JP2017197512A - Novel antibody-conjugates with improved therapeutic index for targeting cd30 tumors and methods for improving therapeutic index of antibody-conjugates - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide antibody-conjugates for targeting CD30.SOLUTION: The invention provides an antibody-conjugate obtainable by the steps of: (i) contacting a glycoprotein having 1-4 core N-acetylglucosamine moieties with a compound of Formula S (F)-P in the presence of a catalyst to obtain a modified antibody, where S(F)is a sugar derivative having x functional groups Fcapable of reacting with a functional group Q, x is 1 or 2, P is a nucleoside mono- or di-phosphate, and the catalyst is capable of transferring the S (F)moiety to the core-GlcNAc moiety; and (ii) reacting the modified antibody with a linker-conjugate comprising a functional group Qcapable of reacting with the functional group Fand a target molecule D linked to Qvia a linker Lto obtain an antibody-conjugate of which linker L comprises S-Z-Lwhere Zis a connecting group resulting from the reaction between Qand F.SELECTED DRAWING: None

Description

  The present invention is in the field of bioconjugation. More particularly, the invention relates to a particular mode of conjugation for preparing bioconjugates that have a beneficial effect on the therapeutic index of the bioconjugate, particularly in targeting CD30 expressing tumors.

  Bioconjugation is the process of linking two or more molecules, at least one of which is a biomolecule. The biomolecule (s) may also be referred to as “target biomolecule (s)” and the other molecule (s) may be referred to as “target molecules” or “target molecules”. Sometimes it is done. Typically, the biomolecule of interest (BOI) consists of a protein (or peptide), glycan, nucleic acid (or oligonucleotide), lipid, hormone or natural drug (or fragment or combination thereof). The other molecule of interest (MOI) may be a biomolecule, thus leading to the formation of a homo- or hetero-dimer (or higher oligomer) or the other molecule is It can have certain features imparted to the molecule. For example, modulation of protein structure and function by covalent modification using chemical probes for detection and / or isolation has evolved as a powerful tool in proteome-based research and biomedical applications. Protein fluorescence or affinity tagging is the key to studying protein transport in the natural habitat of the protein. Vaccines based on protein-carbohydrate conjugates have been active in the fight against HIV, cancer, malaria and pathogenic bacteria. On the other hand, the carbohydrates fixed to the microarray are useful for elucidation of glycome. Synthetic DNA and RNA oligonucleotides (ONs) introduce suitable functional groups for diagnostic and therapeutic applications such as microarray technology, antisense and gene silencing therapy, nanotechnology, and various materials science applications. I need. For example, cell-permeable ligand binding is the most commonly applied strategy to address the low internalization rate of ON encountered during oligonucleotide-based therapy (antisense, siRNA). Similarly, preparation of oligonucleotide-based microarrays requires selective immobilization of ON to a suitable solid surface, such as glass.

  There are many examples of chemical reactions that are suitable for covalently linking two (or more) molecular structures. However, the labeling of biomolecules imposes high limits on the reaction conditions (solvent, concentration, temperature) that can be applied, while the selection of reactive groups can be avoided if chemoselective labeling is desired. Limited. For obvious reasons, biological systems generally perform best in aqueous environments, meaning that bioconjugation reagents should be suitable for application in aqueous systems. In general, two strategic concepts can be recognized in the field of bioconjugation science and technology: (a) conjugation based on functional groups already present in the biomolecule of interest, eg thiol, amine, alcohol or hydroxyphenol units, Or (b) a two-step process involving modifying one (or more) unique reactive groups to BOI prior to the actual conjugation process.

  The first approach is typically a reactive amino acid side chain on a protein (eg cysteine, lysine, serine and tyrosine), or a functional group on a glycan (eg amine, aldehyde) or nucleic acid (eg purine or pyrimidine functional group or alcohol). Accompanying. In particular, G.M. T. T. As summarized in Hermanson “Bioconjugate Technologies”, Elsevier, 3rd edition, 2013, over the years, a number of reactive functional groups have been used for chemoselective targeting of one of these functional groups. For example, maleimides, haloacetamides, activated esters, activated carbonates, sulfonyl halides, activated thiol derivatives, alkenes, alkynes, allenamides, etc. are becoming available, each of which identifies itself for conjugation Conditions (pH, concentration, stoichiometry, light, etc.). Most notably, cysteine-maleimide conjugation is superior in protein conjugation due to high reaction rate and chemoselectivity. However, as is the case with many proteins, and of course other biomolecules, if cysteine is not available for conjugation, other methods are often required, each suffering from its own drawbacks.

  An accurate and widely applicable solution for bioconjugation involves the above two-step approach. Although more troublesome, two-step conjugation via a modified functional group typically yields higher selectivity (site specificity) than conjugation at a natural functional group. Moreover, complete stability can be achieved by appropriate selection of the construct. This can be an important drawback of one-step conjugation at the native functional group, especially in the case of cysteine-maleimide conjugation. Typical examples of functional groups that can be imparted to BOI include (strained) alkyne, (strained) alkene, norbornene, tetrazine, azide, phosphine, nitrile oxide, nitrone, nitrileimine, diazo compound, carbonyl compound, (O- Alkyl) hydroxylamine and hydrazine, which can be achieved by either chemical or molecular biology techniques. Each of the above functional groups is known to have at least one reaction partner, often with full interreactivity. For example, cyclooctynes react selectively and exclusively with 1,3-dipoles, strained alkenes with tetrazine, and phosphines with azides, leading to fully stable covalent bonds. However, some of the above functional groups have the disadvantage of being highly lipophilic, which can compromise conjugation efficiency, especially in combination with the desired lipophilic molecule (see below). I want to be)

  The final linking unit between the biomolecule and the other molecule of interest should also be preferentially completely compatible with the aqueous environment in terms of solubility, stability and biocompatibility. For example, highly lipophilic linkers can lead to aggregation (during and / or after conjugation), which can significantly increase the reaction time and / or conjugation yield, especially if the MOI is also hydrophobic in nature. May be reduced. Similarly, highly lipophilic linker-MOI combinations can lead to non-specific binding of the same or other biomolecules to the surface or specific hydrophobic patches. If the linker is sensitive to aqueous hydrolysis or other water-induced cleavage reactions, the components comprising the original bioconjugate are separated by diffusion. For example, certain ester moieties are not suitable because they are saponified, while β-hydroxycarbonyl or γ-dicarbonyl compounds can lead to retroaldol reactions or retromichael reactions, respectively. Finally, the linker should be inert to the functional groups present in the bioconjugate or to any other functional group that may be encountered during application of the bioconjugate. In particular, linkers featuring, for example, ketone or aldehyde moieties (which can lead to imine formation), α, β-unsaturated carbonyl compounds (Michael addition), thioesters or other activated esters (amide bond formation) The use of is excluded.

  Compounds made with linear oligomers of ethylene glycol, so-called polyethylene glycol (PEG) linkers, have recently become particularly popular in biomolecular conjugation processes. PEG linkers are highly water-soluble, non-toxic, non-antigenic and produce little or no aggregation. For this reason, a wide variety of linear bifunctional PEG linkers are commercially available from various sources that can be selectively modified at either end with the (bio) molecule of interest. The PEG linker is the product of the ethylene oxide polymerization process, so that it can be partially broken down into PEG constructs with an average weight distribution centered around 1 kDa, 2 kDa, 4 kDa or more (up to 60 kDa) Typically obtained as a long stochastic mixture. Homogeneous individual PEG (dPEG) with a molecular weight of up to 4 kDa is also known, and its branched version is up to 15 kDa. Interestingly, the PEG unit itself confers special features on biomolecules. In particular, protein PEGylation can lead to prolonged residence in vivo, reduced degradation by metabolic enzymes, and reduced or eliminated protein immunogenicity. Some PEGylated proteins are FDA approved and are currently on the market.

  PEG linkers are quite suitable for bioconjugation of small and / or water soluble moieties under aqueous conditions due to their high polarity. However, in the case of conjugation of the desired hydrophobic water-insoluble molecule, the polarity of the PEG units is insufficient to offset the hydrophobicity, a significant reduction in reaction rate, lower yield and aggregation induction challenges Can lead to In such cases, long PEG linkers and / or significant amounts of organic co-solvents may be required to solubilize the reagents. For example, in the field of antibody-drug conjugates, control of the binding of a discrete number of toxic payloads to a monoclonal antibody is key, where the payload is auristatin E or F, maytansinoid, duocarmycin, calicheamicin or Typically selected from the group of pyrrolobenzodiazepines (PBD), many others are under consideration. With the exception of auristatin F, all toxic payloads are poorly water-insoluble and require an organic co-solvent such as 25% dimethylacetamide (DMA) or 50% propylene glycol (PG) to achieve successful conjugation And In the case of a hydrophobic payload, despite the use of the above-mentioned co-solvents, large stoichiometric reagents may be required during conjugation, while efficiency and yield are, for example, by reference Nat. Biotechn. 2014, 24, 1256-1263, as described by Senter et al., May be significantly impaired by aggregation (during process or after product isolation). Although the use of long PEG spacers (12 units or more) can partially enhance solubility and / or conjugation efficiency, long PEG spacers can lead to more rapid in vivo clearance, and hence ADC It has been shown to negatively affect the pharmacokinetic profile.

  Using conventional linkers (eg PEG), effective conjugation is often due to the relatively low solubility of the linker-conjugate in aqueous media, especially when relatively water insoluble or hydrophobic target molecules are used. Be disturbed. In our quest for short polar spacers that allow fast and effective conjugation of hydrophobic moieties, we improved the solubility of the linker-conjugate, thereby conjugating efficiency. A sulfamide linker has been developed that has been found to significantly improve and reduce both aggregation and product aggregation during the process. This sulfamide linker is disclosed in international application PCT / NL2015 / 0505067, which is incorporated herein in its entirety.

  Linkers are known in the art and are disclosed, for example, in WO 2008/070291, incorporated by reference. WO 2008/070291 discloses linkers for the coupling of targeting agents to anchoring components. The linker contains a hydrophilic region represented by polyethylene glycol (PEG) and an extension lacking a chiral center coupled to the targeting agent.

WO 01/88535, incorporated by reference, discloses linker systems for surfaces for bioconjugation, in particular linker systems with novel hydrophilic spacer groups. The hydrophilic atom or hydrophilic group for use in the linker system is selected from the group consisting of O, NH, C═O (keto group), O—C═O (ester group) and CR ³ R ⁴ ; Here, R ³ and R ⁴ are independently selected from the group consisting of H, OH, C ₁ -C ₄ alkoxy and C ₁ -C ₄ acyloxy.

  WO 2014/100762 incorporated by reference describes compounds having hydrophilic self-sacrificial linkers that are cleavable under appropriate conditions and that incorporate a hydrophilic group to provide better solubility of the compound. ing. The compound contains a drug moiety, a targeting moiety capable of targeting a selected cell population, and an acyl unit, an optional spacer unit to provide a distance between the drug moiety and the targeting moiety. Includes a linker, a peptide linker that may be cleavable under appropriate conditions, a hydrophilic self-sacrificial linker, and an optional second self-sacrificial spacer or cyclized self-elimination linker. A hydrophilic self-sacrificing linker is, for example, a benzyloxycarbonyl group.

  The present invention relates to a method or use for increasing the therapeutic index of a bioconjugate, ie a conjugate of a biomolecule and a target molecule. The inventors surprisingly found that bioconjugates prepared via a particular mode of conjugation are the same bioconjugate obtained via different modes of conjugation, i.e. the same biomolecule, the same. It has been found that it exhibits a higher therapeutic index compared to the target molecule (eg active substance) and the same biomolecule drug ratio. The mode of conjugating the biomolecule to the target molecule is exposed at the linker itself and / or at the point of attachment of the linker to the biomolecule. It cannot be recalled based on current knowledge that linkers and / or points of attachment can have an effect on the therapeutic index of bioconjugates such as antibody-drug-conjugates. In the above field, linkers are considered inert for treatment and are only present as a consequence of the preparation of bioconjugates. The selection of a particular mode of conjugation has no effect on the therapeutic index and is a breakthrough in the field of bioconjugates, particularly antibody-drug-conjugates.

  The bioconjugate according to the invention, on the other hand, is more effective than the same bioconjugate obtained through different modes of conjugation, ie the same biomolecule, the same target molecule (eg active substance) and the same biomolecule / target molecule ratio. (Therapeutically effective) and / or on the other hand is more tolerated. This finding has dramatic implications for the treatment of subjects using the bioconjugates according to the invention as the therapeutic window is expanded. As a result of the expansion of the treatment window, the treatment dose can be reduced, resulting in a reduction in potential unwanted side effects.

（式中、Ｓ（Ｆ^１）_ｘ及びｘは、上で定義されている通りであり；ＡＢは抗体を表し；ＧｌｃＮＡｃは、Ｎ−アセチルグルコサミンであり；Ｆｕｃは、フコースであり；ｂは０又は１であり；ｙは１、２、３又は４である）に従った修飾抗体を得ることができる、ステップ；並びに
（ｉｉ）修飾抗体を、官能基Ｆ^１と反応できる官能基Ｑ^１及びリンカーＬ^２を介してＱ^１に接続されている標的分子Ｄを含むリンカー−コンジュゲートと反応させることで抗体−コンジュゲートを得るステップであって、リンカーＬはＳ−Ｚ^３−Ｌ^２を含み、Ｚ^３は、Ｑ^１とＦ^１との間の反応から生じる接続基である、ステップ。 In one embodiment, the modes of conjugation according to the invention include:
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine substituents with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x Is a sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is a nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) By transferring the _x moiety to the core-GlcNAc moiety, formula (24):

Where S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or it is a 1; y can be obtained a modified antibody according to 1, 2, 3 or 4), the step; and (ii) a modified antibody, functional groups ^{Q 1} capable of reacting with functional groups ^{F 1} and Obtaining an antibody-conjugate by reacting with a linker-conjugate comprising a target molecule D connected to Q ¹ via a linker L ² , wherein the linker L comprises SZ ³ -L ² , Z ³ is a connecting group resulting from the reaction between Q ¹ and F ¹ .

