use of any of items

[21] -

[22] or

[24] -

[80] or

[82] -

[84] or

[86] or

[88] , wherein the at least one functional moiety comprises a nucleic acid.

[0624]

[90] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[88] -

[89] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[88] -

[89] , or the use of any of items

[21] -

[22] or

[24] -

[79] or

[88] -

[89] , wherein the at least one functional moiety is a nucleic acid.

[0625]

[91] The molecule of any of items [2]-[3], [5]-[6],

[27] -

[80] ,

[82] -

[84] ,

[86] , or

[88] -

[90] , the method of any of items [8]-[9],

[11] -

[12] ,

[14] -

[15] ,

[17] -

[19] ,

[27] -

[80] ,

[82] -

[84] ,

[86] ,

[88] -

[90] , or the use of any of items

[21] -

[22] ,

[24] -

[80]

[82] -

[84]

[86]

[88] -

[90] , wherein the at least one functional moiety comprises an oligonucleotide.

[0626]

[92] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[88] -

[91] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[88] -

[91] or the use of any of items

[21] -

[22] or

[24] -

[79] or

[88] -

[91] , wherein the at least one functional moiety is an oligonucleotide.

[0627]

[93] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] -

[89] or

[91] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] -

[89] or

[91] or the use of any of items

[21] -

[22] or

[24] -

[79] , wherein the at least one functional moiety comprises a small molecule.

[0628]

[94] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[93] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[93] or the use according to any of items

[21] -

[22] or

[24] -

[79] or

[93] , wherein the at least one functional moiety is a small molecule.

[0629]

[95] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] -

[89] or

[91] or

[93] -

[94] , or item [8]-[9] or

[11] -

[12] or

[14] -[1 5] or

[17] to

[19] or

[27] to

[80] or

[82] to

[84] or

[86] or

[88] to

[89] or

[91] or

[93] to

[94] or use of any of the methods or items

[21] to

[22] or

[24] to

[80] or

[82] to

[84] or

[86] or

[88] to

[89] or

[91] or

[93] to

[94] .

[0630]

[96] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[80] or

[82] -

[84] or

[86] or

[88] -

[89] or

[91] or

[93] -

[95] , or item [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] - Use of any of the methods or items

[19] or

[27] to

[80] or

[82] to

[84] or

[86] or

[88] to

[89] or

[91] or

[93] to

[95] or any of the methods or items

[21] to

[22] or

[24] to

[80] or

[82] to

[84] or

[86] or

[88] to

[89] or

[91] or

[93] to

[95] .

[0631]

[97] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[79] or

[93] -

[96] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[79] or

[93] -

[96] or the use of any of items

[21] -

[22] or

[24] -

[79] or

[93] -

[96] , wherein the at least one functional moiety is a small molecule.

[0632]

[98] The molecule of any of items [2]-[3], [5]-[6], or

[27] -

[97] , the method of any of items [8]-[9],

[11] -

[12] ,

[14] -

[15] ,

[17] -

[19] , or

[27] -

[97] , or the use of any of items

[21] -

[22] , or

[24] -

[97] , wherein the at least one functional moiety is not a sugar.

[0633]

[99] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[98] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[98] or the use of any of items

[21] -

[22] or

[24] -

[98] , wherein the at least one functional moiety does not comprise a sugar.

[0634]

[0100] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[99] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[99] , or use of any of items

[21] -

[22] or

[24] -

[99] , wherein at least one functional moiety has a molecular weight of at least 100 Da.

[0635]

[0101] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0100] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0100] , or use of any of items

[21] -

[22] or

[24] -

[0100] , wherein the at least one functional moiety has a molecular weight of at least 500 Da.

[0636]

[0102] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0101] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0101] , or use of any of items

[21] -

[22] or

[24] -

[0101] , wherein the at least one functional moiety has a molecular weight of at least 1000 Da.

[0637]

[0103] For the molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0102] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0102] , or the use of any of items

[21] -

[22] or

[24] -

[0102] , wherein the at least one functional moiety has a molecular weight of at least 2000 Da.

[0638]

[0104] The molecule of any of items [2] to [3] or [5] to [6] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0103] , the method of any of items [8] to [9] or

[11] to

[12] or

[14] to

[15] or

[17] to

[19] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0103] or the use of any of items

[21] to

[22] or

[24] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0103] , wherein the at least one functional moiety has a molecular weight of at least 10 kDa.

[0639]

[0105] The molecule of any of items [2] to [3] or [5] to [6] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0104] , the method of any of items [8] to [9] or

[11] to

[12] or

[14] to

[15] or

[17] to

[19] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0104] or the use of any of items

[21] to

[22] or

[24] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0104] , wherein the at least one functional moiety has a molecular weight of at least 50 kDa.

[0640]

[0106] The molecule of any of items [2] to [3] or [5] to [6] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0105] , the method of any of items [8] to [9] or

[11] to

[12] or

[14] to

[15] or

[17] to

[19] or

[27] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0105] or the use of any of items

[21] to

[22] or

[24] to

[84] or

[86] to

[91] or

[93] or

[95] to

[0105] , wherein the at least one functional moiety has a molecular weight of at least 100 kDa.

[0641]

[0107] The molecule of any of items [2] to [3], [5] to [6], or

[27] to

[0102] , the method of any of items [8] to [9],

[11] to

[12] ,

[14] to

[15] ,

[17] to

[19] , or

[27] to

[0102] , or the use of any of items

[21] to

[22] , or

[24] to

[0102] , wherein the at least one functional moiety has a molecular weight of up to 1000 Da.

[0642]

[0108] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0103] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0103] , or use of any of items

[21] -

[22] or

[24] -

[0103] , wherein the at least one functional moiety has a molecular weight of up to 2000 Da.

[0643]

[0109] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0104] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0104] or use of any of items

[21] -

[22] or

[24] -

[0104] , wherein the at least one functional moiety has a molecular weight of at most 10 kDa.

[0644]

[0110] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0105] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0105] or use of any of items

[21] -

[22] or

[24] -

[0105] , wherein the at least one functional moiety has a molecular weight of at most 50 kDa.

[0645]

[0111] A molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0106] , a method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0106] or use of any of items

[21] -

[22] or

[24] -

[0106] , wherein the at least one functional moiety has a molecular weight of up to 200 kDa.

[0646]

[0112] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0106] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0106] or the use of any of items

[21] -

[22] or

[24] -

[0106] , wherein the at least one functional moiety has a molecular weight of at most 1 MDa.

[0647]

[0113] The molecule of any of items [2]-[3] or [5]-[6] or

[27] -

[0106] , the method of any of items [8]-[9] or

[11] -

[12] or

[14] -

[15] or

[17] -

[19] or

[27] -

[0106] or the use of any of items

[21] -

[22] or

[24] -

[0106] , wherein the at least one functional moiety has a molecular weight of up to 5 MDa.

