Trans-cyclooctene formulations

Abstract

Disclosed herein include compositions comprising a buffer, and trans-cyclooctenes (TCOs) that may have improved properties, for example in clinical use. In certain embodiments, the TCOs may have improved in vitro and in vivo properties as compared to other TCOs. In certain embodiments, the compositions disclosed herein provide improved stability and / or shelf life. The disclosure also pertains to in vivo methods of using said compositions, as well as medical uses thereof, combinations comprising said TCOs, and / or methods of preparing said compositions.

Description

[0001] Title: TRANS-CYCLOOCTENE FORMULATIONS

[0002] Technological field

[0003] The disclosure disclosed herein relates to trans-cyclooctenes (TCOs), and formulations thereof, with improved properties. Compositions and combinations comprising the TCOs of the disclosure, as well as methods for using or preparing same are provided as well.

[0004] Background

[0005] In the field of bioorthogonal chemistry the ligation between TCOs and dienes, in particular tetrazines, has been studied in depth. While the ligation works well both in vitro as in vivo, identifying optimal compounds for clinical use remains a research focus.

[0006] Along these lines, it is desired to identify new TCOs with overall good in vitro and in vivo properties, e.g. one or more of: fast blood clearance rate, high uptake at the target site (e.g. a tumor), low off-target uptake, a good metabolism profile, good stability, good reactivity with tetrazines, and / or high payload release (especially in vivo).

[0007] There is thus a need for new TCOs that address one or more of the abovementioned problems and / or desires.

[0008] Furthermore, the stability of formulations comprising TCOs and / or conjugates thereof has not been studied. Therefore, it is desired to identify new compositions comprising TCOs and / or conjugates thereof, with overall good storage properties, e.g. one or more of: little to no aggregation of the TCO and / or the conjugate thereof, chemical stability of the TCO and / or the conjugate thereof (i.e. low payload release during storage, low trans-cis isomerization during storage, and / or little to no decoupling of the TCO from the protein when a conjugate is stored), a good shelflife, and in particular a good shelflife over a wide range of storage temperatures.

[0009] Summary

[0010] The disclosure relates to at least the following embodiments:

[0011] Embodiment 1. A composition comprising (i) a buffer; and (ii) a compound having a structure according to Formula (1) and / or a conjugate thereof; wherein Formula (1) is: wherein L1is selected from the group consisting of linear or branched C4-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene; L2a, L2b, and L2dare each independently a linker; L2cis selected from the group consisting of C1-C8(hetero)alkanetriyl, C5-C6(hetero)arenetriyl, C3-C7cycloalkanetriyl, and C2-C7heterocycloalkanetriyl; T1is selected from the group consisting of -OT1A, hydrogen, C2-C6alkyl, C6aryl, C4-C5heteroaryl, C3-C6cycloalkyl, C5-C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3; each T1Ais independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue; T2is a bioconjugation moiety or a group -L3-CB; wherein L3is a residue of a bioconjugation moiety, and CBis selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof; T3is a polymer; and R48is selected from the group consisting of -OH, -O-acetyl, -O-C1-4alkyl, halogen, active carbonate, and a releasable group; wherein the conjugate comprises a protein conjugated to at least one compound according to Formula (1), wherein in the conjugate T2is a residue of a bioconjugation moiety, and in the conjugate said protein and said compound are conjugated via T2; and preferably L1is linear or branched C4-C12alkylene, more preferably L1is linear or branched C4-C10alkylene, and most preferably L1is linear C5-C6alkylene; preferably L2a, L2b, and L2dare each independently a linker containing at most twenty atoms; more preferably L2a, L2b, and L2dare each independently selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl, preferably L2Tis hydrogen; preferably L2cis C1-C8(hetero)alkanetriyl, more preferably L2cis C1-C8alkanetriyl, and most preferably L2cis C4-C6alkanetriyl; preferably T1is -OT1A; and most preferably T1is -OH; preferably T1is in an axial position; preferably T1Ais hydrogen or methyl, more preferably T1Ais hydrogen; preferably T2is maleimidyl, N-hydroxysuccinimidyl, or -L3-CB; preferably L3is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety; preferably CBis a protein, more preferably CBis an antibody or a diabody, even more preferably CBis a diabody, and most preferably CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably T3is a polymer comprising a polyethylene glycol moiety; preferably R48is a releasable group; preferably R48is in an axial position; preferably in the conjugate the protein is a diabody or an antibody; more preferably in the conjugate the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably in the conjugate the protein is conjugated to at most 12 of said compounds; more preferably in the conjugate the protein is conjugated to at most 8 of said compounds, most preferably in the conjugate the protein is conjugated to at most 4 of said compounds; preferably in said conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein, a residue of a hydroxyl of said protein, or a residue of an amine of said protein; more preferably in the conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein; preferably in the conjugate T2is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety; more preferably T2is a residue of a maleimidyl moiety; preferably the buffer is an aqueous solution, and more preferably the composition is an aqueous solution of the compound and / or conjugate in the buffer; most preferably the composition is an aqueous solution of the conjugate in the buffer.

[0012] Embodiment 2. The composition according to Embodiment 1, wherein the buffer has a pH of at most 6.7; preferably the buffer has a pH in a range of from 4.6 to 6.7.

[0013] Embodiment 3. The composition according to any one of the preceding Embodiments, wherein the buffer has a pH of at most 6.3.

[0014] Embodiment 4. The composition according to any one of the preceding Embodiments, wherein the buffer comprises a buffer compound; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; more preferably the buffer compound is citrate; even more preferably the buffer compound is sodium citrate. Embodiment 5. The composition according to Embodiment 4, wherein the buffer compound is selected from the group consisting of citrate, succinate, and histidine.

[0015] Embodiment 6. The composition according to any one of the preceding Embodiments, wherein the buffer comprises a carbohydrate; preferably the carbohydrate is sucrose, or trehalose; most preferably the carbohydrate is sucrose.

[0016] Embodiment 7. The composition according to any one of the preceding Embodiments, wherein the buffer comprises a chelator; preferably the chelator is ethylenediaminetetraacetic acid (EDTA).

[0017] Embodiment 8. The composition according to any one of the preceding Embodiments, wherein the buffer comprises a surfactant; preferably the surfactant is polysorbate 20 or polysorbate 40; most preferably the surfactant is polysorbate 20.

[0018] Embodiment 9. The composition according to any one of the preceding Embodiments, wherein the buffer comprises:

[0019] (i) a buffer compound;

[0020] (ii) a carbohydrate;

[0021] (iii) a chelator; and

[0022] (iv) a surfactant; and wherein the buffer has has a pH of at most 6.7; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; preferably the carbohydrate is sucrose, or trehalose; preferably the chelator is ethylenediaminetetraacetic acid (EDTA); preferably the surfactant is polysorbate 20 or polysorbate 40; and preferably the buffer has a pH in a range of from 4.6 to 6.7.

[0023] Embodiment 10. The composition according to any one of the preceding Embodiments, wherein the composition comprises (i) the buffer; and (ii) the conjugate; preferably the composition essentially consists of (i) the buffer; and (ii) the conjugate.

[0024] Embodiment 11. The composition according to any one of the preceding Embodiments, wherein the conjugate is according to Formula (C1):

[0025] wherein CBis a protein; T2is a residue of a bioconjugation moiety; CJ is in a range of from 1 to 12; R48is a releasable group; T1, T3, L1, L2a, L2b, L2c, and L2dare as defined in Embodiment 1; preferably the protein is a diabody or an antibody; more preferably the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4.

[0026] Embodiment 12. The composition according to Embodiment 11, wherein said conjugate is according to Formula (C2): wherein y is an integer in a range of from 10 to 50; preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, and most preferably in a range of from 23 to 25. Embodiment 13. The composition according to Embodiment 12, wherein said conjugate is according to Formula (C3): wherein x is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6.

[0027] Embodiment 14. The composition according to any one of the preceding Embodiments, wherein R48is a releasable group, and said releasable group is -O-C(=O)-CA; wherein CAis a drug; preferably the drug is linked to the moiety -O-C(=O)- via a secondary or tertiary nitrogen atom that is part of the drug, forming a carbamate; preferably the drug is monomethyl auristatin E (MMAE).

[0028] Embodiment 15. The composition according to Embodiment 13, wherein x is 5; y is 24; T1is OH; R48is -O-C(=O)-CA; CAis monomethyl auristatin E; CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; and T2is wherein the asterisk indicates a bond to AVP0458, and the wiggly line denotes a bond to the rest of the conjugate.

[0029] Embodiment 16. The composition according to Embodiment 12, wherein L1is unsubstituted linear C5alkylene; T1is OH; T1is in an axial position; R48is -O-C(=O)-CA; CA is monomethyl auristatin E; R48is in an axial position; y is 24; L2ais -C(O)NH- or -NHC(O)-; L2bis -C(O)NH- or -NHC(O)-; L2cis >CH-(CH2)4-; L2dis -C(O)NH- or -NHC(O)-; CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; and

[0030] T2is wherein the asterisk indicates a bond to

[0031] AVP0458, and the wiggly line denotes a bond to the rest of the conjugate.

[0032] Embodiment 17. The composition according to any one of Embodiments 13, 15, and 16, wherein the composition comprises (i) the buffer; and (ii) the conjugate; and wherein the composition has a pH of at most 6.3, and / or wherein the buffer comprises a carbohydrate; a chelator; a surfactant; and / or a buffer compound selected from the group consisting of citrate, succinate, and histidine; and preferably the composition essentially consists of (i) the buffer; and (ii) the conjugate.

[0033] Embodiment 18. The composition according to Embodiment 17, wherein the conjugate is according to Embodiment 13.

[0034] Embodiment 19. The composition according to Embodiment 17, wherein the conjugate is according to Embodiment 15.

[0035] Embodiment 20. The composition according to Embodiment 17, wherein the conjugate is according to Embodiment 16.

[0036] Embodiment 21. The composition according to any one of Embodiments 1 to 14, wherein the conjugate is wherein CJ is in a range of from 1 to 12; wherein CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4; preferably CBis linked to each maleimidyl group via a sulfur atom, preferably the sulfur atom is part of a cysteine.

[0037] Embodiment 22. The composition according to Embodiment 21, wherein the conjugate is or

[0038] Embodiment 23. The composition according to any one of Embodiments 1 to 17, wherein the conjugate is

[0039] Embodiment 24. The composition according to any one of the preceding Embodiments, wherein the composition is a pharmaceutical composition; preferably a pharmaceutical composition suitable for intravenous injection.

[0040] Embodiment 25. A combination of

[0041] (A1) a conjugate as defined in any one of Embodiments 1 to 23; and / or

[0042] (A2) a compound as defined in Embodiment 1; with

[0043] (A3) a buffer according to any one of Embodiments 1 to 9; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3).

[0044] Embodiment 26. The combination according to Embodiment 25, wherein the combination is of

[0045] (A1) a conjugate as defined in any one of Embodiments 1 to 23; with

[0046] (A3) a buffer according to any one of Embodiments 1 to 9; preferably the combination is a kit wherein (A1) is physically separated from (A3); and preferably the combination essentially consists of (A1) and (A3) .

[0047] Embodiment 27. The combination of any one of Embodiments 25 and 26 wherein the combination further comprises: (B) a diene; preferably the diene is a tetrazine; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3) and (B), and wherein (A3) is physically separated from (B).

[0048] Embodiment 28. The combination according to Embodiment 27, wherein the diene is selected from the group consisting of: Embodiment 29. The composition according to any one of Embodiments 1 to 24; or the combination according to any one of Embodiments 25 to 28; for use as a medicament.

[0049] Embodiment 30. The composition according to any one of Embodiments 1 to 24; or the combination according to any one of Embodiments 25 to 28; for use in the treatment of a disease in a subject; preferably the subject is a human; preferably the disease is cancer.

[0050] Embodiment 31. A method of treating a disease in a subject, wherein said method comprises the step of administering to said subject:

[0051] (a) the composition according to any one of Embodiments 1 to 24; and / or

[0052] (b) the combination according to any one of Embodiments 25 to 28; preferably the subject is a human; preferably the disease is cancer.

[0053] Embodiment 32. A method for preparing a dried composition, wherein said method comprises the step of drying a composition according to any one of Embodiments 1 to 24; preferably the drying is carried out by lyophilization.

[0054] Embodiment 33. A dried composition obtainable by the method of Embodiment 32; wherein preferably the dried composition is a lyophilized composition.

[0055] Embodiment 34. A method for preparing a composition according to any one of Embodiments 1 to 24; wherein said method comprises the step of:

[0056] (a) contacting a buffer as defined in any one of Embodiments 1 to 9 with a compound and / or a conjugate as defined in any one of Embodiments 1, and 10 to 24; and / or the step of

[0057] (b) reconstituting the dried composition of Embodiment 33 with water; preferably the method comprises the step of contacting a buffer as defined in any one of Embodiments 1 to 9 with a conjugate as defined in any one of Embodiments 1, and 10 to 24.

[0058] Embodiment 35. A method of storing the composition according to any one of Embodiments 1 to 24, wherein the method comprises the steps of:

[0059] (a) providing a composition according to any one of Embodiments 1 to 24;

[0060] (b) maintaining said composition at a temperature of at most 40°C; preferably the temperature is at most 35 °C; more preferably, the temperature is in a range of from -100°C to 35°C.

[0061] Embodiment 36. Use of a buffer as defined in any one of Embodiments 1 to 9 to store a compound and / or a conjugate as defined in any one of Embodiments 1, and 10 to 24; preferably the use is to store said conjugate.

[0062] Embodiment 37. Use of a buffer as defined in any one of Embodiments 1 to 9 to stabilize a compound and / or a conjugate as defined in any one of Embodiments 1, and 10 to 24, during storage; preferably the use is to stabilize said conjugate; preferably during storage the buffer and the compound and / or conjugate are maintained at a temperature of at most 40°C; more preferably the temperature is at most 35 °C; even more preferably, the temperature is in a range of from -100°C to 35°C.

[0063] Detailed Description

[0064] As an example from the field of bioorthogonal chemistry, WO 2022 / 197182 describes AVP0458-22-PEG24, herein referred to as compound 1. Compound 1 is an AVP0458 diabody modified with four TCO-containing moieties, and has the following structure:

[0065] However, the inventors have identified, for the first time, that certain properties of compound 1 may be improved.

[0066] First, it was found that while compound 1 has a good clearance from blood, further improvements are desired. Compound 1 has a half-life in the blood of healthy mice of 4.22 hours, and 48 hours after injection in healthy mice 1.14% ID / g of compound 1 in the blood of said mice was observed. Based on this, it is desired that new TCOs be provided that show faster clearance rates as compared to compound 1.

[0067] Second, it was found that while compound 1 shows good tumor uptake of 18.42 %ID / g in LS174T xenograft bearing mice, further improvements are desired. It is therefore also desired that TCOs with a higher tumor uptake be provided. Third, it was observed that compound 1 shows uptake at off-target sites, e.g. nontumour sites such as the heart, lung, etc. It is also desired that TCOs be provided with a lower off-target uptake.

[0068] Fourth, it was observed that compound 1 shows a metabolism profile that can be improved. Thus, it is also desired that TCOs be provided showing a better metabolism profile.

[0069] Some aspects and embodiments of the disclosure are therefore, in a broad sense, based on the judicious insight that TCOs of the disclosure may meet one or more of the aforementioned desires. In particular, it was surprisingly found that replacing the PEG4 moiety of compound 1 may result in a higher clearance rate, higher tumor uptake, lower off- target uptake, a better metabolism profile, and / or further improved in vitro and in vivo properties.

[0070] Especially the combination of a faster clearance rate and a higher tumor uptake is surprising, since this means that the faster clearance from blood is not due to e.g. excretion from the body. Instead, the compounds of the disclosure may be quickly taken up in the tumour. Even more advantageously the uptake of compounds of the disclosure in off-target tissues may be much lower as compared to the uptake in the tumour. This means that a higher percentage of payload may be released at the desired target site, and that the trigger to activate this payload release (typically a diene, e.g. a tetrazine) may be administered at an earlier point in time, shortening the entire procedure. Thus, the compounds of the disclosure may also result in a higher convenience for patients, as fewer and / or less severe side-effects may be expected as well as a shorter treatment time.

[0071] Furthermore, some aspects and embodiments of the disclosure are therefore, in a broad sense, based on the judicious insight that compositions of the disclosure may meet one or more of the aforementioned desires regarding formulations. In particular, it was surprisingly found that the compositions as disclosed herein may have overall good storage properties. In particular, the buffers as disclosed herein may chemically and / or physically stabilize the compounds and / or conjugates of the disclosure. For example, the buffers as disclosed herein may lead to little to no aggregation of the TCO and / or the conjugate thereof. Additionally or alternatively, the buffers of the disclosure may lead to low payload release during storage, low trans-cis isomerization during storage, and / or little to no decoupling of the TCO from the protein when a conjugate is stored. Moreover, the buffer of the disclosure may lead to a good shelflife, and in particular a good shelflife over a wide range of storage temperatures. Preferred embodiments of the disclosure are further described below. All of these embodiments, regardless of whether said embodiments are disclosed in the general part of the description, can be combined as long as said embodiments are not mutually exclusive.

[0072] Compositions of the disclosure

[0073] The compositions of the disclosure comprise a buffer, and a compound of Formula (1) and / or a conjugate as defined herein. Preferably, the composition of the disclosure comprises a buffer and a conjugate as defined herein. More preferably, the composition of the disclosure essentially consists of a buffer and a conjugate as defined herein.

[0074] Preferably, the composition of the disclosure is an aqueous solution. In that light, it is also preferred that the buffer as disclosed herein is an aqueous solution.

[0075] Preferably, in the composition of the disclosure the compound and / or conjugate are dissolved in said buffer. It will be understood that the dissolution of the compound and / or the conjugate in said buffer will typically not significantly affect the concentration of the other components in said buffer, in particular the concentration(s) of the buffer compound, carbohydrate, chelator, and / or surfactant. Thus, the preferred concentrations for the the buffer compound, carbohydrate, chelator, and surfactant in the buffer, are also the preferred concentrations of said components in the composition of the disclosure.

[0076] Preferably, the composition of the disclosure is a pharmaceutical composition.

[0077] Buffers of the disclosure

[0078] Preferably, the buffer in the composition of the disclosure comprises one or more components selected from the group consisting of buffer compounds, carbohydrates, chelators, surfactants, and water. Preferably, said buffer comprises a buffer compound and water. More preferably, said buffer comprises a buffer compound, water, and a carbohydrate. More preferably, said buffer comprises a buffer compound, water, a carbohydrate, and a surfactant. More preferably still, said buffer comprises a buffer compound, water, a carbohydrate, a surfactant, and a chelator.

[0079] Most preferably, said buffer essentially consists of a buffer compound, a carbohydrate, a surfactant, a chelator, and water. It will be understood that the phrase “essentially consists of" leaves room for low amounts of other components being present, such as minor contaminants, possible counterions of the buffer compound, and / or salt ions that result from adjusting the pH with e.g. HC1 and NaOH. Preferably, the phrase “essentially consists of" indicates that at most 5 wt%, more preferably at most 4 wt%, more preferably still at most 3 wt%, even more preferably at most 2 wt%, even more preferably at most 1 wt%, and most preferably at most 0.5 wt%, of the buffer is not a buffer compound, a carbohydrate, a surfactant, a chelator, or water, wherein the wt% is as compared to the total weight of the buffer including water. pH

[0080] Preferably, the buffer has a pH of at most at most 6.7, more preferably at most 6.6, more preferably at most 6.5, more preferably at most 6.4, more preferably at most 6.3, more preferably at most 6.2, more preferably at most 6.1, more preferably at most 6.0, more preferably at most 5.9, more preferably at most 5.8, more preferably at most 5.7, and most preferably at most 5.6.

[0081] Preferably, the buffer has a pH of at least 4.3, more preferably at least 4.4, more preferably at least 4.5, more preferably at least 4.6, more preferably at least 4.7, more preferably at least 4.8, more preferably at least 4.9, more preferably at least 5.0, more preferably at least 5.1, more preferably at least 5.2, more preferably at least 5.3, and most preferably at least 5.4.

[0082] Preferably, the buffer has a pH in a range of from 4.3 to 6.7, more preferably of from 4.4 to 6.6, more preferably of from 4.5 to 6.5, more preferably of from 4.6 to 6.4, more preferably of from 4.7 to 6.3, more preferably of from 4.8 to 6.2, more preferably of from 4.9 to 6.1, more preferably of from 5.0 to 6.0, more preferably of from 5.1 to 5.9, more preferably of from 5.2 to 5.8, more preferably of from 5.3 to 5.7, and most preferably of from 5.4 to 5.6.

[0083] Preferably, the buffer has a pH of about 4.3, about 4.4, or about 4.5. More preferably , the buffer has a pH of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about

[0084] 5.2, about 5.3, about 5.4, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about

[0085] 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7. Most preferably, the buffer has a pH of about 5.5.

[0086] It will be understood that the pH values as mentioned herein in relation to the buffer preferably also apply to the composition of the disclosure as a whole. Consequently, the following preferences apply to the composition of the disclosure.

[0087] Preferably, the composition has a pH of at most at most 6.7, more preferably at most 6.6, more preferably at most 6.5, more preferably at most 6.4, more preferably at most 6.3, more preferably at most 6.2, more preferably at most 6.1, more preferably at most 6.0, more preferably at most 5.9, more preferably at most 5.8, more preferably at most 5.7, and most preferably at most 5.6.

[0088] Preferably, the composition has a pH of at least 4.3, more preferably at least 4.4, more preferably at least 4.5, more preferably at least 4.6, more preferably at least 4.7, more preferably at least 4.8, more preferably at least 4.9, more preferably at least 5.0, more preferably at least 5.1, more preferably at least 5.2, more preferably at least 5.3, and most preferably at least 5.4.

[0089] Preferably, the composition has a pH in a range of from 4.3 to 6.7, more preferably of from 4.4 to 6.6, more preferably of from 4.5 to 6.5, more preferably of from 4.6 to 6.4, more preferably of from 4.7 to 6.3, more preferably of from 4.8 to 6.2, more preferably of from 4.9 to 6.1, more preferably of from 5.0 to 6.0, more preferably of from 5.1 to 5.9, more preferably of from 5.2 to 5.8, more preferably of from 5.3 to 5.7, and most preferably of from 5.4 to 5.6.

[0090] Preferably, the composition has a pH of about 4.3, about 4.4, or about 4.5. More preferably , the composition has a pH of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, or about 6.7. Most preferably, the composition has a pH of about 5.5.

[0091] The skilled person is aware of common techniques available to accurately measure the pH of a solution. Preferably, a pH meter is used to determine the pH of a composition as disclosed herein. Preferably, the pH is determined when the composition as disclosed herein has a temperature in a range of from 15 °C to 37 °C, more preferably of from 17 °C to 30 °C, more preferably still of from 18 °C to 25 °C, yet more preferably of from 20 °C to 25 °C, and most preferably of from 20 °C to 22 °C.

[0092] Buffer compounds

[0093] Preferably, the buffer comprises a buffer compound. Preferably, the buffer compound is selected from the group consisting of citrate, succinate, histidine, sodium phosphate, potassium phosphate, Tris (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2- (N-morpholino)ethanesulfonic acid), acetate, bicarbonate, borate, glycine, PIPES (piperazine- N,N’-bis(2-ethanesulfonic acid)), CAPS (3-(cyclohexylamino)propanesulfonic acid), HEPPS (N-(2-Hydroxyethyl)piperazine-N’-(3-propanesulfonic acid)), Bis-Tris, Bis-Tris propane, cacodylate, imidazole, and tartrate. More preferably, the buffer compound is selected from the group consisting of citrate, succinate, and histidine. Most preferably, the buffer compound is citrate.

[0094] It will be understood that if herein reference is made to citrate as a buffer compound, it may refer to citrate per se, monosodium citrate, disodium citrate, trisodium citrate, or combinatins thereof. However, as sodium citrate salts typically dissociate when dissolved in water, for the final concentration of the buffer compound it does not matter which citrate form was used. Consequently, if the buffer compound used herein is citrate, the concentration refers to the concentration of the citrate per se.