に従った基又はその塩を含むリンカーＬを含有することを確実にする。 In one embodiment, the mode of conjugation according to the invention is that the bioconjugate is of formula (1):

To ensure that it contains a linker L comprising a group according to

  The inventors surprisingly found that bioconjugates prepared to contain a linker L comprising a group according to formula (1) or a salt thereof are free of groups according to formula (1). It has been found that it exhibits a higher therapeutic index compared to the same bioconjugate containing the linker, ie the same biomolecule, the same target molecule (eg active substance) and the same biomolecule drug ratio.

In the group according to formula (1):
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ is independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is further target molecule D, where D is a spacer moiety It is connected optionally to N through.

In the context of the present invention, the above mode of conjugation is used to connect biomolecule B with target molecule D via linker L. Conjugation refers to a specific mode of connecting a biomolecule to a target molecule. The bioconjugate according to the invention has the formula (A):
BLD
(A)
(Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of "-" is independently a bond or a spacer moiety).

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ若しくはＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）に従った基又はその塩を含むように調製するステップを含む、バイオコンジュゲートの治療指数を増加させるための方法。
２．バイオコンジュゲートを、それを必要とする対象に投与するステップをさらに含む、実施形態１による方法。
３．対象が癌患者である、実施形態２による方法。
４．生体分子が抗体であり、バイオコンジュゲートが抗体−薬物−コンジュゲートである、先行する実施形態のいずれかによる方法。
５．標的分子Ｄが活性物質、好ましくは細胞毒である、先行する実施形態のいずれかによる方法。
６．バイオコンジュゲートが式Ｂ−Ｚ^３−Ｌ−Ｄを有し、ここでＺ^３は、反応性基Ｑ^１を官能基Ｆ^１と反応させることによって得られる、先行する実施形態のいずれかによる方法。
７．Ｚ^３が、式Ｑ^１−Ｌ−Ｄ（式中、Ｌは、式（１）：

に従った基又はその塩を含む）を有するリンカー−コンジュゲートを、式Ｂ−Ｆ^１を有する生体分子と反応させることによって得られ、ここでＢ、Ｄ、ａ及びＲ^１は、実施形態１に定義されている通りである、実施形態６による方法。
８．リンカー−コンジュゲートが、式（４ａ）若しくは（４ｂ）：

（式中：
ａは、独立して０又は１であり；
ｂは、独立して０又は１であり；
ｃは、０又は１であり；
ｄは、０又は１であり；
ｅは、０又は１であり；
ｆは、１〜１５０の範囲における整数であり；
ｇは、０又は１であり；
ｉは、０又は１であり；
Ｄは、標的分子であり；
Ｑ^１は、生体分子に存在する官能基Ｆ^１と反応できる反応性基であり；
Ｓｐ^１は、スペーサー部分であり；
Ｓｐ^２は、スペーサー部分であり；
Ｓｐ^３は、スペーサー部分であり；
Ｓｐ^４は、スペーサー部分であり；
Ｚ^１は、Ｑ^１又はＳｐ^３を、Ｓｐ^２、Ｏ若しくはＣ（Ｏ）又はＮ（Ｒ^１）に接続する接続基であり、；
Ｚ^２は、Ｄ又はＳｐ^４を、Ｓｐ^１、Ｎ（Ｒ^１）、Ｏ又はＣ（Ｏ）に接続する接続基であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される；又はＲ^１は、Ｄ、−［（Ｓｐ^１）_ｂ（Ｚ^２）_ｅ（Ｓｐ^４）_ｉＤ］若しくは−［（Ｓｐ^２）_ｃ（Ｚ^１）_ｄ（Ｓｐ^３）_ｇＱ^１］であり、ここでＤは標的分子であり、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２、Ｄ、Ｑ^１、ｂ、ｃ、ｄ、ｅ、ｇ及びｉは、上で定義されている通りである）又はその塩に従った、実施形態７によるプロセス。
９．Ｓｐ^１、Ｓｐ^２、Ｓｐ^３及びＳｐ^４が、直鎖又は分枝のＣ_１〜Ｃ_２００アルキレン基、Ｃ_２〜Ｃ_２００アルケニレン基、Ｃ_２〜Ｃ_２００アルキニレン基、Ｃ_３〜Ｃ_２００シクロアルキレン基、Ｃ_５〜Ｃ_２００シクロアルケニレン基、Ｃ_８〜Ｃ_２００シクロアルキニレン基、Ｃ_７〜Ｃ_２００アルキルアリーレン基、Ｃ_７〜Ｃ_２００アリールアルキレン基、Ｃ_８〜Ｃ_２００アリールアルケニレン基及びＣ_９〜Ｃ_２００アリールアルキニレン基からなる群から独立して選択され、アルキレン基、アルケニレン基、アルキニレン基、シクロアルキレン基、シクロアルケニレン基、シクロアルキニレン基、アルキルアリーレン基、アリールアルキレン基、アリールアルケニレン基及びアリールアルキニレン基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３群から選択される１個又は複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_２〜Ｃ_２４アルケニル基、Ｃ_２〜Ｃ_２４アルキニル基及びＣ_３〜Ｃ_２４シクロアルキル基からなる群から独立して選択され、アルキル基、アルケニル基、アルキニル基及びシクロアルキル基は任意選択で置換されている、実施形態８によるプロセス。
１０．Ｚ^１及びＺ^２が、−Ｏ−、−Ｓ−、−ＮＲ^２−、−Ｎ＝Ｎ−、−Ｃ（Ｏ）−、−Ｃ（Ｏ）ＮＲ^２−、−ＯＣ（Ｏ）−、−ＯＣ（Ｏ）Ｏ−、−ＯＣ（Ｏ）ＮＲ^２、−ＮＲ_２Ｃ（Ｏ）−、−ＮＲ^２Ｃ（Ｏ）Ｏ−、−ＮＲ^２Ｃ（Ｏ）ＮＲ^２−、−ＳＣ（Ｏ）−、−ＳＣ（Ｏ）Ｏ−、−ＳＣ（Ｏ）ＮＲ^２−、−Ｓ（Ｏ）−、−Ｓ（Ｏ）_２−、−ＯＳ（Ｏ）_２−、−ＯＳ（Ｏ）_２Ｏ−、−ＯＳ（Ｏ）_２ＮＲ^２−、−ＯＳ（Ｏ）−、−ＯＳ（Ｏ）Ｏ−、−ＯＳ（Ｏ）ＮＲ^２−、−ＯＮＲ^２Ｃ（Ｏ）−、−ＯＮＲ^２Ｃ（Ｏ）Ｏ−、−ＯＮＲ^２Ｃ（Ｏ）ＮＲ^２−、−ＮＲ^２ＯＣ（Ｏ）−、−ＮＲ^２ＯＣ（Ｏ）Ｏ−、−ＮＲ^２ＯＣ（Ｏ）ＮＲ^２−、−ＯＮＲ^２Ｃ（Ｓ）−、−ＯＮＲ^２Ｃ（Ｓ）Ｏ−、−ＯＮＲ^２Ｃ（Ｓ）ＮＲ^２−、−ＮＲ^２ＯＣ（Ｓ）−、−ＮＲ^２ＯＣ（Ｓ）Ｏ−、−ＮＲ^２ＯＣ（Ｓ）ＮＲ^２−、−ＯＣ（Ｓ）−、−ＯＣ（Ｓ）Ｏ−、−ＯＣ（Ｓ）ＮＲ^２−、−ＮＲ^２Ｃ（Ｓ）−、−ＮＲ^２Ｃ（Ｓ）Ｏ−、−ＮＲ^２Ｃ（Ｓ）ＮＲ^２−、−ＳＳ（Ｏ）_２−、−ＳＳ（Ｏ）_２Ｏ−、−ＳＳ（Ｏ）_２ＮＲ^２−、−ＮＲ^２ＯＳ（Ｏ）−、−ＮＲ^２ＯＳ（Ｏ）Ｏ−、−ＮＲ^２ＯＳ（Ｏ）ＮＲ^２−、−ＮＲ^２ＯＳ（Ｏ）_２−、−ＮＲ^２ＯＳ（Ｏ）_２Ｏ−、−ＮＲ^２ＯＳ（Ｏ）_２ＮＲ^２−、−ＯＮＲ^２Ｓ（Ｏ）−、−ＯＮＲ^２Ｓ（Ｏ）Ｏ−、−ＯＮＲ^２Ｓ（Ｏ）ＮＲ^２−、−ＯＮＲ^２Ｓ（Ｏ）_２Ｏ−、−ＯＮＲ^２Ｓ（Ｏ）_２ＮＲ^２−、−ＯＮＲ^２Ｓ（Ｏ）_２−、−ＯＰ（Ｏ）（Ｒ^２）_２−、−ＳＰ（Ｏ）（Ｒ^２）_２−、−ＮＲ^２Ｐ（Ｏ）（Ｒ^２）_２−及びそれらの２つ以上の組合せからなる群から独立して選択され、ここでＲ^２は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_２〜Ｃ_２４アルケニル基、Ｃ_２〜Ｃ_２４アルキニル基及びＣ_３〜Ｃ_２４シクロアルキル基からなる群から独立して選択され、アルキル基、アルケニル基、アルキニル基及びシクロアルキル基は任意選択で置換されている、実施形態８又は９によるプロセス。
１１．Ｓｐ^１、Ｓｐ^２、Ｓｐ^３及びＳｐ^４が、存在するならば、直鎖又は分枝のＣ_１〜Ｃ_２０アルキレン基からなる群から独立して選択され、アルキレン基は、任意選択で置換され、Ｏ、Ｓ又はＮＲ^３からなる群から選択される１個又は複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択され、Ｑ^１が、式（９ａ）、（９ｑ）、（９ｎ）、（９ｏ）又は（９ｐ）、（９ｔ）又は（９ｚｈ）：

（式中：
Ｕは、Ｏ又はＮＲ^９であり、Ｒ^９は、水素、直鎖若しくは分枝のＣ_１〜Ｃ_１２アルキル基又はＣ_４〜Ｃ_１２（ヘテロ）アリール基であり；
Ｒ^１０は、（チオ）エステル基であり；
Ｒ^１８は、任意選択で置換されているＣ_１〜Ｃ_１２アルキル基及びＣ_４〜Ｃ_１２（ヘテロ）アリール基からなる群から選択される）に従う、実施形態８〜１０のいずれか１つによるプロセス。
１２．反応性基Ｑ^１と官能基Ｆ^１との間の反応が、（１０ａ）又は（１０ｂ）として表すことができる接続部分Ｚ^３を形成するためのチオール−アルケンコンジュゲーション、（１０ｃ）として表すことができる接続部分Ｚ^３を形成するためのアミノ−（活性化）カルボン酸コンジュゲーション、Ｙ＝ＮＨである（１０ｄ）として表すことができる接続部分Ｚ^３を形成するためのケトン−ヒドラジノコンジュゲーション、Ｙ＝Ｏである（１０ｄ）として表すことができる接続部分Ｚ^３を形成するためのケトン−オキシアミノコンジュゲーション、（１０ｅ）又は（１０ｇ）として表すことができる接続部分Ｚ^３を形成するためのアルキン−アジドコンジュゲーション、及びＮ_２が脱離する（１０ｈ）として表すことができる接続部分Ｚ^３を形成するためのアルケン−１，２，４，５−テトラジンコンジュゲーション又はアルキン−１，２，４，５−テトラジンコンジュゲーションから選択されるコンジュゲーション反応であり、ここで、部分（１０ａ）、（１０ｂ）、（１０ｃ）、（１０ｄ）、（１０ｅ）、（１０ｇ）及び（１０ｈ）は、

（式中、Ｚは、水素、メチル及びピリジルから選択される）によって表される、先行する実施形態のいずれかによる方法。
１３．ａ＝０である、先行する実施形態のいずれかによる方法。
１４．式（Ａ）：
Ｂ−Ｌ−Ｄ
（Ａ）
［式中：
Ｂは、生体分子であり；
Ｌは、Ｂ及びＤを連結するリンカーであり；
Ｄは、標的分子であり；
「−」の各出現は、独立して、結合又はスペーサー部分であり、
ここでＬは、式（１）：

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）に従った基又はその塩を含む］によって表される、それを必要とする対象の処置における使用のためのバイオコンジュゲート。
１５．癌の処置における使用のための、実施形態１４に従った使用のためのバイオコンジュゲート。 For embodiments in which the mode of conjugation is referred to as “sulfamide linkage”, the following embodiments are preferred:
1. Formula (A):
BLD
(A)
(Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or spacer moiety) and the reactive group Q ¹ of the target molecule (D) is reacted with the functional group F ¹ of the biomolecule (B). Thus, L is the formula (1):