[0648]

[0114] A molecule according to any one of items [1] to [6] or

[27] to

[0113] , a method according to any one of items [7] to

[19] or

[27] to

[0113] , or the use of any one of items

[20] to

[0113] , wherein a comparison molecule lacking the soluble tag but having the same structure as the molecule has an isoelectric point (pI) of 5 to 9.

[0649]

[0115] A molecule of any of items [1] to [6] or

[27] to

[0114] , a method of any of items [7] to

[19] or

[27] to

[0114] , or a use of any of items

[20] to

[0114] , wherein a comparison molecule having the same structure as the molecule but lacking the solubility tag has a solubility (expressed as g of compound per ml) measured at 25°C in PBS (phosphate buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) within ±50%, preferably ±30%, of the solubility of a compound consisting of the antibody trastuzumab and two copies of an auristatin covalently bound to its Fc region, measured under the same conditions.

[0650]

[0116] A molecule of any of items [1] to [6] or

[27] to

[0115] , a method of any of items [7] to

[19] or

[27] to

[0115] , or a use of any of items

[20] to

[0115] , wherein the number of functional moieties per molecule is in the range of 1 to 15.

[0651]

[0117] A molecule of any of items [1] to [6] or

[27] to

[0116] , a method of any of items [7] to

[19] or

[27] to

[0116] , or a use of any of items

[20] to

[0116] , wherein the number of functional moieties per molecule is in the range of 1 to 10.

[0652]

[0118] Any of the molecules of items [1] to [6] or

[27] to

[0117] , any of the methods of items [7] to

[19] or

[27] to

[0117] , or any of the uses of items

[20] to

[0117] , wherein the number of functional moieties per molecule is in the range of 1 to 8.

[0653]

[0119] Any of the molecules of items [1] to [6] or

[27] to

[0118] , any of the methods of items [7] to

[19] or

[27] to

[0118] , or any of the uses of items

[20] to

[0118] , wherein the number of functional moieties per molecule is in the range of 1 to 4.

[0654]

[0120] Any of the molecules of items [1] to [6] or

[27] to

[0119] , any of the methods of items [7] to

[19] or

[27] to

[0119] , or any of the uses of items

[20] to

[0119] , wherein the molecule contains one or less functional moieties.

[0655]

[0121] Any of the molecules of items [1] to [6] or

[27] to

[0119] , any of the methods of items [7] to

[19] or

[27] to

[0119] , or any of the uses of items

[20] to

[0119] , wherein the number of functional moieties per molecule is within the range of 2 to 8.

[0656]

[0122] Any of the molecules of items [1] to [6] or

[27] to

[0119] , any of the methods of items [7] to

[19] or

[27] to

[0119] , or any of the uses of items

[20] to

[0119] , wherein the number of functional moieties per molecule is within the range of 4 to 8.

[0657]

[0123] Per molecule Targeting Any numerator of items [1] to [6] or

[27] to

[0122] , any method of items [7] to

[19] or

[27] to

[0122] , or any use of items

[20] to

[0122] , wherein the number of moieties is in the range of 1 to 15.

[0658]

[0124] Per molecule Targeting Any numerator of items [1] to [6] or

[27] to

[0123] , any method of items [7] to

[19] or

[27] to

[0123] , or any use of items

[20] to

[0123] , wherein the number of moieties is in the range of 1 to 10.

[0659]

[0125] per molecule Targeting Any numerator of items [1] to [6] or

[27] to

[0124] , any method of items [7] to

[19] or

[27] to

[0124] , or any use of items

[20] to

[0124] , wherein the number of moieties is in the range of 1 to 8.

[0660]

[0126] per molecule Targeting Any numerator of items [1] to [6] or

[27] to

[0125] , any method of items [7] to

[19] or

[27] to

[0125] , or any use of items

[20] to

[0125] , wherein the number of moieties is in the range of 1 to 4.

[0661]

[0127] No more than one molecule Targeting Any molecule of items [1] to [6] or

[27] to

[0126] , any method of items [7] to

[19] or

[27] to

[0126] , or any use of items

[20] to

[0126] , including a moiety.

[0662]

[0128] Per molecule Targeting The number of moieties is in the range of 2 to 8, and the numerator of any of items [1] to [6] or

[27] to

[0126] , the method of any of items [7] to

[19] or

[27] to

[0126] , or the use of any of items

[20] to

[0126] .

[0663]

[0129] Per molecule Targeting The number of moieties is in the range of 4 to 8, and the numerator of any of items [1] to [6] or

[27] to

[0126] , the method of any of items [7] to

[19] or

[27] to

[0126] , or the use of any of items

[20] to

[0126] .

[0664] or the molecule (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag The molecule of any of items [1] to [6] or

[27] to

[0129] , or the method of any of items [7] to

[19] or

[27] to

[0129] .

[0665]

[0131] The molecule (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag The molecule of any of items [1] to [6] or

[27] to

[0130] , or the method of any of items [7] to

[19] or

[27] to

[0130] , including:

[0666]

[0132] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag An antibody-drug conjugate comprising:

[0667]

[0133] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag An antibody-drug conjugate comprising:

[0668] A method for increasing the solubility of an antibody-drug conjugate, comprising: (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently connects the payload and the antibody component wherein said antibody-drug conjugate is covalently attached to at least one soluble tag.

[0669] A method for increasing the solubility of an antibody-drug conjugate, comprising: (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; covalently attaching at least one soluble tag to said antibody-drug conjugate.

[0670] A method for increasing the solubility of an antibody-drug conjugate, comprising: (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; and preparing the antibody-drug conjugate wherein the antibody-drug conjugate is covalently attached to at least one solubility tag.

[0671] A method for increasing the solubility of an antibody-drug conjugate, comprising: (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; and preparing the antibody-drug conjugate in a form in which the antibody-drug conjugate is covalently attached to at least one solubility tag.

[0672]

[0138] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; 1. A method for increasing the solubility of a compound, comprising preparing a molecule in which the compound is covalently linked to at least one solubility tag; wherein the molecule is an antibody-drug conjugate.

[0673]

[0139] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; 1. A method for increasing the solubility of a compound comprising preparing a molecule in which the compound is covalently linked to at least one solubility tag; wherein the molecule is an antibody-drug conjugate.

[0674]

[0140] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; 1. A method for increasing the solubility of a molecule comprising preparing the molecule in a form in which the molecule is covalently attached to at least one solubility tag, whereby (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag The method provides an antibody-drug conjugate comprising:

[0675]

[0141] (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently attaches the payload to the antibody component; 1. A method for increasing the solubility of a molecule comprising preparing the molecule in a form in which it is covalently attached to at least one solubility tag, whereby (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker that covalently attaches the payload or the antibody component; and (iv) at least one soluble tag The method provides an antibody-drug conjugate comprising:

[0676]

[0142] Use of soluble tags to increase the solubility of antibody-drug conjugates.