[0095] Preferably, the buffer comprises a buffer compound in a concentration of at least 0.5 mM, more preferably at least 1.0 mM, more preferably at least 1.5 mM, more preferably at least 2.0 mM, more preferably at least 2.5 mM, more preferably at least 3.0 mM, more preferably at least 3.5 mM, more preferably at least 4.0 mM, more preferably at least 4.5 mM, more preferably at least 5.0 mM, more preferably at least 5.5 mM, more preferably at least 6.0 mM, more preferably at least 6.5 mM, more preferably at least 7.0 mM, more preferably at least 7.5 mM, more preferably at least 8.0 mM, more preferably at least 8.5 mM, more preferably at least 9.0 mM, more preferably at least 9.5 mM, more preferably at least 10.0 mM, more preferably at least 10.5 mM, more preferably at least 11.0 mM, more preferably at least 11.5 mM, more preferably at least 12.0 mM, more preferably at least 12.5 mM, more preferably at least 13.0 mM, more preferably at least 13.5 mM, more preferably at least 14.0 mM, more preferably at least 14.5 mM, more preferably at least 15.0 mM, more preferably at least 15.5 mM, more preferably at least 16.0 mM, more preferably at least 16.5 mM, more preferably at least 17.0 mM, more preferably at least 17.5 mM, more preferably at least 18.0 mM, more preferably at least 18.5 mM, more preferably at least 19.0 mM, and most preferably at least 19.5 mM.

[0096] Preferably, the buffer comprises a buffer compound in a concentration of at most 200 mM, more preferably at most 190 mM, more preferably at most 180 mM, more preferably at most 170 mM, more preferably at most 160 mM, more preferably at most 150 mM, more preferably at most 140 mM, more preferably at most 130 mM, more preferably at most 120 mM, more preferably at most 110 mM, more preferably at most 100 mM, more preferably at most 95 mM, more preferably at most 90 mM, more preferably at most 85 mM, more preferably at most 80 mM, more preferably at most 75 mM, more preferably at most 70 mM, more preferably at most 65 mM, more preferably at most 60 mM, more preferably at most 55 mM, more preferably at most 50 mM, more preferably at most 45 mM, more preferably at most 40 mM, more preferably at most 35 mM, more preferably at most 34.5 mM, more preferably at most 34 mM, more preferably at most 33.5 mM, more preferably at most 33 mM, more preferably at most 32.5 mM, more preferably at most 32 mM, more preferably at most 31.5 mM, more preferably at most 31 mM, more preferably at most 30.5 mM, more preferably at most 30 mM, more preferably at most 29.5 mM, more preferably at most 29 mM, more preferably at most 28.5 mM, more preferably at most 28 mM, more preferably at most 27.5 mM, more preferably at most 27 mM, more preferably at most 26.5 mM, more preferably at most 26 mM, more preferably at most 25.5 mM, more preferably at most 25 mM, more preferably at most 24.5 mM, more preferably at most 24 mM, more preferably at most 23.5 mM, more preferably at most 23 mM, more preferably at most 22.5 mM, more preferably at most 22 mM, more preferably at most 21.5 mM, more preferably at most 21 mM

[0097] Preferably, the buffer comprises a buffer compound in a concentration in a range of from 0.5 mM to 200 mM, more preferably of from 1.0 mM to 190 mM, more preferably of from 1.5 mM to 180 mM, more preferably of from 2.0 mM to 170 mM, more preferably of from 2.5 mM to 160 mM, more preferably of from 3.0 mM to 150 mM, more preferably of from 3.5 mM to 140 mM, more preferably of from 4.0 mM to 130 mM, more preferably of from 4.5 mM to 120 mM, more preferably of from 5.0 mM to 110 mM, more preferably of from 5.5 mM to 100 mM, more preferably of from 6.0 mM to 95 mM, more preferably of from 6.5 mM to 90 mM, more preferably of from 7.0 mM to 85 mM, more preferably of from 7.5 mM to 80 mM, more preferably of from 8.0 mM to 75 mM, more preferably of from 8.5 mM to 70 mM, more preferably of from 9.0 mM to 65 mM, more preferably of from 9.5 mM to 60 mM, more preferably of from 10.0 mM to 55 mM, more preferably of from 10.5 mM to 50 mM, more preferably of from 11.0 mM to 45 mM, more preferably of from 11.5 mM to 40 mM, more preferably of from 12.0 mM to 35 mM, more preferably of from 12.5 mM to 34 mM, more preferably of from 13.0 mM to 33 mM, more preferably of from 13.5 mM to 32 mM, more preferably of from 14.0 mM to 31 mM, more preferably of from 14.5 mM to 30 mM, more preferably of from 15.0 mM to 29 mM, more preferably of from 15.5 mM to 28 mM, more preferably of from 16.0 mM to 27 mM, more preferably of from 16.5 mM to 26 mM, more preferably of from 17.0 mM to 25 mM, more preferably of from 17.5 mM to 24 mM, more preferably of from 18.0 mM to 23 mM, more preferably of from 18.5 mM to 22 mM, more preferably of from 19.0 mM to 21 mM, more preferably of from 19.5 mM to 20.5 mM,

[0098] Preferably, the buffer comprises a buffer compound in a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM, about 93 mM, about 94 mM, about 95 mM, about 96 mM, about 97 mM, about 98 mM, about 99 mM, or about 100 mM.

[0099] Most preferably, the buffer comprises a buffer compound in a concentration of about 20 mM.

[0100] Carbohydrates

[0101] Preferably, the buffer comprises a carbohydrate. Preferably, the carbohydrate is selected from the group consisting of sucrose, trehalose, glucose, maltose, lactose, dextran, cyclodextrin, maltodextrin, mannitol, and sorbitol. More preferably, the carbohydrate is sucrose or trehalose. Most preferably, the carbohydrate is sucrose.

[0102] Preferably, the buffer comprises a carbohydrate at a concentration of at least 0.5 g / L, more preferably at least 1 g / L, more preferably at least 2 g / L, more preferably at least 3 g / L, more preferably at least 4 g / L, more preferably at least 5 g / L, more preferably at least 7 g / L, more preferably at least 10 g / L, more preferably at least 15 g / L, more preferably at least 20 g / L, more preferably at least 25 g / L, more preferably at least 30 g / L, more preferably at least 35 g / L, more preferably at least 40 g / L, and most preferably at least 45 g / L

[0103] Preferably, the buffer comprises a carbohydrate at a concentration of at most 150 g / L, more preferably at most 140 g / L, more preferably at most 130 g / L, more preferably at most 125 g / L, more preferably at most 120 g / L, more preferably at most 110 g / L, more preferably at most 100 g / L, more preferably at most 90 g / L, more preferably at most 85 g / L, more preferably at most 80 g / L, more preferably at most 75 g / L, more preferably at most 70 g / L, more preferably at most 65 g / L, more preferably at most 60 g / L, more preferably at most 55 g / L

[0104] Preferably, the buffer comprises a carbohydrate at a concentration in a range of from 0.5 to 150 g / L, more preferably of from 1 to 140 g / L, more preferably of from 2 to 130 g / L, more preferably of from 3 to 125 g / L, more preferably of from 4 to 120 g / L, more preferably of from 5 to 110 g / L, more preferably of from 7 to 100 g / L, more preferably of from 10 to 90 g / L, more preferably of from 15 to 85 g / L, more preferably of from 20 to 80 g / L, more preferably of from 25 to 75 g / L, more preferably of from 30 to 70 g / L, more preferably of from 35 to 65 g / L, more preferably of from 40 to 60 g / L, and most preferably of from 45 to 55

[0105] Preferably, the buffer comprises a carbohydrate at a concentration of about 30 g / L, about 31 g / L, about 32 g / L, about 33 g / L, about 34 g / L, about 35 g / L, about 36 g / L, about 37 g / L, about 38 g / L, about 39 g / L, about 40 g / L, about 41 g / L, about 42 g / L, about 43 g / L, about 44 g / L, about 45 g / L, about 46 g / L, about 47 g / L, about 48 g / L, about 49 g / L, about 51 g / L, about 52 g / L, about 53 g / L, about 54 g / L, about 55 g / L, about 56 g / L, about 57 g / L, about 58 g / L, about 59 g / L, about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, or about 70 g / L. Most preferably, the buffer comprises a carbohydrate at a concentration of about 50 g / L.

[0106] Chelator

[0107] Preferably, the buffer comprises a chelator. Preferably, the chelator is selected from the group consisting of EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol- bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), DTP A (diethylenetriaminepentaacetic acid), NT A (nitrilotriacetic acid), citrate, BAPTA (l,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid), TETA (triethylenetetramine), phosphates, imidazole, and TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine). Most preferably, the chelator is EDTA.

[0108] Preferably, the buffer comprises a chelator at a concentration of at least 0.001 mM, more preferably at least 0.005 mM, more preferably at least 0.0075 mM, more preferably at least 0.01 mM, more preferably at least 0.015 mM, more preferably at least 0.02 mM, more preferably at least 0.03 mM, more preferably at least 0.04 mM, more preferably at least 0.05 mM, more preferably at least 0.06 mM, more preferably at least 0.07 mM, more preferably at least 0.08 mM, and most preferably at least 0.09 mM. Preferably, the buffer comprises a chelator at a concentration of at most 20 mM, more preferably at most 15 mM, more preferably at most 10 mM, more preferably at most 5 mM, more preferably at most 2.5 mM, more preferably at most 1.0 mM, more preferably at most 0.5 mM, more preferably at most 0.4 mM, more preferably at most 0.3 mM, more preferably at most 0.2 mM, more preferably at most 0.15 mM, more preferably at most 0.12 mM, and most preferably at most 0.11 mM.

[0109] Preferably, the buffer comprises a chelator at a concentration in a range of from 0.001 to 20 mM, more preferably of from 0.005 to 15 mM, more preferably of from 0.0075 to 10 mM, more preferably of from 0.01 to 5 mM, more preferably of from 0.015 to 2.5 mM, more preferably of from 0.02 to 1.0 mM, more preferably of from 0.03 to 0.5 mM, more preferably of from 0.04 to 0.4 mM, more preferably of from 0.05 to 0.3 mM, more preferably of from 0.06 to 0.2 mM, more preferably of from 0.07 to 0.15 mM, more preferably of from 0.08 to 0.12 mM, and most preferably of from 0.09 to 0.11 mM.

[0110] Preferably, the buffer comprises a chelator at a concentration of about 0.01 mM, about 0.02 mM, about 0.03 mM, about 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 0.08 mM, about 0.09 mM, about 0.11 mM, about 0.12 mM, about 0.13 mM, about 0.14 mM, about 0.15 mM, about 0.16 mM, about 0.17 mM, about 0.18 mM, about 0.19 mM, or about 0.2 mM. Most preferably, the buffer comprises a chelator at a concentration of about 0.10 mM.

[0111] Surfactant

[0112] Preferably, the buffer comprises a surfactant. Preferably, the surfactant is a nonionic surfactant. Preferably, the surfactant is selected from the group consisting of polysorbate 20, polysorbate 80, sodium dodecyl sulfate, octyl phenol ethoxylate, CHAPS (3-[(3- cholamidopropyl)dimethylammonio]- 1 -propanesulfonate), nonyl phenoxypolyethoxylethanol, sodium deoxycholate, digitonin, polyoxyethylene lauryl ether, and octyl β-D- glucopyranoside. More preferably, the surfactant is polysorbate 20 or polysorbate 80. Most preferably, the surfactant is polysorbate 20.

[0113] It will be understood that “polysorbate 20” denotes polyoxyethylene (20) sorbitan monolaurate, and that “polysorbate 80” refers to polyoxyethylene (20) sorbitan monooleate.

[0114] The structure of polysorbate 20 is:

[0115] The structure of polysorbate 80 is:

[0116] For the various surfactants, typically preferred upper limits for the concentration in the buffer are: sodium dodecyl sulfate (up to 0.1-1% w / v), octyl phenol ethoxylate (up to 1-2% v / v), polysorbate 20 (up to 5% v / v), polysorbate 80 (up to 5% v / v), CHAPS (up to 0.1-1% w / v), nonyl phenoxypolyethoxylethanol (up to 1-2% v / v), sodium deoxycholate (up to 0.5% w / v), digitonin (up to 0.1-1% w / v), polyoxyethylene lauryl ether (up to 0.1-1% v / v), and octyl β-D-glueopyranoside (up to 0.5-1% w / v); wherein the w / v or v / v is as compared to the total volume of the buffer.

[0117] Preferably, the buffer comprises a surfactant at a concentration of at least 0.001 mg / mL, more preferably at least 0.005 mg / mL, more preferably at least 0.01 mg / mL, more preferably at least 0.02 mg / mL, more preferably at least 0.03 mg / mL, more preferably at least 0.04 mg / mL, more preferably at least 0.05 mg / mL, more preferably at least 0.07 mg / mL, more preferably at least 0.09 mg / mL, more preferably at least 0.11 mg / mL, more preferably at least 0.13 mg / mL, more preferably at least 0.15 mg / mL, more preferably at least 0.17 mg / mL, and most preferably at least 0.19 mg / mL.

[0118] Preferably, the buffer comprises a surfactant at a concentration of at most 5 mg / mL, more preferably at most 4.5 mg / mL, more preferably at most 4 mg / mL, more preferably at most 3.5 mg / mL, more preferably at most 3 mg / mL, more preferably at most 2.5 mg / mL, more preferably at most 2 mg / mL, more preferably at most 1.5 mg / mL, more preferably at most 1 mg / mL, more preferably at most 0.8 mg / mL, more preferably at most 0.6 mg / mL, more preferably at most 0.4 mg / mL, more preferably at most 0.3 mg / mL, and most preferably at most 0.25 mg / mL.

[0119] Preferably, the buffer comprises a surfactant at a concentration in a range of from from 0.001 to 5 mg / mL, more preferably of from 0.005 to 4.5 mg / mL, more preferably of from 0.01 to 4 mg / mL, more preferably of from 0.02 to 3.5 mg / mL, more preferably of from 0.03 to 3 mg / mL, more preferably of from 0.04 to 2.5 mg / mL, more preferably of from 0.05 to 2 mg / mL, more preferably of from 0.07 to 1.5 mg / mL, more preferably of from 0.09 to 1 mg / mL, more preferably of from 0.11 to 0.8 mg / mL, more preferably of from 0.13 to 0.6 mg / mL, more preferably of from 0.15 to 0.4 mg / mL, more preferably of from 0.17 to 0.3 mg / mL, and most preferably of from 0.19 to 0.25 mg / mL.

[0120] Preferably, the buffer comprises a surfactant at a concentration of about 0.001 mg / mL, about 0.005 mg / mL, about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.07 mg / mL, about 0.09 mg / mL, about 0.11 mg / mL, about 0.13 mg / mL, about 0.15 mg / mL, about 0.17 mg / mL, about 0.19 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, or about 5 mg / mL. Most preferably, the buffer comprises a surfactant at a concentration of about 0.2 mg / mL.

[0121] Preferred buffers

[0122] Preferably, the buffer of the disclosure has a pH in range of from 5.0 to 5.9, and said buffer comprises: (i) citrate in a concentration of from 5 to 100 mM; (ii) sucrose in a concentration of from 20 to 100 g / L; (iii) polysorbate 20 in a concentration of from 0.05 to 1 mg / mL; and (iv) EDTA in a concentration of from 0.01 to 1.0 mM.

[0123] More preferably, the buffer of the disclosure has a pH in range of from 5.3 to 5.7, and said buffer comprises: (i) citrate in a concentration of from 10 to 30 mM; (ii) sucrose in a concentration of from 40 to 60 g / L; (iii) polysorbate 20 in a concentration of from 0.10 to 0.30 mg / mL; and (iv) EDTA in a concentration of from 0.05 to 0.20 mM.

[0124] Most preferably, the buffer of the disclosure has a pH of about 5.5, and said buffer comprises: (i) citrate in a concentration of about 20 mM; (ii) sucrose in a concentration of about 50 g / L; (iii) polysorbate 20 in a concentration of about 0.20 mg / mL; and (iv) EDTA in a concentration of about 0.10 mM.

[0125] Compounds and conjugates of the disclosure

[0126] Preferably, the composition of the disclosure comprises the compound and / or conjugate, preferably the conjugate, in a concentration of at least 0.01 mg / mL, more preferably at least 0.1 mg / mL, more preferably at least 0.2 mg / mL, more preferably at least 0.3 mg / mL, more preferably at least 0.5 mg / mL, more preferably at least 1.0 mg / mL, more preferably at least 1.5 mg / mL, more preferably at least 2.0 mg / mL, more preferably at least 2.5 mg / mL, more preferably at least 3.0 mg / mL, more preferably at least 3.5 mg / mL, more preferably at least 4.0 mg / mL, and most preferably at least 4.5 mg / mL.

[0127] Preferably, the composition of the disclosure comprises the compound and / or conjugate, preferably the conjugate, in a concentration of at most 50 mg / mL, more preferably at most 45 mg / mL, more preferably at most 40 mg / mL, more preferably at most 35 mg / mL, more preferably at most 30 mg / mL, more preferably at most 25 mg / mL, more preferably at most 20 mg / mL, more preferably at most 15 mg / mL, more preferably at most 10 mg / mL, more preferably at most 8 mg / mL, more preferably at most 7 mg / mL, more preferably at most 6 mg / mL, and most preferably at most 5.5 mg / mL.

[0128] Preferably, the composition of the disclosure comprises the compound and / or conjugate, preferably the conjugate, in a concentration in a range of from 0.01 to 50 mg / mL, more preferably of from 0.1 to 45 mg / mL, more preferably of from 0.2 to 40 mg / mL, more preferably of from 0.3 to 35 mg / mL, more preferably of from 0.5 to 30 mg / mL, more preferably of from 1.0 to 25 mg / mL, more preferably of from 1.5 to 20 mg / mL, more preferably of from 2.0 to 15 mg / mL, more preferably of from 2.5 to 10 mg / mL, more preferably of from 3.0 to 8 mg / mL, more preferably of from 3.5 to 7 mg / mL, more preferably of from 4.0 to 6 mg / mL, and most preferably of from 4.5 to 5.5 mg / mL.

[0129] Preferably, the composition of the disclosure comprises the compound and / or conjugate, preferably the conjugate, in a concentration of about 0.5 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, about 2.0 mg / mL, about 2.5 mg / mL, about 3.0 mg / mL, about 3.5 mg / mL, about 4.0 mg / mL, about 4.5 mg / mL, about 5.5 mg / mL, about 6.0 mg / mL, about 6.5 mg / mL, about 7.0 mg / mL, about 7.5 mg / mL, about 8.0 mg / mL, about 8.5 mg / mL, about 9.0 mg / mL, about 9.5 mg / mL, about 10.0 mg / mL, about 10.5 mg / mL, about 11.0 mg / mL, about 11.5 mg / mL, about 12.0 mg / mL, about 12.5 mg / mL, about 13.0 mg / mL, about 13.5 mg / mL, about 14.0 mg / mL, about 14.5 mg / mL, about 15.0 mg / mL, about 15.5 mg / mL, about 16.0 mg / mL, about 16.5 mg / mL, about 17.0 mg / mL, about 17.5 mg / mL, about 18.0 mg / mL, about 18.5 mg / mL, about 19.0 mg / mL, about 19.5 mg / mL, or about 20.0 mg / mL

[0130] Most preferably, the composition of the disclosure comprises the compound and / or conjugate, preferably the conjugate, in a concentration of about 5.0 mg / mL.

[0131] Preferred compositions

[0132] Preferably, the composition of the disclosure has a pH in range of from 5.0 to 5.9, and said composition comprises: (i) citrate in a concentration of from 5 to 100 mM; (ii) sucrose in a concentration of from 20 to 100 g / L; (iii) polysorbate 20 in a concentration of from 0.05 to 1 mg / mL; (iv) EDTA in a concentration of from 0.01 to 1.0 mM; and (v) of from 0.1 to 20.0 mg / mL of a compound and / or conjugate of the disclosure, preferably of from 0.1 to 20.0 mg / mL of a conjugate of the disclosure..

[0133] More preferably, the composition of the disclosure has a pH in range of from 5.3 to 5.7, and said composition comprises: (i) citrate in a concentration of from 10 to 30 mM; (ii) sucrose in a concentration of from 40 to 60 g / L; (iii) polysorbate 20 in a concentration of from 0.10 to 0.30 mg / mL; (iv) EDTA in a concentration of from 0.05 to 0.20 mM; and (v) of from 1.0 to 10.0 mg / mL of a compound and / or conjugate of the disclosure, preferably of from 1.0 to 10.0 mg / mL of a conjugate of the disclosure.

[0134] Most preferably, the composition of the disclosure has a pH in range of about 5.5, and said composition comprises: (i) citrate in a concentration of about 20 mM; (ii) sucrose in a concentration of about 50 g / L; (iii) polysorbate 20 in a concentration of about 0.20 mg / mL; (iv) EDTA in a concentration of about 0.10 mM; and (v) about 5.0 mg / mL of a compound and / or conjugate of the disclosure, preferably about 5.0 mg / mL of a conjugate of the disclosure.

[0135] Compounds of the disclosure

[0136] The compounds of the disclosure comprise an eight-membered non-aromatic cyclic mono-alkenylene moiety, wherein said moiety comprises a non-vinylic carbon atom, wherein said non-vinylic carbon atom is substituted with at least one structure according to Formula (A):

[0137] Formula (A); wherein L1and L2are each independently a linker; and T2and T3are organic moieties.

[0138] Preferably, the eight-membered non-aromatic cyclic mono-alkenylene moiety is a trans-cyclooctene. Preferably, the eight-membered non-aromatic cyclic mono-alkenylene moiety has an allylic carbon, and the allylic carbon is substituted with an R48moiety as defined herein, preferably the R48moiety is a releasable group.

[0139] Preferably, compounds of the disclosure are according to Formula (1) as defined herein. It is understood that any compounds as provided herein may be in a form, formulation or solution in which the compound is present as a salt, solvate or hydrate of the compound. Accordingly, whereever herein a compound or genus of compounds are provided, or reference is made to “compound of the disclosure” or “compounds of the disclosure”, it will be understood that also the salt, hydrate, or solvate of said compound(s) are included by such a statement even if the terms salt, hydrate or solvate are not specifically mentioned in each instance. In certain embodiments, a compound of the disclosure is purified or in a form or state in which it is not present as a salt, or as a hydrate, or as a solvate of the compound; however, unless specifically indicated as such it is intended to be assumed that any compound herein may be in the form of a salt, hydrate or solvate.

[0140] Preferred embodiments of the compounds of the disclosure are further described below in relation to several Formulae and variables.

[0141] Formulae for compounds

[0142] Preferably, the compound of the disclosure is according to a Formula selected from the group consisting of Formula (1), Formula (2), Formula (3), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (I), Formula (J), Formula (K), Formula (L), Formula (M), Formula (N), Formula (O), Formula (P), and Formula (Q).

[0143] Preferably, the compound of the disclosure is according to Formula (B). More preferably, the compound of the disclosure is according to Formula (C). Even more preferably, the compound of the disclosure is according to Formula (1). More preferably still, the compound of the disclosure is according to Formula (D). Even more preferably, the compound of the disclosure is according to Formula (E). Yet more preferably, the compound of the disclosure is according to Formula (F). Still more preferably, the compound of the disclosure is according to Formula (G). More preferably still, the compound of the disclosure is according to Formula (2). Even more preferably still, the compound of the disclosure is according to Formula (H). Yet more preferably still, the compound of the disclosure is according to Formula (I). Even more preferably, the compound of the disclosure is according to Formula (J). More preferably still, the compound of the disclosure is according to Formula (K). Even more preferably still, the compound of the disclosure is according to Formula (L). Yet more preferably, the compound of the disclosure is according to Formula (M). Even more preferably still, the compound of the disclosure is according to Formula (N). Still more preferably, the compound of the disclosure is according to Formula (O). Yet more preferably, the compound of the disclosure is according to Formula (3). Still more preferably, the compound of the disclosure is according to Formula (P). Even more preferably, the compound of the disclosure is according to Formula (Q).