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I where target molecule, space Comprising the step of preparing to include group or a salt thereof according to) is connected optionally to N via a moiety, methods for increasing the therapeutic index of bioconjugate.
2. The method according to embodiment 1, further comprising administering the bioconjugate to a subject in need thereof.
3. The method according to embodiment 2, wherein the subject is a cancer patient.
4). The method according to any of the previous embodiments, wherein the biomolecule is an antibody and the bioconjugate is an antibody-drug-conjugate.
5. The method according to any of the previous embodiments, wherein target molecule D is an active substance, preferably a cytotoxin.
6). Bioconjugate has the formula B-Z 3 ^-L-D, wherein Z ³ is a reactive group Q ¹ is obtained by reacting with a functional group F ^1, the method according to any of the preceding embodiments .
7). Z ³ represents the formula Q ¹ -LD (wherein L represents the formula (1):

Is obtained by reacting a biomolecule having the formula B-F ¹ with B, D, a and R ¹ in the embodiment 1. The method according to embodiment 6, wherein the method is as defined in
8). The linker-conjugate is of formula (4a) or (4b):

(Where:
a is independently 0 or 1;
b is independently 0 or 1;
c is 0 or 1;
d is 0 or 1;
e is 0 or 1;
f is an integer in the range of 1 to 150;
g is 0 or 1;
i is 0 or 1;
D is the target molecule;
Q ¹ is a reactive group that can react with the functional group F ¹ present in the biomolecule;
Sp ¹ is a spacer moiety;
Sp ² is a spacer moiety;
Sp ³ is a spacer moiety;
Sp ⁴ is a spacer moiety;
Z ¹ is a connecting group that connects Q ¹ or Sp ³ to Sp ² , O or C (O) or N (R ¹ );
Z ² is a connecting group that connects D or Sp ⁴ to Sp ¹ , N (R ¹ ), O or C (O);
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in ^{R 3} are independently selected from the group consisting of hydrogen and _C 1 -C ₄ alkyl group; or ^{R 1 _{is, D, - [(Sp 1}} ) b (Z 2) e (Sp 4) i D Or ^- a _{^{[(Sp 2) c (Z}} 1) d (Sp 3) g Q 1], where D is the target ^{molecule, Sp 1, Sp 2, Sp} 3, Sp 4, Z 1, Z 2 , D, Q ¹ , b, c, d, e, g and i are as defined above) or a salt thereof.
9. Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are linear or branched C ₁ -C ₂₀₀ alkylene groups, C ₂ -C ₂₀₀ alkenylene groups, C ₂ -C ₂₀₀ alkynylene groups, C ₃ -C ₂₀₀ cycloalkylenes. _group, C _{5 -C 200} cycloalkenylene _group, C _{8 -C 200} cycloalkynylene _groups, C _{7 -C 200} alkyl arylene _group, C _{7 -C 200} arylalkylene _group, C _{8 -C 200} aryl alkenylene group and _{C 9} -C ₂₀₀ are independently selected from the group consisting of aryl alkynylene group, an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, aryl alkenylene group And arylalkynylene groups are optionally It is, O, is interrupted optionally by one or more heteroatoms selected from S and NR ³ group, wherein ^{R 3} is _hydrogen, C 1 _{-C 24} alkyl _group, C 2 _{-C 24} alkenyl group is independently selected from C ₂ -C ₂₄ alkynyl and C ₃ -C ₂₄ group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group is optionally substituted, Process according to embodiment 8.
10. Z ¹ and Z ² are —O—, —S—, —NR ² —, —N═N—, —C (O) —, —C (O) NR ² —, —OC (O) —, — _{^{OC (O) O -, -}} OC (O) NR 2, -NR 2 C (O) -, - NR 2 C (O) O -, - NR 2 C (O) NR 2 -, - SC (O) ^{-, - SC (O) O} -, - SC (O) NR 2 -, - S (O) -, - S (O) 2 -, - OS (O) 2 -, - OS (O) 2 O- ^{_{, -OS (O) 2 NR 2}} -, - OS (O) -, - OS (O) O -, - OS (O) NR 2 -, - ONR 2 C (O) -, - ONR 2 C (O ) O—, —ONR ² C (O) NR ² —, —NR ² OC (O) —, —NR ² OC (O) O—, —NR ² OC (O) NR ² —, —ONR ² C ( S)-, -ONR ² C (S) O-, -ONR ² C (S) NR ^2- , —NR ² OC (S) —, —NR ² OC (S) O—, —NR ² OC (S) NR ² —, —OC (S) —, —OC (S) O—, —OC (S) NR ² —, —NR ² C (S) —, —NR ² C (S) O—, —NR ² C (S) NR ² —, —SS (O) ₂ —, —SS (O) ₂ O—, —SS (O) ₂ NR ² —, —NR ² OS (O) —, —NR ² OS (O) O—, —NR ² OS (O) NR ² —, —NR ² OS (O ) ₂ −, —NR ² OS (O) ₂ O—, —NR ² OS (O) ₂ NR ² —, —ONR ² S (O) —, —ONR ² S (O) O—, —ONR ² S (O) NR ² —, —ONR ² S (O) ₂ O—, —ONR ² S (O) ₂ NR ² —, —ONR ² S (O) ₂ —, —OP (O) (R ² ) ₂ -, -SP (O) (R _{^{2) 2 -, - NR 2}} P (O) (R 2) 2 - and it is independently selected from the group consisting of combinations of two or more thereof, wherein ^{R 2} is _hydrogen, C 1 _{-C 24} alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group optionally Embodiment 10. The process according to embodiment 8 or 9, which is replaced with a selection.
11. Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are independently selected from the group consisting of linear or branched C ₁ -C ₂₀ alkylene groups, wherein the alkylene groups are optionally substituted Optionally interrupted by one or more heteroatoms selected from the group consisting of O, S, or NR ³ , wherein R ³ is independent of the group consisting of hydrogen and a C ₁ -C ₄ alkyl group. Q ¹ is selected from formulas (9a), (9q), (9n), (9o) or (9p), (9t) or (9zh):

(Where:
U is O or NR ⁹ and R ⁹ is hydrogen, a linear or branched C ₁ -C ₁₂ alkyl group or a C ₄ -C ₁₂ (hetero) aryl group;
R ¹⁰ is a (thio) ester group;
R ¹⁸ is selected from the group consisting of an optionally substituted C ₁ -C ₁₂ alkyl group and a C ₄ -C ₁₂ (hetero) aryl group) according to any one of embodiments 8-10. process.
12 The reaction between the reactive group Q ¹ and the functional group F ¹ is represented as (10c), a thiol-alkene conjugation to form a connecting moiety Z ³ that can be represented as (10a) or (10b) amino to form a connecting portion Z ^3, which may - (activated) carboxylic acid conjugation, Y = is NH ketone to form a connecting portion Z ³ which may be represented as (10d) - hydrazino conjugation , ketone for forming a connecting portion ^{Z 3} which may be represented as a Y = O (10d) - oxyamino conjugation, to form a connecting portion ^{Z 3} which may be represented as (10e) or (10 g) alkyne - azide conjugation, and N ₂ is eliminated (10h) form a connecting portion Z ³ which may be represented as A conjugation reaction selected from alkene-1,2,4,5-tetrazine conjugation or alkyne-1,2,4,5-tetrazine conjugation, wherein moiety (10a), (10b), (10c), (10d), (10e), (10g) and (10h)

A method according to any of the previous embodiments, wherein Z is selected from hydrogen, methyl and pyridyl.
13. The method according to any of the previous embodiments, wherein a = 0.
14 Formula (A):
BLD
(A)
[Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or a spacer moiety;
Where L is the formula (1):

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I wherein the target molecule is a spacer Min represented by containing group or a salt thereof according to the connection are) optionally] to N via a bioconjugate for use in the treatment of a subject in need thereof.
15. A bioconjugate for use according to embodiment 14, for use in the treatment of cancer.

  The invention thus relates, in a first aspect, to a method or use for increasing the therapeutic index of a bioconjugate, wherein the mode of conjugation according to the invention is comprised in preparing the bioconjugate. Or used to prepare. In one embodiment, the mode of conjugation comprises steps (i) and (ii) as defined herein. In an alternative embodiment, the mode of conjugation comprises preparing a bioconjugate of formula (A) such that a linker L as defined above is included in the bioconjugate. In one embodiment, the method or use according to the invention further comprises administering the bioconjugate to a subject in need thereof. The invention according to the first aspect uses the mode of conjugation as defined above in a bioconjugate to increase the therapeutic index of the bioconjugate or increases the therapeutic index of the bioconjugate Can also be expressed as the use of linker L as defined above in the bioconjugate.

  In a further aspect, the invention relates to the treatment of a subject in need thereof comprising administration of a bioconjugate according to the invention. Typically, the bioconjugate is administered in a therapeutically effective amount. In view of increased therapeutic efficacy, administration may occur less frequently and / or at lower doses than in treatments using conventional bioconjugates. Alternatively, taking into account increased tolerability, administration may occur more frequently and / or at higher doses than in treatments using conventional bioconjugates. Administration can be a single dose or can be performed, for example, 1 to 4 times a month, preferably 1 to 2 times a month, more preferably every 3 or 4 weeks, most preferably 4 weeks Once every time. As will be appreciated by those skilled in the art, the dose of the bioconjugate according to the present invention may depend on many factors, and the optimal dose can be determined by one skilled in the art through routine experimentation. The bioconjugate has a target body weight of 0.01-50 mg / kg, more precisely 0.03-25 mg / kg or most accurately 0.05-10 mg / kg, or alternatively 0.1-25 mg / kg Alternatively, it is typically administered at a dose of 0.5-10 mg / kg.

In one embodiment, the administration of the bioconjugate according to the invention is a lower dose than TD ₅₀ which is the same bioconjugate but does not contain the mode of conjugation according to the invention, and the dose is the same bioconjugate Preferably at most 99-90%, more preferably at most 89-60%, more preferably 59-30%, most preferably at most 29-10% of the TD ₅₀ which does not contain the mode of conjugation according to the invention It is. Alternatively, the administration of the bioconjugate according to the invention is a higher dose than TD ₅₀ which is the same bioconjugate but does not contain the mode of conjugation according to the invention, and the dose is the same bioconjugate but Preferably at most 10-29%, more preferably at most 30-59%, more preferably at 60-89%, most preferably at most 90-99% of the TD ₅₀ not including the mode of conjugation according to the invention .

In one embodiment, the administration of the bioconjugate according to the invention is a lower dose than the ED ₅₀ which is the same bioconjugate but does not comprise the mode of conjugation according to the invention, and the dose is the same bioconjugate Is preferably at most 99-90%, more preferably at most 89-60%, more preferably 59-30%, most preferably at most 29-10% of the ED ₅₀ which does not contain the mode of conjugation according to the invention It is. Alternatively, the administration of the bioconjugate according to the invention is a higher dose than TD ₅₀ which is the same bioconjugate but does not contain the mode of conjugation according to the invention, and the dose is the same bioconjugate but Preferably at most 1.1 to 1.49 times higher, more preferably at most 1.5 to 1.99 times higher, more preferably 2 to 4.99 times higher than the TD ₅₀ without mode of conjugation according to the invention. High, most preferably at most 5 to 10 times higher.

The preparation of the bioconjugate typically comprises reacting a linker-conjugate having the formula Q ¹ -LD, where L and D are as defined above, Q ¹ is A reactive group capable of reacting with the functional group F ¹ , the biomolecule has the formula BF ¹ , where B is as defined above, and F ¹ is a functional group capable of reacting with Q ¹ It is a group. Herein, Q ¹ and F ¹ react to form a connecting group Z ³ located in the spacer portion between B and L in the bioconjugate according to formula (A).

FIG. 1 describes the general concept of biomolecule conjugation: a target biomolecule (BOI) containing one or more functional groups F ¹ is covalently bonded to a reactive group Q ¹ via a specific linker Incubating with the surplus (excess) target molecule D (also referred to as the molecule of interest or MOI). In the process of bioconjugation, a chemical reaction between F ¹ and Q ¹ takes place, thereby forming a bioconjugate that includes a covalent connection between BOI and MOI. The BOI can be, for example, a peptide / protein, glycan or nucleic acid. For example, at the 2-position using a 3-mercaptopropionyl group (11a), an azidoacetyl group (11b) or an azidodifluoroacetyl group (11c), or at the 6-position of N-acetylgalactosamine (11d) using an azidoazidoacetyl group FIG. 2 shows some structures of derivatives of UDP saccharides of galactosamine that can be modified. Any of the UDP-sugars 11a-d binds to a glycoprotein containing a GlcNAc moiety 12 (eg, a monoclonal antibody in which the glycan is trimmed by endoglycosidase) under the action of a galactosyltransferase mutant or GalNAc-transferase. FIG. 2 schematically represents a method that can be used to thereby generate a β-glycoside 1-4 linkage between a GalNAc derivative and a GlcNAc (compounds 13a-d, respectively). Modified antibodies 13a-d are by means of nucleophilic addition to maleimide (for conjugation to 3-mercaptopropionyl-galactosamine modified 13a leading to thioether conjugate 14 or modified cysteine residues leading to thioether conjugate 17) Or a method that can undergo a bioconjugation process (for 13b, 13c, or 13d leading to triazole 15a, 15b, or 16, respectively) with strain-enhanced cycloaddition using a cyclooctyne reagent. A representative series of functional groups (F ¹ ) in a biomolecule, either naturally occurring or introduced by modification, which react with a reactive group Q ¹ to form a linking group Z is a diagram showing the functional group ^{(F 1)} leading to ^3. The functional group F ¹ can also be artificially introduced (modified) into the biomolecule at any selected position. FIG. 2 shows a preferred bioconjugate according to the present invention. In the examples, conjugates 52-57 and 59 are prepared and their therapeutic index is investigated. Conjugates 52-57 are conjugated to brentuximab as an antibody, and conjugate 59 is conjugated to iratumumab. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. Examples for control antibody-conjugate Adcetris (FIG. 7A) and antibody-conjugates 57 (FIG. 7B), 56 (FIG. 7C), 52 (FIG. 7D), 54 (FIG. 7E), 53 (FIG. 7F) according to the invention Figure 3 is a graph illustrating the results of 38 tolerability studies. Based on 100% at the start of treatment (Day 1), the percentage body weight change (ΔBW) over time is illustrated. Weight loss indicates that the conjugate is not tolerated at a particular dose. C = vehicle treated. FIG. 4 is a graph illustrating the results of the efficacy study of Example 37 for a control antibody-conjugate Adpetris and an antibody-conjugate 56 according to the present invention. Efficacy is expressed as a change in tumor volume over time, where greater effectiveness leads to greater reduction or less increase in tumor volume. C = vehicle treated. It is a graph which illustrates the same result. The effectiveness of antibody-conjugates 53, 55 and 57 according to the present invention has been presented at various doses. Efficacy is expressed as a change in tumor volume over time, where greater effectiveness leads to greater reduction or less increase in tumor volume. C = vehicle treated. FIG. 40 is a graph showing the reversal of drug antibody ratio (DAR) over time for control antibody-conjugate Adcetris and for antibody-conjugates 56 and 57 according to the present invention, corresponding to Example 39. The antibody-conjugate according to the present invention has a theoretical DAR of 4 that hardly decreases over time, while the control antibody-conjugate decreases rapidly below the DAR of the antibody-conjugate according to the present invention. Start with a slightly higher DAR.