[0677]

[0143] The use of item

[0142] , wherein the use comprises a step of covalently binding the soluble tag to the antibody-drug conjugate.

[0678]

[0144] The antibody-drug conjugate (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently connects the payload and the antibody component Use of items

[0142] to

[0143] , including:

[0679]

[0145] The antibody-drug conjugate (i) antibody component, (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently connects the payload and the antibody component Use of items

[0142] to

[0144] .

[0680]

[0146] Any of the molecules of items

[39] to

[40] or

[54] to

[0131] , any of the antibody-drug conjugates of items

[0132] to

[0133] , any of the methods of items

[39] to

[40] or

[54] to

[0131] or

[0134] to

[0141] , or any of the uses of items

[39] to

[40] or

[54] to

[0131] or

[0144] to

[0145] , wherein the antibody component is an intact antibody or an antigen-binding fragment thereof.

[0681]

[0147] The antibody component is an intact antibody. Any molecule of items

[39] to

[40] or

[54] to

[0131] or

[0146] , or any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] , or any method of items

[39] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] , or use of any of items

[39] to

[40] or

[54] to

[0131] or

[0144] to

[0146] .

[0682]

[0148] The molecule of any of items

[39] to

[40] or

[54] to

[0131] or

[0146] or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] , wherein the antibody component is an antigen-binding fragment of an intact antibody. The method of any of items

[39] to

[40] ,

[54] to

[0131] ,

[0134] to

[0141] ,

[0146] or the use of any of items

[39] to

[40] or

[54] to

[0131] or

[0144] to

[0146] .

[0683]

[0149] The antibody component (respectively the antibody of item

[38] ) is a monoclonal antibody or a polyclonal antibody, the molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0147] , the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0147] , the method of any of items

[0132] to

[0133] or

[0146] to

[0147] , or the use of any of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0147] .

[0684]

[0150] The molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0147] or

[0149] or the antibody-drug conjugate of item

[0132] to

[0133] or

[0146] to

[0147] or

[0149] , or the method of any of items

[0132] to

[0133] or

[0146] to

[0147] or

[0149] or the use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0147] or

[0149] , wherein the antibody component (respectively the antibody of item

[38] ) is a monoclonal antibody.

[0685]

[0151] The antibody component (respectively, the antibody of item

[38] ) is a monospecific antibody or a bispecific antibody, and any molecule of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0147] or

[0149] to

[0150] or any molecule of items

[0132] to

[0133] or

[0146] to

[0147] or

[0149] to

[0150] any antibody-drug conjugate or any method of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0147] or

[0149] to

[0150] or use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0147] or

[0149] to

[0150] .

[0686]

[0152] , the molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0151] or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0151] , the method of any of items

[0132] to

[0133] or

[0146] to

[0151] or the use of any of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0151] , wherein the antibody component (respectively the antibody of item

[38] ) is a bispecific antibody or antigen-binding fragment thereof capable of binding to both antigens for which the bispecific antibody is specific.

[0687]

[0153] The molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0147] or

[0149] to

[0152] , or the method of any of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0147] or

[0149] to

[0152] , or the use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0147] or

[0149] to

[0152] , wherein the antibody of item

[38] is a bispecific antibody.

[0688]

[0154] The molecule or antibody-drug conjugate of any of items

[38] to

[40] ,

[54] to

[0131] ,

[0146] to

[0147] , and

[0149] to

[0153] , or the molecule or antibody-drug conjugate of any of items

[0132] to

[0133] ,

[0146] to

[0147] , or the antibody component (respectively, the antibody of item

[38] ) is an antibody selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody. or use of any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0147] or

[0149] to

[0153] or any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0147] or

[0149] to

[0153] .

[0689]

[0155] Any of the molecules or antibody-drug conjugates of items

[38] to

[40] ,

[54] to

[0131] ,

[0146] to

[0147] , and

[0149] to

[0154] , or items

[0132] to

[0133] , wherein the antibody component (respectively, the antibody of item

[38] ) is an antibody selected from the group consisting of an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, an IgA antibody, an IgM antibody, and hybrids thereof. or use of any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0147] or

[0149] to

[0154] or any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0147] or

[0149] to

[0154] .

[0690]

[0156] The molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] , the antibody-drug conjugate of any of items

[0132] to

[0133] , or item

[0132] to

[0133] , wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, scFv, diabody and VHH. Any of the methods of

[0146] or

[0148] or

[0152] or the use of any of the items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or the use of any of the items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] .

[0691]

[0157] The molecule of any of items

[38] to

[40] ,

[54] to

[0131] ,

[0146] ,

[0148] ,

[0152] , and

[0156] or the antibody-drug conjugate of any of items

[0132] to

[0133] ,

[0146] ,

[0148] ,

[0152] , and

[0156] , wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, and Fv. 1. Use of any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] .

[0692]

[0158] The molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0157] or the antibody-drug of any of items

[0132] to

[0133] or

[0146] or

[0148] or

[0152] or

[0156] to

[0157] , wherein the antigen-binding fragment is selected from an scFv, a diabody, or a VHH. Conjugates or any of the methods or use of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0157] .

[0693]

[0159] Any molecule of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0158] or any antibody of items

[0132] to

[0133] ,

[0146] ,

[0148] ,

[0152] ,

[0156] to

[0158] , wherein the antigen-binding fragment is an antigen-binding fragment of a monoclonal antibody or a polyclonal antibody. - a drug conjugate or any of the methods of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0158] or the use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] or

[0156] to

[0158] .

[0694]

[0160] The molecule of any of items

[38] to

[40] ,

[54] to

[0131] ,

[0146] ,

[0148] ,

[0152] , or

[0156] to

[0159] , wherein the antigen-binding fragment is an antigen-binding fragment of a monoclonal antibody. Any antibody-drug conjugate of items

[0132] to

[0133] ,

[0146] ,

[0148] ,

[0152] ,

[0156] to

[0159] , or any method of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0159] , or use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] or

[0156] to

[0159] .

[0695]

[0161] The molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0160] or the antibody-drug complex of any of items

[0132] to

[0133] or

[0146] or

[0148] or

[0152] or

[0156] to

[0160] , wherein the antigen-binding fragment is a monospecific antibody or a bispecific antibody. Conjugate or any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0160] .

[0696]

[0162] A molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0161] or items

[38] to

[40] , wherein the antigen-binding fragment of item

[38] is an antigen-binding fragment of a bispecific antibody capable of binding to both antigens for which the bispecific antibody is specific. ] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0161] or the use of any of the methods or items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] or

[0156] to

[0161] .

[0697]

[0163] A molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0162] , or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] or

[0148] or

[0152] or

[0156] to

[0162] , wherein the antigen-binding fragment is an antibody selected from the group consisting of a chimeric antibody, a humanized antibody, and a human antibody. Any method of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or

[0156] to

[0162] or use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] or

[0156] to

[0162] .