[0144] Formula (1) is:

[0145] Formula (2) is:

[0146] Formula (3) is: Formula (G) is:

[0147] Formula (H) is:

[0148] Formula (I) is:

[0149] Formula (J) is:

[0150] Formula (K) is:

[0151] Formula (L) is: Formula (M) is: Formula (N) is:

[0152] Formula (O) is:

[0153] Formula (P) is:

[0154] Formula (Q) is: Conjugates of the disclosure

[0155] The disclosure also relates to a conjugate, wherein the conjugate comprises a protein conjugated to at least one compound according to the disclosure, wherein T2is a residue of a bioconjugation moiety, and said protein and said compound are conjugated via T2. Thus, the conjugate of the disclosure is to be understood as a compound of the disclosure (wherein T2was originally a bioconjugation moiety) linked to a protein via T2, wherein due to the coupling of said compound and said protein, T2in the conjugate of the disclosure is the residue of a bioconjugation moiety, preferably the residue of an N-maleimidyl group, viz.: wherein the asterisk indicates a bond to the protein, and the wiggly line denotes a bond to the rest of the compound of the disclosure.

[0156] In the conjugate of the disclosure, the protein is preferably a diabody or an antibody, more preferably a diabody, and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1.

[0157] Preferably, in the conjugate of the disclosure the protein and the compound of the disclosure are conjugated via T2and a residue of a sulfhydryl of said protein, a residue of a hydroxyl of said protein, or a residue of an amine of said protein; more preferably via T2and a residue of a sulfhydryl of said protein. Preferably, the residue of the sulfhydryl group of said protein is part of a cysteine residue of said protein.

[0158] It is understood that any conjugates as provided herein may be in a form, formulation or solution in which the compound is present as a salt, solvate or hydrate of the compound. Accordingly, whereever herein a compound or genus of conjugates are provided, or reference is made to “conjugate of the disclosure” or “conjugates of the disclosure”, it will be understood that also the salt, hydrate, or solvate of said conjugate(s) are included by such a statement even if the terms salt, hydrate or solvate are not specifically mentioned in each instance. In certain embodiments, a conjugate of the disclosure is purified or in a form or state in which it is not present as a salt, or as a hydrate, or as a solvate of the conjugate; however, unless specifically indicated as such it is intended to be assumed that any conjugate herein may be in the form of a salt, hydrate or solvate. Formulae of conjugates of the disclosure

[0159] Below, preferred embodiments of the conjugates of the disclosure are shown in the form of formulae. Therein, parameter CJ is shown. In relation to conjugates of the disclosure, CJ is in a range of from 1 to 12, preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, and most preferably of from 3.5 to 4. It will be understood that for individual conjugates, CJ is typically an integer, and is most preferably about 4. When measuring CJ for multiple conjugates, however, an average number may be obtained, which is not necessarily an integer. As C J is commonly determined for multiple conjugates, CJ in relation to the disclosure typically refers to an average number.

[0160] Preferably, the conjugate of the disclosure is according to a Formula selected from the group consisting of Formula (C1), Formula (C2), Formula (C3), Formula (C4), Formula (C5), Formula (C6), Formula (C7), Formula (C8), Formula (C9), Formula (CIO), Formula (C11), Formula (C12), Formula (C13), Formula (C14), Formula (C15), Formula (C16), Formula (C16A), Formula (C16B), Formula (C17), Formula (C18), Formula (C19), Formula (C19A), Formula (C19B), Formula (C20), Formula (C21), Formula (C22), Formula (C23), Formula (C24), and Formula (C25).

[0161] Preferably, the conjugate of the disclosure is according to Formula (C4). More preferably, the conjugate of the disclosure is according to Formula (C5). Even more preferably, the conjugate of the disclosure is according to Formula (C1). More preferably still, the conjugate of the disclosure is according to Formula (C6). Even more preferably, the conjugate of the disclosure is according to Formula (C7). Yet more preferably, the conjugate of the disclosure is according to Formula (C8). Still more preferably, the conjugate of the disclosure is according to Formula (C9). More preferably still, the conjugate of the disclosure is according to Formula (C2). Even more preferably still, the conjugate of the disclosure is according to Formula (CIO). Yet more preferably still, the conjugate of the disclosure is according to Formula (C11). Even more preferably, the conjugate of the disclosure is according to Formula (C12). More preferably still, the conjugate of the disclosure is according to Formula (C13). Even more preferably still, the conjugate of the disclosure is according to Formula (C14). Yet more preferably, the conjugate of the disclosure is according to Formula (C15). Even more preferably still, the conjugate of the disclosure is according to Formula (C16). More preferably still, the conjugate of the disclosure is according to Formula (C16A). Even more preferably, the conjugate of the disclosure is according to Formula (C16B). Still more preferably, the conjugate of the disclosure is according to Formula (C17). Yet more preferably, the conjugate of the disclosure is according to Formula (C3). Still more preferably, the conjugate of the disclosure is according to Formula (C18). Even more preferably still, the conjugate of the disclosure is according to Formula (C19) or (C20). More preferably still, the conjugate of the disclosure is according to Formula (C19A). Even more preferably, the conjugate of the disclosure is according to Formula (C19B). Still more preferably, the conjugate of the disclosure is according to any one of Formulae (C21), (C22), (C23), (C24), and (C25). Even more preferably still, the conjugate of the disclosure is according to Formula (C19). Most preferably, the conjugate of the disclosure is according to Formula (C23), or (C25).

[0162] Formula (C4) is

[0163] Formula (C5) is Formula (C6) is

[0164] Formula (C7) is

[0165] Formula (C8) is

[0166] Formula (C9) is

[0167] Formula (C10) is

[0168] Formula (C11) is Formula (C12) is

[0169] Formula (C13) is

[0170] Formula (C14) is

[0171] Formula (C15) is

[0172] Formula (C16) is

[0173] Formula (C16 A) is wherein Hais hydrogen, and T1is positioned cis relative to Ha; and preferably R48is positioned trans relative to Ha.

[0174] Formula (C16B) is wherein Hais hydrogen, and T1is positioned cis relative to Ha; and preferably R48is positioned trans relative to Ha.

[0175] Formula (C17) is

[0176] Formula (C18) is

[0177] Formula (C19) is Formula (C19A) is wherein Hais hydrogen; T1is OH; R48is and T1is positioned cis relative to Ha; and preferably R48is positioned trans relative to Ha; preferably CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; and preferably CJ is of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4.

[0178] Formula (C19B) is wherein Hais hydrogen; T1is OH; R48is and T1is positioned cis relative to Ha; and preferably R48is positioned trans relative to Ha; preferably CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; and preferably CJ is of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4.

[0179] Formula (C20) is

[0180] wherein E1is -H or -CH3, preferably E1is -H.

[0181] Formula (C21) is wherein E1is -H or -CH3, preferably E1is -H. Formula (C22) is wherein E1is -H or -CH3, preferably E1is -H.

[0182] Formula (C23) is

[0183] Formula (C24) is

[0184] Formula (C25) is

[0185] Variables in formulae of the compounds and conjugates of the disclosure

[0186] It will be understood that the variables indicated below relate to both the compounds of the disclosure and the conjugates of the disclosure. As explained herein, only for T2and CBthe definitions may differ between the compounds of the disclosure and the conjugates of the disclosure.

[0187] L1

[0188] L1is a linker. Preferably, L1is according to Radical Group 2 as defined herein.

[0189] Preferably, L1contains of from 1 to 100 atoms, preferably of from 2 to 75 atoms, more preferably of from 3 to 60 atoms, even more preferably of from 4 to 50 atoms, more preferably still of from 5 to 40 atoms, yet more preferably of from 6 to 35 atoms, even more preferably of from 7 to 30 atoms, more preferably still of from 8 to 25 atoms, even more preferably of from 9 to 22 atoms, and most preferably of from 10 to 20 atoms. Preferably, L contains about 15 atoms. More preferably, L1is selected from the group consisting of linear or branched C1-C12(hetero)alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene. More preferably than the foregoing, L1is selected from the group consisting of linear or branched C1-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene. More preferably than the foregoing, L1is selected from the group consisting of linear or branched C2-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene. More preferably than the foregoing, L1is selected from the group consisting of linear or branched C3-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene. More preferably than the foregoing, L1is selected from the group consisting of linear or branched C4-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene. More preferably than the foregoing, L1is a linear or branched C1-C12alkylene. More preferably than the foregoing, L1is a linear or branched C2-C12alkylene, viz. linear C2alkylene, linear or branched C3alkylene, linear or branched C4alkylene, linear or branched C5alkylene, linear or branched C6alkylene, linear or branched C7alkylene, linear or branched C8alkylene, linear or branched C9alkylene, linear or branched C10alkylene, linear or branched C11alkylene, or linear or branched C12alkylene. More preferably than the foregoing, L1is a linear or branched C3-C12alkylene. More preferably than the foregoing, L1is a linear or branched C4-C12alkylene. More preferably than the foregoing, L1is a linear or branched C4- C11alkylene. More preferably than the foregoing, L1is a linear or branched C4-C10alkylene. More preferably than the foregoing, L1is a linear or branched C4-C9alkylene. More preferably than the foregoing, L1is a linear or branched C4-C8alkylene. More preferably than the foregoing, L1is a linear or branched C4-C7alkylene. More preferably than the foregoing, L1is a linear or branched C4-C6alkylene. More preferably than the foregoing, L1is a linear or branched C5alkylene. More preferably than the foregoing, L1is a linear C1-C12alkylene. More preferably than the foregoing, L1is a linear C2-C12alkylene, viz. linear C2alkylene, linear C3alkylene, linear C4alkylene, linear C5alkylene, linear C6alkylene, linear C7alkylene, linear C8alkylene, linear C9alkylene, linear C10alkylene, linear C11alkylene, or linear C12alkylene. More preferably than the foregoing, L1is a linear C3-C12alkylene. More preferably than the foregoing, L1is a linear C4-C12alkylene. More preferably than the foregoing, L1is a linear C4-C11alkylene. More preferably than the foregoing, L1is a linear C4-C10alkylene. More preferably than the foregoing, L1is a linear C4-C9alkylene. More preferably than the foregoing, L1is a linear C4-C8alkylene. More preferably than the foregoing, L1is a linear C4-C7alkylene. More preferably than the foregoing, L1is a linear C4- C6alkylene. Most preferably, L1is a linear C5alkylene. L1can be substituted or unsubistuted. Preferably, L1is unsubstituted. Most preferably, L1is an unsubstituted, linear C5alkylene.

[0190] Without wishing to be bound by theory, the inventors believe that the linker L1of compounds of Formula (1) of the present disclosure may provide a faster blood clearance rate, while maintaining a high uptake at the target site of the compound of Formula (1). Still without wishing to be bound by theory, an advantage of linkers L1having a length as defined in claim 1, in particular linear C4-C12alkylene, may be that sufficient distance between the moiety CBand the trans-cyclooctene can be achieved, so that the double bond of the trans- cyclooctene may readily react with a diene. Still without wishing to be bound by theory, an advantage of linkers L1having a length as defined in claim 1, in particular linear C4-C12alkylene, may be that they are not too long, so that they may still be shielded by moiety CBwhich may prevent e.g. deactivation. An advantage of relatively short alkylene linkers, such as C4-C6alkylene, may be that their solubility is also higher than for relatively long alkylene linkers.

[0191] L2

[0192] L2is a linker. Preferably, L2is according to Radical Group 2 as defined herein.

[0193] Preferably, L2contains of from 1 to 200 atoms, preferably of from 2 to 150 atoms, more preferably of from 3 to 100 atoms, even more preferably of from 4 to 90 atoms, more preferably still of from 5 to 80 atoms, yet more preferably of from 6 to 70 atoms, even more preferably of from 7 to 60 atoms, more preferably still of from 8 to 50 atoms, even more preferably of from 9 to 45 atoms, and most preferably of from 10 to 35 atoms.

[0194] Preferably, L2is selected from the group consisting of linear or branched C1-C12(hetero)alkanetriyl, C3-C8(hetero)cycloalkanetriyl, C6-C12arenetriyl, and C4-C11heteroarenetriyl. More preferably than the foregoing, L2is a linear or branched C1-C12(hetero)alkanetriyl. More preferably than the foregoing, L2is a linear or branched C1-C12heteroalkanetriyl. More preferably than the foregoing, L2is a branched C1-C12(hetero)alkanetriyl. More preferably than the foregoing, L2is a branched C1-C12heteroalkanetriyl. More preferably than the foregoing, L2is a branched C3-C11heteroalkanetriyl. More preferably than the foregoing, L2is a branched C6-Cio heteroalkanetriyl. More preferably than the foregoing, L2is a branched C8heteroalkanetriyl. More preferably than the foregoing, L2is a branched C8heteroalkanetriyl substituted with up to five =O groups. More preferably than the foregoing, L2is a branched C8heteroalkanetriyl substituted with three =O groups. More preferably than the foregoing, L2is a branched C8 heteroalkanetriyl containing up to five -NH- groups. More preferably than the foregoing, L2is a branched C8heteroalkanetriyl containing three -NH- groups. More preferably than the foregoing, L2is a branched C8heteroalkanetriyl containing three -NH- groups, and wherein the C8heteroalkanetriyl is substituted with three =O groups.

[0195] More preferably, L2is:

[0196] Even more preferably, L2is:

[0197] More preferably still, L2is:

[0198] Most preferably, L2is:

[0199] In preferred embodiments, L2has the following structure: . Herein, L2a, L2b, L2c, and L2dare each independently a linker. Preferably, L2a, L2b, L2c, and L2dare each independently according to Radical Group 2 as defined herein. L2a

[0200] L2ais a linker. Preferably, L2ais according to Radical Group 2 as defined herein. More preferably, L2ais a linker containing at most twenty atoms. More preferably than the foregoing, L2ais a linker containing at most fifteen atoms. More preferably than the foregoing, L2ais a linker containing at most ten atoms. More preferably than the foregoing, L2ais a linker containing at most five atoms. More preferably than the foregoing, L2ais selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl. More preferably than the foregoing, L2ais selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2ais selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2ais -NHC(O)-. L2b

[0201] L2bis a linker. Preferably, L2bis according to Radical Group 2 as defined herein. More preferably, L2bis a linker containing at most twenty atoms. More preferably than the foregoing, L2bis a linker containing at most fifteen atoms. More preferably than the foregoing, L2bis a linker containing at most ten atoms. More preferably than the foregoing, L2bis a linker containing at most five atoms. More preferably than the foregoing, L2bis selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl. More preferably than the foregoing, L2bis selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2bis selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2bis -NHC(O)-. L2c

[0202] L2cis a linker. Preferably, L2cis according to Radical Group 2 as defined herein. More preferably than the foregoing, L2cis a linker comprising at most 50 atoms. More preferably than the foregoing, L2cis a linker comprising at most 40 atoms. More preferably than the foregoing, L2cis a linker comprising at most 30 atoms. More preferably than the foregoing, L2cis a linker comprising at most 20 atoms. More preferably than the foregoing, L2cis a linker comprising at most 15 atoms. More preferably than the foregoing, L2cis selected from the group consisting of C1-C8(hetero)alkanetriyl, C5-C6(hetero)arenetriyl. C3-C7cycloalkanetriyl, and C2-C7heterocycloalkanetriyl. More preferably than the foregoing, L2cis C1-C8(hetero)alkanetriyl. More preferably than the foregoing, L2cis C1-C8alkanetriyl. More preferably than the foregoing, L2cis C2-C7alkanetriyl. More preferably than the foregoing, L2cis C3-C6alkanetriyl. More preferably than the foregoing, L2cis C4-C5alkanetriyl. More preferably than the foregoing, L2cis C5alkanetriyl. Most preferably, L2cis >CH-CH2-CH2- CH2-CH2-.

[0203] L2dis a linker. Preferably, L2dis according to Radical Group 2 as defined herein. More preferably than the foregoing, L2dis a linker containing at most twenty atoms. More preferably than the foregoing, L2dis a linker containing at most fifteen atoms. More preferably than the foregoing, L2dis a linker containing at most ten atoms. More preferably than the foregoing, L2dis a linker containing at most five atoms. More preferably than the foregoing, L2dis selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl. More preferably than the foregoing, L2dis selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2dis selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2dis -C(O)NH-.

[0204] T1

[0205] T1is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5, as defined herein. Preferably, each T1is independently according to Radical Group 1 as defined herein.

[0206] More preferably, each T1is independently selected from the group consisting of - OT1A, hydrogen, C1-C12(hetero)alkyl, C6aryl, C4-C5heteroaryl, C3-C6(hetero)cycloalkyl, C5- C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, - SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3. Even more preferably, each T1is independently selected from the group consisting of -OT1A, hydrogen, C2-C6alkyl, C6aryl, C4-C5heteroaryl, C3-C6cycloalkyl, C5-C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, - C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3. Yet more preferably, each T1is independently selected from the group consisting of -OT1A, C2-C6alkyl, C6aryl, C4-C5heteroaryl, C3-C6cycloalkyl, C5-C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, - N(T1A)2-CO-T1A, and -Si(T1A)3. More preferably still, T1is -OT1A.

[0207] Most preferably, T1is -OH.

[0208] As used herein, each T1Ais independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue. More preferably, each T1Ais independently selected from the group consisting of hydrogen, C1-C6(hetero)alkyl, C1-C6(hetero)alkenyl, C1-C6(hetero)alkynyl, C2-C5heteroaryl, phenyl, and an amino acid residue. Even more preferably, each T1Ais independently selected from the group consisting of hydrogen, C1-C4(hetero)alkyl, C1-C4(hetero)alkenyl, C1-C4 (hetero)alkynyl, C3-C5heteroaryl, phenyl, an aspartic acid residue, a glutamic acid residue, and a glycine residue. Even more preferably, each T1Ais independently selected from the group consisting of hydrogen, C1-C3alkyl, an aspartic acid residue, a glutamic acid residue, and a glycine residue. Most preferably, T1Ais hydrogen.

[0209] Preferably, T1is in an axial position. Without wishing to be bound by theory, the inventors believe that in that case and when R48is a releasable group, when the compound of the disclosure reacts with a diene, T1aids in releasing the payload. This results in optimal release yields and / or release kinetics.

[0210] T2

[0211] T2is an organic moiety. Preferably, T2is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein, or wherein T2is a group -L3-CB. More preferably, T2is a bioconjugation moiety, a residue of a bioconjugation moiety, or a group -L3-CB. More preferably, T2is a bioconjugation moiety, or a group -L3-CB.

[0212] In preferred embodiments, T2is a bioconjugation moiety. These embodiments typically relate to compounds that can be coupled to e.g. a protein. More preferably, T2is according to Radical Group If as defined herein. Residues of these bioconjugation moieties are known in the art. More preferably, T2is N-maleimidyl. In these embodiments, it is most preferred that T2is:

[0213] In other preferred embodiments, T2is a residue of a bioconjugation moiety. These embodiments typically relate to conjugates of the disclosure, wherein T2links to e.g. a protein. Such residues are well-known to the skilled person. In these embodiments, it is most preferred that T2is: wherein the asterisk indicates a bond to the protein, and the wiggly line denotes a bond to the rest of the compound of the disclosure.

[0214] In other preferred embodiments, T2is a group -L3-CB. These embodiments relate to when T2itself comprises a Construct B (CB), which is usually a protein. CBis as defined herein. L3is according to Radical Group 2. Preferably, L3is a residue of a bioconjugation moiety. More preferably, L3is a residue of an N-maleimidyl moiety or a residue of an N- hydroxy-succinimidyl moiety. In these embodiments, it is preferred that T2is selected from the group consisting of In these embodiments, it is most preferred that T2is:

[0215] For the moiety -L3-CB, it is preferred that L3and a sulfur atom, secondary nitrogen atom, or tertiary nitrogen atom, preferably a sulfur atom, of CBtogether form any one of the following structures -L3-CB: wherein CB1indicates S, secondary N, or tertiary N that is part of CB, preferably S; the wiggly lines indicates a bond to moiety L1, and the asterisk indicates a bond to the remainder of CB, preferably AVP0458.

[0216] CB

[0217] CBis according to Radical Group 4 or Radical Group 5, as defined herein. Preferably, CBis a targeting agent as defined herein. Preferably, CBis selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof. More preferably, CBis a protein. Even more preferably, CBis an antibody or a diabody. More preferably still, CBis a diabody.

[0218] For conjugates of the disclosure, CBis a protein; more preferably, CBis in that case an antibody or a diabody; and more preferably still, in that case CBis a diabody.

[0219] An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this disclosure, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgGl, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, antiidiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis- scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as Nanobody™), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART™), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and [Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331-343], the contents of which are hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Other embodiments use antibody mimetics as Drug DDor Targeting Agent TT, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference. For the avoidance of doubt, in the context of this disclosure the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.

[0220] Preferably, an antibody is selected from the group consisting of AVP0458, CC49, 3F8, abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, ansuvimab, anrukinzumab, apolizumab, aprutumab ixadotin, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atidortoxumab, atinumab, atoltivimab, atoltivimab, maftivimab, odesivimab, atorolimumab, avelumab, azintuxizumab vedotin, bamlanivimab, bapineuzumab, basiliximab, bavituximab, BCD- 100, bebtelovimab, bectumomab, bedinvetmab, begelomab, belantamab mafodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab tesirine, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, casirivimab, capromab, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, cergutuzumab amunaleukin, certolizumab pegol, cetrelimab, cetuximab, cibisatamab, cilgavimab, cirmtuzumab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab pelidotin, coltuximab ravtansine, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab, crotedumab, CR6261, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dezamizumab, dinutuximab, dinutuximab beta, diridavumab, divozilimab, domagrozumab, donanemab, dorlimomab aritox, dostarlimab, drozitumab, DS-8201, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epcoritamab, epitumomab cituxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frovocimab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab ozogamicin, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab vedotin, glofitamab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab, sintilimab, ibritumomab tiuxetan, icrucumab, idarucizumab, ifabotuzumab, igovomab, iladatuzumab vedotin, imalumab, imaprelimab, imciromab, imdevimab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab vedotin, lampalizumab, lanadelumab, landogrozumab, laprituximab emtansine, larcaviximab, lebrikizumab, lecanemab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, loncastuximab tesirine, losatuxizumab vedotin, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, lupartumab, lupartumab amadotin, lutikizumab, maftivimab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, narnatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEODOO 1, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, pozelimab, prezalumab, plozalizumab, pogalizumab, polatuzumab vedotin, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, regdanvimab, relatlimab, remtolumab, reslizumab, retifanlimab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab pegol, robatumumab, Rmab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirine, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samalizumab, samrotamab vedotin, sarilumab, satralizumab, satumomab pendetide, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, SGN-CD19A, SHP647, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab vedotin, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, sotrovimab, spartalizumab, spesolimab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talacotuzumab, talizumab, talquetamab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, teclistamab, tefibazumab, telimomab aritox, telisotuzumab, telisotuzumab vedotin, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragol, umab, tiragotumab, tislelizumab, tisotumab vedotin, tixagevimab, TNX-650, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talirine, vanalimab, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, vilobelimab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab mafodotin, votumumab, vunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab aritox.

[0221] Preferably, CBis selected from the group consisting of AVP0458, CC49, insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrin-like repeat proteins, interferons, e.g. alpha, beta, and gamma interferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Examples of peptides as targeting agents include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin. Other examples of targeting agents include lipocalins, such as anticalins. One particular embodiment uses Affibodies™ and multimers and derivatives.

[0222] More preferably, CBis AVP0458 or CC49. Most preferably, CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1.

[0223] Preferably, CBis linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S or N that is part of CB. More preferably, CBis linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S that is part of CB.