In the context of the present invention, the conjugation reaction is on the one hand a biomolecule (BOI) containing the functional group F ¹ and on the other hand a target molecule (MOI) containing the reactive group Q ¹ , or as used herein. With a “linker-conjugate” as defined, where Q ¹ reacts with F ¹ to form a connecting group that connects BOI and MOI in the bioconjugate. In this specification, the reactive group Q ¹ is linked to the MOI via a linker, which includes a sulfamide moiety according to formula (1).

The reactive group Q ¹ may be bound to either end of the moiety of formula (1), in which case the MOI is bound to the opposite end of the moiety of formula (1). In one embodiment, the reactive group Q ¹ is attached to the moiety of formula (1) via the carbonyl end and the MOI is attached via the sulfamide end of the moiety of formula (1). . In one embodiment, the reactive group Q ¹ is attached to the moiety of formula (1) via the sulfamide end and the MOI is attached via the carbonyl end of the moiety of formula (1). .
Definition

  The verb “include” and its use, as used in this specification and claims, includes items that follow the word, but does not exclude items that are not specifically described. Used in a non-limiting sense to mean.

  In addition, reference to an element by the indefinite article “a” or “an” means that more than one of the elements is required unless the context clearly requires that one and only one of the elements be present. It does not remove the possibility that it exists. The indefinite article “a” or “an” thus usually means “at least one”.

  The compounds disclosed in the specification and claims can contain one or more asymmetric centers and different diastereomers and / or enantiomers of the compounds can exist. Reference to any compound in the specification and claims is meant to include all diastereomers and mixtures thereof, unless otherwise specified. In addition, the recitation of any compound in the specification and claims is meant to include both the individual enantiomers, as well as any mixture of enantiomers, racemic or otherwise, unless otherwise specified. Where the structure of a compound is depicted as a particular enantiomer, it should be understood that the invention of this application is not limited to that particular enantiomer.

  The compounds may be in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms unless otherwise specified. Where the structure of a compound is depicted as a particular tautomer, it should be understood that the invention of this application is not limited to that particular tautomer.

  The compounds disclosed herein and in the claims may further exist as exo and endo diastereoisomers. Unless stated otherwise, the description of any compound in the specification and claims is meant to include both the individual exo and individual endo diastereoisomers of the compound, and mixtures thereof. Where the structure of a compound is depicted as a particular endo or exo diastereomer, it is meant that the invention of this application is not limited to that particular endo or exo diastereomer.

  Furthermore, the compounds disclosed herein and in the claims may exist as cis and trans isomers. Unless stated otherwise, the description of any compound in the specification and claims is meant to include both the individual cis and individual trans isomers of the compounds, and mixtures thereof. By way of example, where the structure of a compound is depicted as a cis isomer, it should be understood that the corresponding trans isomer or mixture of cis and trans isomers is not excluded from the invention of the present application. Where the structure of a compound is depicted as a particular cis or trans isomer, it should be understood that the invention of this application is not limited to that particular cis or trans isomer.

Unsubstituted alkyl groups have the general formula C _n H _{2n + 1} and can be straight or branched. Optionally, the alkyl group is substituted with one or more substituents as further specified in this document. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl and the like.

A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkyl group contains at least 3 carbon atoms, having the general formula C _{n H 2n-1.} Optionally, the cycloalkyl group is substituted with one or more substituents as further specified herein. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

An alkenyl group contains one or more carbon-carbon double bonds and can be straight or branched. An unsubstituted alkenyl group containing one C—C double bond has the general formula C _n H _2n-1 . An unsubstituted alkenyl group containing two C—C double bonds has the general formula C _n H _2n-3 . Alkenyl groups may contain terminal carbon-carbon double bonds and / or internal carbon-carbon double bonds. A terminal alkenyl group is an alkenyl group in which the carbon-carbon double bond is located at the terminal position of the carbon chain. An alkenyl group may contain two or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, t-butenyl, 1,3-butadienyl, 1,3-pentadienyl and the like. Unless otherwise specified, an alkenyl group may be optionally substituted with one or more independently selected substituents as defined below. Unless otherwise specified, an alkenyl group may be optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.

Alkynyl groups contain one or more carbon-carbon triple bonds and can be straight or branched. An unsubstituted alkynyl group containing one C—C triple bond has the general formula C _n H _2n-3 . Alkynyl groups may contain terminal carbon-carbon triple bonds and / or internal carbon-carbon triple bonds. A terminal alkynyl group is an alkynyl group in which the carbon-carbon triple bond is located at the terminal position of the carbon chain. An alkynyl group may contain two or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group may be optionally substituted with one or more independently selected substituents as defined below. Examples of alkynyl groups include ethynyl, propynyl, isopropynyl, t-butynyl and the like. Unless otherwise specified, an alkynyl group may be optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N, and S.

  Aryl groups contain 6-12 carbon atoms and can include monocyclic and bicyclic structures. Optionally, the aryl group may be substituted with one or more substituents as further specified in this document. Examples of aryl groups are phenyl and naphthyl.

  Arylalkyl groups and alkylaryl groups contain at least 7 carbon atoms and can include monocyclic and bicyclic structures. Optionally, the arylalkyl group and the alkylaryl may be substituted with one or more substituents as further specified in this document. An arylalkyl group is for example benzyl. An alkylaryl group is, for example, 4-t-butylphenyl.

A heteroaryl group contains at least 2 carbon atoms (ie, at least C ₂ ) and one or more heteroatoms N, O, P, or S. A heteroaryl group may have a monocyclic or bicyclic structure. Optionally, the heteroaryl group may be substituted with one or more substituents as further specified herein. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phosphoryl and oxazolyl.

Heteroarylalkyl groups and alkylheteroaryl groups contain at least 3 carbon atoms (ie, at least C ₃ ) and can include monocyclic and bicyclic structures. Optionally, the heteroaryl group may be substituted with one or more substituents as further specified herein.

Where an aryl group is shown as a (hetero) aryl group, the notation is meant to include aryl groups and heteroaryl groups. Similarly, an alkyl (hetero) aryl group is meant to include alkylaryl groups and alkylheteroaryl groups, and (hetero) arylalkyl is meant to include arylalkyl groups and heteroarylalkyl groups. _Thus, C 2 _{-C 24} (hetero) aryl groups _should be construed to include C 2 _{-C 24} heteroaryl group, and _C 6 _{-C 24} aryl group. _Similarly, C 3 _{-C 24} alkyl (hetero) aryl group _is meant to include C 7 _{-C 24} alkylaryl group and _C 3 _{-C 24} alkyl _heteroaryl, C 3 _{-C 24} (hetero) aryl Alkyl is meant to include C ₇ -C ₂₄ arylalkyl groups and C ₃ -C ₂₄ heteroarylalkyl groups.

A cycloalkynyl group is a cyclic alkynyl group. An unsubstituted cycloalkynyl group containing one triple bond has the general formula C _n H _2n-5 . Optionally, the cycloalkynyl group is substituted with one or more substituents as further specified in this document. An example of a cycloalkynyl group is cyclooctynyl.

  A heterocycloalkynyl group is a cycloalkynyl group interrupted by a heteroatom selected from the group of oxygen, nitrogen and sulfur. Optionally, the heterocycloalkynyl group is substituted with one or more substituents as further specified herein. An example of a heterocycloalkynyl group is azacyclooctynyl.

  The (hetero) aryl group includes an aryl group and a heteroaryl group. The alkyl (hetero) aryl group includes an alkylaryl group and an alkylheteroaryl group. The (hetero) arylalkyl group includes an arylalkyl group and a heteroarylalkyl group. (Hetero) alkynyl groups include alkynyl and heteroalkynyl groups. The (hetero) cycloalkynyl group includes a cycloalkynyl group and a heterocycloalkynyl group.

  A (hetero) cycloalkyne compound is defined herein as a compound containing a (hetero) cycloalkynyl group.

  Some of the compounds disclosed in this specification and claims are fused (hetero) cycloalkyne compounds, ie, the second ring structure is fused to a (hetero) cycloalkynyl group, ie cyclized. It can be described as a (hetero) cycloalkyne compound. For example, in a fused (hetero) cyclooctyne compound, a cycloalkyl (eg, cyclopropyl) or arene (eg, benzene) can be cyclized to a (hetero) cyclooctynyl group. The triple bond of the (hetero) cyclooctynyl group in the fused (hetero) cyclooctyne compound has three possible positions: the 2-position, the 3-position or the 4-position of the cyclooctyne moiety ("IUPAC Nomenclature of Organic Chemistry", Rule A31. (Numbering according to 2). Reference to any fused (hetero) cyclooctyne compound in the present specification and claims is meant to include all three individual positional isomers of the cyclooctyne moiety.

Unless otherwise specified, alkyl groups, cycloalkyl groups, alkenyl groups, alkynyl groups, (hetero) aryl groups, (hetero) arylalkyl groups, alkyl (hetero) aryl groups, alkylene groups, alkenylene groups, alkynylene groups, Cycloalkylene group, cycloalkenylene group, cycloalkynylene group, (hetero) arylene group, alkyl (hetero) arylene group, (hetero) arylalkylene group, (hetero) arylalkenylene group, (hetero) arylalkynylene group, alkenyl group , an alkoxy group, an alkenyloxy group, (hetero) aryloxy group, an alkynyloxy group and cycloalkyl group _is, C 1 _{-C 12} alkyl _groups, C 2 _{-C 12} alkenyl _groups, C 2 _{-C 12} alkynyl groups, C _{3 ~C 12} cycloalkyl _Group, C 5 _{-C 12} cycloalkenyl _group, C 8 _{-C 12} cycloalkynyl _group, C 1 _{-C 12} alkoxy _group, C 2 _{-C 12} alkenyloxy _group, C 2 _{-C 12} alkynyloxy _groups, C 3 -C It may be substituted with one or more substituents independently selected from the group consisting of ₁₂ cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl group has the formula (R ²⁰ ) ₃ Si—, where R ²⁰ is a C ₁ -C ₁₂ alkyl group, a C ₂ -C ₁₂ alkenyl group, a C ₂ -C ₁₂ alkynyl group, a C ₃ -C ₁₂ cycloalkyl group, a C _{1 to} C ₁₂ alkoxy groups, C _{2 to} C ₁₂ alkenyloxy groups, C _{2 to} C ₁₂ alkynyloxy groups, and C _{3 to} C ₁₂ cycloalkyloxy groups. Wherein an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an alkoxy group, an alkenyloxy group, an alkynyloxy group and a cycloalkyloxy group are optionally substituted, an alkyl group, Alkoxy, cycloalkyl and cycloalkoxy groups are optionally interrupted by one or more heteroatoms selected from the group consisting of O, N and S.

The general term “sugar” is used herein to denote simple sugars such as glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term “sugar derivative” is used herein to denote a monosaccharide sugar derivative, ie, a monosaccharide sugar containing substituents and / or functional groups. Examples of sugar derivatives include amino sugars and sugar acids such as glucosamine (GlcNH ₂ ), galactosamine (GalNH ₂ ) N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), N-acetylneuraminic acid (NeuNAc) And sialic acid (Sia), also referred to as N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA), and iduronic acid (IdoA). Examples of sugar derivatives also include compounds shown herein as S (F ¹ ) _x , where S is a sugar or sugar derivative, and S contains x functional groups F ¹ .

  The core N-acetylglucosamine substituent (core-GlcNAc substituent) or the core N-acetylglucosamine moiety is defined herein as N on the amide nitrogen atom in the side chain of the asparagine amino acid of the antibody, preferably via C1. -Defined as GlcNAc linked via a glycosidic bond. The core-GlcNAc substituent can be present at the native glycosylation site of the antibody, but the core-GlcNAc substituent can also be introduced at a different site of the antibody. In this specification, the core-N-acetylglucosamine substituent is a monosaccharide substituent, or a disaccharide core further referred to as GlcNAc (Fuc) if the core-GlcNAc substituent is fucosylated. GlcNAc- (α1-6-Fuc) substituent. As used herein, “core-GlcNAc substituent” should not be confused with “core-GlcNAc”. Core-GlcNAc is defined herein as an internal GlcNAc that is part of a polysaccharide or oligosaccharide containing more than two saccharides, ie, a GlcNAc through which a poly or oligosaccharide is attached to an antibody. Has been.