[0698]

[0164] Any molecule of items

[38] to

[40] or

[54] to

[0131] or

[0146] or

[0148] or

[0152] or

[0156] to

[0163] or any of items

[0132] to

[0133] or

[0146] or

[0148] or

[0152] or

[0156] to

[0163] , wherein the antigen-binding fragment is an antibody selected from the group consisting of IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, IgA antibodies, IgM antibodies, and hybrids thereof. or any of the antibody-drug conjugates of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] or

[0148] or

[0152] or any of the methods of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0146] or

[0148] or

[0152] or any of the methods of items

[0156] to

[0163] .

[0699]

[0165] A molecule according to any one of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0164] or an antibody-drug conjugate according to any one of items

[0132] to

[0133] or

[0146] to

[0164] or a method of any of items [7] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0164] or a use of any of items

[20] to

[0131] or

[0144] to

[0164] , wherein the targeting moiety / the antibody component (respectively the antibody of item

[38] ) is capable of specifically binding to an antigen present on the surface of a target cell.

[0700]

[0166] A molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0165] or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0165] or a method of any of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0165] or a use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0165] , wherein the antibody component (respectively the antibody of item

[38] ) is an antibody to an antigen present on the surface of a target cell or an antigen-binding fragment of such an antibody.

[0701]

[0167] A molecule or antibody-drug conjugate or method or use of any of items

[0165] to

[0166] , wherein the antigen present on the surface of the target cells is present in greater abundance on the surface of the target cells than on the surface of other cell types.

[0702]

[0168] A molecule or antibody-drug conjugate or method or use of any of items

[0165] to

[0167] , wherein the antigen present on the surface of the target cell is present on the surface of the target cell but is substantially absent on the surface of other cell types.

[0703]

[0169] A molecule or antibody-drug conjugate or method or use of any of items

[0165] to

[0168] , wherein the antigen present on the surface of the target cell is present on the surface of the target cell but not on the surface of other cell types.

[0704]

[0170] A molecule or antibody-drug conjugate or method or use of any of items

[0165] to

[0169] , wherein the binding of the antibody component (respectively, the antibody of item

[38] ) enables the antigen present on the surface of the target cell to specifically recruit the antibody-drug conjugate to the target cell.

[0705]

[0171] A molecule or antibody-drug conjugate or method or use of any of items

[0165] to

[0170] , wherein the antigen present on the surface of the target cell is selected from the group consisting of a tumor antigen and an immune cell antigen.

[0706]

[0172] A molecule or antibody-drug conjugate or method or use according to any of items

[0165] to

[0171] , wherein the antigen present on the surface of the target cell is a tumor antigen.

[0707]

[0173] Applicable Targeting A molecule according to any one of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0172] or an antibody-drug conjugate according to any one of items

[0132] to

[0133] or

[0146] to

[0172] or a method according to any of items [7] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0172] or a use of any of items

[20] to

[0131] or

[0144] to

[0172] , wherein the portion / the antibody component (respectively the antibody of item

[38] ) is capable of specifically binding to an antigen selected from the group consisting of a tumor antigen and an immune cell antigen.

[0708]

[0174] Applicable TargetingA molecule according to any one of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0173] or an antibody-drug conjugate according to any one of items

[0132] to

[0133] or

[0146] to

[0173] or a method according to any of items [7] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0173] or a use of any of items

[20] to

[0131] or

[0144] to

[0173] , wherein the portion / the antibody component (respectively the antibody of item

[38] ) is capable of specifically binding to a tumor antigen.

[0709]

[0175] A molecule or antibody-drug conjugate or method or use according to any one of items

[0171] to

[0174] , wherein the tumor antigen is an antigen present on the surface of a tumor cell.

[0710]

[0176] The tumor antigen is CD11a, CD4, CD19, CD20, CD21, CD22, CD23, CD25, CD52, CD30, CD33, CD37, CD40L, CD52, CD56, CD70, CD72, CD74, CD79a, CD79b, CD138, CD163, Her2, Her3, EGFR, Mucl8, integrin, PSMA, CEA, BLys, ROR1, NaPi2b, NaPi3b, CEACAM5, Muc1, integrin avb6, Met, Trop2, BCMA, disialoganglioside GD2, B-PR1B, E16, STRAP1, 0772P, Sema 5b, ETBR, MSG783, STRAP2, Trp4, CRIPTO, FcRH1, FcRH2, NCA, IL20R-α, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CXCR5, HLA-DOB, P2X5, LY6 4, IRTA2, TENB2, PSMA, FOLH1, STR5, SSTR1, SSTR2, SSTR3, SSTR4, TGAV, ITGB6, CA9, EGFRvIII, IL2RA, AXL, CD3Q, TNFRSF8, TNFRSF17, CTAG, CTA; A molecule or antibody-drug conjugate, or a method or use according to any of items

[0171] to

[0175] , selected from the group consisting of CD174 / fucosyltransferase 3 (Lewis blood group), CLEC14A, GRP78, HSPA5, ASG-5, ENPP3, PRR4, GCC, GUCY2C, Liv-1, SLC39A8, 5T4, NCMA1, CanAg, FOLR1, GPN B, TIM-1, HAVCR1, Mindin / RG-1, B7-H4, VTCN1, PTK7, SDC1, claudins (preferably claudin 18.2), RON, MST1R, EPHA2, MS4A1, TNC (tenascin C), FAP, DKK-1, CS1 / SLAMF7, ENG (endoglin), ANXA1 (annexin A1), VCAM-1 (CD106), and folate receptor alpha.

[0711]

[0177] The tumor antigen is xCT, gpNMB, carbonic anhydrase IX (CAIX), cKIT, c-MET, tumor-associated glycoprotein 72 (TAG-72), TROP-2, TRA-1-60, TRA, TNF-α, TM4SF1, TIM-1, TAA, TA-MUC1 (tumor-specific antigen of mucin-1), sortilin (SORT1), STn, STING, STAP1, SSTR2, SSEA-4, SLITRK6, SLC44A4, SLAMF7, SAIL, receptor tyrosine kinase (RTK), ROR2, ROR1, RNF43, prolactin receptor (PRLR), polymorphic epithelial mucin (PEM), phosphatidylserine (PS), phosphatidylserine, PTK7, PSMA, PD-L1, P-cadherin, OX001L, OAcGD2, Nectin-4, NaPi2b, NOTCH3, mesothelin (MSLN), MUC16, MTX5, MTX3, MT1-MMP, MRC2, MET, MAGE, Ly6E, Lewis Y anti- Hara, LRRC15, LRP-1, LIV-1, LHRH, LGR5, LGALS3BP.LAMP-1, KLK2, KAAG-1, IL4R, IL7R, IL1RAP, IL-4, IL -3, IL-2, IL-13R, IGF-1R, HSP90, HLA-DR, HER-3, HER-2, Globo H, GPR20, GPC3.GPC-1, GD3, GD2, GCC, FS H, FOLRα, FOLR, FLT3, FGFR3, FGFR2, FCRH5, EphA3, EphA2, EpcAM, ETB, ENPP3, EGFRviii, EGFR, EFNA4 , Dysadherin, DR5 (Death receptor 5), DPEP3, DLL3, DLK-1, DCLK1, Cripto, Cathepsin D, CanAg, CXCR5, CSP-1, CLL-1, CLDN6, CLDN18. 2, CEACAM6, CEACAM5, CEA, CDH6, CD79b, CD74, CD71, CD70, CD56, CD51, CD48, CD46, CD45, CD44v6, CD40L, CD38, CD37, CD352, CD33 , CD317, CD30, CD300f, CD3, CD25, CD248, CD22, CD205, CD20, CD19, CD184, CD166, CD147, CD142, CD138, CD123, CCR7, CA9, CA6, C4.The molecule or antibody-drug conjugate, or the method or use of any of items