[0224] AVP0458

[0225] As used herein, AVP0458 refers to a TAG72-binding diabody derived from the CC49 antibody. AVP0458 is a diabody consisting of two monomers, each monomer having an amino acid sequence according to SEQ ID NO:1:

[0226] SEQ ID NO:1 (amino acid sequence of AVP0458 diabody monomer): SVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDD

[0227] FKYNERFKGKATLTADKSSSTAYLQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTV

[0228] SSGGGGSDIVMTQSCSSCPVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSP

[0229] KLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVETEDLAVYYCQQYYSYPLTFGAGT KLVLKR

[0230] Herein, the underlining indicates the cysteines that are preferably modified with or linked to a compound of the disclosure or the remainder thereof if AVP0458 is itself part of the compound of the disclosure.

[0231] Thus, in SEQ ID NO: 1 it is preferred that at least one of the underligned cysteines, more preferably both underlined cysteines, is modified with or linked to a compound according to the disclosure. In other words: it is preferred that the sulfur atom of the underlined cysteines is coupled to a moiety T2as defined herein, preferably T2is the residue of an N-maleimidyl group.

[0232] Targeting Agent

[0233] A Targeting Agent, TT, binds to a Primary Target. A "primary target" as used in the present disclosure can be any molecule, which is present in an organism, tissue or cell. Preferably, a “primary target” relates to a target for a targeting agent for therapy, imaging, theranostics, diagnostics, or in vitro studies.

[0234] In order to allow specific targeting of the above-listed Primary Targets, the Targeting Agent TTcan comprise compounds including but not limited to antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, cell penetrating peptide, membrane translocation moiety, bombesin, elastin, peptide mimetics, organic compounds, inorganic compounds, carbohydrates, monosaccharides, oligosacharides, polysaccharides, oligonucleotides, aptamers, viruses, whole cells, phage, drugs, polymers, liposomes, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, toxins. Examples of organic compounds envisaged within the context of the present disclosure are, or are derived from, dyes, compounds targeting CAIX and PSMA, estrogens, e.g. estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol. Examples of Targeting Agents of protein nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, Affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrin-like repeat proteins, interferons, e.g. alpha, beta, and gamma interferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet-derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Examples of peptides as targeting agents include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin. Other examples of targeting agents include lipocalins, such as anticalins. One particular embodiment uses Affibodies™ and multimers and derivatives.

[0235] In one embodiment antibodies are used as the TT. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this disclosure, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgGl, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies, recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem / bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as Nanobody™), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART™), and multimers and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri-scFvs) including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and [Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331-343], the contents of which are hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Other embodiments use antibody mimetics as TT, such as but not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference. For the avoidance of doubt, in the context of this disclosure the term "antibody" is meant to encompass all of the antibody variations, fragments, derivatives, fusions, analogs and mimetics outlined in this paragraph, unless specified otherwise.

[0236] Preferably the TTis selected from antibodies and antibody derivatives such as antibody fragments, fragment fusions, proteins, peptides, peptide mimetics, organic molecules, dyes, fluorescent molecules, enzyme substrates.

[0237] Preferably the TTbeing an organic molecule has a molecular weight of less than 2000 Da, more preferably less than 1500 Da, more preferably less than 1000 Da, even more preferably less than 500 Da.

[0238] In another preferred embodiment the TTis selected from antibody fragments, fragment fusions, and other antibody derivatives that do not contain a Fc domain.

[0239] In another embodiment the TTis a polymer and accumulates at the Primary Target by virtue of the EPR effect. Typical polymers used in this embodiment include but are not limited to polyethyleneglycol (PEG), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF). Other examples are copolymers of a polyacetal / polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Other examples are oligopeptides, polypeptides, glycopolysaccharides, and polysaccharides such as dextran and hyaluronan. In addition reference is made to [G. Pasut, F.M. Veronese, Prog. Polym. Sei. 2007, 32, 933-961]. In some embodiments the TTcan be a cell penetrating moiety, such as cell penetrating peptide. In other embodiments, the TTis a polymer, particle, gel, biomolecule or another above listed TTmoiety and is locally injected to create a local depot of Prodrug, which can subsequently be activated by the Activator. In another embodiment the targeting agent TTis a solid material such as but not limited to polymer, metal, ceramic, wherein this solid material is or is comprised in a cartridge, reservoir, depot, wherein preferably said cartridge, reservoir, depot is used for drug release in vivo. In some embodiments, the targeting agent TTalso acts as a Drug, which may be denoted as DD. T3

[0240] T3is an organic moiety. Preferably, T3is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein. More preferably, T3is according to Radical Group 3, as defined herein. Even more preferably, T3is a polymer. More preferably still, T3is a polymer comprising a polyethylene glycol moiety.

[0241] More preferably, T3comprises a moiety -(CH2CH2-O-)y-T4. Herein, y is an integer in a range of from 1 to 50, preferably y is an integer in a range of from 2 to 45, more preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, even more preferably in a range of from 23 to 25, and most preferably y is 24. This definition and these preferences for y also apply to compounds of Formula (2), Formula (3), Formula (G), Formula (O), Formula (P), and Formula (Q), wherein y is used as well.

[0242] T4is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5 as defined herein. Preferably, T4is according to Radical Group 1. More preferably, T4is according to Radical Group la. More preferably, T4is according to Radical Group lb. More preferably, T4is according to Radical Group 1c. More preferably, T4is according to Radical Group Id. Even more preferably, T4is according to Radical Group le. Most preferably, T4is methyl.

[0243] Even more preferably, T3is a moiety -(CH2CH2-O-)y-T4. Most preferably, T3is a moiety -(CH2CH2-O-)24-CH3.

[0244] Variables of Formula (B)

[0245] In Formula (B), R48and T1are as defined herein. In Formula (B), TL is a structure according to Formula (A) as defined herein.

[0246] In Formula (B), y1 is an integer of from 0 to 4, preferably an integer of from 1 to 2, most preferably y1 is 1.

[0247] In Formula (B), y2 is an integer of from 0 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y2 is 1.

[0248] In Formula (B), y3 is an integer of from 1 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y3 is 1..

[0249] In Formula (B), each of X1, X2, X3, X4, X5, and X6is independently selected from the group consisting of a substituted or unsubstituted carbon atom, a nitrogen atom, or an oxygen atom, provided that if one of X1, X2, X3, X4, X5, and X6is a nitrogen atom or an oxygen atom, an adjacent X1, X2, X3, X4, X5, and X6is not a nitrogen atom or an oxygen atom.

[0250] Preferably, each of X1, X2, X3, X4, X5, and X6is independently a substituted or unsubstituted carbon atom. More preferably, X1and / or X6are independently a carbon atom substituted with R48. Even more preferably, X1is a carbon atom substituted with R48, and most preferably, X1is -CHR48-. More preferably still, X1is -CHR48-, and X4is -CT1TL-. Preferably, X2, X3, X5, and X6are unsubstituted carbon atoms, more preferably -CH2-.

[0251] Variable x in Formulae (3). (O), (P). (Q). (C16). (C16A). (C16B). (C17). and (C18)

[0252] In Formula (3), Formula (O), Formula (P), Formula (Q), Formula (C16), Formula (C16A), Formula (C16B), Formula (C17), and Formula (C18), x is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6, and most preferably x is 5. R48R48is selected from the group consisting of -OH, -O-acetyl, -O-C1-4alkyl, halogen, active carbonate, and a releasable group.

[0253] Preferably, R48is a substituent on an allylic carbon of a compound of the disclosure.

[0254] Preferably, R48is in the axial position. This preference holds especially when R48is a releasable group. Having the releasable group in an axial position results in better release of the payload as compared to having the releasable group in an equatorial position.

[0255] Preferably, group R48is a releasable group. Such releasable groups are well-known and have a clear meaning in the art. Especially in the context of click-to-release reactions, as in the present disclosure, the skilled person would immediately recognize that a releasable group on the allylic carbon of a trans-cyclooctene (viz. R48in Formula (1)) refers to a group that may be released from the trans-cyclooctene upon contacting the trans-cyclooctene with an activator such as a diene.

[0256] In preferred embodiments, the releasable group is -(Y1-C(=Y2))i-(SP)j-CA. Therein, each of Y1and Y2are independently selected from O, and S; preferably Y1and Y2are O. For the releasable group, j is 0 or 1 ; preferably j is 0; and i is 0 or 1 ; preferably i is 1. If i is 0, -(SP)j-CAis connected to the remainder of the compound via O or S, that is part of -(SP)j-CA. On the other hand, if i is 1, -(SP)j-CAis connected to -C(=Y2)- via O, S, a secondary N, or a tertiary N, that is part of -(SP)j-CA. Preferably, if i is 1, -(SP)j-CAis connected to -C(=Y2)- via a secondary N, or a tertiary N, that is part of -(SP)j-CA.

[0257] More preferably, the releasable group is -(O-C(=Y2))i-(SP)j-CA. More preferably, the releasable group is -(Y1-C(=O))i-(SP)j-CA. More preferably, the releasable group is -(O- C(=O))i-(SP)j-CA. More preferably, the releasable group is -O-C(=O)-(SP)j-CA.

[0258] Most preferably, the releasable group is -O-C(=O)-CA.

[0259] CAis Construct A, which is a payload. Preferably, CAis an organic molecule or an inorganic molecule. More preferably, CAis a drug. Preferably, CAis monomethyl auristatin E (MMAE), exatecan, or an exatecan derivative. Most preferably, CAis monomethyl auristatin E (MMAE).

[0260] Preferably, CAis linked to the moiety -(Y1-C(=Y2))i-, preferably -O-C(=O)-, via a secondary or tertiary nitrogen atom that is part of CA, forming a carbamate. Preferably, CAis monomethyl auristatin E (MMAE) linked to the moiety -(Y1-C(=Y2))i-, preferably -O- C(=O)-, via a secondary or tertiary nitrogen atom that is part of MMAE, forming a carbamate; or CAis exatecan or an exatecan derivative linked to the moiety -(Y1-C(=Y2))i-, preferably - O-C(=O)-, via a primary or secondary nitrogen atom that is part of exatecan, forming a carbamate. More preferably, CAis monomethyl auristatin E (MMAE) linked to the moiety - (Y1-C(=Y2))i-, preferably -O-C(=O)-, via a secondary or tertiary nitrogen atom that is part of MMAE, forming a carbamate.

[0261] Preferably, group R48is: wherein E1is -H or -CH3, preferably E1is -H. More preferably, group R48is: wherein E1is -H or -CH3, preferably E1is -H.

[0262] Most preferably, group R48is:

[0263] SPis a spacer, of which preferred embodiments are defined below. Preferably, when SPis part of a releasable group, SPis a self-immolative linker, which is herein also referred to as LC. Such self-immolative linkers are well-known in the art, and preferred embodiments of self-immolative linkers are defined below. If the spacer in the releasable group is a self- immolative linker, upon reaction of a compound of the disclosure with a diene, initially a construct -LC-CAis released. Thereafter, the self-immolative linker self-immolates and releases the payload CA.

[0264] Drugs

[0265] Drugs that can be used in a compound of Formula (1) are pharmaceutically active compounds. Preferably the pharmaceutically active compound is selected from the group consisting of cytotoxins, antiproliferative / antitumor agents, antiviral agents, antibiotics, antiinflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, and enzyme inhibitors. Preferably these pharmaceutically active compounds are selected from the group consisting of antibodies, antibody derivatives, antibody fragments, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, cytokines, and chemokines. Most preferably, the drug is a protein, a toxin, a chelating moiety, monomethyl auristatin E, or doxorubicin; wherein preferably the chelating moiety comprises a radionuclide. Preferably these drugs are low to medium molecular weight compounds, preferably organic compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da, more preferably about 300 to about 1000 Da). Exemplary cytotoxic drug types for use as conjugates to the Trigger and to be released upon IEDDA reaction with the Activator, for example for use in cancer therapy, include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors. Examples include but are not limited to colchinine, vinca alkaloids, anthracyclines (e.g. doxorubicin, epirubicin, idarubicin, daunorubicin), camptothecins, taxanes, taxols, vinblastine, vincristine, vindesine, calicheamycins, tubulysins, tubulysin M, cryptophycins, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, deBouganin, dactinomycines, CC1065 and its analogs, duocarmycins, maytansines, maytansinoids, dolastatins, auristatins, pyrrolobenzodiazepines and dimers (PBDs), indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins (e.g. mitomycin C, mitomycin A, caminomycin), melphalan, leurosine, leurosideine, actinomycin, tallysomycin, lexitropsins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporin, esperamicin, the pteridine family of drugs, SN- 38 and its analogs, platinum-based drugs, cytotoxic nucleosides. Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HD AC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling pathway inhibitors, and RNA polymerase inhibitors. In some embodiments, the drug is an auristatin. Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HP A), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ. MMAE is a preferred auristatin. Suitable auristatins are also described in U.S. Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248; PCT Application Publication Nos. WO09 / 117531, W02005 / 081711, WO04 / 010957; W002 / 088172 and WOOl / 24763, and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety. Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5,138,036), dolastatin 15 (U.S. Pat No. 4,879,278), dolastatin 16 (U.S. Pat No. 6,239,104), dolastatin 17 (U.S. Pat No. 6,239,104), and dolastatin 18 (U.S. Pat No. 6,239,104), each patent incorporated herein by reference in their entirety. Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441,163; 6,716,821 and 7,276,497. Other examples include mertansine and ansamitocin.

[0266] Pyrrolobenzodiazepines (PBDs), which expressly include dimers and analogs, include but are not limited to those described in [Denny, Exp. Opin. Ther. Patents, 10(4):459-474 (2000)], [Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44], Antonow et al., Chem Rev. 2011, 111(4), 2815-64], Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin, CPI, CBI. Other examples include those described in, for example, US Patent No. 5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530; 6,586,618; 6,660,742; 6,756,397; 7,049,316; 7,553,816; 8,815,226; US20150104407; 61 / 988,011 filed may 2, 2014 and 62 / 010,972 filed June 11, 2014; the disclosure of each of which is incorporated herein in its entirety. Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos. 2002 / 0103136 and 2010 / 0305149, and in U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety. Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO97 / 19086; WO98 / 08849; WO98 / 22461; WO98 / 25929; WO98 / 38192; WO99 / 01124; WO99 / 02514; WO99 / 03848; WO99 / 07692; WO99 / 27890; and WO99 / 28324; the disclosures of which are incorporated herein by reference in their entirety. Exemplary cryptophycin compounds are described in U.S. Patent Nos. 6,680,311 and 6,747,021; the disclosures of which are incorporated herein by reference in their entirety. Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin. Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines and the like. Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins. Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflometotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used in the present disclosure include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30:1774 (1987). Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure. Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZDI 152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI 2536, BI6727, GSK461364, ON-01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520. Exemplary P13K / m- TOR / AKT signalling pathway inhibitors include phosphoinositide 3-kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors. Exemplary P13 kinases are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765. Exemplary AKT inhibitors include, but are not limited to AT7867. Exemplary MAPK signaling pathway inhibitors include MEK, Ras, INK, B-Raf and p38 MAPK inhibitors. Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,944 and include GDC-0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD8330 and GDC-0973. Exemplary B-raf inhibitors include CDC- 0879, PLX-4032, and SB590885. Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190. Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171, CHR- 258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib. Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478. Exemplary HDAC inhibitors include Belinostat (PR48101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MCI 568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3 -aminobenzamide, A- 966492, and AZD2461. Exemplary Wnt / Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV-939. Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.

[0267] Exemplary immunomodulators are APRIL, cytokines, including IL-2, IL-7, IL-10, IL12, IL- 15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including TLR1 / 2, TLR3, TLR4, TLR7 / 8, TLR9, TLR12, agonists and antagonists of GITR, CD3, CD28, CD40, CD74, CTLA4, 0X40, PD1, PDL1, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A. Other exemplary drugs include puromycins, topetecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cemadotin, discodermolide, eleutherobin, mitoxantrone, pyrrolobenzimidazoles (PBI), gamma-interferon, Thialanostatin (A) and analogs, CDK11, immunotoxins, comprising e.g. ricin A, diphtheria toxin, cholera toxin. In exemplary embodiments of the disclosure, the drug moiety is a mytomycin compound, a vinca alkaloid compound, taxol or an analogue, an anthracycline compound, a calicheamicin compound, a maytansinoid compound, an auristatin compound, a duocarmycin compound, SN38 or an analogue, a pyrrolobenzodiazepine compound, a indolinobenzodiazepine compound, a pyridinobenzodiazepine compound, a tubulysin compound, a non-natural camptothecin compound, a DNA binding drug, a kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a P13 kinase inhibitor, a topoisomerase inhibitor, or analogues thereof. In one preferred embodiment the drug is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor, (e.g. P13 kinase inhibitor: GDC-0941 and PI- 103), MEK inhibitor, KSP inhibitor, RNA polymerase inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, dolastatin, calicheamicins, SN38, pyrrolobenzodiazepines, pyridinobenzodiazepines, indolinobenzodiazepines, DNA binding drugs, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs. In another preferred embodiment the drug is selected from DNA binding drugs and microtubule agents, including pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines, maytansinoids, maytansines, auristatins, tubulysins, duocarmycins, anthracyclines, taxanes. In another preferred embodiment the drug is selected from colchinine, vinca alkaloids, tubulysins, irinotecans, an inhibitory peptide, amanitin and deBouganin. In another preferred embodiment the drug is a radioactive moiety, said moiety comprising a radioactive isotope for radiation therapy. A radionuclide used for therapy is preferably an isotope selected from the group consisting of24Na,32P,33P,47Sc,59Fe,67Cu,76As,77As,80Br,82Br,89Sr,90Nb,90Y,103Ru,105Rh,109Pd,111Ag,111In,121Sn,127Te,1311,140La,141Ce,142Pr,143Pr,144Pr,149Pm,149Tb,151Pm,153Sm,159Gd,161Tb,165Dy,166Dy,166Ho,169Er,172Tm,175Yb,177Lu,186Re,188Re,198Au,199Au,211At,211Bi,212Bi,212Pb,213Bi,214Bi,223Ra,224Ra,225Ac, and227Th. When the radioactive moiety is intended to comprise a metal, such as177Lu, such radiometal is preferably provided in the form of a chelate. In such a case the radioactive moiety preferably comprises a structural moiety capable of forming a coordination complex with such a metal. A good example hereof are macrocylic lanthanide(III) chelates derived from 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (H4dota). Preferably, the structural moiety capable of forming a coordination complex with such a metal is a chelating moiety as defined herein. In other embodiments the radioactive moiety comprises a prosthetic group (i.e. a phenol) that is bound by a non-metal radionuclide, such as131I. Drugs optionally include a (portion of a) membrane translocation moiety (e.g. adamantine, poly-lysine / arginine, TAT, human lactoferrin) and / or a targeting agent (against e.g. a tumor cell receptor) optionally linked through a stable or labile linker. Exemplary references include: Trends in Biochemical Sciences, 2015,. 40, 12, 749; J. Am. Chem. Soc. 2015, 137, 12153-12160; Pharmaceutical Research, 2007, 24, 11, 1977. It will further be understood that, in addition to one or more targeting agents (or CB) that may be attached to the Trigger or Linker LCa targeting agent TT may optionally be attached to a drug, optionally via a spacer SP. Alternatively, it will be further understood that the targeting agent (or CB) may comprise one or more additional drugs which are bound to the targeting agent by other types of linkers, e.g. cleavable by proteases, pH, thiols, or by catabolism. It will be understood that chemical modifications may also be made to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the disclosure. Drugs containing an amine functional group for coupling to the Trigger include mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, l-(2 chloroethyl) 1,2-dimethanesulfonyl hydrazide, tallysomycin, cytarabine, dolastatins (including auristatins) and derivatives thereof. Drugs containing a hydroxyl function group for coupling to the Trigger include etoposide, camptothecin, taxol, esperamicin, l,8-dihydroxy-bicyclo[7.3. l]trideca-4-9-diene-2, 6-diyne- 13-one (U.S. Pat No. 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n- (5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof. Drugs containing a sulfhydryl functional group for coupling to the Trigger include esperamicin and 6-mecaptopurine, and derivatives thereof.

[0268] Log P

[0269] In preferred embodiments the Log P of compounds of Formula (1) have a value in a range of from 2.0 and -2.0, more preferably in a range of from 1.0 and -1.0.

[0270] In embodiments where it is required that a compound as disclosed herein, in particular a diene, has an extracellular volume of distribution it is preferred that the Log P of said compound is at most 2, preferably at most 1, more preferably at most 0, even more preferably at most -1. In embodiments where it is required that a compound as disclosed herein, in particular a diene, has an intracellular volume of distribution it is preferred that the Log P of the Activator is at least -1, preferably at least 0, more preferably at least 1, even more preferably at least 2.

[0271] Molecular weight

[0272] For a compound of Formula (1) wherein T2is a bioconjugation moiety, it is preferred that the molecular weight of said compound is at most 5 kDa, more preferably at most 4 kDa, even more preferably at most 3.5 kDa, more preferably stil at most 3 kDa, and most preferably at most 2.5 kDa. For a compound of Formula (1) wherein T2is a group -L3-CB, it is preferred that the molecular weight of said compound is at most 100 kDa, more preferably at most 85 kDa, even more preferably at most 75 kDa, more preferably stil at most 65 kDa, and most preferably at most 62.5 kDa.

[0273] Spacers SP

[0274] All linkers as used herein may each independently be a spacer SP. As the skilled person is aware, the specific structure of a spacer used in either a dienophile or diene as described herein does not typically influence whether the payload is released. However, in some cases specific spacers are preferred. For example, if a payload is to be released, the spacer between e.g. the allylic carbon of the eight-membered non-aromatic cyclic mono-alkenylene moiety and the payload is preferably a self-immolative linker. Such a linker, which is typically referred to as LCherein, ensures that upon release of the end of the linker connected to said allylic carbon, a further rearrangement or reaction takes place, after which the payload is decoupled from the linker LC. Below, first spacers in general are discussed, and thereafter the more specific self-immolative linkers.

[0275] In general, a spacer SPas used herein is a moiety according to RG2, more preferably any one of the preferred and / or specific embodiments thereof.

[0276] Preferably, a spacer SPconsists of one or multiple Spacer Units SUarranged linearly and / or branched and may be connected to one or more CBmoieties and / or one or more LCor TRmoieties. The Spacer may be used to connect CBto one TR(Example A below; with reference to Formula 5 a and 5b: f, e, a = 1) or more TR(Example B and C below; with reference to Formula 5 a and 5b: f, e = 1, a ≥ 1), but it can also be used to modulate the properties, e.g. pharmacokinetic properties, of the CB-TR-CAconjugate (Example D below; with reference to Formula 5a and 5b: one or more of c,e,g,h ≥ 1). Thus a Spacer unit does not necessarily connect two entities together, it may also be bound to only one component, e.g. the TRor LC. Alternatively, the Spacer may comprise a Spacer Unit linking CBto TRand in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F below; with reference to Formula 5a and 5b: e ≥ 1). The Spacer may also consist of two different types of SUconstructs, e.g. a PEG linked to a peptide, or a PEG linked to an alkylene moiety (Example E below; with reference to Formula 5a and 5b: e ≥ 1). For the sake of clarity, Example B depicts a SUthat is branched by using a multivalent branched SU. Example C depicts a SUthat is branched by using a linear SUpolymer, such as a peptide, whose side chain residues serve as conjugation groups.

[0277]

[0278] The Spacer may be bound to the Activator in similar designs such as depicted in above examples A- F.