Thus, an antibody comprising a core-N-acetylglucosamine substituent as defined herein is an antibody comprising a monosaccharide core-GlcNAc substituent as defined above, or the core An antibody comprising a disaccharide core-GlcNAc (Fuc) substituent if the GlcNAc substituent is fucosylated. If the GlcNAc in the core-GlcNAc substituent or GlcNAc-S (F ¹ ) _x substituent is fucosylated, fucose is most commonly linked to O6 of the core-GlcNAc substituent at α-1,6. ing. The fucosylated core-GlcNAc substituent is designated as core-GlcNAc (Fuc) and the fucosylated GlcNAc-S (F ¹ ) _x substituent is designated as GlcNAc (Fuc) -S (F ¹ ) _x .

  The term “nucleotide” is used herein in the usual scientific sense. The term “nucleotide” refers to a molecule composed of a nucleobase, a five-carbon sugar (either ribose or 2-deoxyribose), and one, two, or three phosphate groups. Without the phosphate group, the nucleobase and sugar constitute the nucleoside. Thus, nucleotides can also be referred to as nucleoside monophosphates, nucleoside diphosphates or nucleoside triphosphates. The nucleobase can be adenine, guanine, cytosine, uracil or thymine. Examples of nucleotides include uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine diphosphate (CDP) and cytidine monophosphate (CMP).

  The term “protein” is used herein in the usual scientific sense. As used herein, a polypeptide comprising about 10 or more amino acids is considered a protein. Proteins may contain non-natural amino acids as well as natural.

  The term “glycoprotein” is used herein in the usual scientific sense to refer to a protein comprising one or more monosaccharide or oligosaccharide chains (“glycans”) covalently linked to the protein. Glycans are linked to the hydroxyl group of proteins (O-linked glycans), for example to the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline, or to the amide functional group of a protein (N-glycoprotein), for example asparagine or arginine. Or it may be bound to carbon of a protein (C-glycoprotein), for example, tryptophan. Glycoproteins may contain more than one glycan, may contain a combination of one or more monosaccharides and one or more oligosaccharide glycans, N-linked glycans, O-linked glycans and C-linked glycans May be included. It is estimated that over 50% of all proteins have some form of glycosylation and are therefore recognized as glycoproteins. Examples of glycoproteins include PSMA (prostate specific membrane antigen), CAL (Candida antalylipase), gp41, gp120, EPO (erythropoietin), antifreeze proteins and antibodies.

  The term “glycan” is used herein in the usual scientific sense to refer to a mono- or oligosaccharide chain that is linked to a protein. Thus, the term glycan refers to the carbohydrate portion of a glycoprotein. Glycans may be free of further substitution (monosaccharides) or may be further substituted at one or more of the sugar's hydroxyl groups (oligosaccharides) via the C-1 carbon of one sugar. It is bound to protein. Naturally occurring glycans typically contain from 1 to about 10 sugar moieties. However, if a longer polysaccharide chain is linked to a protein, the polysaccharide chain is also considered herein as a glycan. The glycoprotein glycan can be a monosaccharide. Typically, glycoprotein monosaccharide glycans consist of a single N-acetylglucosamine (GlcNAc), glucose (Glc), mannose (Man) or fucose (Fuc) covalently bound to the protein. Glycans can also be oligosaccharides. The oligosaccharide chain of a glycoprotein can be linear or branched. In oligosaccharides, sugars that are directly bound to proteins are called core sugars. In an oligosaccharide, a sugar that is not directly bound to a protein but bound to at least two other sugars is called an internal sugar. In an oligosaccharide, a sugar that is not directly attached to a protein but directly attached to a single other sugar, i.e., a sugar that does not carry an additional sugar substituent at one or more of the other hydroxyl groups of the sugar. Are called terminal sugars. To avoid doubt, there may be only one core sugar, although there are multiple terminal sugars in the oligosaccharides of glycoproteins. The glycan can be an O-linked glycan, an N-linked glycan, or a C-linked glycan. In O-linked glycans, mono- or oligosaccharide glycans are attached to the O atom in the amino acid of the protein, typically via the hydroxyl group of serine (Ser) or threonine (Thr). In N-linked glycans, mono- or oligosaccharide glycans bind to proteins via N atoms in the amino acids of the protein, typically via the amide nitrogen in the side chain of asparagine (Asn) or arginine (Arg). ing. In C-linked glycans, mono- or oligosaccharide glycans are bound to C atoms in the amino acids of proteins, typically to the C atom of tryptophan (Trp).

The term “antibody” is used herein in the usual scientific sense. An antibody is a protein produced by the immune system that can recognize and bind to a specific antigen. An antibody is an example of a glycoprotein. The term antibody is used herein in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies), antibody fragments. And double chain antibodies and single chain antibodies. The term “antibody” is also meant herein to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind cancer antigens. The term “antibody” refers to not only whole antibodies but also antigen-binding fragments of antibodies, such as antibody Fab fragments, F (ab ′) ₂ , Fv fragments or Fc fragments from cleaved antibodies, scFv-Fc fragments, miniforms , Bispecific antibody, bispecific antibody or scFv. Furthermore, the term includes genetically modified antibodies and antibody derivatives. Antibodies, antibody fragments and genetically modified antibodies can be obtained by methods known in the art. Typical examples of antibodies include, among others, abciximab, rituximab, basiluximab, paclizumab, paclizumab, infliximab, trastuzumab, alemtuzumab, adalimumab, tositumomab-I131, cetuximab, ibritumumab, pivazumab, pivazumab, pivazumab Zumab Pegor, Golimumab, Canakinumab, Katuximab, Ustekinumab, Tocilizumab, Ofatumumab, Denosumab, Belimumab, ipilimumab and brentuximab. In a preferred embodiment, the antibody comprising a core-N-acetylglucosamine substituent (core-GlcNAc substituent) is a monoclonal antibody (mAb). Preferably, the antibody is selected from the group consisting of IgA, IgD, IgE, IgG and IgM antibodies. The antibody is more preferably an IgG antibody, and the antibody is most preferably an IgG1 antibody. Where the antibody is an entire antibody, the antibody preferably comprises one or more, more preferably one core-GlcNAc substituent in each heavy chain, wherein the core-GlcNAc substituent is optionally fucosylated. Has been. Thus, the entire antibody preferably comprises two or more, preferably two, optionally fucosylated core-GlcNAc substituents. Where the antibody is a single chain antibody or an antibody fragment, such as a Fab fragment, the antibody preferably comprises one or more core-GlcNAc substituents that are optionally fucosylated. In an antibody comprising a core-GlcNAc substituent, the core-GlcNAc substituent can be located anywhere in the antibody, provided that the substituent does not interfere with the antigen binding site of the antibody. In a preferred embodiment, the core N-acetylglucosamine substituent is present at the native N-glycosylation site of the antibody.

A linker is defined herein as a moiety that connects two or more elements of a compound. For example, in a bioconjugate, the biomolecule and the target molecule are covalently connected to each other via a linker; in the linker-conjugate, the reactive group Q ¹ is covalently attached to the target molecule via the linker. In the linker-construct, the reactive group Q ¹ is covalently connected to the reactive group Q ² via the linker. The linker can include one or more spacer moieties.

  A spacer moiety is defined herein as a moiety that is spaced apart (ie, provides a distance between) and covalently links two (or more) parts of the linker together. The linker may be part of a linker-construct, linker-conjugate or bioconjugate, for example, as defined below.

  A bioconjugate is defined herein as a compound in which a biomolecule is covalently connected to a target molecule via a linker. The bioconjugate comprises one or more biomolecules and / or one or more target molecules. The linker can include one or more spacer moieties. Antibody-conjugate refers to a bioconjugate in which the biomolecule is an antibody.

  Biomolecules as used herein are any molecule that can be isolated from nature, or smaller molecular structures that are components of macromolecular structures derived from nature, in particular nucleic acids, proteins, glycans and lipids. Defined as any molecule composed of blocks. In the present specification, a biomolecule can also be referred to as a target biomolecule (BOI). Examples of biomolecules include enzymes, (non-catalytic) proteins, polypeptides, peptides, amino acids, oligonucleotides, monosaccharides, oligosaccharides, polysaccharides, glycans, lipids and hormones.

  A target molecule, also referred to as a molecule of interest (MOI), is defined herein as a molecular structure having the desired properties conferred on a biomolecule upon conjugation.

  The term “salt thereof” means a compound formed when an acidic proton, typically an acid proton, is replaced by a cation such as a metal cation or an organic cation. Where applicable, the salt is a pharmaceutically acceptable salt, but 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 can be protonated with an inorganic acid or an organic acid to form a cation, and a conjugate base of the inorganic acid or organic acid is used as the anionic constituent of the salt.

  The term “pharmaceutically acceptable” salt refers to a salt that is acceptable for administration to a patient, such as a mammal (a salt with a counterion that has acceptable mammalian safety for a given dosage regimen). Means. Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. “Pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt of a compound, which is derived from various organic and inorganic counterions known in the art, eg, sodium , Potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and organic or inorganic acid salts such as hydrochloride, hydrobromide, formate, tartaric acid if the molecule contains a basic functional group Examples thereof include salts, besylate salts, mesylate salts, acetate salts, maleate salts, and oxalate salts.

In the present specification, a sulfamide linker and a conjugate of the sulfamide linker are disclosed. The term “sulfamide linker” refers to a sulfamide group, more particularly an acylsulfamide group [—C (O) —N (H) —S (O) ₂ —N (R ¹ ) —] and / or carbamoyl. It refers to a linker comprising a sulfamide group [—O—C (O) —N (H) —S (O) ₂ —N (R ¹ ) —].

As used herein, the term “therapeutic index” (TI) has the conventional meaning well known to those skilled in the art, and is the dose that results in the desired pharmacological effect in 50% of the population (effective amount or ED ₅₀ ). Refers to the ratio of the dose (TD ₅₀ ) of a drug that is toxic to 50% of the population divided by (ie, causes an adverse effect at an incidence or severity that is incompatible with the targeted indication). Therefore, TI = TD ₅₀ / ED ₅₀ . The therapeutic index can be determined by clinical trials or for example by plasma exposure studies. See also Muller et al., Nature Reviews Drug Discovery 2012, 11, 751-761.

As used herein, the term “therapeutic efficacy” refers to the ability of a substance to achieve a particular therapeutic effect, eg, a reduction in tumor volume. The therapeutic effect can be measured by determining the extent to which a substance achieves the desired effect, typically in comparison to another substance under the same circumstances. A suitable criterion for therapeutic efficacy is an ED ₅₀ value that can be determined, for example, during clinical trials or by plasma exposure studies. In the case of preclinical therapeutic efficacy determination, the therapeutic effect of a bioconjugate (eg, ADC) can be verified by patient-induced tumor xenografts in mice, in which case efficacy is a beneficial effect. Refers to the ADC's ability to provide. Alternatively, the tolerability of the ADC in rodent safety studies can also be a measure of therapeutic efficacy.

As used herein, the term “tolerated” refers to the maximum dose of a particular substance that does not cause adverse effects at an incidence or severity that is incompatible with the targeted indication. A suitable criterion for tolerability for a particular substance is a TD ₅₀ value that can be determined, for example, during clinical trials or by plasma exposure studies.
Conjugation modes

  In the context of the present invention, “mode of conjugation” refers to the process used to conjugate target molecule D to biomolecule B, in particular antibody AB, as well as the resulting bioconjugate, in particular conjugation. Refers to the structural feature of the linker that connects the target molecule to the biomolecule, which is a direct consequence of the process. Thus, in one embodiment, the mode of conjugation refers to a process for conjugation of a target molecule to a biomolecule, particularly an antibody. In an alternative embodiment, the mode of conjugation is a direct consequence of the process for conjugation of the target molecule to a biomolecule, in particular an antibody, the structural features of the linker and / or the attachment point of the linker to the biomolecule. Point to.

In the context of the present invention, the mode of conjugation further comprises at least one of “core-GlcNAc functionalization” and “sulfamide linkage” as defined below. The mode of conjugation preferably further includes both “core-GlcNAc functionalization” and “sulfamide linkage” as defined below.
Core-GlcNAc functionalization

（式中、Ｓ（Ｆ^１）_ｘ及びｘは、上で定義されている通りであり；ＡＢは抗体を表し；ＧｌｃＮＡｃはＮ−アセチルグルコサミンであり；Ｆｕｃはフコースであり；ｂは０又は１であり；ｙは１、２、３又は４である）に従った修飾抗体を得ることができる、ステップ；並びに
（ｉｉ）修飾抗体を、官能基Ｆ^１と反応できる官能基Ｑ^１及びリンカーＬ^２を介してＱ^１に接続されている標的分子Ｄを含むリンカー−コンジュゲートと反応させることで抗体−コンジュゲートを得るステップであって、リンカーＬはＳ−Ｚ^３−Ｌ^２を含み、Ｚ^３は、Ｑ^１とＦ^１との間の反応から生じる接続基である、ステップ。 In one embodiment, the mode of conjugation according to the present invention is referred to as “core-GlcNAc functionalization” which refers to a process comprising:
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is , A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) By transferring the _x moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 And y is 1, 2, 3 or 4), and (ii) a functional group Q ¹ and a linker L that can react the modified antibody with the functional group F ^1. Obtaining an antibody-conjugate by reacting with a linker-conjugate comprising a target molecule D connected to Q ¹ via ^{2 wherein} the linker L comprises SZ ³ -L ² and Z ³ is a connecting group resulting from the reaction between Q ¹ and F ¹ .