[0171] to

[0176] , is selected from the group consisting of 4a, BCMA, B7-H4, B7, -H3, Axl, ASCT2, AMHRII, ALK, AG-7, ADAM-9, 5T4, and 4-1BB.

[0712]

[0178] A molecule of any of items

[38] to

[40] or

[54] to

[0131] or

[0146] to

[0177] , or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0177] , or a method of any of items

[38] to

[40] or

[54] to

[0131] or

[0134] to

[0141] or

[0146] to

[0177] , or a use of any of items

[38] to

[40] or

[54] to

[0131] or

[0144] to

[0177] , wherein the antibody component (respectively the antibody of item

[38] ) has a first and a second antigen-binding site.

[0713]

[0179] A molecule or antibody-drug conjugate or method or use of item

[0178] , wherein the first antigen-binding site is capable of specifically binding to a tumor antigen and the second antigen-binding site is capable of specifically binding to a tumor antigen.

[0714]

[0180] A molecule or antibody-drug conjugate or method or use of any of items

[0178] to

[0179] , wherein the first and second antigen-binding sites are capable of binding to different antigens.

[0715]

[0181] A molecule, antibody-drug conjugate, or method or use of any of items

[0165] to

[0171] , wherein the antigen present on the surface of the target cell is an immune cell antigen.

[0716]

[0182] The targeting moiety / antibody component (respectively, the antibody of item

[38] ) is capable of specifically binding to an immune cell antigen, and is selected from the group consisting of any of the molecules of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0178] or

[0180] to

[0181] or items

[0132] to

[0133] or

[0146] to

[0178] or [01

[0146] to

[0178] or

[0180] to

[0181] , or any of the methods of items [7] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0178] or

[0180] to

[0181] , or the use of any of items

[20] to

[0131] or

[0144] to

[0178] or

[0180] to

[0181] .

[0717] The immune cell antigen - antigens present on the surface of immune cells, -Antigens, or molecules secreted by immune cells - Antigens are molecules that interact with receptors on immune cells A molecule or antibody-drug conjugate or method or use of any of items

[0171] or

[0173] or

[0178] or

[0180] to

[0182] .

[0718]

[0184] A molecule or antibody-drug conjugate, or method or use of any of items

[0171] or

[0173] or

[0178] or

[0180] to

[0183] , wherein the immune cell antigen is selected from the group consisting of CD80, CD86, B7H3, TNF-α, TGF-β, TGF-β2, TGF-1, IL-1, IL-4, IL-5, IL-6, IL-12, IL-13, IL-22, IL-23, interferon receptor, PD-1, PD-L1, CTLA4, MSR1, and folate receptor beta.

[0719]

[0185] A molecule or antibody-drug conjugate or method or use according to any of items

[0183] to

[0184] , wherein the immune cells are B cells, T cells or dendritic cells.

[0720]

[0186] A molecule or antibody-drug conjugate or method or use of any of items

[0183] to

[0185] , wherein the immune cells are T cells.

[0721]

[0187] A molecule or antibody-drug conjugate or method or use of any of items

[0171] or

[0173] or

[0178] or

[0180] to

[0186] , wherein the antibody component against the immune cell antigen (respectively the antibody of item

[38] ) has an immunostimulatory or immunosuppressive effect.

[0722]

[0188] Any molecule of items [2] to [3] or [5] to [6] or

[27] to

[0131] or

[0146] to

[0187] , or any method of items [8] to [9] or

[11] to

[12] or

[14] to

[15] or

[17] to

[19] or

[27] to

[0131] or

[0146] to

[0187] , or use of any of items

[21] to

[22] or

[24] to

[0131] or

[0146] to

[0187] , wherein the molecule contains only one type of functional moiety.

[0723]

[0189] Any of the molecules of items

[0130] to

[0131] or

[0146] to

[0188] , or any of the antibody-drug conjugates of items

[0132] to

[0133] or

[0146] to

[0188] , or any of the methods of items

[0134] to

[0141] or

[0146] to

[0188] , or any of the use of items

[0144] to

[0188] , wherein the antibody-drug conjugate is composed of only one type of payload.

[0724]

[0190] Any molecule of items

[0130] to

[0131] or

[0146] to

[0189] or any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0189] , any method of items

[0134] to

[0141] or

[0146] to

[0189] or use of any of items

[0142] to

[0189] , wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 1 to 15.

[0725]

[0191] Any molecule of items

[0130] to

[0131] or

[0146] to

[0190] , or any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0190] , any method of items

[0134] to

[0141] or

[0146] to

[0190] , or use of any of items

[0142] to

[0190] , wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 1 to 10.

[0726]

[0192] Any molecule of items

[0130] to

[0131] or

[0146] to

[0191] , any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0191] , any method of items

[0134] to

[0141] or

[0146] to

[0191] , or use of any of items

[0142] to

[0191] , wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 1 to 8.

[0727]

[0193] Any molecule of items

[0130] to

[0131] or

[0146] to

[0192] , or any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0192] , any method of items

[0134] to

[0141] or

[0146] to

[0192] , or use of any of items

[0142] to

[0192] , wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 1 to 4.

[0728]

[0194] Any molecule of items

[0130] to

[0131] or

[0146] to

[0193] , any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0193] , any method of items

[0134] to

[0141] or

[0146] to

[0193] , or use of any of items

[0142] to

[0193] , wherein the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 2 to 8.

[0729]

[0195] Any molecule of items

[0130] to

[0131] or

[0146] to

[0194] or any antibody-drug conjugate of items

[0132] to

[0133] or

[0146] to

[0194] , any method of items

[0134] to

[0141] or

[0146] to

[0194] or use of any of items

[0142] to

[0194] , wherein the drug-antibody ratio (DAR) of the antibody-drug conjugate is in the range of 4 to 8.