[0279] Each individual spacer unit SUmay be independently selected from the group of radicals according to RG2. The Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. Preferred biopolymer SUare peptides. Preferably each SUcomprises at most 50 carbon atoms, more preferably at most 25 carbon atoms, more preferably at most 10 carbon atoms. In some embodiments the SUis independently selected from the group consisting of (CH2)r, (C3-C8carbocyclo), O-(CH2)r, arylene, (CH2)r-arylene, arylene-(CH2)r, (CH2)r-(C3-C8carbocyclo), (C3-C8carbocyclo)-(CH2)r, (C3-C8heterocyclo), (CH2)r-(C3-C8heterocyclo), (C3-C8heterocyclo)-(CH2)r, -(CH2)rC(O)NR’(CH2)r, (CH2CH2O)r, (CH2CH2O)rCH2,(CH2)rC(O)NR’(CH2CH2O)r, (CH2)rC(O)NR’(CH2CH2O)rCH2, (CH2CH2O)rC(O)NR’(CH2CH2O)r, (CH2CH2O)rC(O)NR’(CH2CH2O)rCH2, (CH2CH2O)rC(O)NR,CH2; wherein r is independently an integer from 1 -10. As used herein, each R’is independently selected from the group consisting of radicals according to RG1. Preferably, R’is hydrogen. Other examples of Spacer Units SUare linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH3, -OCH2CH3, OCH2CH2CO2H. Other polymeric Spacer Units are polymers and copolymers such as poly-(2-oxazoline), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF). Other exemplary polymers are polysaccharides, glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring polysaccharides that can be used as SUare cellulose, amylose, dextran, dextrin, levan, fucoidan, carrageenan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymeric SUcomprises a copolymer of a polyacetal / polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Preferred polymeric SUare PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides. In some embodiments, polymers used in a SUhave a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 dalton to 5 kDa. Other exemplary SUare dendrimers, such as polypropylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol-based dendrimers. The SUof the disclosure expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DIME, BMB, BMDB, BMH, BMOE, BM(PEO)3and BM(PEO)4. To construct a branching Spacer one may use a SUbased on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required functionality for branching. For example serine has three functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching SU. Other exemplary amino acids are lysine and tyrosine. In some embodiments, the Spacer consists of one Spacer Unit, therefore in those cases SPequals SU. Preferably the Spacer consists of two, three or four Spacer Units. In some embodiments, SPhas a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, or from 500 dalton to 5 kDa. In some embodiments, the SPhas a mass of no more than 5000 daltons, no more than 4000 daltons, no more than 3000 daltons, no more than 2000 daltons, no more than 1000 daltons, no more than 800 daltons, no more than 500 daltons, no more than 300 daltons, no more than 200 daltons. In some aspects the SPhas a mass from 100 daltons, from 200 daltons, from 300 daltons to 5000 daltons. In some aspects of the SPhas a mass from 30, 50, or 100 daltons to 1000 daltons, from about 30, 50, or 100 daltons to 500 daltons.

[0280] Preferably, SPcomprises a moiety RG2a, RG2b, RG2c, or a residue of RGlf, as described herein. Preferably, said RG2a, RG2b, RG2c, or a residue of RGlf connects the S to CB, LC, or TR.

[0281] Self-immolative linkers LCLCis an optional self-immolative linker, which may consist of multiple units arranged linearly and / or branched. The possible LCstructures, their use, position and ways of attachment of linkers LC, CAand the TR(the Trigger, i.e. the trans-cyclooctene moiety) are known to the skilled person, see for example [Papot et al., Anticancer Agents Med. Chem., 2008, 8, 618-637]. Nevertheless, preferred but non-limiting examples of self-immolative linkers LCare benzyl-derivatives, such as those drawn below. There are two main self- immolation mechanisms: electron cascade elimination and cyclization-mediated elimination. The preferred example below on the left functions by means of the cascade mechanism, wherein the bond between the allylic carbon of the Trigger and the -O- or -S- attached to said carbon is cleaved, and an electron pair of YC1, for example an electron pair of NR6, shifts into the benzyl moiety resulting in an electron cascade and the formation of 4-hydroxybenzyl alcohol, CO2and the liberated payload. The preferred example in the middle functions by means of the cyclization mechanism, wherein cleavage of the bond to the NR6on the side of the Trigger leads to nucleophilic attack of the amine on the carbonyl, forming a 5-ring 1,3- dimethylimidazolidin-2-one and liberating the payload. The preferred example on the right combines both mechanisms. This linker will degrade not only into CO2and one unit of 4- hydroxybenzyl alcohol (when YC1is O), but also into one l,3-dimethylimidazolidin-2-one unit. wherein the wiggly line indicates a bond to -O- or -S- on the allylic position of the transcyclooctene, and the double dashed line indicates a bond to CA.

[0282] By substituting the benzyl groups of aforementioned self-immolative linkers LC, it is possible to tune the rate of release of the payload, caused by either steric and / or electronic effects on the cyclization and / or cascade release. Synthetic procedures to prepare such substituted benzyl-derivatives are known to the skilled person (see for example [Greenwald et al, J. Med. Chem., 1999, 42, 3657-3667] and [Thomthwaite et al, Polym. Chem., 2011, 2, 773-790]. Some preferred substituted benzyl-derivatives with different release rates are drawn below.

[0283] Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately substituted bicyclo[2.2.1] and bicyclo [2.2.2] ring system, 2-aminophenylpropionic acid amides, and trimethyl lock-based linkers, see e.g. [Chem. Biol. 1995, 2, 223], [J. Am. Chem. Soc. 1972, 94, 5815], [J. Org. Chem. 1990, 55, 5867], the contents of which are hereby incorporated by reference. Further preferred examples of LCcan be found in W02009017394(A1), US7375078, WO2015038426A1, W02004043493, Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509, the contents of which are hereby incorporated by reference.

[0284] Preferably the LChas a mass of no more than 1000 daltons, no more than 500 daltons, no more than 400 daltons, no more than 300 daltons, or from 10, 50 or 100 to 1000 daltons, from 10, 50, 100 to 400 daltons, from 10, 50, 100 to 300 daltons, from 10, 50, 100 to 200 daltons, e.g., 10-1000 daltons, such as 50-500 daltons, such as 100 to 400 daltons.

[0285] A person skilled in the art will know that one LCmay be connected to another LCthat is bound to CA, wherein upon reaction of the Activator with the Trigger TR, LC-LC-CAis released from the TR, leading to self-immolative release of both LCmoietes and the payload. With respect to the LCformulas disclosed herein, the LClinking the TRto the other LCthen does not release the payload but an LCthat is bound via YC1and further links to CA. The skilled person will acknowledge that this principle also holds for further linkers LClinked to LC, e.g. LC-LC-LC-LC-CA.

[0286] Preferably, if the releasable group contains a self-immolative linker, the releasable group is according to any one of Group I, Group II, Group III, and Group IV as shown below. In the structures depicted for said Groups, only bonds to Construct A and an atom (typically oxygen) on the allylic position of the eight-membered non-aromatic cyclic mono-alkenylene moiety (preferably a trans-cyclooctene ring) are shown for reasons of clarity, but said Construct A and said atom are part of the releasable group.

[0287] Releasable groups according to Group I are

[0288] , wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene, wherein U, V, W, Z are each independently selected from the group consisting of -CR7-, and -N-, wherein e is 0 or 1, wherein X is selected from the group consisting of -O-, -S- and -NR6-, wherein preferably each R8and R9are independently selected from the group consisting of hydrogen, C1-C4(hetero)alkyl, C2-C4(hetero)alkenyl, and C4-6(hetero)aryl; wherein for R8and R9the (hetero)alkyl, (hetero)alkenyl, and (hetero)aryl are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H, -PO4H2and -NO2and preferably contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. Preferably, for releasable groups of Group I both R8and R9are hydrogen.

[0289] The releasable group according to Group II is

[0290] , wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene, wherein m is an integer between 0 and 2, preferably m is 0, wherein e is 0 or 1. Preferably, for releasable groups of Group II both R8and R9are hydrogen. Preferably, for releasable groups of Group II R7is methyl or isopropyl. Optionally, R6, R7, R8, R9comprised in said Group I, and II, are -(SP)i-CB.

[0291] For all releasable groups according to Group I and Group II YC1is selected from the group consisting of -O-, -S-, and -NR6-, preferably -NR6-. For all linkers according to Group I, and Group II, YC2is selected from the group consisting of O and S, preferably O.

[0292] Releasable groups according to Group III are

[0293] , wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene.

[0294] Releasable groups according to Group IV are

[0295] , wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene.

[0296] Preferably, R6, R7, R8, R9are according to RG1 or any preferred embodiment thereof. Preferably, R6, R7, R8, R9as used herein are not substituted. Most preferably, R6, R7, R8, R9as used herein are hydrogen.

[0297] Preferably, the self-immolative linker consists of one or more self-immolative units, wherein the one or more self-immolative units are independently a group according to Formula (SL1), or a group according to Formula (SL2): wherein the asterisk indicates a bond to a moiety CB; the wiggly line indicates a bond to the moiety -(Y1-C(=Y2))i- of a releasable group, or to a preceding self-immolative unit; the double dashed line indicates a bond to CAor a further self-immolative unit; A is a 5- membered or 6-membered (hetero)aromatic ring; Y3and Y5are independently O, S, a secondary amine, or a tertiary amine; Y4, Y6, and Y7are independently O or S; e is 0, 1 or 2; f is 0 or 1; A1 is 1 or 2; A2 is 0, 1, 2, or 3; each B1 is an optionally substituted carbon; each C1 is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (hetero)aromatic ring, then -Y3- is at an ortho or para position relative to - (B1)AI-Y6-C(=Y7)-; and provided that if A is a 5 -membered (hetero)aromatic ring and -(B1)AI-Y6-C(=Y7)- is at the 1 -position of said 5-membered (hetero)aromatic ring, then -Y3- is at the 3- or 4-position; wherein the wiggly line indicates a bond to the moiety -(Y1-C(=Y2))i- of a releasable group, or to a preceding self-immolative unit; the double dashed line indicates a bond to CAor a further self-immolative unit; the asterisk indicates a bond to a moiety CB; g is 0 or 1, preferably g is 0; R2Bis is a bond or - C(=Y8)-; Y8and Y10are independently O or S; and Y9is independently O, S, a secondary amine, or a tertiary amine. Preferably, Y8is O. Preferably, Y10is O. Preferably, Y9is -NH-. Combinations of the disclosure

[0298] The disclosure also relates to a combination of (A1) a conjugate as defined herein; and / or (A2) a compound as defined herein claim 1; with (A3) a buffer as defined herein. Preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3).

[0299] Preferably, the combination is a combination of (A1) and (A3); more preferably the combination is a kit wherein (A1) is physically separated from (A3). Most preferably, the combination essentially consists of (A1) and (A3).

[0300] Optionally, the combination of the disclosure further comprises (B) a diene.

[0301] Preferably, the diene is a tetrazine. If the combination comprises (B), it is preferred that the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3) and (B), and wherein (A3) is physically separated from (B).

[0302] It is preferred that the combination comprises (A1) rather than (A2).

[0303] It will be understood that herein, a compound according to the disclosure is a dienophile and / or comprises a dienophile moiety, and may be called a “Trigger”. The diene may be referred to as an “Activator”.

[0304] Preferably, the combination of the disclosure is a kit. More preferably, the combination of the disclosure is a kit wherein (A1), (A2), and / or (A3) is / are physically separated from (B).

[0305] Preferably the diene is a tetrazine. More preferably, the diene is selected from the group consisting of:

[0306] or a salt, hydrate, and / or solvate thereof.

[0307] Preferably, the diene is (TZ1) or a salt, hydrate, and / or solvate thereof. More preferably, the diene is (TZ2) or a salt, hydrate, and / or solvate thereof. More preferably, the diene is (TZ3) or a salt, hydrate, and / or solvate thereof. More preferably, the diene is (TZ4) or a salt, hydrate, and / or solvate thereof. Most preferably, the diene is (TZ5) or a salt, hydrate, and / or solvate thereof.

[0308] (TZ1) is the best-studied tetrazine for in vivo use in literature, and the most promising candidate for clinical use. Reference is made to, inter alia, Rossin et at, Angew. Chem. Int. Ed. 2010, volume 49, pages 3375-3378; Rossin et al., J. Nucl. Med. 2013, volume 54; pages 1989-1995; Rossin et al., Bioconjugate Chem. 2013, volume 24, pages 1210-1217; Rossin et al., Mol. Pharm. 2014, volume 11, pages 3090-3096; Van Duijnhoven et al., J. Nucl. Med. 2015, volume 56, pages 1422-1428; Edem et al. Molecules 2020, volume 25, page 463; Rossin et al., Bioconjugate Chem. 2016, volume 27, pages 1697-1706; Rossin et al., Nature Commun. 2018, volume 9, article 1484; WO 2020 / 256546 (in particular Example 5 at pages 294-296). However, the inventors have identified several hitherto unknown disadvantages of (TZ1). These problems mainly arise when using (TZ1) in vivo as an activator for the payload release from an eight-membered non-aromatic cyclic mono-alkenylene moiety (such as a trans-cyclooctene), which require higher doses than when using (TZ1) for radioimaging and / or radiotherapy.

[0309] First, it was found that compound (TZ1) strongly inhibits the physiologically relevant enzymes cyclooxygenase (COX-1), acetyl cholinesterase (ACES), monoamine oxidase (MAO-B), and calcium channel L-type, dihydropyridine. Each of these proteins is important in maintaining health in a subject, and undesired inhibition of these enzymes and / or transporter may lead to side-effects.

[0310] Second, it was found that (TZ1) has a relatively low maximum tolerated dose (MTD) in mice of about 39 μmol / kg.

[0311] Furthermore, the synthesis of (TZ1) comprises many steps, while it is preferred that tetrazines are used that can be synthesized in fewer steps.

[0312] Finally, it is desired that tetrazines with overall good in vitro and in vivo properties be provided, i.e. one or more of: good stability, good reactivity with and / or high payload release from trans-cyclooctenes (especially in vivo), low membrane permeability, low cell toxicity, and low genotoxicity.

[0313] It was found that (TZ2), (TZ3), (TZ4), and in particular (TZ5) overcome one or more of these disadvantages of (TZ1). Therefore, combinations with at least one of (TZ2), (TZ3), (TZ4), and (TZ5) are preferred over combinations comprising (TZ1), and combinations with (TZ5) are most preferred.

[0314] Medical use

[0315] The disclosure also relates to the composition of the disclosure; or the combination of the disclosure; for use in the treatment of a disease in a subject.

[0316] The disclosure also pertains to a method of treating a disease in a subject, wherein said method comprises the step of administering to said subject: (a) the composition according to the disclosure; and / or (b) the combination according to the disclosure.

[0317] Use of (a) a composition according to the disclosure; and / or (b) a combination according to the disclosure; for the manufacture of a medicament for the treatment of a disease in a subject. In relation to the medical use, preferably the subject is a human. Preferably, the disease is cancer.

[0318] Dried compositions

[0319] The disclosure also relates to a method for obtaining a dried composition, wherein said method comprises the step of drying a composition of the disclosure. Drying techniques are well-known to the skilled person, such as lyophilization, drying by air, or mild heating. Preferably, the drying is carried out by lyophilization. Thus, the disclosure also relates to a dried composition obtainable by said method.

[0320] Moreover, the disclosure relates to a lyophilized composition obtainable by lyophilizing a composition of the disclosure.

[0321] Preferably, the dried composition and the lyophilized composition of the disclosure comprise water in an amount of at most 10 wt%, more preferably at most 5 wt%, even more preferably at most 1 wt%, more preferably still at most 0.5 wt%, and most preferably at most 0.1 wt%, as compared to the total weight of the composition.

[0322] Methods for preparing a composition of the disclosure

[0323] The disclosure also pertains to a method for preparing a composition of the disclosure; wherein said method comprises the step of

[0324] (a) contacting a buffer as defined herein with a compound and / or a conjugate as defined herein; and / or the step of

[0325] (b) reconstituting a dried composition as disclosed herein with water.

[0326] Preferably, the method comprises the step of contacting a buffer as defined herein with a conjugate as defined herein.

[0327] In the contacting step (a) the compound or conjugate can be provided first and added to a buffer, or vice versa. Both the contacting step and the reconstituting step may be performed by mixing, dissolving, dispersing, and / or any other technique known to the skilled person. If required, shaking or ultrasound may be applied to ensure full dissolution of the compound or conjugate in the buffer.

[0328] Alternatively, the compound or conjugate of the disclosure can first be dissolved in a solvent of buffer, after which buffer exchange is performed using a buffer of the disclosure, so as to obtain the composition of the disclosure. For reconstitution, the skilled person can readily determine the amount of water that is required to obtain a composition with the desired concentration of e.g. the buffer compound, carbohydrate, chelator, and the like.

[0329] Methods of storing a composition of the disclosure

[0330] The disclosure also pertains to a method of storing the composition of the disclosure wherein the method comprises the steps of:

[0331] (a) providing a composition of the disclosure; and

[0332] (b) maintaining said composition at a temperature of at most 40°C.

[0333] Preferably, the temperature is at most 35 °C, more preferably at most 32 °C, more preferably at most 30 °C, even more preferably at most 25 °C, even more preferably at most 20 °C, even more preferably at most 15 °C, even more preferably at most 10 °C, and most preferably at most 5 °C.

[0334] Preferably, the temperature is in a range of from -200 to 35 °C, more preferably of from -175 to 32 °C, more preferably of from -150 to 30 °C, even more preferably of from -125 to 25 °C, even more preferably of from -100 to 20 °C, even more preferably of from -85 to 15 °C, even more preferably of from -25 to 10 °C, and most preferably of from -5 to 5 °C.

[0335] Preferably, the temperature is about 4 °C. In other embodiments, however, the tempterature is about -80 °C. In further embodiments, the temperature is about 30°C.

[0336] Preferably, the composition is shielded from light during storage. Preferably, the composition is not perturbed during storage.

[0337] Use of a buffer of the disclosure for storage and / or stabilization during storage

[0338] Furthermore, the disclosure relates to a use of a buffer as defined herein to store a compound as defined herein and / or a conjugate as defined herein; preferably the use is to store said conjugate.

[0339] Additionally, the disclosure pertains to a use of a buffer as defined herein to stabilize a compound as defined herein and / or a conjugate as defined herein, during storage; and preferably the use is to stabilize said conjugate.

[0340] Both these uses may require contacting said buffer with said compound and / or conjugate.

[0341] During storage, it is preferred that the buffer and the compound and / or conjugate are maintained at a temperature of at most 40°C. Preferably, the temperature is at most 35 °C, more preferably at most 32 °C, more preferably at most 30 °C, even more preferably at most 25 °C, even more preferably at most 20 °C, even more preferably at most 15 °C, even more preferably at most 10 °C, and most preferably at most 5 °C.

[0342] Preferably, the temperature is in a range of from -200 to 35 °C, more preferably of from -175 to 32 °C, more preferably of from -150 to 30 °C, even more preferably of from -125 to 25 °C, even more preferably of from -100 to 20 °C, even more preferably of from -85 to 15 °C, even more preferably of from -25 to 10 °C, and most preferably of from -5 to 5 °C.

[0343] Preferably, the temperature is about 4 °C. In other embodiments, however, the tempterature is about -80 °C. In further embodiments, the temperature is about 30°C.

[0344] Preferably, the buffer and the compound and / or conjugate are shielded from light during storage. Preferably, the buffer and the compound and / or conjugate are not perturbed during storage.

[0345] Further points

[0346] The present disclosure is herein described with respect to particular embodiments, but the disclosure is not limited thereto but only by the claims. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

[0347] The term “about” as used herein before a value typically indicates a deviation from that value of ±10% of said value, preferably ±5% of said value, more preferably ±2% of said value, and most preferably ±1% of said value.

[0348] The verb "to comprise", and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

[0349] In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0350] Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present disclosure, the only relevant components of the device are A and B.

[0351] The compounds herein may occur in different tautomeric forms. The compounds according to the disclosure are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the disclosure of the present application is not limited to that specific tautomer, unless stated otherwise.

[0352] The compounds herein may occur in different enantiomeric forms. The compounds according to the disclosure are meant to include all enantiomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the disclosure of the present application is not limited to that specific enantiomer, unless stated otherwise.

[0353] Unless stated otherwise, the compounds of the disclosure and / or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is deprotonated.

[0354] Unless stated otherwise, if in this disclosure reference is made to a molecular structure, such as “compound”, “diene”, “tetrazine”, and the like, it will be understood that such a molecular structure may also be in its salt, hydrate, and / or solvate form.

[0355] In several formulae, groups or substituents are indicated with reference to letters such as “A”, “B”, “X”, “Y”, and various (numbered) “R” groups. In addition, the number of repeating units may be referred to with a letter, e.g. n in -(CH2)n-. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.

[0356] Herein, reference is made to "alkyl", and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents according to Radical Group 1, can be indicated by a designation preceding such terms (e.g. “C1-C8alkyl” means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a -OCH3group is designated as a C4alkyl, because the carbon atom in the substituent is not included in the carbon count.

[0357] A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula CnH2n-1. Optionally, the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

[0358] An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula CnH2n-1. Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula CnH2n-3. An alkenyl group may comprise a terminal carbon-carbon double bond and / or an internal carbon-carbon double bond. A terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain. An alkenyl group may also comprise two or more carbon-carbon double bonds. Examples of an alkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl, 1,3- butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, an alkenyl group may optionally be substituted with one or more, independently selected, substituents according to Radical Group 1.

[0359] A cycloalkenyl group is a cyclic alkenyl group. An unsubstituted cycloalkenyl group comprising one double bond has the general formula CnH2n-3. Optionally, a cycloalkenyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkenyl group is cyclopentenyl.

[0360] An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula CnH2n-3. An alkynyl group may comprise a terminal carbon-carbon triple bond and / or an internal carbon-carbon triple bond. A terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain. An alkynyl group may also comprise two or more carbon-carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents according to Radical Group 1. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc.

[0361] A cycloalkynyl group is a cyclic alkynyl group. An unsubstituted cycloalkynyl group comprising one triple bond has the general formula CnH2n-5. Optionally, a cycloalkynyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkynyl group is cyclooctynyl.

[0362] An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl. Preferably, an aryl group is phenyl.

[0363] Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures. Optionally, the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document. An arylalkyl group is for example benzyl. An alkylaryl group is for example 4-tert-butylphenyl.

[0364] Preferably, heteroaryl groups comprise five to sixteen carbon atoms and contain between one to five heteroatoms. Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.

[0365] Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C3) and may include monocyclic and bicyclic structures. Optionally, the heteroaryl groups may be substituted by one or more substituents further specified in this document.

[0366] Where an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group. Similarly, an alkyl(hetero)aryl group is meant to include an alkylaryl group and an alkylheteroaryl group, and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroarylalkyl group. A C2-C24(hetero)aryl group is thus to be interpreted as including a C2-C24heteroaryl group and a C6-C24aryl group. Similarly, a C3- C24alkyl(hetero)aryl group is meant to include a C7-C24alkylaryl group and a C3-C24alkylheteroaryl group, and a C3-C24(hetero)arylalkyl is meant to include a C7-C24arylalkyl group and a C3-C24heteroarylalkyl group.

[0367] In general, when (hetero) is placed before a group, it refers to both the variant of the group without the prefix hetero- as well as the group with the prefix hetero-. Herein, the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si. Preferably, the one or more heteroatoms is selected from the group consisting of O, N, S, and P. It will be understood that for any compound containing a heteroatom, the N, S, and P atoms are optionally oxidized and the N atoms are optionally quatemized. Preferably, up to two heteroatoms are consecutive, such as in for example -CH2-NH-OCH3and -CH2-O-Si(CH3)3. More preferably, however, the heteroatoms are not directly bound to one another.

[0368] Examples of heteroalkyls include -CH2CH2-O-CH3, -CH2CH2-NH-CH3, -CH2CH2- S(O)-CH3, -CH=CH-O-CH3, CH2CH2-NH2, CH2CH2-SH, -CH2CH2-OH, -CH2CH2-COOH, - CH2C(O)H, -C(O)HCH3, and -Si(CH3)3. Preferably, a C1-C4heteroalkyl contains at most 2 heteroatoms.

[0369] Herein, it will be understood that when the prefix hetero- is used for combinations of groups, the prefix hetero- only refers to the one group before it is directly placed. For example, heteroarylalkyl denotes the combination of a heteroaryl group and an alkyl group, not the combination of a heteroaryl and a heteroalkyl group.