In this embodiment, the antibody is conjugated to the core-GlcNAc residue (optionally substituted with fucose) via a trimmed glycan. The residue is functionalized with one or two sugar derivatives S (F ¹⁾ containing a functional group F ¹ _x, the functional group F ¹ is subsequently linker containing the target molecule D - conjugated It is reacted with the functional group Q ¹ present. The structural features of the resulting linker L that link the antibody to the target molecule are a direct consequence of the conjugation process and include the following:
(A) The point of attachment of linker L to antibody AB is a specific amino acid residue that is glycosylated in a naturally occurring antibody, or is artificially introduced by mutation of a specific amino acid residue in the antibody. Glycosylation site. Thus, the point of attachment of the linker to the antibody giving a highly predictable target molecule to antibody ratio (or DAR: "drug antibody ratio") can be specifically selected.
(B) the linker L is conjugated to the core -GlcNAc portion of an antibody, having the general structure _{^{-S- (M) pp -Z 3 -L}} 2 (D) r. As used herein, S is, the core -GlcNAc moiety through an O4, and through any one of C2, C3, C4 and C6, preferably in ^{Z 3} through C6, optionally A sugar derivative typically connected via a spacer M (ie pp = 0 or 1). Z ³ is a connecting group obtained by a reaction between Q ¹ and F ¹ . The choices for Q ¹ , F ¹ and Z ³ are known to those skilled in the art and are discussed in further detail below. Z ³ is connected to at least one target molecule D via the linker L ² (ie r ≧ 1).
(C) In a preferred embodiment, the linker L, in particular the linker L ² includes group or a salt thereof according to "sulfamide linked" and conjugation of as defined for modes formula called (1). It has been found that when the mode of conjugation according to this embodiment is combined with the “sulfamide linkage” mode of conjugation, the best results have been obtained in terms of improving the therapeutic index of the resulting bioconjugate. It was done.

  The inventors have surprisingly found that using the above process to conjugate the target molecule to the antibody has a beneficial effect on the therapeutic index of the antibody-conjugate. In other words, the therapeutic index of an antibody-conjugate having a mode of conjugation according to this embodiment has an improved therapeutic index over an antibody-conjugate not having a mode of conjugation according to this embodiment.

  The use of the mode of conjugation according to the present invention differs from the process of preparing antibody-conjugates, where the mode of conjugation is used to prepare antibody-conjugates. There are many modes of conjugation to prepare antibody-conjugates, but we keep the antibody and target molecule (s) constant while choosing a particular mode of conjugation. Has been found to beneficially affect the therapeutic index of the conjugate.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ若しくはＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含むように調製するステップを含む。 Within the context of this mode of conjugation termed “core-GlcNAc functionalization”, in one embodiment, the mode of conjugation is of formula (A):
BLD
(A)
(Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or spacer moiety) and the reactive group Q ¹ of the target molecule (D) is reacted with the functional group F ¹ of the biomolecule (B). Wherein L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I where target molecule, space Comprising the step of preparing to include are connected optionally) the N through a moiety.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ若しくはＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含むように調製するステップを含まない。
ステップ（ｉ） Within the context of this mode of conjugation termed “core-GlcNAc functionalization”, in one embodiment, the mode of conjugation is of formula (A):
BLD
(A)
(Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or spacer moiety) and the reactive group Q ¹ of the target molecule (D) is reacted with the functional group F ¹ of the biomolecule (B). Wherein L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I where target molecule, space It does not include the step of preparing to include are connected optionally) the N through a moiety.
Step (i)

In step (i), a glycoprotein comprising 1-4 core N-acetylglucosamine moieties is contacted with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, where S (F ¹ ) _x Is a sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is a nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) The _x moiety can be transferred to the core-GlcNAc moiety. As used herein, a glycoprotein is typically an antibody, such as an antibody trimmed to a core-GlcNAc residue, as further described below.

（式中、Ｓ（Ｆ^１）_ｘ及びｘは、上で定義されている通りであり；ＡＢは抗体を表し；ＧｌｃＮＡｃはＮ−アセチルグルコサミンであり；Ｆｕｃはフコースであり；ｂは０又は１であり；ｙは１、２、３又は４である）に従った修飾抗体を与える。 Step (i) is represented by equation (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 And y is 1, 2, 3 or 4).

  In preferred embodiments, y = 1, 2 or 4, more preferably y = 1 or 2 (eg when AB is a single chain antibody), or alternatively y = 2 or 4 (eg where AB is A double chain antibody). Most preferably y = 2.

In one embodiment, the antibody comprising an optionally fucosylated core-GlcNAc substituent is the antibody of formula (21), wherein AB represents an antibody, GlcNAc is N-acetylglucosamine, and Fuc is It is fucose, b is 0 or 1, y is preferably 1 to 4, and y is preferably 1 or 2.

  Such antibodies comprising a core-GlcNAc substituent are known in the art and can be prepared by methods known by those skilled in the art. In one embodiment, the process according to the invention further comprises deglycosylation of an antibody glycan having core N-acetylglucosamine in the presence of endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein Wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Depending on the nature of the glycan, an appropriate endoglycosidase can be selected. The endoglycosidase is preferably selected from the group consisting of EndoS, EndoA, EndoF, EndoM, EndoD, EndoH and EndoSH enzymes and / or combinations thereof, and this selection depends on the nature of the glycan. EndoSH is further defined below in the fourth aspect of the invention. In a further preferred embodiment, the endoglycosidase is EndoS, EndoS49, EndoF, EndoSH or a combination thereof. In a further preferred embodiment, the endoglycosidase is EndoS, EndoF or a combination thereof. In a further preferred embodiment, the endoglycosidase is EndoS. In another preferred embodiment, the endoglycosidase is EndoS49. In another preferred embodiment, the endoglycosidase is EndoSH.

In step (i), modification of the antibody (21a) with y = 1 leads to a modified antibody (22) containing ^one GlcNAc-S (F ¹ ) _x substituent and the antibody (21b) with y = 2 ) Modification leads to a modified antibody (23) containing two GlcNAc-S (F ¹ ) _x substituents. In one embodiment, it is preferred that y = 2 or 4 when AB is a double chain antibody. In one embodiment, it is preferred that y = 1 or 2 when AB is a single chain antibody.

  In a preferred embodiment, antibody AB is 5T4 (TPBG), αv-integrin / ITGAV, BCMA, C4.4a, CA-IX, CD19, CD19b, CD22, CD25, CD30, CD33, CD37, CD40, CD56, CD70, CD74, CD79b, c-KIT (CD117), CD138 / SDC1, CEACAM5 (CD66e), crypto, CS1, DLL3, EFNA4, EGFR, EGFRvIII, endothelin B receptor (ETBR), ENPP3 (AGS-16), EpCAM, EphA2 , FGFR2, FGFR3, FOLR1 (folate receptor a), gpNMB, guanylcyclase C (GCC), HER2, Erb-B2, Lamp-1, Lewis Y antigen, LIV-1 (SLC39A6, ZIP6) Mesothelin (MSLN), MUC1 (CA6, huDS6), MUC16 / EA-125, NaPi2b, Nectin-4, Notch3, P-cadherin, PSMA / FOLH1, PTK7, SLITRK6 (SLC44A4), STEAP1, TF (CD142), Trop 1, tumors expressing an antigen selected from Trop-2 / EGP-1, Trop-3, Trop-4, preferably CD30 expressing tumors can be targeted.

  In a preferred embodiment, antibody AB can target CD30 expressing tumors, more preferably, antibody AB is Ki-2, Ki-2, Ki-4, Ki-6, Ki-7, HRS-1, HRS -4, Ber-H8, Ber-H2, 5F11 (MDX-060, Iratumumab), Ki-1, Ki-5, M67, Ki-3, M44, HeFi-1, AC10, cAC10 (brentuximab) and its Selected from the group consisting of functional analogues. More preferably, the antibody AB capable of targeting a CD30 expressing tumor is iratumumab or brentuximab, most preferably brentuximab.

S (F ¹ ) _x is defined as a sugar derivative containing x functional groups F ¹ , where x is 1 or 2 and F ¹ is a linker-to form the connecting moiety Z ³ it is a functional group capable of reacting with Q ¹ that is present in the conjugate. The sugar derivative S (F ¹ ) _x can contain one or two functional groups F ¹ . When S (F ¹ ) _x contains two functional groups F ¹ , each functional group F ¹ is independently selected, ie one S (F ¹ ) _x contains different functional groups F ¹ . be able to. In one embodiment, x = 1. In one embodiment, x = 2. The sugar derivative S (F ¹ ) _x is derived from a sugar or sugar derivative S, such as an amino sugar or other derivatized sugar. Examples of saccharides and sugar derivatives include galactose (Gal), mannose (Man), glucose (Glc), glucuronic acid (GlucA) and fucose (Fuc). An amino sugar is a sugar in which a hydroxyl (OH) group is replaced by an amino group, and examples include N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Examples of other derivatized sugars include glucuronic acid (GlucA) and N-acetylneuraminic acid (sialic acid). The sugar derivative S (F ¹ ) _x is preferably galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), glucose (Glc), N-acetylgalactosamine (GalNAc), glucuronic acid (GlucA), fucose It is derived from (Fuc) and N-acetylneuraminic acid (sialic acid), preferably from the group consisting of GlcNAc, Glc, Gal and GalNAc. S ^(F _{1) x} is more preferably derived from Gal or GalNAc, ^{S (F} _{1) x} is most preferably derived from GalNAc.

Compounds of the formula S (F ¹ ) _x -P in which a nucleoside monophosphate or nucleoside diphosphate P is linked to a sugar derivative S (F ¹ ) _x are known in the art. See, for example, Wang et al., Chem. Eur. J. et al. 2010, 16, 13343-13345, Piller et al., ACS Chem. Biol. 2012, 7, 753, Piller et al., Bioorg. Med. Chem. Lett. 2005, 15, 5459-5462 and WO 2009/102820 disclose a number of compounds S (F ¹ ) _x -P and their synthesis. In a preferred embodiment, the nucleoside monophosphate or diphosphate P in S (F ¹ ) _x -P is uridine diphosphate (UDP), guanosine diphosphate (GDP), thymidine diphosphate (TDP), cytidine. Selected from the group consisting of diphosphate (CDP) and cytidine monophosphate (CMP), wherein P is a group consisting of uridine diphosphate (UDP), guanosine diphosphate (GDP) and cytidine diphosphate (CDP) Is more preferably selected, and most preferably P = UDP.

One or two functional groups F ¹ in S (F ¹ ) _x may be linked to the sugar or sugar derivative S in several ways. One or two functional groups F ¹ may be attached to C2, C3, C4 and / or C6 of the sugar or sugar derivative instead of the hydroxyl (OH) group. Since fucose lacks an OH group in C6, if F ¹ is attached to C6 of Fuc, F ¹ is to be noted that replace the H atom. When F ¹ is an azide group, F ¹ is preferably bonded to C2, C4 or C6. As described above, the one or more azide substituents in S (F ¹ ) _x may be substituted with a hydroxyl (OH) group or in the case of 6-azidofucose (6-AzFuc) Alternatively, it may be bound to C2, C3, C4 or C6 of the sugar or sugar derivative S. Alternatively or additionally, the N-acetyl substituent of the amino sugar derivative may be substituted with an azidoacetyl substituent. In a preferred embodiment, S (F ¹ ) _x is 2-azidoacetamidogalactose (GalNAz), 2-azidodifluoroacetamido-2-deoxy-galactose (F ₂ -GalNAz), 6-azido-6-deoxygalactose (6- AzGal), 6- azido-6-deoxy-2-acetamide galactose (6-AzGalNAc or _6-N 3 -GalNAc), 4- azido-4-deoxy-2-acetamide-galactose (4-AzGalNAc), 6- azido - 6-deoxy-2-azidoacetamidogalactose (6-AzGalNAz), 2-azidoacetamidoglucose (GlcNAz), 6-azido-6-deoxyglucose (6-AzGlc), 6-azido-6-deoxy-2-acetamidoglucose ( 6-AzGlcNAc), 4-azido-4-deoxy-2-acetamidoglucose (4-AzGlcNAc) and 6-azido-6-deoxy-2-azidoacetamidoglucose (6-AzGlcNAz), more preferably GalNAz , 4-AzGalNAc and 6-AzGalNAc. Examples of S (F ¹ ) _x -P where F ¹ is an azide group are shown below. When F ¹ is a keto group, F ¹ is preferably bonded to C 2 instead of the OH group of S. Alternatively, F ¹ may be bound to the N atom of an amino sugar derivative, preferably a 2-amino sugar derivative. The sugar derivative then contains a —NC (O) R ³⁶ substituent. R ³⁶ is preferably an optionally substituted C ₂ -C ₂₄ alkyl group. R ³⁶ is more preferably an ethyl group. In a preferred embodiment, S (F ¹ ) _x is 2-deoxy- (2-oxopropyl) galactose (2-keto Gal), 2-N-propionylgalactosamine (2-N-propionylGalNAc), 2-N— (4-Oxopentanoyl) galactosamine (2-N-LevGal) and 2-N-butyrylgalactosamine (2-N-butyryl GalNAc), more preferably from the group consisting of 2-keto GalNAc and 2-N-propionyl GalNAc Selected. Examples of S (F ¹ ) _x -P where F ¹ is a keto group are shown below. When F ¹ is an alkynyl group, preferably a terminal alkynyl group or a (hetero) cycloalkynyl group, the alkynyl group is preferably present in the 2-amino sugar derivative. An example of S (F ¹ ) _x where F ¹ is an alkynyl group is 2- (but-3-ionic acid amide) -2-deoxy-galactose. Examples of S (F ¹ ) _x -P where F ¹ is an alkynyl group are shown below.