[0730]

[0196] Any molecule of items [2] to [3], [5] to [6],

[27] to

[73] ,

[75] to

[76] ,

[78] to

[0131] , or

[0146] to

[0195] , any antibody-drug conjugate of items

[0132] to

[0133] , or

[0146] to

[0195] , or any antibody-drug conjugate of items [8] to [9], wherein the payload (respectively the functional moiety) is a therapeutic agent. ],

[11] -

[12] ,

[14] -

[15] ,

[17] -

[19] ,

[27] -

[73] ,

[75] -

[76] ,

[78] -

[0131] ,

[0134] -

[0141] ,

[0146] -

[0195] or the use of any of the methods or items

[21] -

[22] or

[24] -

[73] or

[75] -

[76] or

[78] -

[0131] or

[0144] -

[0195] .

[0731]

[0197] The molecule or antibody-drug conjugate or method or use of item

[0196] , wherein the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent or an immunosuppressant.

[0732]

[0198] A molecule or antibody-drug conjugate or method or use according to any of items

[0196] to

[0197] , wherein the therapeutic agent is a cytotoxic agent.

[0733]

[0199] A molecule or antibody-drug conjugate or method or use of any of items

[0197] to

[0198] , wherein the cytotoxic agent is selected from the group consisting of a microtubule formation inhibitor, an EG5 inhibitor and a DNA damaging agent.

[0734]

[0200] The molecule or antibody-drug conjugate or method or use of item

[0199] , wherein the microtubule formation inhibitor is selected from the group consisting of auristatins, maytansinoids and tubulysins.

[0735]

[0201] The molecule or antibody-drug conjugate or method or use of item

[0200] , wherein the auristatin is auristatin E, MMAE or MMAF.

[0736]

[0202] A molecule or antibody-drug conjugate or method or use according to any of items

[0200] to

[0201] , wherein the auristatin is auristatin E or MMAE.

[0737]

[0203] The molecule or antibody-drug conjugate or method or use of item

[0200] , wherein the maytansinoid is selected from the group consisting of maytansine, DM1, DM2, DM3 and DM4.

[0738]

[0204] The molecule or antibody-drug conjugate or method or use of any of items

[0200] or

[0203] , wherein the maytansinoid is selected from the group consisting of maytansine, DM2 and DM4.

[0739]

[0205] The molecule or antibody-drug conjugate or method or use of item

[0199] , wherein the EG5 inhibitor is selected from the group consisting of ispenisib, filanesib, ritronesib and K858.

[0740]

[0206] The molecule or antibody-drug conjugate or method or use of item

[0199] , wherein the DNA damaging agent is selected from the group consisting of a topoisomerase I inhibitor, a topoisomerase II inhibitor and a DNA alkylating agent.

[0741]

[0207] A molecule or antibody-drug conjugate or method or use of any of items

[0200] or

[0206] , wherein the cytotoxic agent is a topoisomerase I inhibitor.

[0742]

[0208] A molecule or antibody-drug conjugate or method or use of any of items

[0206] or

[0207] , wherein the topoisomerase I inhibitor is selected from the group consisting of exatecan, camptothecin, SN38, Dxd, preferably the topoisomerase I inhibitor is exatecan, SN38 or Dxd or a mutant thereof.

[0743]

[0209] A molecule or antibody-drug conjugate or method or use of any of items

[0200] or

[0206] , wherein the cytotoxic agent is a topoisomerase II inhibitor.

[0744]

[0210] The molecule or antibody-drug conjugate or method or use of any of items

[0206] or

[0209] , wherein the topoisomerase II inhibitor is doxorubicin or a variant thereof, preferably doxorubicin.

[0745]

[0211] A molecule or antibody-drug conjugate or method or use of any of items

[0200] or

[0206] , wherein the cytotoxic agent is a DNA alkylating agent.

[0746]

[0212] A molecule or antibody-drug conjugate or method or use of any of items

[0206] or

[0211] , wherein the DNA alkylating agent is selected from the group consisting of duocarmycin, CBI dimer, pyrrolobenzodiazepine and variants thereof, preferably the DNA alkylating agent is selected from the group consisting of duocarmycin, CBI dimer and diazepine (preferably pyrrolobenzodiazepine or indolinebenzodiazepine).

[0747]

[0213] A molecule or antibody-drug conjugate or method or use of any of items

[0196] to

[0198] , wherein the therapeutic agent is selected from the group consisting of auristatin, MMAE (monomethylauristatin E), duocarmycin, CBI (cyclopropanebenzo[e]indoline) dimer, maytansine, pyrrolobenzodiazepine, and indolinobenzodiazepine.

[0748]

[0214] A molecule, antibody-drug conjugate, or method or use of any of items

[0196] to

[0198] , wherein the cytotoxic agent is exatecan, duocarmycin, or a CBI dimer.

[0749]

[0215] A molecule or antibody-drug conjugate or method or use of any of items

[0196] to

[0198] , wherein the therapeutic agent is selected from the group consisting of auristatins, duocarmycins, CBI (cyclopropanebenzo[e]indoline) dimers and maytansinoids.

[0750]

[0216] A molecule or antibody-drug conjugate or method or use of any of items

[0196] to

[0198] , wherein the therapeutic agent is selected from the group consisting of MMAE (monomethylauristatin E), CBI (cyclopropanebenzo[e]indoline) dimer and maytansinoid DM4, duocarmycin.

[0751]

[0217] A molecule or antibody-drug conjugate, or a method or use according to any of items

[0196] to

[0198] , wherein the therapeutic agent is selected from the group consisting of dolastatin, auristatin, MMAE, MMAF, amberstatin 269, auristatin 101, auristatin f, auristatin w, CEN-106, CM1, DGN462, DGN549, DM1, DM2, DM4, doxorubicin, duocarmycin, exatecan, OX-4235, PNU-159682, rapamycin, SG3199, SG1882, SN-38, tublysin, amanitin, aminopterin, anthracycline, calicheamicin, camptothecin, fujimycin, hemiasterlin, maytansinoid, PBD, rapamycin, and vinblastine.

[0752]

[0218] A molecule, antibody-drug conjugate, or method or use of any of items

[0196] to

[0197] , wherein the therapeutic agent is an anti-inflammatory agent.

[0753]

[0219] A molecule or antibody-drug conjugate or method or use of any of items

[0197] or

[0218] , wherein the anti-inflammatory agent is a glucocorticoid receptor agonist.

[0754]

[0220] A molecule or antibody-drug conjugate or method or use according to any of items

[0197] or

[0218] to

[0219] , wherein the anti-inflammatory agent is a steroid.

[0755]

[0221] A molecule or antibody-drug conjugate, method, or use of any of items

[0197] or

[0218] to

[0220] , wherein the anti-inflammatory agent is selected from the group consisting of cortisol, cortisone acetate, beclomethasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, and mometasone.

[0756]

[0222] A molecule or antibody-drug conjugate or method or use according to any of items

[0197] or

[0218] to

[0219] , wherein the anti-inflammatory agent is a non-steroidal anti-inflammatory agent.