[0370] Herein, the prefix cyclo- denotes that groups are cyclic. It will be understood that when the prefix cyclo- is used for combinations of groups, the prefix cyclo- only refers to the one group before it is directly placed. For example, cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group. In general, when (cyclo) is placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.

[0371] Herein, the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties. An example of an alkylene is propylene (-CH2-CH2-CH2-), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix. For example, an alkylene attached to an -H is identical to an alkyl group. I.e. propylene, - CH2-CH2-CH2-, attached to an -H at one terminus, -CH2-CH2-CH2-H, is logically identical to propyl, -CH2-CH2-CH3.

[0372] Herein, when combinations of groups are listed with the suffix -ene, it refers to a divalent group, i.e. that the group is linked to at least two other moieties, wherein each group of the combination contains one linkage to one of these two moieties. As such, for example alkylarylene is understood as a combination of an arylene group and an alkylene group. An example of an alkylarylene group is -phenyl-CH2-, and an example of an arylalkylene group is -CH2-phenyl-.

[0373] Herein, the suffix -triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties. An example of an arenetriyl is depicted below: wherein the wiggly lines denote bonds to different groups of the main compound. It is understood that if a group with the suffix -triyl is substituted at one position with - H, then this group is identical to a divalent group with the suffix -ene. For example, an arenetriyl substituted with -H is identical to an arylene group. Similarly, it is understood that if a group with the suffix -triyl is substituted at two positions with -H, then this group is identical to a monovalent group. For example, an arenetriyl substituted with two -H is identical to an aryl group.

[0374] Unless indicated otherwise, a hetero group may contain a heteroatom at non-terminal positions or at one or more terminal positions. In this case, “terminal” refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, C2heteroalkylene may refer to -NH-CH2-CH2-, -CH2-NH-CH2-, and -CH2-CH2- NH-. For example, C2heteroalkyl may refer to -NH-CH2-CH3, -CH2-NH-CH3, and -CH2- CH2-NH2.

[0375] Herein, it is understood that cyclic compounds (i.e. aryl, cycloalkyl, cycloalkenyl, etc.) are understood to be monocyclic, polycyclic or branched. It is understood that the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring. For example, C10aryl optionally containing heteroatoms may refer to inter alia a naphthyl group (fused rings) or to e.g. a bipyridyl group (substituted rings, both containing an N atom).

[0376] Unless stated otherwise, any group disclosed herein that is not cyclic is understood to be linear or branched. In particular, (hetero)alkyl groups, (hetero)alkenyl groups, (hetero)alkynyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, and the like are linear or branched, unless stated otherwise.

[0377] As used herein, unless stated otherwise all of the following groups: (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, (hetero )alkylene, (hetero)alkenylene, (hetero)alkynylene, (hetero)cycloalkylene, (hetero)cycloalkenylene, (hetero)cycloalkynylene, (hetero)arylene, (hetero)alkanetriyl, (hetero)cycloalkanetriyl, arenetriyl, heteroarenetriyl, combinations thereof, and the like, can be substituted or unsubstituted; preferably these groups are unsubstituted. If said groups are substituted, said groups preferably contain up to 4, more preferably up to 3, more preferably still up to 2, and most preferably 1 substituent according to Radical Group 1 as defined herein.

[0378] The general term "sugar" is herein used to indicate a monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and / or functional groups. Examples of a sugar derivative include amino sugars and sugar acids, e.g. glucosamine (GlcNH2), galactosamine (GalNH2) N- acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). A sugar may be without further substitution, and then it is understood to be a monosaccharide. A sugar may be further substituted with at one or more of its hydroxyl groups, and then it is understood to be a disaccharide or an oligosaccharide. A disaccharide contains two monosaccharide moieties linked together. An oligosaccharide chain may be linear or branched, and may contain from 3 to 10 monosaccharide moieties.

[0379] The term “amino acid” is used herein in its normal scientific meaning. In particular, amino acids in relation to the disclosure comprise both natural and unnatural amino acids. Preferably, amino acids as used herein are selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, azidolysine, beta-alanine (bAla), 4-aminomethyl phenylalanine (Amf), 4- guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl phenylalanine (laf), 3 -pyridyl alanine (Pya), 4-piperidyl alanine (Ppa), 4-aminomethyl cyclohexyl alanine (Ama), 4- aminocyclohexyl alanine (Aca), ornithine (Om), citrulline, hydroxylysine (Hyl), allohydroxylysine (aHyl), 6-N-methyllysine (MeLys), desmosine (Des), isodesmosine (Ide), 2- aminoadipic acid (Aad), 3-aminoadipic acid (bAad), 2-aminobutyric acid (Abu), 4- aminobutyric acid (4Abu), 6-aminohexonic acid (Acp), 2-aminoheptanoic acid (Ahe), 2- aminoisobutyric acid (Aib), 3 -aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2,4- diaminobutyric acid (Dbu), 2,2’-diaminopimelic acid (Dpm), 2-3 -diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), 3 -hydroxyproline (3Hyp), 4- hydroxyproline (4Hyp), allo-isoleucine (Alle), sarcosine (MeGly), N-methylisoleucine (Melle), N-methylvaline (MeVal), norvaline (Nva), and norleucine (Nle).

[0380] The term "protein" is herein used in its normal scientific meaning. Herein, polypeptides comprising about 10 or more amino acids are considered proteins. A protein may comprise natural, but also unnatural amino acids. The term “protein” herein is understood to comprise antibodies and antibody fragments.

[0381] The term “peptide” is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acids in a range of from 2 to 9. The term “peptoid” is herein used in its normal scientific meaning.

[0382] A spacer is herein defined as a moiety that connects two or more elements of a compound. The terms “spacer” and “linker” are used herein interchangeably. Typically, a spacer is herein denoted as SP, and the more specific self-immolative linkers as LC. It will be understood that when herein, it is stated that “each individual SPis linked at all ends to the remainder of the structure” this refers to the fact that the spacer SPconnects multiple moieties within a structure, and therefore the spacer has multiple ends by defintion. The spacer SPmay be linked to each individual moiety via different or identical moieties that may be each individually selected. Typically, these linking moieties are to be seen to be part of spacer S itself. In case the spacer SPlinks two moieties within a structure, “all ends” should be interpreted as “both ends”. As an example, if the spacer connects a trans-cylooctene moiety to a Construct B, then “the remainder of the molecule” refers to the trans-cylooctene moiety and Construct B, while the connecting moieties between the spacer and the trans-cyclooctene moiety and Construct B (i.e. at both ends) may be individually selected.

[0383] As used herein, a bioconjugation moiety is defined as a moiety that allows conjugation to a biomolecule, preferably to a protein, peptide, amino acid, or amino acid residue. Preferably, a bioconjugation moiety, for example T2in a compound of the disclosure, is selected from the group consisting of N-maleimidyl, halogenated N-alkylamido, sulfonyloxy N-alkylamido, vinyl sulfone, (activated) carboxylic acids, active ester, benzenesulfonyl halides, ester, carbonate, sulfonyl halide, thiol or derivatives thereof, C2-6alkenyl, C2-6alkynyl, C7-18cycloalkynyl, C5-18heterocycloalkynyl, bicyclo [6.1.0]non-4-yn-9-yl], C3-12cycloalkenyl, azido, phosphine, nitrile oxide, nitrone, nitrile imine, isonitrile, diazo, ketone, (O-alkyl)hydroxylamino, hydrazine, halogenated N-maleimidyl, aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2,5-dibromohexanediamide, alkynone, 3-arylpropiolonitrile, l,l-bis(sulfonylmethyl)-methylcarbonyl or elimination derivatives thereof, carbonyl halide, allenamide, 1,2-quinone, isothiocyanate, isocyanate, aldehyde, triazine, squaric acids, 2-imino-2-methoxyethyl, (oxa)norbomene, (oxa)norbomadiene, (imino)sydnones, methylsulfonyl phenyloxadiazole, aminooxy, 2-amino benzamidoxime, ethynylphosphonamidates, reactive in the Pictet-Spengler ligation and hydrazine- Pictet-Spengler (HIPS) ligation, DNA intercalators, tetrazine, trans-cyclooctene, and photocrosslinkers. More preferably, the bioconjugation moiety is selected from the group consisting of N-maleimidyl, halogenated N-alkylamido, sulfonyloxy N-alkylamido, vinyl sulfone, carboxylic acids, benzenesulfonyl halides, ester, carbonate, sulfonyl halide, thiol, C2-6alkenyl, C2-6alkynyl, C7-18cycloalkynyl, C5-18heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9- yl], C3-12cycloalkenyl, azido, phosphine, nitrile oxide, nitrone, nitrile imine, isonitrile, diazo, ketone, (O-alkyl)hydroxylamino, hydrazine, halogenated N-maleimidyl, aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2,5-dibromohexanediamide, alkynone, 3-arylpropiolonitrile, l,l-bis(sulfonylmethyl)-methylcarbonyl, carbonyl halide, allenamide, 1,2-quinone, isothiocyanate, isocyanate, aldehyde, triazine, squaric acids, 2- imino-2-methoxyethyl, (oxa)norbomene, (oxa)norbomadiene, (imino)sydnones, methylsulfonyl phenyloxadiazole, aminooxy, 2-amino benzamidoxime, and ethynylphosphonamidates. Most preferably, the bioconjugation moiety is N-maleimidyl. Residues of these bioconjugation moieties are known to the skilled person.

[0384] As used herein, an organic molecule is defined as a molecule comprising a C-H bond. Organic compound and organic molecule are used synonymously.

[0385] As used herein, an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond. It will be understood that “inorganic molecule” typically also comprises hydrogen, -COOH, etc.

[0386] As used herein, a “small molecule” is preferably a small organic molecule. In general, a small molecule has a molecular weight of at most 2 kDa, more preferably at most 1 kDa, more preferably at most 750 Da, more preferably at most 500 Da, and most preferably at most 300 Da. Preferably, a small molecule has a molecular weight of at least 15 Da, more preferably at least 50 Da, more preferably at least 75 Da, and most preferably at least 100 Da.

[0387] As used herein, “particle” is preferably defined as a microparticle or a nanoparticle. The term "salt thereof" means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. The term "salt thereof" also means a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

[0388] The term "pharmaceutically accepted salt” means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.

[0389] "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc.

[0390] As used herein, the term “solvate” refers to a compound that apart from a main molecule (e.g. a compound of the disclosure, a diene, and the like) further includes a stoichiometric or non-stoichiometric amount of solvent bound to said main molecule by non- covalent intermolecular forces. In particular, the term “solvate” may refer to a crystalline compound, the crystal lattice structure of which contains one or more molecules of the solvent.

[0391] As used herein, the term “hydrate” refers to a compound that apart from a main molecule (e.g. a compound of the disclosure, a diene, and the like) further includes a stoichiometric or non-stoichiometric amount of water bound to said main molecule by non- covalent intermolecular forces. In particular, the term “hydrate” may refer to a crystalline compound, the crystal lattice structure of which contains one or more molecules of water.

[0392] The logarithm of the partition-coefficient, i.e. Log P, is herein used as a measure of the hydrophobicity of a compound. Typically, the Log P is defined as

[0393] The skilled person is aware of methods to determine the partition-coefficient of compounds without undue experimentation. Alternatively, the skilled person knows that software is available to reliably estimate the Log P value, for example as a function within ChemDraw® software or online available tools.

[0394] The unified atomic mass unit or Dalton is herein abbreviated to Da. The skilled person is aware that Dalton is a regular unit for molecular weight and that 1 Da is equivalent to 1 g / mol (grams per mole).

[0395] It will be understood that herein, the terms “moiety” and “group” are used interchangeably when referring to a part of a molecule.

[0396] It will be understood that when a heteroatom is denoted as -X(R’)2-, wherein X is the heteroatom and R’ is a certain moiety, then this denotes that two moieties R’ are attached to the heteroatom.

[0397] It will be understood that when a group is denoted as, for example, -((R51)2-R52)2- or a similar notation, in which R51and R52are certain moieties, then this denotes that first, it should be written as -R51-R51-R52-R51-R51-R52- before the individual R51 and R52moieties are selected, rather than first selecting moieties R51and R52and then writing out the formula.

[0398] As used herein, “activated carboxylic acid” and “active ester” may be used interchangeably. As the skilled person is aware, an “activated carboxylic acid” or an “active ester” is a derivative of a carboxylic acid (-C(O)OH) of which the -OH moiety has been exchanged for a better leaving group. Preferred activated carboxylic acids or active esters are selected from the group consisting of -C(O)O-N-succinimidyl, -C(O)O-pentafluorophenyl, - C(O)O-tetrafluorophenyl, -C(O)O-4-nitrophenyl, and -C(O)Cl. More preferably, the activated carboxylic acid or active ester is -C(O)O-N-succinimidyl, or -C(O)O-pentafluorophenyl.

[0399] As the skilled person is aware, an “active carbonate” is a derivative of a carbonate (-O- C(O)-OH) of which the -OH moiety has been exchanged for a better leaving group. Preferred active carbonates are -OC(O)O-N-succinimidyl, -OC(O)O-pentafluorophenyl, -OC(O)O- tetrafluorophenyl, -OC(O)O-4-nitrophenyl, and -OC(O)C1. More preferably, the active carbonate is -OC(O)O-N-succinimidyl, or -OC(O)O-pentafluorophenyl.

[0400] As used herein, a “drug” refers to a pharmaceutical agent. As such, “drug”, “pharmaceutical agent”, “therapeutic agent”, and “medicine” can typically be used interchangeably. Preferred drugs in relation to the disclosure are monomethyl auristatin E (MMAE), exatecan, and exatecan derivatives. Preferably, exatecan and exatecan derivatives have the following structure: wherein E1is -H, or an optionally substituted C1-C4alkyl group. It will be understood that when E1is -H, said structure is exatecan. Preferably, E1is -H, -CH3, or -C(O)- CH2-OH. If E1is - H or -CH3, then the exatecan or exatecan derivative is preferably linked to the remainder of R48via the nitrogen atom to which E1is attached. If E1is C(O)-CH2-0H, then the exatecan derivative is preferably linked to the remainder of R48via the oxygen atom that is part of the hydroxyl group of E1. More preferably, E1is -H or -CH3. Most preferably, E1is -H. Most preferably, the drug is monomethyl auristatin E (MMAE).

[0401] Radical groups (RG)

[0402] Radical Group 1: terminal groups

[0403] For Radical Group 1 (RG1), the radical is selected from the group consisting of -H, - Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -CN, -N3, -NCS, -SCN, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, -CF2H, -CFH2, =O, =NH, -SH, -SO2H, -(SP)i-CB, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, and combinations thereof. Herein, SPis a spacer as defined herein, CBis Construct B as defined herein, and i is an integer in a range of from 0 to 4, preferably i is 0 or 1.

[0404] For RG1, “combinations thereof" in particular refers to (hetero)alkylcycloalkyl, (hetero)alkylcycloalkenyl, (hetero)alkylcycloalkynyl, (hetero)cycloalkylalkyl, (hetero)cycloalkenylalkyl, (hetero)cycloalkynylalkyl, (hetero)alkenylcycloalkyl, (hetero)alkenylcycloalkenyl, (hetero)alkenylcycloalkynyl, (hetero)cycloalkylalkenyl, (hetero)cycloalkenylalkenyl, (hetero)cycloalkynylalkenyl, (hetero)alkynylcycloalkyl, (hetero)alkynylcycloalkenyl, (hetero)alkynylcycloalkynyl, (hetero)cycloalkylalkynyl, (hetero)cycloalkenylalkynyl, (hetero)cycloalkynylalkynyl, (hetero)arylalkyl, (hetero)arylalkenyl, (hetero)arylalkynyl, alkyl(hetero)aryl, alkenyl(hetero)aryl, alkynyl(hetero)aryl, cycloalkyl(hetero)aryl, cycloalkenyl(hetero)aryl, cycloalkynyl(hetero)aryl, (hetero)arylcycloalkyl, (hetero)arylcycloalkenyl, and (hetero)arylcycloalkynyl. In addition, “combinations thereof" in relation to RG1 also refers to e.g. an alkyl group substituted with one or more -Cl and / or -OH groups. As such, RG1 also comprises radicals such as -NH-CH2-C00H (a glycine residue), which is a combination of a heteroalkyl and -COOH.

[0405] Preferably, for RG1 the radical is selected from the group RGla consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C24(hetero)alkyl, C2-C24(hetero)alkenyl, C2-C24(hetero)alkynyl, C3-C24cycloalkyl, C2-C24heterocycloalkyl, C5-C24cycloalkenyl, C3-C24heterocycloalkenyl, C7-C24cycloalkynyl, C5-C24(hetero)cycloalkynyl, C6-C24aryl, C2-C24heteroaryl, and combinations thereof.

[0406] More preferably, for RG1 the radical is selected from the group RGlb consisting of - H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C12(hetero)alkyl, C2-C12(hetero)alkenyl, C2-C12(hetero)alkynyl, C3- C12cycloalkyl, C2-C12heterocycloalkyl, C5-C12cycloalkenyl, C3-C12heterocycloalkenyl, C7- C12cycloalkynyl, C5-C12(hetero)cycloalkynyl, C6-C12aryl, C2-C12heteroaryl, and combinations thereof.

[0407] Even more preferably, for RG1 the radical is selected from the group RGlc consisting of-H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C8(hetero)alkyl, C2-C8(hetero)alkenyl, C2-C8(hetero)alkynyl, C3-C8cycloalkyl, C2-C8heterocycloalkyl, C5-C8cycloalkenyl, C3-C8heterocycloalkenyl, C7-C8cycloalkynyl, C5-C8(hetero)cycloalkynyl, C6-C8aryl, C2-C8heteroaryl, and combinations thereof.

[0408] More preferably still, for RG1 the radical is selected from the group RGld consisting of-H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C6(hetero)alkyl, C2-C6(hetero)alkenyl, C2-C6(hetero)alkynyl, C3-C6cycloalkyl, C2-C6heterocycloalkyl, C5-C7cycloalkenyl, C3-C5heterocycloalkenyl, C8cycloalkynyl, C6-C7(hetero)cycloalkynyl, phenyl, C3-C5heteroaryl, and combinations thereof.

[0409] Most preferably, for RG1 the radical is selected from the group RGle consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H, -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C3(hetero)alkyl, C3-C6cycloalkyl, C2-C5heterocycloalkyl, phenyl, C4-C5heteroaryl, and combinations thereof.

[0410] In some embodiments, for RG1 the radical is a conjugation moiety, which is a chemical group that can be used for binding, conjugation or coupling of a Construct, such as Construct-B, or a Spacer, or another molecule or construct of interest. The person skilled in the art is aware of the myriad of strategies that are available for the chemoselective or -unselective or enzymatic coupling or conjugation of one molecule or construct to another.

[0411] In some embodiments, RG1 is a moiety that allows conjugation to a protein comprising natural and / or non-natural amino acids. Moieties suitable for conjugation are known to the skilled person. Conjugation strategies are for example found in [O. Boutureira, G.J.L. Bemardes, Chem. Rev., 2015, 115, 2174-2195].

[0412] If RG1 is a conjugation moiety, it is preferably selected from the group RGlf consisting of N-maleimidyl, halogenated N-alkylamido, sulfonyloxy N-alkylamido, vinyl sulfone, (activated) carboxylic acids, active ester, benzenesulfonyl halides, ester, carbonate, sulfonyl halide, thiol or derivatives thereof, C2-6alkenyl, C2-6alkynyl, C7-18cycloalkynyl, C5-18heterocycloalkynyl, bicyclo [6.1.0]non-4-yn-9-yl], C3-12cycloalkenyl, azido, phosphine, nitrile oxide, nitrone, nitrile imine, isonitrile, diazo, ketone, (O-alkyl)hydroxylamino, hydrazine, halogenated N-maleimidyl, aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2,5-dibromohexanediamide, alkynone, 3-arylpropiolonitrile, l,l-bis(sulfonylmethyl)-methylcarbonyl or elimination derivatives thereof, carbonyl halide, allenamide, 1,2-quinone, isothiocyanate, isocyanate, aldehyde, triazine, squaric acids, 2- imino-2-methoxyethyl, (oxa)norbomene, (oxa)norbomadiene, (imino)sydnones, methylsulfonyl phenyloxadiazole, aminooxy, 2-amino benzamidoxime, ethynylphosphonamidates, reactive in the Pictet-Spengler ligation and hydrazine- Pictet-Spengler (HIPS) ligation, DNA intercalators, tetrazine, trans-cyclooctene, and photocrosslinkers. More preferably, RGlf is N-maleimidyl.

[0413] In other embodiments RGlf is selected from the group consisting of hydroxyl, amine, halogens, vinyl pyridine, disulfide, pyridyl disulfide, sulfonyloxy, mercaptoacetamide, anhydride, sulfonylated hydroxyacetamido, sulfonyl chlorides, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In yet other embodiments RGlf is a group that can be connected to another group by means of an enzyme, for example sortase or Tubulin tyrosine ligase.

[0414] Radical Group 2: connecting groups

[0415] For Radical Group 2 (RG2), the radical is selected from the group consisting of (hetero)alkylene, (hetero)alkenylene, (hetero)alkynylene, (hetero)cycloalkylene, (hetero)cycloalkenylene, (hetero)cycloalkynylene, (hetero)arylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.

[0416] The radicals from RG2 are optionally attached to one or more radicals according to RG1. Thus, RG2 also covers e.g. -NH-CH(CH2OH)-C(O)- (i.e. a serine residue), which is a heteroalkylene attached to -OH and =O.

[0417] For RG2, “combinations thereof" in particular, but not exclusively, refers to alkyl(hetero)arylene, (hetero)arylalkylene, (hetero)arylalkenylene, (hetero)arylalkynylene, alkenyl(hetero)arylene, and alkynyl(hetero)arylene.

[0418] Preferably, for RG2 the radical is selected from the group consisting of C1-C24(hetero)alkylene, C2-C24(hetero)alkenylene, C2-C24(hetero)alkynylene, C3-C24cycloalkylene, C2-C24heterocycloalkylene, C5-C24cycloalkenylene, C3-C24heterocycloalkenylene, C7-C24cycloalkynylene, C5-C24(hetero)cycloalkynylene, C6-C24arylene, C2-C24heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof. More preferably, for RG2 the radical is selected from the group consisting of C1-C12(hetero)alkylene, C2-C12(hetero)alkenylene, C2-C12(hetero)alkynylene, C3-C12cycloalkylene, C2-C12heterocycloalkylene, C5-C12cycloalkenylene, C3-C12heterocycloalkenylene, C7-C12cycloalkynylene, C5-C12(hetero)cycloalkynylene, C6-C12arylene, C2-C12heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.

[0419] Even more preferably, for RG2 the radical is selected from the group consisting of C1- C8(hetero)alkylene, C2-C8(hetero)alkenylene, C2-C8(hetero)alkynylene, C3-C8cycloalkylene, C2-C8heterocycloalkylene, C5-C8cycloalkenylene, C3-C8heterocycloalkenylene, C7-C8cycloalkynylene, C5-C8(hetero)cycloalkynylene, C6-C8arylene, C2-C8heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.

[0420] More preferably still, for RG2 the radical is selected from the group consisting of C1- C6(hetero)alkylene, C2-C6(hetero)alkenylene, C2-C6(hetero)alkynylene, C3-C6cycloalkylene, C2-C6heterocycloalkylene, C5-C7cycloalkenylene, C3-C5heterocycloalkenylene, C8cycloalkynylene, C6-C7(hetero)cycloalkynylene, phenylene, C3-C5heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.

[0421] Even more preferably still, for RG2 the radical is selected from the group consisting of C1-C3(hetero)alkylene, C3-C6cycloalkylene, C2-C5heterocycloalkylene, phenylene, C4-C5heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.