In one embodiment, F ¹ is selected from the group consisting of an azide group, a keto group, and an alkynyl group. The azide group is the azide functional group —N ₃ . The keto group is a — [C (R ³⁷ ) ₂ ] _o C (O) R ³⁶ group, wherein R ³⁶ is a methyl group or an optionally substituted C ₂ -C ₂₄ alkyl group, and R ³⁷ is hydrogen, are independently selected from the group consisting of halogen and ^{R 36,} o is 0-24, preferably 0-10, and more preferably 0,1,2,3,4,5 or 6. R ³⁷ is preferably hydrogen. The alkynyl group is preferably a terminal alkynyl group or a (hetero) cycloalkynyl group as defined above. In one embodiment, an alkynyl group is ^{- _{[C (R 37) 2]}} o C≡C-R 37 group, wherein ^{R 37} and o are as defined ^{above; R 37} is Preferably it is hydrogen.

In one embodiment, F ¹ is an azide or alkyne moiety. Most preferably, F ¹ is an azide group (—N ₃ ). In one embodiment, F ¹ is an azide moiety, Q ¹ is a (cyclo) alkyne moiety, and Z ³ is a triazole moiety.

Some examples (25-28) of S (F ¹ ) _x -UDP, uridine diphosphate linked to azide or alkynyl substituted saccharides and sugar derivatives are shown below.

S (F ¹ ) _x -P is GalNAz-UDP (25), 6-AzGal-UDP (26), 6-AzGalNAc-UDP (6-azido-6-deoxy-N-acetylgalactosamine-UDP) (27) 4-AzGalNAz-UDP, 6-AzGalNAz-UDP, 6-AzGlc-UDP, 6-AzGlcNAz-UDP and 2- (but-3-ionic acid amide) -2-deoxy-galactose-UDP (28) Is preferably selected from. Most preferably, S (F ¹ ) _x -P is GalNAz-UDP (25) or 6-AzGalNAc-UDP (27).

Suitable catalysts that can transfer the S (F ¹ ) _x moiety to the core-GlcNAc moiety are known in the art. Suitable catalysts are those in which the particular sugar derivative nucleotide S (F ¹ ) _x -P in the particular process is a substrate. More specifically, the catalyst catalyzes the formation of β (1,4) -glycoside bonds. The catalyst is preferably from the group of galactosyltransferase and N-acetylgalactosaminyltransferase, more preferably β (1,4) -N-acetylgalactosaminyltransferase (GalNAcT) and β (1,4) -galactosyltransferase ( Selected from the group of β (1,4) -N-acetylgalactosaminyltransferase having a mutant catalytic domain. Suitable catalysts and mutants thereof are disclosed in WO2014 / 066561, WO2016 / 022027 and International application PCT / EP2016 / 059194, all of which are incorporated herein by reference. In one embodiment, the catalyst is wild type galactosyltransferase or N-acetylgalactosaminyltransferase, preferably N-acetylgalactosaminyltransferase. In an alternative embodiment, the catalyst is a mutant galactosyltransferase or an N-acetylgalactosaminyltransferase, preferably a mutant N-acetylgalactosaminyltransferase. The mutant enzymes described in WO 2016/022027 and in international application PCT / EP2016 / 059194 are particularly preferred.

These galactosyltransferase (mutant) enzyme catalysts can recognize internal sugars and sugar derivatives as acceptors. Therefore, the sugar derivative S (F ¹ ) _x is linked to the core-GlcNAc substituent in step (i), regardless of whether the GlcNAc is fucosylated or not.

Step (i) is preferably carried out in a suitable buffer solution such as phosphate, buffered saline (eg phosphate-buffered saline, Tris-buffered saline), citrate, HEPES, Tris and glycine. . Suitable buffers are known in the art. The buffer solution is preferably phosphate-buffered saline (PBS) or Tris buffer. Step (i) is preferably in the range of about 4 ° C to about 50 ° C, more preferably in the range of about 10 ° C to 45 ° C, more preferably in the range of about 20 ° C to about 40 ° C, and most preferably about 30 ° C. Performed at a temperature in the range of ~ 37 ° C. Step (i) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (i) is carried out at a pH in the range of about 7 to about 8.
Step (ii)

In step (ii), the modified antibody is reacted with a linker-conjugate comprising a functional group Q ¹ capable of reacting with functional group F ¹ and a target molecule D connected to Q ¹ via a linker L ² , An antibody-conjugate is obtained in which the linker L comprises SZ ³ -L ² and Z ³ is the connecting group resulting from the reaction between Q ¹ and F ¹ . Linker-conjugates and preferred embodiments thereof are further defined below. The linker L ² preferably comprises a formula group or a salt thereof according to (1), the linker is further defined below.

The complementary functional group Q ¹ of the functional group F ¹ of the modified antibody is known in the art. For example, when F ¹ is an azide group, the linkage of the azido-modified antibody and the linker-conjugate is preferably performed via a cycloaddition reaction. The functional group Q ¹ is then preferably selected from the group consisting of alkynyl groups, preferably terminal alkynyl groups, and (hetero) cycloalkynyl groups. For example, when F ¹ is a keto group, the linking of the keto-modified antibody to the linker-conjugate is preferably done via selective conjugation with a hydroxylamine derivative or hydrazine, resulting in an oxime or hydrazone, respectively. . The functional group Q ¹ is then preferably a primary amino group such as —NH ₂ group, an aminooxy group such as —O—NH ₂ , or a hydrazinyl group such as —N (H) NH ₂ . The linker-conjugate is then preferably H ₂ N—L ² (D) _r , H ₂ N—O—L ² (D) _r or H ₂ N—N (H) —L ² (D) _{r respectively.} It is. For example, when F ¹ is an alkynyl group, the linking of the alkyne-modified antibody to the linker-conjugate is preferably performed via a cycloaddition reaction, preferably 1,3-dipolar cycloaddition. The functional group Q ¹ is then preferably a 1,3-dipole such as an azide, nitrone or nitrile oxide. Then, the linker - conjugate is preferably _{^{N 3 -L 2 (D) r}} .

  In a preferred embodiment, in step (ii), the azide of the azide-modified antibody according to the invention is subjected to a cycloaddition reaction via an alkynyl group, preferably a terminal alkynyl group, or a (hetero) cycloalkynyl group of the linker-conjugate. React with. The above cycloaddition reaction of a molecule containing an azide with a molecule containing a terminal alkynyl group or (hetero) cycloalkynyl group is one of the reactions known in the art as “click chemistry”. In the case of linker-conjugates containing terminal alkynyl groups, the cycloaddition reaction needs to be performed in the presence of a suitable catalyst, preferably a Cu (I) catalyst. However, in preferred embodiments, the linker-conjugate comprises a (hetero) cycloalkynyl group, more preferably a strained (hetero) cycloalkynyl group. When (hetero) cycloalkynyl is a strained (hetero) cycloalkynyl group, the presence of a catalyst is not required and the reaction occurs spontaneously by a reaction called strain-promoted azido-alkyne cycloaddition (SPAC). There is even a thing. The above reaction is one of the reactions known in the art as “metal free click chemistry”. Strained (hetero) cycloalkynyl groups are known in the art and are described in more detail below.

Thus, in a preferred embodiment, step (ii) comprises reacting the modified antibody with a linker-conjugate, wherein the linker-conjugate comprises a (hetero) cycloalkynyl group and one of the purposes. Or a plurality of molecules, wherein the modified antibody is an antibody containing a GlcNAc-S (F ¹ ) _x substituent, GlcNAc is N-acetylglucosamine, and S (F ¹ ) _x is F ¹ is an azide group And x is a sugar derivative containing x functional groups F ^{1 wherein} 1 or 2 and the GlcNAc-S (F ¹ ) _x substituent is N of the GlcNAc-S (F ¹ ) _x substituent. -Conjugated to the antibody via C1 of acetylglucosamine and the GlcNAc is optionally fucosylated. In a further preferred embodiment, the (hetero) cycloalkynyl group is a strained (hetero) cycloalkynyl group.

  The target molecule D can be selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, a nanoparticle, a microparticle, and a biomolecule.

  In the context of D, the term “active substance” means a pharmacological and / or biological substance, ie a substance that is biologically and / or pharmaceutically active, eg a drug, a prodrug, a diagnostic agent, It relates to a protein, peptide, polypeptide, peptide tag, amino acid, glycan, lipid, vitamin, steroid, nucleotide, nucleoside, polynucleotide, RNA or DNA. Examples of peptide tags include cell penetrating peptides such as human lactoferrin or polyarginine. An example of a glycan is oligomannose. An example of an amino acid is lysine.

  When the target molecule is an active substance, the active substance is preferably selected from the group consisting of drugs and prodrugs. More preferably, the active substance is selected from the group consisting of pharmaceutically active compounds, especially low to medium molecular weight compounds (eg about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In a further preferred embodiment, the active substance is selected from the group consisting of cytotoxins, antiviral agents, antibacterial agents, peptides and oligonucleotides. Examples of cytotoxins include colchicine, vinca alkaloid, anthracycline, camptothecin, doxorubicin, daunorubicin, taxane, calicheamicin, tubricin, irinotecan, enediyne, inhibitory peptide, amanitin, deBouganin, duocarmycin, maytansin, auristatin, Indolinobenzodiazepine or pyrrolobenzodiazepine (PBD). In view of the poor water solubility of the active substance, preferred active substances include vinca alkaloid, anthracycline, camptothecin, taxane, tubulin, enediyne, duocarmycin, maytansine, auristatin, indolinobenzodiazepine and pyrrolobenzodiazepine, especially vinca alkaloid. , Anthracyclines, camptothecins, taxanes, tubulin, enediyne, maytansine, pyrrolobenzodiazepines and auristatins.

  The term “reporter molecule” as used herein refers to a molecule whose presence is readily detected, such as a diagnostic agent, dye, fluorophore, radioisotope label, contrast agent, magnetic resonance imaging agent or mass label.

  A wide variety of fluorophores, also referred to as fluorescent probes, are known to those skilled in the art. Several fluorophores are described in, for example, G.M. T. T. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013, chapter 10: “Fluorescent probes”, pages 395-463. Examples of fluorophores include all types of Alexa Fluor (eg Alexa Fluor 555), cyanine dyes (eg Cy3 or Cy5) and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, Examples include pyrene derivatives, naphthalimide derivatives, phycobilin protein derivatives (eg allophycocyanin), chromomycin, lanthanide chelates, and quantum dot nanocrystals. Considering the poor water solubility of fluorophores, preferred fluorophores include cyanine dyes, coumarin derivatives, fluorescein and derivatives thereof, pyrene derivatives, naphthalimide derivatives, chromomycin, lanthanide chelates, and quantum dot nanocrystals, particularly coumarin derivatives, Fluorescein, pyrene derivatives and chromomycin.

Examples of radioisotope labeling include, for example, DTPA (diethylenetriaminepentaacetic anhydride), DOTA (1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid) , NOTA (1,4,7-triazacyclononane N, N ′, N ″ -triacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-N, N ′, N ″, N ′ '' - tetraacetic ^{acid), DTTA (N 1 - (} p- isothiocyanatobenzyl) - diethylenetriamine ^{-N 1, N 2, N 3} , N 3 - tetraacetic acid), deferoxamine or DFA (N '- [5- [ [4-[[5- (acetylhydroxyamino) pentyl] amino] -1,4-dioxobutyl] hydroxyamino] pentyl] -N- (5-aminopentyl) -N-hydroxybutanediamide) or HYN ^{99m Tc,} 111 ^In via a chelating moiety such as C (hydrazino nicotinamide) is connected ^{optionally, 114m In, 115 In, 18} F, 14 C, 64 Cu, 131 I, 125 I, ¹²³ I, ²¹² Bi, ⁸⁸ Y, ⁹⁰ Y, ⁶⁷ Cu, ¹⁸⁶ Rh, ¹⁸⁸ Rh, ⁶⁶ Ga, ⁶⁷ Ga and ¹⁰ B. Isotope labeling techniques are known to those skilled in the art, see eg GT Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd Edition, 2013, Chapter 12: “Isotopic labeling techniques”, pages 507-534.

  Suitable polymers for use as the target molecule D in the compounds according to the invention are known to the person skilled in the art and several examples are for example incorporated by reference in G.C. T. T. Hermanson, “Bioconjugate Technologies”, Elsevier, 3rd Edition, 2013, Chapter 18: “PEGylation and synthetic polymer modification”, pages 787-838, are described in more detail. When the target molecule D is a polymer, the target molecule D is polyethylene glycol (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1, xx-diaminoalkane polymer (where xx is Xx is the number of carbon atoms in the alkane, preferably xx is an integer in the range 2 to 200, preferably 2 to 10), (poly) ethylene glycol diamine (e.g. 1,8-diamino-3, Preferably from the group consisting of 6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (eg dextran), poly (amino acids) (eg poly (L-lysine)) and poly (vinyl alcohol) Independently selected. In view of the poor water solubility of the polymer, preferred polymers include 1, xx-diaminoalkane polymers and poly (vinyl alcohol).