[0757]

[0223] The molecule or antibody-drug conjugate or method or use of item

[0222] , wherein the non-steroidal anti-inflammatory agent is a Cox2 inhibitor.

[0758]

[0224] A molecule, antibody-drug conjugate or method or use of any of items

[0196] to

[0197] , wherein the therapeutic agent is an immunostimulant.

[0759]

[0225] A molecule or antibody-drug conjugate or method or use of item

[0222] or

[0224] , wherein the immune stimulant is selected from a TLR7 agonist, a TLR8 agonist, a TLR7 antagonist, a TLR8 antagonist, a Sting inhibitor, a TGFβ inhibitor, an a2A inhibitor and an a2B inhibitor.

[0760]

[0226] A molecule, antibody-drug conjugate, or method or use of any of items

[0196] to

[0197] , wherein the therapeutic agent is an immunosuppressant.

[0761]

[0227] A molecule or antibody-drug conjugate or method or use of any of items

[0197] or

[0226] , wherein the immunosuppressant is selected from the group consisting of IMDH (inosine monophosphate dehydrogenase) inhibitors, mTor (mechanistic target of rapamycin) inhibitors, SYK (spleen tyrosine kinase) inhibitors, JAK (Janus kinase) inhibitors and calcineurin inhibitors.

[0762]

[0228] any molecule of items [2] to [3], [5] to [6],

[27] to

[72] ,

[74] to

[75] , or

[77] to

[0131] , or any antibody-drug conjugate of items

[0132] to

[0133] , or

[0146] to

[0195] , or any antibody-drug conjugate of items [8] to [9], [ Use of any of the methods or items

[21] to

[22] or

[24] to

[72] or

[74] to

[75] or

[77] to

[0131] or

[0144] to

[0195] .

[0763]

[0229] The molecule or antibody-drug conjugate or method or use of item

[0228] , wherein the detectable label is a radioisotope, a fluorescent compound or an enzyme.

[0764]

[0230] Detectable labels include cyanine dyes, sulfocyanine dyes, Alexa Fluor® dyes (Molecular Probes / Thermo Fisher Scientific), DyLight® Fluor dyes (Dyomics / Thermo Fisher Scientific), FluoProbes® dyes (Interchim), Seta® dyes (SETA BioMedicals), and IRIS. TM A molecule or antibody-drug conjugate, method, or use according to any one of items

[0228] to

[0229] , wherein the molecule or antibody-drug conjugate is selected from the group consisting of: dyes (Cyanine Technologies).

[0765]

[0231] A molecule or antibody-drug conjugate or method or use according to any of items

[0228] to

[0230] , wherein the detectable label is a cyanine dye selected from the group consisting of Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, and Cy7.

[0766]

[0232] A molecule or antibody-drug conjugate or method or use according to any of items

[0228] to

[0231] , wherein the detectable label is a sulfo-cyanine dye selected from the group consisting of sulfo-Cy2, sulfo-Cy3, sulfo-Cy3B, sulfo-Cy3.5, sulfo-Cy5, sulfo-Cy5.5, and sulfo-Cy7.

[0767]

[0233] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0232] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0232] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0232] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0232] , wherein each of the linkers has a molecular weight of at most 1,500 Da.

[0768]

[0234] Any molecule of item [3] or [6] or

[27] to

[0131] or

[0146] to

[0234] or any antibody-drug conjugate of item

[0132] to

[0133] or

[0146] to

[0234] or any method of item [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0234] or use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0234] , wherein the / each linker has a molecular weight of up to 1,000 Da.

[0769]

[0235] Any molecule of item [3] or [6] or

[27] to

[0131] or

[0146] to

[0235] or any antibody-drug conjugate of item

[0132] to

[0133] or

[0146] to

[0235] or any method of item [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0235] or use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0235] , wherein the / each linker has a molecular weight of up to 500 Da.

[0770]

[0236] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0236] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0236] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0236] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0236] , wherein the linker is stable in an extracellular environment.

[0771]

[0237] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0236] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0236] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0236] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0236] , wherein the linker is stable in an intracellular environment.

[0772]

[0238] A molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0236] , or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0236] , or a method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0236] , or a use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0236] , wherein the linker is cleaved upon exposure to the intracellular environment.

[0773]

[0239] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0238] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0238] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0238] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0238] , wherein the linker is cleavable by enzymatic or chemical cleavage.

[0774]

[0240] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0239] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0239] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0239] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0239] , wherein the linker is cleavable by enzymatic cleavage.

[0775]

[0241] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0239] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0239] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0239] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0239] , wherein the linker is cleavable by chemical cleavage.

[0776]

[0242] The molecule or antibody-drug conjugate or method or use of item

[0239] or

[0240] , wherein the enzymatic cleavage is cleavage by exposure to a glycosidase, protease or esterase.

[0777]

[0243] A molecule or antibody-drug conjugate or method or use of any of items

[0239] to

[0242] , wherein the enzymatic cleavage is due to exposure to a tumor-specific enzyme, preferably a tumor-specific protease or esterase.

[0778]

[0244] The molecule, antibody-drug conjugate, or method or use of any of items

[0242] to

[0243] , wherein the protease is a cathepsin.

[0779]

[0245] The molecule or antibody-drug conjugate or method or use of item

[0242] , wherein the glycosidase is glucuronidase.

[0780]

[0246] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0245] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0245] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0245] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0245] , wherein the linker comprises a protease cleavage site, preferably a cathepsin B cleavage site.

[0781]

[0247] Any molecule of item [3] or [6] or

[27] to

[0131] or

[0146] to

[0246] , or any antibody-drug conjugate of item

[0132] to

[0133] or

[0146] to

[0246] , or any method of item [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0246] , or use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0246] , wherein the linker comprises a glucuronide.

[0782]

[0248] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0239] or

[0241] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0239] or

[0241] , or the method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0239] or

[0241] , or the use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0239] or

[0241] , wherein the linker cleavable by chemical cleavage is a pH-sensitive linker.

[0783]

[0249] The molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0239] or

[0241] or

[0248] , or the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0239] or

[0241] or

[0248] , or item [9] or or the use of any of the methods or items

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0239] or

[0241] or

[0248] .

[0784]

[0250] A molecule of any of items

[0239] or

[0241] , an antibody-drug conjugate of any of items

[0239] or

[0241] , a method of any of items

[0239] or

[0241] or a use of any of items

[0239] or

[0241] , wherein the linker cleavable by chemical cleavage is cleavable under reducing conditions.

[0785]

[0251] A molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0239] or

[0241] or

[0250] , or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0239] or

[0241] or

[0250] , or item [9] or or use of any of the methods or items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0239] or

[0241] or

[0250] .