[0422] RG2a is selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N=N-, -C(O)-, - C(O)NR4-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, - NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -S(O)-, - S(O)2-, -OS(O)2-, -S(O2)O-, -OS(O)2O-, -OS(O)2NR4-, -NR4S(O)2O-, -C(O)NR4S(O)2NR4-, - OC(O)NR4S(O)2NR4-, -OS(O)-, -OS(O)O-, -OS(O)NR4-, -ONR4C(O)-, -ONR4C(O)O-, - ONR4C(O)NR4-, -NR4OC(O)-, -NR4OC(O)O-, -NR4OC(O)NR4-, -ONR4C(S)-, -ONR4C(S)O- , -ONR4C(S)NR4-, -NR4OC(S)-, -NR4OC(S)O-, -NR4OC(S)NR4-, -OC(S)-, -C(S)O-, - OC(S)O-, -OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, -SS(O)2-, -S(O)2S-, -OS(O2)S-, -SS(O)2O-, - NR4OS(O)-, -NR4OS(O)O-, -NR4OS(O)NR4-, -NR4OS(O)2-, -NR4OS(O)2O-, - NR4OS(O)2NR4-, -ONR4S(O)-, -ONR4S(O)O-, -ONR4S(O)NR4-, -ONR4S(O)2O-, - ONR4S(O)2NR4-, -ONR4S(O)2-, -OP(O)(R4)2-, -SP(O)(R4)2-, and -NR4P(O)(R4)2-. Herein, R4is according to RG1, preferably R4is hydrogen or methyl, more preferably R4is hydrogen.

[0423] Preferably, RG2a is selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N=N- , -C(O)-, -C(O)NR4-, -OC(O)-, -C(O)O-, -0C(O)NR4-, -NR4C(O)-, -NR4C(O)O-, - NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -S(O)-, - S(O)2-, -C(O)NR4S(O)2NR4-, -OC(O)NR4S(O)2NR4-, -OC(S)-, -C(S)O-, -OC(S)O-, - OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, and -SS(O)2-.

[0424] More preferably, for RG2 the radical is RG2b or RG2c, most preferably RG2b.

[0425] RG2b is selected from the group consisting of

[0426] Therein, R' is a radical according to RG1, preferably R’ is hydrogen or C1-3alkyl. The dashed and wiggly lines denote bonds to the other parts of the molecule.

[0427] RG2c is selected from the group consisting of

[0428] Therein, R' is a radical according to RG1, preferably R’ is hydrogen or C1-3alkyl. The dashed and wiggly lines denote bonds to the other parts of the molecule.

[0429] Radical Group 3: organic molecule

[0430] For Radical Group 3 (RG3) the radical is an organic molecule selected from the group consisting of a nucleic acid, a peptide, a protein, a carbohydrate, an aptamer, a hormone, a toxin, a steroid, a cytokine, a lipid, a small organic molecule as defined herein, a polymer, LNA, PNA, an amino acid, a peptoid, a chelating moiety, a molecule comprising a radionuclide, a fluorescent dye, a phosphorescent dye, a drug, a resin, a bead, an organic particle, a gel, an organic surface, an organometallic compound, a cell, and combinations thereof.

[0431] Preferably, for RG3 the radical is a a nucleic acid, a peptide, a protein, a carbohydrate, a lipid, a polymer, an amino acid, a chelating moiety, a drug, or a gel.

[0432] As used herein, a nucleic acid is preferably selected from the group consisting of an oligonucleotide, a polynucleotide, DNA, and RNA.

[0433] As used herein, a protein is preferably an antibody or a diabody. A preferred antibody is CC49, and a preferred diabody is AVP0458.

[0434] As used herein, a carbohydrate is preferably selected from the group consisting of a monosaccharide, an oligosaccharide, and a polysaccharide.

[0435] As used herein, a polymer is typically selected from the group consisting of polyethyleneglycol (PEG), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF), copolymers of a polyacetal / polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof, oligopeptides, polypeptides, glycopolysaccharides, and polysaccharides such as dextran and hyaluronan. Preferably, a polymer as used herein is polyethylene glycol (PEG).

[0436] As used herein, a resin is preferably a polystyrene resin or an agarose resin.

[0437] As used herein, an organic particle is preferably a liposome or a polymersome.

[0438] As used herein, a chelating moiety is preferably selected from the group consisting of DTP A (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane- N,N\N",N" -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid), TETA (1, 4, 8, l l-tetraazacyclotetradecane-N,N',N",N' -tetraacetic acid), OTTA (Nl-(p- isothiocyanatobenzyl)-diethylenetriamine-Ni,N2,N3,N3-tetraacetic acid), deferoxamine or DFA (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-l ,4- dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide) or HYNIC (hydrazinonicotinamide), EDTA (ethylenediaminetetraacetic acid), OTAM, TACN, sarcophagine, and 3,4-HOPO-based chelators.

[0439] More preferably, herein a chelating moiety is selected from the group consisting of wherein the wiggly line denotes a bond to the remaining part of the molecule, optionally bound via -C(O)NH-, wherein the chelator moieties according to said group optionally chelate a metal, wherein the metal is preferably selected from the group consisting of44Sc,62Cu,64Cu,66Ga,67Ga,67Cu,68Ga,86Y,89Zr,90Y,99mTc,111In,166Ho,177Lu,186Re,188Re,211Bi,212Bi,212Pb,213Bi,214Bi, and225Ac. Radical Group 4: inorganic molecule

[0440] For Radical Group 4 (RG4), the radical is an inorganic molecule selected from the group consisting of an inorganic surface, an inorganic particle, an allotrope of carbon, an inorganic drug, a radionuclide, and combinations thereof.

[0441] As used herein, an inorganic surface is preferably selected from the group consisting of chips, wafers, metal such as gold, and silica-based surfaces such as glass.

[0442] As used herein, an inorganic particle is preferably selected from the group consisting of beads, silica-based particles, polymer-based materials, and iron oxide particles. Preferably, a bead is a magnetic bead or a gold bead.

[0443] As used herein, an allotrope of carbon is preferably selected from the group consisting of fullerenes such as Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q- carbon, lineam acetylenic carbon, amorphous carbon, and carbon nanotubes.

[0444] As used herein, an inorganic drug is preferably cisplatin.

[0445] Radical group 5: further terminal groups

[0446] For RG5 the radical is: wherein the dashed line indicates a bond to the remaining part of the dienophile or diene.

[0447] For RG5, each R10is independently selected from RG2, preferably from RG2a.

[0448] For RG5, each R11is independently selected from RG2, preferably not being RG2a, RG2b, or RG2c.

[0449] For RG5, R12is selected from RG1 or RG3, preferably RG3, more preferably a protein, polymer, or chelating moiety.

[0450] Preferably, z is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4. Preferably, z is 0. In case the compound according to the disclosure comprises more than one moiety RG5, each z is independently selected.

[0451] Preferably, h is 0 or 1. In case the compound according to the disclosure comprises more than one moiety RG5, each h, z, and n is independently selected. Preferably, each n belonging to RG5 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3. Preferably, n is 1. In other preferred embodiments n is an integer in the range from 12 to 24. Preferably, z is 0, and n is 1. In other embodiments, z is 1, and n is 1. Preferably, the moiety RG5 has a molecular weight in a range of from 100 Da to 3000 Da, preferably, in a range of from 100 Da to 2000 Da, more preferably, in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, the moiety RG5 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.

[0452] Preferably, RG5 is selected from the group RG5a consisting of:

[0453]

[0454] , wherein the wiggly line denotes a bond to the remainder of the molecule.

[0455] It is understood that when n is more than 1, -((R10)h-R11)n-(R10)h-R12may be preceded by a group -(R10)h-R11- so as to form a group -(R10)h-R11-((R10)h-R11)n-(R10)h-R12. It is understood that this follows from the definition of how to write out the repeating units, i.e. -((R10)h-R11)2- would first be written as -(R10)h-Rii-(R10)h-R11- before R10, h, and R11are independently selected.

[0456] List of Clauses

[0457] The disclosure also relates to the following Clauses, which may be combined with any of the embodiments, preferences, and the like, as disclosed herein:

[0458] Clause 1. A composition comprising

[0459] (i) a buffer; and

[0460] (ii) a compound having a structure according to Formula (1) and / or a conjugate thereof; wherein Formula (1) is: wherein L1is selected from the group consisting of linear or branched C4-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4- C11heteroarylene; L2a, L2b, and L2dare each independently a linker; L2cis selected from the group consisting of C1-C8(hetero)alkanetriyl, C5-C6(hetero)arenetriyl, C3-C7cycloalkanetriyl, and C2-C7heterocycloalkanetriyl; T1is selected from the group consisting of -OT1A, hydrogen, C2-C6alkyl, C6aryl, C4-C5heteroaryl, C3-C6cycloalkyl, C5-C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, - C(O)T1A, -C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3; each T1Ais independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue; T2is a bioconjugation moiety or a group -L3-CB; wherein L3is a residue of a bioconjugation moiety, and CBis selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof; T3is a polymer; and R48is selected from the group consisting of -OH, -O-acetyl, -O-C1-4alkyl, halogen, active carbonate, and a releasable group; wherein the conjugate comprises a protein conjugated to at least one compound according to Formula (1), wherein in the conjugate T2is a residue of a bioconjugation moiety, and in the conjugate said protein and said compound are conjugated via T2; and preferably L1is linear or branched C4-C12alkylene, more preferably L1is linear or branched C4-C10alkylene, and most preferably L1is linear C5-C6alkylene; preferably L2a, L2b, and L2dare each independently a linker containing at most twenty atoms; more preferably L2a, L2b, and L2dare each independently selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl, preferably L2Tis hydrogen; preferably L2cis C1-C8(hetero)alkanetriyl, more preferably L2cis C1-C8alkanetriyl, and most preferably L2cis C4-C6alkanetriyl; preferably T1is -OT1A; and most preferably T1is -OH; preferably T1Ais hydrogen or methyl, more preferably T1Ais hydrogen; preferably T2is maleimidyl, N-hydroxysuccinimidyl, or -L3-CB; preferably L3is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety; preferably CBis a protein, more preferably CBis an antibody or a diabody, even more preferably CBis a diabody, and most preferably CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably T3is a polymer comprising a polyethylene glycol moiety; preferably R48is a releasable group; preferably in the conjugate the protein is a diabody or an antibody; more preferably in the conjugate the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably in the conjugate the protein is conjugated to at most 12 of said compounds; more preferably in the conjugate the protein is conjugated to at most 8 of said compounds, most preferably in the conjugate the protein is conjugated to at most 4 of said compounds; preferably in said conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein, a residue of a hydroxyl of said protein, or a residue of an amine of said protein; more preferably in the conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein; preferably in the conjugate T2is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety; more preferably T2is a residue of a maleimidyl moiety; preferably the buffer is an aqueous solution, and more preferably the composition is an aqueous solution of the compound and / or conjugate in the buffer; most preferably the composition is an aqueous solution of the conjugate in the buffer.

[0461] Clause 2. The composition according to Clause 1, wherein the buffer has a pH of at most 6.7; preferably the buffer has a pH in a range of from 4.6 to 6.7.

[0462] Clause 3. The composition according to any one of the preceding Clauses, wherein the buffer comprises a buffer compound; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; more preferably the buffer compound is citrate; even more preferably the buffer compound is sodium citrate.

[0463] Clause 4. The composition according to any one of the preceding Clauses, wherein the buffer comprises a carbohydrate; preferably the carbohydrate is sucrose, or trehalose; most preferably the carbohydrate is sucrose. Clause 5. The composition according to any one of the preceding Clauses, wherein the buffer comprises a chelator; preferably the chelator is ethylenediaminetetraacetic acid (EDTA).

[0464] Clause 6. The composition according to any one of the preceding Clauses, wherein the buffer comprises a surfactant; preferably the surfactant is polysorbate 20 or polysorbate 40; most preferably the surfactant is polysorbate 20.

[0465] Clause 7. The composition according to any one of the preceding Clauses, wherein the buffer comprises:

[0466] (i) a buffer compound;

[0467] (ii) a carbohydrate;

[0468] (iii) a chelator; and

[0469] (iv) a surfactant; and wherein the buffer has has a pH of at most 6.7; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; preferably the carbohydrate is sucrose, or trehalose; preferably the chelator is ethylenediaminetetraacetic acid (EDTA); preferably the surfactant is polysorbate 20 or polysorbate 40; and preferably the buffer has a pH in a range of from 4.6 to 6.7.

[0470] Clause 8. The composition according to any one of the preceding Clauses, wherein the composition comprises (i) the buffer; and (ii) the conjugate; preferably the composition essentially consists of (i) the buffer; and (ii) the conjugate.

[0471] Clause 9. The composition according to any one of the preceding Clauses, wherein the conjugate is according to Formula (C1):

[0472] wherein CBis a protein; T2is a residue of a bioconjugation moiety; CJ is in a range of from 1 to 12; R48is a releasable group; T1, T3, L1, L2a, L2b, L2c, and L2dare as defined in Clause 1; preferably the protein is a diabody or an antibody; more preferably the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4.

[0473] Clause 10. The composition according to Clause 9, wherein said conjugate is according to Formula (C2): wherein y is an integer in a range of from 1 to 50; preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, and most preferably in a range of from 23 to 25. Clause 11. The composition according to Clause 10, wherein said conjugate is according to Formula (C3): wherein x is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6.

[0474] Clause 12. The composition according to any one of the preceding Clauses, wherein R48is a releasable group, and said releasable group is -O-CO-CA; wherein CAis a drug; preferably the drug is linked to the moiety -O-CO- via a secondary or tertiary nitrogen atom that is part of the drug, forming a carbamate; preferably the drug is monomethyl auristatin E (MMAE).

[0475] Clause 13. The composition according to any one of the preceding Clauses, wherein the conjugate is wherein CJ is in a range of from 1 to 12; wherein CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4; preferably CBis linked to each maleimidyl group via a sulfur atom, preferably the sulfur atom is part of a cysteine.

[0476] Clause 14. The conjugate according to Clause 13, wherein the conjugate is or

[0477] Clause 15. The composition according to any one of the preceding Clauses, wherein the composition is a pharmaceutical composition; preferably a pharmaceutical composition suitable for intravenous injection.

[0478] Clause 16. A combination of

[0479] (A1) a conjugate as defined in any one of Clauses 1 to 14; and / or

[0480] (A2) a compound as defined in Clause 1; with

[0481] (A3) a buffer according to any one of Clauses 1 to 6; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3).

[0482] Clause 17. The combination according to Clause 16, wherein the combination is of

[0483] (A1) a conjugate as defined in any one of Clauses 1 to 14; with

[0484] (A3) a buffer according to any one of Clauses 1 to 7; preferably the combination is a kit wherein (A1) is physically separated from (A3); and preferably the combination essentially consists of (A1) and (A3) .

[0485] Clause 18. The combination of any one of Clauses 16 and 17 wherein the combination further comprises: (B) a diene; preferably the diene is a tetrazine; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3) and (B), and wherein (A3) is physically separated from (B).

[0486] Clause 19. The combination according to Clause 18, wherein the diene is selected from the group consisting of:

[0487] Clause 20. The composition according to any one of Clauses 1 to 15; or the combination according to any one of Clauses 16 to 19; for use as a medicament.

[0488] Clause 21. The composition according to any one of Clauses 1 to 15; or the combination according to any one of Clauses 16 to 19; for use in the treatment of a disease in a subject, preferably the subject is a human; preferably the disease is cancer.

[0489] Clause 22. A method of treating a disease in a subject, wherein said method comprises the step of administering to said subject:

[0490] (a) the composition according to any one of Clauses 1 to 15; and / or

[0491] (b) the combination according to any one of Clauses 16 to 19; preferably the subject is a human; preferably the disease is cancer.

[0492] Clause 23. A method for preparing a dried composition according to any one of Clauses 1 to 15, wherein said method comprises the step of drying said composition; preferably the drying is carried out by lyophilization.

[0493] Clause 24. A dried composition obtainable by the method of Clause 23; wherein preferably the dried composition is a lyophilized composition.

[0494] Clause 25. A method for preparing a composition according to any one of Clauses 1 to 15; wherein said method comprises the step of:

[0495] (a) contacting a buffer as defined in any one of Clauses 1 to 7 with a compound and / or a conjugate as defined in any one of Clauses 1, and 8 to 15; and / or the step of (b) reconstituting the dried composition of Clause 24 with water; preferably the method comprises the step of contacting a buffer as defined in any one of Clauses 1 to 7 with a conjugate as defined in any one of Clauses 1, and 8 to 15.

[0496] Clause 26. A method of storing the composition according to any one of Clauses 1 to 15, wherein the method comprises the steps of:

[0497] (a) providing a composition according to any one of Clauses 1 to 15;

[0498] (b) maintaining said composition at a temperature of at most 40°C; preferably the temperature is at most 35 °C; more preferably, the temperature is in a range of from -100°C to 35°C.

[0499] Clause 27. Use of a buffer as defined in any one of Clauses 1 to 7 to store a compound and / or a conjugate as defined in any one of Clauses 1, and 8 to 15; preferably the use is to store said conjugate.

[0500] Clause 28. Use of a buffer as defined in any one of Clauses 1 to 7 to stabilize a compound and / or a conjugate as defined in any one of Clauses 1, and 8 to 15, during storage; preferably the use is to stabilize said conjugate; preferably during storage the buffer and the compound and / or conjugate are maintained at a temperature of at most 40°C; more preferably the temperature is at most 35 °C; even more preferably, the temperature is in a range of from -100°C to 35°C.

[0501] Examples

[0502] The following Examples do not limit the scope of the claims. It will be understood that embodiments as disclosed herein, which may differ from those shown in the Examples, are still within the claimed scope.

[0503] Example 1: Synthesis

[0504] Example 1a: compounds 1 and 2

[0505] Intermediate compound 3 was obtained in three steps with an overall yield of 29% using commercially available starting materials:

[0506] Compound 3.

[0507] Compounds 4 and 5 were synthesized by coupling compound 3 to maleimide-PEG4-COOH or maleimide-C5-COOH, respectively, using conventional coupling reagents. This afforded the desired products, viz. compounds 4 and 5, in high yield.

[0508] Compound 4 or 5, respectively, was conjugated to diabody AVP0458 to afford compound 1 or 2 following the optimized procedure by Rossin et al., Nature Communications (2018)9:1484.

[0509] Briefly, 10 mg AVP0458 was reacted with 6 mM DTT for 3 h at room temperature on a roller bench, followed by purification via PD-10 pre-equilibrated with 0.1 M phosphate buffer pH 6.8, containing 2 mM EDTA (PB-EDTA buffer). The purified diabody solution was then split in two aliquots which were then added with 30 eq of compound 4 or compound 5, respectively, dissolved in dry DMSO at 10 mM concentration. The two reaction mixtures were incubated for Ih at room temperature on a roller bench followed by overnight incubation at +4°C. The two resulting conjugates ADCs (compound 1, conjugate of AVP0458 and compound 4; and compound 2, conjugate of AVP0458 and compound 5) were then purified from the crude mixtures by SEC (Superdex75 10 / 300 column eluted with PBS at 0.8 mL / min) followed by concentration via Amicon Ultra-4 (30 kDa MW cut-off). UV measurements on the final solutions showed 75-80% recovery of diabody. SDS-PAGE analysis of the two ADC solutions showed the presence of one species with the expected increase in MW with respect to that of the monomer in AVP0458. Further analysis using mass spectrometry showed a complete reaction between the four cysteine residues in AVP0458 with the respective TCOs, confirming the production of two conjugates with a DAR of 4.

[0510] Compound 1 has the following structure:

[0511] Compound 2 has the following structure:

[0512] Example 1b: reference compounds 14a and 14b and claimed compounds 15a and 15b

[0513] Reference compounds 14a and 14b were synthesized by conjugating compound Ila or 11b, respectively, to the diabody AVP0458. Likewise, claimed compounds 15a and 15b were synthesized by conjugating compound 13a or 13b, respectively, to the diabody AVP0458. Below, first the synthesis of Ila and 11b is described, and then the synthesis of 13a and 13b. Thereafter, the conjugation is described of Ila, 11b, 13a, or 13b to AVP0458 to yield 14a, 14b, 15a, or 15b.

[0514] Example 1b-i: synthesis of compounds 11a and 11b

[0515] Compound Ila was prepared in several steps in situ. To a suspension of exatecan mesylate (7) (287 mg, 0.54 mmol, MedChemExpress) in 6 mL of anhydrous dimethylformamide (DMF) in a glass vial was added compound 8 (506 mg, 0.90 mmol) and diethylamine (DIEA; 313 μL, 1.80 mmol). Compound 8 is a bis-axial variant, viz. wherein both the -OH group and the -OC(O)-O-pentafluorophenyl group at the allylic position are in an axial position. Compound 8, and diastereomers thereof, can for example be synthesized and isolated as disclosed by WO 2022 / 197182, in particular Examples 2 and 3 thereof.

[0516] The mixture was stirred at room temperature in the dark for 2 h, at which point LC- MS analysis indicated complete consumption of 7a and formation of intermediate 9a. The excess of compound 8 was quenched by addition of N-isopropylmethylamine (73 mg, 1.0 mmol) and stirring at room temperature in the dark for 2 h. To this reaction mixture was added a solution of compound 10 (trifluoroacetic acid salt, 1555 mg, 0.90 mmol) and DIEA (312 μL, 1.80 mmol) in 4 mL of anhydrous DMF. The reaction mixture was stirred at room temperature in the dark for 2 h. The mixture was purified by preparative RP-HPLC (HPLC conditions: solvent A (0.05% TFA in water), solvent B (acetonitrile), 10 to 65% of B over 30 min at a rate of 50 mL / min, elution time 24 min). The collected fractions were analyzed by HPLC / LC-MS. The pure fractions were lyophilized in the dark to give compound Ila. (234 mg, 19 % yield from 7). LCMS m / z 1122.7 ((M+2H) / 2).

[0517] Compound 11b is prepared in several steps in situ. To N-methyl exatecan (7b, 1 eq) in 6 mL of anhydrous dimethylformamide (DMF) in a glass vial is added compound 8 (1 eq) and diethylamine (4 eq). The mixture is stirred at room temperature in the dark for 11 days. To this reaction mixture is added a solution of compound 10 (trifluoroacetic acid salt, 1 eq) and DIEA (4 eq) in 4 mL of anhydrous DMF. The reaction mixture is stirred at room temperature in the dark for 2 h. The mixture is purified by preparative RP-HPLC (HPLC conditions: solvent A (0.05% TFA in water), solvent B (acetonitrile), 10 to 65% of B over 30 min at a rate of 50 mL / min. The collected fractions are analyzed by HPLC / LC-MS and pure fractions are lyophilized to give compound 11b.

[0518] Example 1b-ii: synthesis of compounds 13a and 13b

[0519] Compound 13a was prepared in several steps in situ. To a suspension of exatecan mesylate (7a) (287 mg, 0.54 mmol, MedChemExpress) in 6 mL of anhydrous DMF in a glass vial was added compound 8 (506 mg, 0.90 mmol) and DIEA (313 μL, 1.80 mmol). Compound 8 is a bis-axial variant, viz. wherein both the -OH group and the -OC(O)-O-pentafluorophenyl group at the allylic position are in an axial position. The mixture was stirred at room temperature in the dark for 2 h, at which point LC-MS analysis indicated complete consumption of 7a and formation of intermediate 9a. The excess of compound 8 was quenched by addition of N-isopropylmethylamine (73 mg, 1.0 mmol) and stirring at room temperature in the dark for 2 h. To this reaction mixture was added a solution of compound 12 (HC1 salt, 1300 mg, 0.90 mmol, Biomatrik) and DIEA (156 μL, 0.90 mmol) in 4 mL of anhydrous DMF. The reaction mixture was stirred at room temperature in the dark for 2 h.

[0520] The mixture was purified by preparative RP-HPLC (HPLC conditions: solvent A (0.05% TFA in water), solvent B (acetonitrile), 10 to 65% of B over 30 min at a rate of 50 mL / min, elution time 26 min). The collected fractions were analyzed by HPLC / LC-MS. The pure fractions were lyophilized in the dark to give compound 13a. (280 mg, 25 % yield from 7). LCMS m / z 1020.3 ((M+2H) / 2).