[0786]

[0252] A molecule of any of items [3] or [6] or

[27] to

[0131] or

[0146] to

[0251] , or an antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0251] , or a method of any of items [9] or

[12] or

[15] or

[18] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0251] , or a use of any of items

[22] or

[25] or

[27] to

[0131] or

[0144] to

[0251] , wherein the linker comprises a cathepsin B cleavage site, a glucuronide bond or a disulfide bond.

[0787]

[0253] The soluble tag is covalently attached Targeting a molecule according to any one of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0252] or an antibody-drug conjugate according to any one of items

[0132] to

[0133] or

[0146] to

[0252] or a method according to any of items [7] to

[19] or

[27] to

[0131] or

[0134] to

[0141] or

[0146] to

[0252] or the use of any of items

[20] to

[0131] or

[0142] to

[0252] , wherein the molecule is linked to a moiety or functional moiety or linker.

[0788]

[0254] A molecule of any of items [1] to [6] or

[27] to

[0131] or

[0146] to

[0253] , or a method of any of items [7] to

[19] or

[27] to

[0131] or

[0146] to

[0253] , or a use of any of items

[20] to

[0131] or

[0146] to

[0253] , wherein the soluble tag is covalently linked to the targeting moiety.

[0789]

[0255] The molecule of any of items [2] to [3] or [5] to [6] or

[27] to

[0131] or

[0146] to

[0254] ; the antibody-drug conjugate of any of items

[0132] to

[0133] or

[0146] to

[0254] ; or the method of any of items [8] to [9] or

[11] to

[12] or

[14] to

[15] or

[17] to

[19] or

[27] to

[0131] or

[0146] to

[0254] ; or the use of any of items

[21] to

[22] or

[24] to

[0131] or [0146...

Claims

1. (i) Targeting portion (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker covalently linking the payload or the targeting moiety; and (iv) at least one soluble tag wherein the soluble tag has a chemical group having a structural formula selected from the following (I) to (IV): 【Chemistry 1】

2. The molecule of claim 1 , wherein the soluble tag is covalently attached to the at least one payload and / or the linker.

3. 3. The molecule of claim 1 or 2, wherein the targeting moiety is selected from the group consisting of proteins, peptides, nucleic acids and oligonucleotides.

4. 4. The molecule of any of claims 1 to 3, wherein the targeting moiety is an antibody directed against an antigen present on the surface of a target cell, or an antigen-binding fragment of such an antibody.

5. The molecule of any one of claims 1 to 4, wherein the targeting moiety is capable of specifically binding to a tumor antigen.

6. The molecule of any one of claims 1 to 5, wherein the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent, or an immunosuppressant.

7. 7. The molecule of any one of claims 1 to 6, wherein the linker / each of the linkers has a molecular weight of up to 1,500 Da.

8. 1. A method for increasing the solubility of a compound, comprising: (i) the targeting portion; (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently connects the payload and the targeting moiety; wherein the compound is covalently attached to at least one soluble tag, wherein the soluble tag has a chemical group having a structural formula selected from the group consisting of (I) to (IV) below, wherein the method comprises the step of covalently attaching the compound to at least one soluble tag: 【Chemistry 2】

9. A pharmaceutical composition comprising a molecule according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

10. A molecule according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 9 for use as a medicament.

11. A compound for use in preparing a molecule according to any one of claims 1 to 7, comprising a soluble tag according to any one of claims 1 or 2 bonded to an activating group selected from maleimide, halogen acetamide, alkyl halogen, a Michael acceptor (wherein the Michael acceptor is vinylpyridine), and a group suitable for cycloaddition (wherein the group suitable for cycloaddition is a ketone, hydrazone, semicarbazone, carboxylic acid, alkene, or alkyne suitable for cycloaddition).

12. The molecule of any one of claims 1 to 7, wherein the molecule comprises an antibody-drug conjugate.

13. The molecule of any one of claims 1 to 7, wherein the molecule comprises a peptide-drug conjugate.

14. (i) Targeting portion (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; (iii) a linker covalently linking the payload or the targeting moiety; and (iv) at least one soluble tag; wherein the soluble tag has a chemical group having a structural formula selected from the following (I) to (IV): 【Transformation 3】

15. The molecule of claim 14 , wherein the soluble tag is covalently attached to the at least one payload and / or the linker.

16. 16. The molecule of claim 14 or 15, wherein the monosaccharide units in the soluble tag are independently selected from the group consisting of aldoses, ketoses and chemically modified forms of the aldoses or ketoses.

17. 17. The molecule of any of claims 14 to 16, wherein the monosaccharide units in the soluble tag are individually selected from the group consisting of tetroses, pentoses, hexoses and chemically modified forms of tetroses, pentoses and hexoses, wherein the tetroses are individually selected from the group consisting of erythrose and threose; the pentoses are individually selected from the group consisting of ribose, arabinose, xylose, and lyxose; The hexoses are individually selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose and talose.

18. The molecule of any of claims 14 to 17, wherein the targeting moiety is selected from the group consisting of proteins, peptides, nucleic acids and oligonucleotides.

19. The molecule of any of claims 14 to 18, wherein the targeting moiety is an antibody directed against an antigen present on the surface of a target cell, or an antigen-binding fragment of such an antibody.

20. The molecule of any of claims 14 to 19, wherein the targeting moiety is capable of specifically binding to a tumor antigen.

21. The molecule of any of claims 14 to 20, wherein the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, an immunostimulatory agent or an immunosuppressant agent.

22. A molecule according to any one of claims 14 to 21, wherein the linker / each of the linkers has a molecular weight of up to 1,500 Da.

23. 1. A method for increasing the solubility of a compound, comprising: (i) the targeting portion; (ii) at least one payload, wherein the at least one payload is a therapeutic agent or a detectable label; and (iii) a linker that covalently connects the payload and the targeting moiety; and 1. A method comprising preparing a molecule wherein the compound is covalently attached to at least one soluble tag, the soluble tag having a chemical group having a structural formula selected from the group consisting of (I) to (IV) below, wherein the method comprises the step of covalently attaching the compound and the at least one soluble tag: 【Chemistry 4】

24. A pharmaceutical composition comprising a molecule according to any one of claims 14 to 22, further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

25. A molecule according to any one of claims 14 to 22 or a pharmaceutical composition according to claim 24 for use as a medicament.

26. A compound for use in preparing a molecule according to any one of claims 14 to 22, comprising a soluble tag according to any one of claims 14 to 17 bonded to an activating group selected from maleimide, halogen acetamide, alkyl halogen, a Michael acceptor (wherein the Michael acceptor is vinylpyridine), and a group suitable for cycloaddition (wherein the group suitable for cycloaddition is a ketone, hydrazone, semicarbazone, carboxylic acid, alkene, or alkyne suitable for cycloaddition).

27. The molecule of any one of claims 14 to 22, wherein the molecule comprises an antibody-drug conjugate.

28. The molecule of any one of claims 14 to 22, wherein the molecule comprises a peptide-drug conjugate.

Images