[0521] Compound 13b is prepared in several steps in situ. To N-methyl exatecan (7b, 1 eq) in 6 mL of anhydrous dimethylformamide (DMF) in a glass vial is added compound 8 (1 eq) and diethylamine (4 eq). The mixture is stirred at room temperature in the dark for 11 days. To this reaction mixture is added a solution of compound 12 (trifluoroacetic acid salt, 1 eq) and diethylamine (4 eq) in 4 mL of anhydrous DMF. The reaction mixture is stirred at room temperature in the dark for 2 h. The mixture is purified by preparative RP-HPLC (HPLC conditions: solvent A (0.05% TFA in water), solvent B (acetonitrile), 10 to 65% of B over 30 min at a rate of 50 mL / min. The collected fractions are analyzed by HPLC / LC-MS and pure fractions are lyophilized to give compound 13b.

[0522] Example 1b-iii: synthesis of compounds 14a, 14b, 15a, and 15b

[0523] Compound Ila, 11b, 13a, or 13b was conjugated to diabody AVP0458 to afford compound 14a, 14b, 15a, or 15b, respectively, following the optimized procedure by Rossin et al., Nature Communications (2018)9:1484.

[0524] Briefly, 2 mg AVP0458 was reacted with 6 mM DTT for 2 h at room temperature on a roller bench, followed by purification via PD-10 pre-equilibrated with 0.1 M phosphate buffer pH 6.8, containing 2 mM ETDA (PB-EDTA buffer). The purified diabody solution was then split in four aliquots which were then added with 24 eq of compound Ila, 11b, 13a, or 13b, respectively, dissolved in dry DMSO at 10 mM concentration. The four reaction mixtures were incubated for Ih at room temperature on a roller bench followed by overnight incubation at +4°C. The four resulting conjugates ADCs (compounds 14a, 14b, 15a, and 15b, conjugates of AVP0458 and compound Ila, 11b, 13a, or 13b, respectively) were then purified from the crude mixtures by SEC (Superdex75 10 / 300 column eluted with PBS at 0.8 mL / min) followed by concentration via Amicon Ultra-4 (30 kDa MW cut-off). UV measurements on the final solutions showed 60-78% recovery of diabody. SDS-PAGE analysis of the two ADC solutions showed the presence of one species with the expected increase in MW with respect to that of the monomer in AVP0458. Further analysis using mass spectrometry showed a complete reaction between the four cysteine residues in AVP0458 with the respective TCOs, confirming the production of two conjugates with a DAR of 4.

[0525] Conjugates 14a, 14b, 15a, and 15b have the following structures:

[0526]

[0527] Example 2; in vivo blood clearance in tumor-free mice

[0528] Six groups of tumor-free mice (n = 3 or 4 per group) were injected125I-labeled compound 1, 2, 14a, 15a, 14b, or 15b in a dosage of 5 mg / kg (for compounds 1, 2, 14a, and 15a) or 1 mg / kg (for compounds 14b and 15b). Blood samples (ca 50 μL) were withdrawn from the vena saphena at various times up to 72h post injection.

[0529] For experiments using compounds 1, 2, 14a, or 15a, after gamma-counting plasma isolated from blood was reacted ex vivo with an excess of tetrazine 6 for at least Ih at 37°C. Then, the samples were analyzed by SEC on a Superdex75 10 / 300 column eluted with PBS at 0.8 mL / min. The eluates were collected in 1 ml fractions which were then measured by gamma-counting using a dual-isotope protocol with crossover correction.

[0530] From the above exeperiments, the amount of compound 1, 2, 14a, 15a, 14b, or 15b in the blood of the mice 48 hours after injection could be determined, as well as the respective halflives in blood for said compounds. The results are shown in Table 1.

[0531] Table 1. In vivo blood clearance in tumor-free mice.

[0532] Thus, compound 2 advantageously and surprisingly has a faster clearance rate than compound 1. Likewise, compounds 15a and 15b advantageously and surprisingly have faster clearance rates than compounds 14a and 14b, respectively. Furthermore, it was observed that at least compounds 1, 2, 14a, and 15a showed high in vivo TCO stability.

[0533] Example 3: in vivo tumor and off-target binding of in tumour-bearing mice

[0534] Below, the in vivo tumor binding and off-target binding in tumor-bearing mice of compounds 1, 2, 14a, and 15a is described. The protocol for compounds 1 and 2 are discussed first, and then the protocol for compounds 14a and 15a. Thereafter, the results are presented in Table 1.

[0535] Example 3-i: protocol for compounds 1 and 2

[0536] Two groups of mice (n=5) bearing LS174T xenografts were injected compound 1 (reference) or compound 2 (2 mg / kg) followed 49h later by111In-labeled tetrazine 6 (10 eq with respect to

[0537] ADC). Three hours post tetrazine 6 injection, the mice were euthanized and blood, tumors and other tissues were harvested, weighed and counted (together with standards) in a gamma counter with dual isotope protocol with crossover correction. The125I counts were used to calculate the amounts of compounds 1 and 2 in the various tissues (as %ID / g).

[0538] Example 3-ii: protocol for compounds 14a and 15a

[0539] Two groups of mice (n=4) bearing LS174T xenografts were injected compound 14a

[0540] (reference) or compound 15a (2 mg / kg) The mice were euthanized 52 h post-ADC injection and blood, tumors and other tissues were harvested, weighed and counted together with standards. The125I counts were used to calculate the amounts of compounds 1 and 2 in the various tissues (as %ID / g).

[0541] The results of Example 3 are shown in Tables 2 and 3.

[0542] Table 2. Biodistribution of compounds in tumor-bearing mice.

[0543] Table 3. Biodistribution of compounds in tumor-bearing mice.

[0544] From Tables 2 and 3 it is clear that compound 2 shows a higher uptake in tumour and a lower off-target uptake than reference compound 1. Likewise, compound 15a shows a higher uptake in tumour and a lower off-target uptake than reference compound 14a. These results are highly advantageous, since the higher the tumour / off target ratio, the lower the extent of unwanted side-effects are usually observed.

[0545] Example 4; further in vitro and in vivo properties of compound 2

[0546] Other properties of compound 2 were tested as well:

[0547] 1. In vitro metabolism studies were carried out using compound 1 or 2 in the presence or absence of acidified human liver S9 fraction for up to 24 hours. From these studies, it was shown that compound 2 has a better metabolism profile than compound 1.

[0548] 2. An in vitro reaction of compound 2 with a standard tetrazine yielded quantitative MMAE release after 24 hours of incubation.

[0549] 3. At most 0.8% MMAE release was detected after 6 days of incubating compound 2 in mouse plasma in the absence of a tetrazine or any other trigger. Example 5: Preparation and stability of formulations of compound 2

[0550] Formulations of compound 2 were prepared and tested. In a first stage (see Example 5.1) certain buffers were prepared, stored, and analyzed. Based on the results from the first stage, several buffers were selected to proceed in the second stage (see Example 5.2), after which an optimal formulation was selected. In a third stage, an optimal formulation was tested for longterm stability.

[0551] Example 5.1 - first stage of storage experiments

[0552] Stock solutions of compound 2 were buffer exchanged into candidate buffers as shown in Table 4, using Cytiva NAP columns prepacked with Sephadex G-25 DNA grade. First solutions of compound 2 in candidate buffers were obtained, and the protein concentration in said first solutions was determined by UV-VIS. The first solutions were diluted using the appropriate candidate buffer to obtain second solutions wherein the target concentration was achieved of 2 mg / mL of compound 2. From these second solutions, 1 mL aliquots were generated in 2 mL microcentrifuge polypropylene tubes compliant with USP Class VI. All steps were performed under sterile conditions.

[0553] Table 4. Compositions of compound 2 tested in a first stage. All compositions comprise the buffer compound in a concentration of 20 mM; compound 2 in a concentration of 2 mg / mL and EDTA in a concentration of 1 mM.

[0554] In a first test, the aliquots were subjected to three freeze-thaw cycles, and analyzed using sizeexclusion chromatography (SEC). Of all buffers, only 1-1 and 1-2 showed increased aggregation as confirmed by the SEC analysis. Consequently, buffers 1-1 and 1-2 were not considered for stage 2.

[0555] In the below tests the aliquots were immediately analyzed, or stored for up to two weeks at a constant temperature of either 30°C, 2-8°C, or -80°C, and analyzed after for example 1 week and / or two weeks of storage.

[0556] When visually inspected immediately or after 1 week or 2 weeks of storage under all conditions, all formulations appeared liquid, clear, with no visible precipitate. The only exception was formulation 1-4 when stored at 30 °C, which appeared cloudy, and after centrifugation a precipitate was observed. Analysis using SEC-HPLC (size exclusion chromatography high-performance liquid chromatography) indicated that a significant decrease in protein monomer concentration was observed for this sample, but not for any of the other formulations (also not for formulation 1-4 stored at 2-8°C, or -80°C). Therefore, it was concluded that formulation 1-4 leads to aggregation of compound 2 when stored at 30 °C, and formulation 1-4 was not considered for stage 2.

[0557] The average drug-to-antibody ratio (avDAR) was monitored by HIC-HPLC (hydrophobic interaction chromatography - high-performance liquid chromatography), which showed no significant change in the avDAR value over the course of the two weeks for any of the formulations at all conditions. Free toxin-linker and toxin was monitored by RP-HPLC (reverse-phase high-performance liquid chromatography) by comparison to the profile determined immediately after preparing the formulations. If new peaks appeared, this would be a strong indication for drug-linker degradation. In fact, no additional peaks were detected. Combined, these tests demonstrated the stability of the toxin linker of compound 2 in the candidate formulations.

[0558] However, mass spectrometry analysis detected changes for compound 2 formulated in buffer 1-4, 1-5, 1-7, or 1-8. A peak corresponding to conjugates with 3 drugs attached went from being a distinct peak (viz. immediately after preparation) to a much broader peak (viz. after two weeks of storage), indicating some structural change or aggregation. Therefore, buffers 1-4, 1-5, 1-7, and 1-8 were not considered for stage 2.

[0559] The relative % area of the acidic species was monitored by cIEF (capillary isoelectric focusing). Buffers 1-3, 1-7, 1-8, and 1-9 were shown to produce a higher % area of acidic species compared to the other buffer systems, over the course of the two-week stability study. This was most noticeable in the samples of buffers 1-3, 1-7, 1-8, and 1-9, stored at +30°C. This was not seen in the other buffers. Therefore, buffers 1-3, 1-7, 1-8, and 1-9 were not considered for stage 2.

[0560] For buffer 1-6 all results were optimal, so therefore buffer 1-6 was selected for stage 2.

[0561] Example 5.2 - second stage of storage experiments

[0562] Stock solutions of compound 2 were prepared, wherein the stock solutions contained 20 mM citrate, and 0.1 mM EDTA, and had a pH of 5.5. The protein concentration in said stock solutions was determined by UV-VIS. The stock solutions were diluted using a combination of a solution containing 200 g / L sucrose or trehalose; and a solution containing 6 g / L polysorbate 20 or polysorbate 80; wherein the solutions contained 20 mM citrate, and 0.1 mM EDTA, and had a pH of 5.5. In this way, the compositions of Table 5 were prepared, wherein the compositions have a concentration of compound 2 of 5 mg / mL. From these second solutions, 1 mL aliquots were generated in 2 mL microcentrifuge polypropylene tubes compliant with USP Class VI. All steps were performed under sterile conditions.

[0563] Table 5. Compositions of compound 2 tested in the second stage. All compositions have a pH of 5.5, and comprise 20 mM citrate, 0.2 mg / mL of surfactant, 0.1 mMEDTA, and 5 mg / mL of compound 2.

[0564] The aliquots were immediately tested, or stored at a constant temperature of either 30°C, 2-8°C, or -80°C. The stability of compound 2 was tested over time using the techniques mentioned in Example 5.1. As confirmed by these tests, compound 2 was stable in all eight formulations (2-1 to 2-8) stored at 2-8°C, or -80°C. The % Monomer, protein concentration, avDAR and acidic species remain stable for at least eight weeks. There were signs of some degradation when formulations 2.1 to 2.8 were stored at 30°C for eight weeks, but the degree of degradation was comparable for all formulations.

[0565] Example 5.3 - third stage of storage experiments: long-term storage

[0566] Compound 2 was formulated at 5 mg / mL in 20 mM citrate, 146 mM (about 50 g / L) sucrose, 0.1 mM EDTA, 0.02% w / v polysorbate 20, pH 5.5 and stored in glass vials with stoppers and caps at -20°C, 5°C and 25°C±2°C / 50% relative humidity (RH). At -20°C and 5°C, the compound was stable for at least 9 months and at 25°C for at least 3 months based on the following analytical parameters: pH stability, polysorbate content, protein concentration, monomer and aggregation by SE-HPLC, main peak, high moleculare weight species and low molecular weight species by reduced CE-SDS and non-reduced CE-SDS, main peak by icIEF, Drug to Diabody ratio, TCO integrity, activity by binding, unconjugated diabody, residual linker-drug and related species, and subvisible particles.

Claims

1. Claims1. A composition comprising(i) a buffer; and(ii) a compound having a structure according to Formula (1) and / or a conjugate thereof; wherein Formula (1) is: whereinL1is selected from the group consisting of linear or branched C4-C12alkylene, C3-C8(hetero)cycloalkylene, C6-C12arylene, and C4-C11heteroarylene;L2a, L2b, and L2dare each independently a linker;L2cis selected from the group consisting of C1-C8(hetero)alkanetriyl, C5-C6(hetero)arenetriyl, C3-C7cycloalkanetriyl, and C2-C7heterocycloalkanetriyl;T1is selected from the group consisting of -OT1A, hydrogen, C2-C6alkyl, C6aryl, C4-C5heteroaryl, C3-C6cycloalkyl, C5-C12alkyl(hetero)aryl, C5-C12(hetero)arylalkyl, C4-C12alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A-C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3; each T1Ais independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue;T2is a bioconjugation moiety or a group -L3-CB; whereinL3is a residue of a bioconjugation moiety, andCBis selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof;T3is a polymer; andR48is selected from the group consisting of -OH, -O-acetyl, -O-C1-4alkyl, halogen, active carbonate, and a releasable group; wherein the conjugate comprises a protein conjugated to at least one compound according to Formula (1), wherein in the conjugate T2is a residue of a bioconjugation moiety, and in the conjugate said protein and said compound are conjugated via T2; and preferably L1is linear or branched C4-C12alkylene, more preferably L1is linear or branched C4-C10alkylene, and most preferably L1is linear C5-C6alkylene; preferably L2a, L2b, and L2dare each independently a linker containing at most twenty atoms; more preferably L2a, L2b, and L2dare each independently selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2Tis hydrogen or methyl, preferably L2Tis hydrogen; preferably L2cis C1-C8(hetero)alkanetriyl, more preferably L2cis C1-C8alkanetriyl, and most preferably L2cis C4-C6alkanetriyl; preferably T1is -OT1A; and most preferably T1is -OH; preferably T1is in an axial position; preferably T1Ais hydrogen or methyl, more preferably T1Ais hydrogen; preferably T2is maleimidyl, N-hydroxysuccinimidyl, or -L3-CB; preferably L3is a residue of a maleimidyl moiety or a residue of an N- hydroxysuccinimidyl moiety; preferably CBis a protein, more preferably CBis an antibody or a diabody, even more preferably CBis a diabody, and most preferably CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably T3is a polymer comprising a polyethylene glycol moiety; preferably R48is a releasable group; preferably R48is in an axial position; preferably in the conjugate the protein is a diabody or an antibody; more preferably in the conjugate the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably in the conjugate the protein is conjugated to at most 12 of saidcompounds; more preferably in the conjugate the protein is conjugated to at most 8 of said compounds, most preferably in the conjugate the protein is conjugated to at most 4 of said compounds; preferably in said conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein, a residue of a hydroxyl of said protein, or a residue of an amine of said protein; more preferably in the conjugate said protein and said compound are conjugated via T2and a residue of a sulfhydryl of said protein; preferably in the conjugate T2is a residue of a maleimidyl moiety or a residue of an N-hydroxysuccinimidyl moiety; more preferably T2is a residue of a maleimidyl moiety; preferably the buffer is an aqueous solution, and more preferably the composition is an aqueous solution of the compound and / or conjugate in the buffer; most preferably the composition is an aqueous solution of the conjugate in the buffer.

2. The composition according to claim 1, wherein the buffer has a pH of at most 6.7; preferably the buffer has a pH in a range of from 4.6 to 6.7.

3. The composition according to any one of the preceding claims, wherein the buffer has a pH of at most 6.3.

4. The composition according to any one of the preceding claims, wherein the buffer comprises a buffer compound; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; more preferably the buffer compound is citrate; even more preferably the buffer compound is sodium citrate.

5. The composition according to claim 4, wherein the buffer compound is selected from the group consisting of citrate, succinate, and histidine.

6. The composition according to any one of the preceding claims, wherein the buffer comprises a carbohydrate;preferably the carbohydrate is sucrose, or trehalose; most preferably the carbohydrate is sucrose.

7. The composition according to any one of the preceding claims, wherein the buffer comprises a chelator; preferably the chelator is ethylenediaminetetraacetic acid (EDTA).

8. The composition according to any one of the preceding claims, wherein the buffer comprises a surfactant; preferably the surfactant is polysorbate 20 or polysorbate 40; most preferably the surfactant is polysorbate 20.

9. The composition according to any one of the preceding claims, wherein the buffer comprises:(i) a buffer compound;(ii) a carbohydrate;(iii) a chelator; and(iv) a surfactant; and wherein the buffer has has a pH of at most 6.7; preferably the buffer compound is selected from the group consisting of citrate, succinate, and histidine; preferably the carbohydrate is sucrose, or trehalose; preferably the chelator is ethylenediaminetetraacetic acid (EDTA); preferably the surfactant is polysorbate 20 or polysorbate 40; and preferably the buffer has a pH in a range of from 4.6 to 6.7.

10. The composition according to any one of the preceding claims, wherein the composition comprises (i) the buffer; and (ii) the conjugate; preferably the composition essentially consists of (i) the buffer; and (ii) the conjugate.

11. The composition according to any one of the preceding claims, wherein the conjugate is according to Formula (C1):whereinCBis a protein;T2is a residue of a bioconjugation moiety;CJ is in a range of from 1 to 12; R48is a releasable group;T1, T3, L1, L2a, L2b, L2c, and L2dare as defined in claim 1; preferably the protein is a diabody or an antibody; more preferably the protein is a diabody; and most preferably the protein is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4.

12. The composition according to claim 11, wherein said conjugate is according to Formula (C2):wherein y is an integer in a range of from 10 to 50; preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, and most preferably in a range of from 23 to 25.

13. The composition according to claim 12, wherein said conjugate is according to Formula (C3): whereinx is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6.

14. The composition according to any one of the preceding claims, wherein R48is a releasable group, and said releasable group is -O-C(=O)-CA; wherein CAis a drug; preferably the drug is linked to the moiety -O-C(=O)- via a secondary or tertiary nitrogen atom that is part of the drug, forming a carbamate; preferably the drug is monomethyl auristatin E (MMAE).

15. The composition according to claim 13, wherein x is 5; y is 24; T1is OH; R48is -O-C(=O)-CA; CAis monomethyl auristatin E; CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; and T2iswherein the asterisk indicates a bond to AVP0458, and the wiggly line denotes a bond to the rest of the conjugate.

16. The composition according to claim 12, wherein L1is unsubstituted linear C5alkylene; T1is OH; T1is in an axial position; R48is -O-C(=O)-CA; CAis monomethyl auristatin E; R48is in an axial position; y is 24; L2ais -C(O)NH- or -NHC(O)-; L2bis -C(O)NH- or -NHC(O)-; L2cis >CH-(CH2)4-; L2dis -C(O)NH- or -NHC(O)-; CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; andT2is wherein the asterisk indicates a bond toAVP0458, and the wiggly line denotes a bond to the rest of the conjugate.

17. The composition according to any one of claims 13, 15, and 16, wherein the composition comprises (i) the buffer; and (ii) the conjugate; and wherein the composition has a pH of at most 6.3, and / or wherein the buffer comprises a carbohydrate; a chelator; a surfactant; and / or a buffer compound selected from the group consisting of citrate, succinate, and histidine;and preferably the composition essentially consists of (i) the buffer; and (ii) the conjugate.

18. The composition according to claim 17, wherein the conjugate is according to claim13.

19. The composition according to claim 17, wherein the conjugate is according to claim15.

20. The composition according to claim 17, wherein the conjugate is according to claim16.

21. The composition according to any one of claims 1 to 14, wherein the conjugate iswherein CJ is in a range of from 1 to 12; wherein CBis AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1; preferably CJ is of from 2 to 10, more preferably of from 2.5 to 8, even more preferably of from 3 to 6, even more preferably still of from 3.5 to 4, and most preferably about 4; preferably CBis linked to each maleimidyl group via a sulfur atom, preferably the sulfur atom is part of a cysteine.

22. The composition according to claim 21, wherein the conjugate is23. The composition according to any one of claims 1 to 17, wherein the conjugate is24. The composition according to any one of the preceding claims, wherein the composition is a pharmaceutical composition; preferably a pharmaceutical composition suitable for intravenous injection.

25. A combination of(A1) a conjugate as defined in any one of claims 1 to 23; and / or(A2) a compound as defined in claim 1; with(A3) a buffer according to any one of claims 1 to 9; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3).

26. The combination according to claim 25, wherein the combination is of(A1) a conjugate as defined in any one of claims 1 to 23; with(A3) a buffer according to any one of claims 1 to 9; preferably the combination is a kit wherein (A1) is physically separated from (A3); and preferably the combination essentially consists of (A1) and (A3) .

27. The combination of any one of claims 25 and 26 wherein the combination further comprises: (B) a diene; preferably the diene is a tetrazine; preferably the combination is a kit wherein the (A1) and / or (A2) are physically separated from (A3) and (B), and wherein (A3) is physically separated from (B).

28. The combination according to claim 27, wherein the diene is selected from the group consisting of:

29. The composition according to any one of claims 1 to 24; or the combination according to any one of claims 25 to 28; for use as a medicament.

30. The composition according to any one of claims 1 to 24; or the combination according to any one of claims 25 to 28; for use in the treatment of a disease in a subject; preferably the subject is a human; preferably the disease is cancer.

31. A method of treating a disease in a subject, wherein said method comprises the step of administering to said subject:(a) the composition according to any one of claims 1 to 24; and / or(b) the combination according to any one of claims 25 to 28; preferably the subject is a human; preferably the disease is cancer.

32. A method for preparing a dried composition, wherein said method comprises the step of drying a composition according to any one of claims 1 to 24; preferably the drying is carried out by lyophilization.

33. A dried composition obtainable by the method of claim 32; wherein preferably the dried composition is a lyophilized composition.

34. A method for preparing a composition according to any one of claims 1 to 24; wherein said method comprises the step of:(a) contacting a buffer as defined in any one of claims 1 to 9 with a compound and / or a conjugate as defined in any one of claims 1, and 10 to 24; and / or the step of(b) reconstituting the dried composition of claim 33 with water; preferably the method comprises the step of contacting a buffer as defined in any one of claims 1 to 9 with a conjugate as defined in any one of claims 1, and 10 to 24.

35. A method of storing the composition according to any one of claims 1 to 24, wherein the method comprises the steps of:(a) providing a composition according to any one of claims 1 to 24;(b) maintaining said composition at a temperature of at most 40°C; preferably the temperature is at most 35 °C; more preferably, the temperature is in a range of from -100°C to 35°C.

36. Use of a buffer as defined in any one of claims 1 to 9 to store a compound and / or a conjugate as defined in any one of claims 1, and 10 to 24; preferably the use is to store said conjugate.

37. Use of a buffer as defined in any one of claims 1 to 9 to stabilize a compound and / or a conjugate as defined in any one of claims 1, and 10 to 24, during storage; preferably the use is to stabilize said conjugate; preferably during storage the buffer and the compound and / or conjugate are maintained at a temperature of at most 40°C; more preferably the temperature is at most 35 °C; even more preferably, the temperature is in a range of from -100°C to 35°C.

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