  Suitable solid surfaces for use as target molecule D are known to those skilled in the art. Solid surfaces can be, for example, functional surfaces (eg nanomaterials, carbon nanotubes, fullerenes or virus capsid surfaces), metal surfaces (eg titanium, gold, silver, copper, nickel, tin, rhodium or zinc surfaces), metals Alloy surface (here, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin , Derived from uranium, zinc and / or zirconium), polymer surface (wherein the polymer is eg polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly (dimethylsiloxane) or polymethyl methacrylate, polyacrylamide), glass surface Silicone table , Chromatographic support surface (where the chromatographic support, for example silica support, agarose support, a cellulose support or alumina support) and the like. When the target molecule D is a solid surface, D is preferably selected independently from the group consisting of a functional surface or a polymer surface.

  Hydrogels are known to those skilled in the art. A hydrogel is a water-swelling network formed by cross-linking between polymeric components. For example, A.B. S. Hoffman, Adv. Drug Delivery Rev. See 2012, 64, 18. When the target molecule is a hydrogel, the hydrogel is preferably composed of poly (ethylene) glycol (PEG) as a polymer reference.

  Suitable micro and nanoparticles for use as the target molecule D are known to those skilled in the art. A variety of suitable micro and nanoparticles are described in, for example, G.M. T. T. Hermanson, “Bioconjugate Technologies”, Elsevier, 3rd edition, 2013, chapter 14: “Microparticles and nanoparticles”, pages 549-587. Micro or nanoparticles can be of any shape, such as spheres, rods, tubes, cubes, triangles and cones. The micro or nano particles are preferably spherical. The chemical composition of the micro- and nanoparticles can vary. When the target molecule D is a micro or nanoparticle, the micro or nanoparticle is, for example, a polymeric micro or nanoparticle, a silica micro or nanoparticle, or a gold micro or nanoparticle. Where the particles are polymeric micro- or nanoparticles, the polymer is preferably polystyrene or a copolymer of styrene (eg, a copolymer of styrene and divinylbenzene, butadiene, acrylate and / or vinyl toluene), polymethyl methacrylate (PMMA), Polyvinyl toluene, poly (hydroxyethyl methacrylate (pHEMA) or polyethylene glycol dimethacrylate / 2-hydroxyethyl methacrylate) [poly (EDGMA / HEMA)]. Optionally, the surface of the micro- or nanoparticles is modified, for example with a detergent, by graft polymerization of a secondary polymer or by covalent bonds such as another polymer or spacer moiety.

  The target molecule D can also be a biomolecule. Biomolecules and preferred embodiments thereof are described in more detail below. When the target molecule D is a biomolecule, the biomolecule includes proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and It is preferably selected from the group consisting of monosaccharides.

  Preferred options for D are described further below for antibody-conjugates according to the third aspect. A bioconjugate in the context of the present invention may contain more than one target molecule D, which may be the same or different.

（式中：
Ｌ^２は、本明細書において定義されている通りのリンカーであり；
Ｄは、標的分子であり；
ｒは、１〜２０であり；
Ｒ^３１は、水素、ハロゲン、−ＯＲ^３５、−ＮＯ_２、−ＣＮ、−Ｓ（Ｏ）_２Ｒ^３５、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され、ここでアルキル基、（ヘテロ）アリール基、アルキル（ヘテロ）アリール基及び（ヘテロ）アリールアルキル基は任意選択で置換されており、２個の置換基Ｒ^３１は一緒に連結されて縮環シクロアルキル又は縮環（ヘテロ）アレーン置換基を形成することができ、Ｒ^３５は、水素、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され；
Ｘは、Ｃ（Ｒ^３１）_２、Ｏ、Ｓ又はＮＲ^３２であり、ここでＲ^３２は、Ｒ^３１又はＬ^２（Ｄ）_ｒであり、Ｌ^２、Ｄ及びｒは、上で定義されている通りであり；
ｑは、０又は１であるが、ｑが０であるならばＸはＮＬ^２（Ｄ）_ｒであることを条件とし；
ａａは、０、１、２、３、４、５、６、７又は８である）を有する。 The linker-conjugate preferably comprises a (hetero) cycloalkynyl group. In a preferred embodiment, the linker-conjugate has the formula (31):

(Where:
L ² is a linker as defined herein;
D is the target molecule;
r is 1-20;
R ³¹ is hydrogen, _{^{halogen, -OR 35, -NO 2, -CN}} , -S (O) 2 R 35, C 1 ~C 24 alkyl _group, C 6 _{-C 24} (hetero) aryl _{group, C} 7 ~ C ₂₄ alkyl (hetero) aryl groups and C ₇ -C 24 independently from the group consisting of _(hetero) aryl group is selected, wherein the alkyl group, (hetero) aryl group, alkyl (hetero) aryl groups and (hetero ) An arylalkyl group is optionally substituted and the two substituents R ³¹ can be joined together to form a fused cycloalkyl or fused (hetero) arene substituent, and R ³⁵ is hydrogen, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl _groups, C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) ants Independently selected from the group consisting of Ruarukiru group;
X is C (R ³¹ ) ₂ , O, S or NR ³² , where R ³² is R ³¹ or L ² (D) _r , L ² , D and r are as defined above As it is;
q is 0 or 1, provided that if q is 0, then X is NL ² (D) _r ;
aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

（式中：
Ｌ^２は、本明細書において定義されている通りのリンカーであり；
Ｄは、標的分子であり；
ｒは、１〜２０であり；
Ｒ^３１は、水素、ハロゲン、−ＯＲ^３５、−ＮＯ_２、−ＣＮ、−Ｓ（Ｏ）_２Ｒ^３５、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され、ここでアルキル基、（ヘテロ）アリール基、アルキル（ヘテロ）アリール基及び（ヘテロ）アリールアルキル基は任意選択で置換されており、２個の置換基Ｒ^３１は一緒に連結されて縮環シクロアルキル又は縮環（ヘテロ）アレーン置換基を形成することができ、Ｒ^３５は、水素、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され；
Ｘは、Ｃ（Ｒ^３１）_２、Ｏ、Ｓ又はＮＲ^３２であり、ここでＲ^３２はＲ^３１又はＬ^２（Ｄ）_ｒであり、Ｌ^２、Ｄ及びｒは、上で定義されている通りであり；
ｑは、０又は１であるが、ｑが０であるならばＸはＮＬ^２（Ｄ）_ｒであることを条件とし；
ａａは、０、１、２、３、４、５、６、７又は８であり；
ａａ’は、０、１、２、３、４、５、６、７又は８であり；
ａａ＋ａａ’＜１０である）を有する。 In another preferred embodiment, the linker-conjugate is of formula (31b):

(Where:
L ² is a linker as defined herein;
D is the target molecule;
r is 1-20;
R ³¹ is hydrogen, _{^{halogen, -OR 35, -NO 2, -CN}} , -S (O) 2 R 35, C 1 ~C 24 alkyl _group, C 6 _{-C 24} (hetero) aryl _{group, C} 7 ~ C ₂₄ alkyl (hetero) aryl groups and C ₇ -C 24 independently from the group consisting of _(hetero) aryl group is selected, wherein the alkyl group, (hetero) aryl group, alkyl (hetero) aryl groups and (hetero ) An arylalkyl group is optionally substituted and the two substituents R ³¹ can be joined together to form a fused cycloalkyl or fused (hetero) arene substituent, and R ³⁵ is hydrogen, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl _groups, C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) ants Independently selected from the group consisting of Ruarukiru group;
X is C (R ³¹ ) ₂ , O, S or NR ³² where R ³² is R ³¹ or L ² (D) _r and L ² , D and r are defined above. Street;
q is 0 or 1, provided that if q is 0, then X is NL ² (D) _r ;
aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa ′ is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa + aa ′ <10).

  In a further preferred embodiment, aa + aa 'is 4, 5, 6 or 7, more preferably aa + aa' is 4, 5 or 6, and most preferably aa + aa 'is 5.

In preferred embodiments, if q is 1, then X is C (R ³¹ ) ₂ , O, S or NR ³¹ .

In another preferred embodiment, a is 5, ie the (hetero) cycloalkynyl group is preferably a (hetero) cyclooctyne group. In another preferred embodiment, X is C (R ³² ) ₂ or NR ³² . When X is C (R ³² ) ₂ , R ³² is preferably hydrogen. When X is NR ³² , R ³² is preferably L ² (D) _r . In yet another preferred embodiment, r is 1 to 10, more preferably r is 1, 2, 3, 4, 5, 67 or 8, and r is 1, 2, 3, 4, 5 or 8. 6 is more preferred, and r is most preferably 1, 2, 3 or 4.

The L ² (D) _r substituent can be present on the C atom in the (hetero) cycloalkynyl group or, in the case of a heterocycloalkynyl group, on the heteroatom of the heterocycloalkynyl group. When the (hetero) cycloalkynyl group contains a substituent, such as a fused-ring cycloalkyl, the L ² (D) _r substituent can also be present in the above substituent.

Linker - in order to obtain a conjugate, the method for connecting to a (hetero) cycloalkynyl group at one end of the linker L ² and the target molecule at the other end, the linker, (hetero) cycloalkynyl groups and Depends on the exact nature of the target molecule. Suitable methods are known in the art.

  It is preferred that the linker-conjugate comprises a (hetero) cyclooctyne group, more preferably a distorted (hetero) cyclooctyne group. Suitable (hetero) cycloalkynyl moieties are known in the art. For example, DIFO, DIFO2, and DIFO3 are disclosed in US Patent Application Publication No. 2009/0068738, which is incorporated by reference. DIBO is disclosed in WO2009 / 066763, which is incorporated by reference. The DIBO is optionally Am. Chem. Soc. Can be sulfation (S-DIBO) as disclosed in 2012, 134, 5381. BARAC is a J.B. Am. Chem. Soc. 2010, 132, 3688-3690 and US Patent Application Publication No. 2011/0207147.

Preferred examples of linker-conjugates containing (hetero) cyclooctyne groups are shown below.

  Other cyclooctyne moieties known in the art are DIBAC (also known as ADIBO or DBCO) and BCN. DIBAC is available from Chem. Commun. 2010, 46, 97-99. BCN is disclosed in WO 2011/136645, which is incorporated by reference.

  In a preferred embodiment, the linker-conjugate has the formula (32), (33), (34), (35) or (36).

（式中：
Ｒ^１、Ｌ^２、Ｄ及びｒは、上で定義されている通りであり；
Ｙは、Ｏ、Ｓ又はＮＲ^３２であり、ここでＲ^３２は、上で定義されている通りであり；
Ｒ^３３は、水素、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され；
Ｒ^３４は、水素、Ｙ−Ｌ^２（Ｄ）_ｒ、−（ＣＨ_２）_ｎｎ−Ｙ−Ｌ^２（Ｄ）_ｒ、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、アルキル基は、Ｏ、Ｎ及びＳからなる群から選択される１個又は複数のヘテロ原子によって任意選択で中断されており、アルキル基、（ヘテロ）アリール基、アルキル（ヘテロ）アリール基及び（ヘテロ）アリールアルキル基は、独立して任意選択で置換されており；並びに
ｎｎは、１、２、３、４、５、６、７、８、９又は１０である）を有する。 In another preferred embodiment, the linker-conjugate is of formula (37):

(Where:
R ¹ , L ² , D and r are as defined above;
Y is O, S or NR ³² , where R ³² is as defined above;
R ³³ is hydrogen, halogen, a C ₁ -C ₂₄ alkyl group, a C ₆ -C ₂₄ (hetero) aryl group, a C ₇ -C ₂₄ alkyl (hetero) aryl group, and a C ₇ -C ₂₄ (hetero) aryl alkyl group. Selected independently from the group consisting of;
R ³⁴ is _{^{hydrogen, Y-L 2 (D)}} r, - (CH 2) nn -Y-L 2 (D) r, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl groups are selected _from C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) the group consisting of arylalkyl group, the alkyl group has from 1 selected from O, the group consisting of N and S Or optionally interrupted by a plurality of heteroatoms, wherein the alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl groups are independently optionally substituted; and nn is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

（式中、Ｙ、Ｌ^２、Ｄ、ｎｎ及びｒは、上で定義されている通りである）を有する。 In a further preferred embodiment, R ³¹ is hydrogen. In another preferred embodiment, R ³³ is hydrogen. In another preferred embodiment, n is 1 or 2. In another preferred embodiment, R ³⁴ is hydrogen, YL ² (D) _r or — (CH ₂ ) _nn —YL ² (D) _r . In another preferred embodiment, R ³² is hydrogen or L ² (D) _r . In a further preferred embodiment, the linker-conjugate is of formula 38:

Wherein Y, L ² , D, nn and r are as defined above.

（式中、Ｌ^２、Ｄ及びｒは、上で定義されている通りである）を有する。 In another preferred embodiment, the linker-conjugate is of formula (39):

Wherein L ² , D and r are as defined above.

（式中、Ｌ^２、Ｄ及びｒは、上で定義されている通りである）を有する。 In another preferred embodiment, the linker-conjugate is of formula (35):

Wherein L ² , D and r are as defined above.

The value of pp and the nature of M depend on the azide-substituted sugar or sugar derivative S (F ¹ ) _x present in the azide-modified antibody according to the invention linked to the linker-conjugate. If the azide in S (F ¹ ) _x is present at the C2, C3 or C4 position (instead of the sugar OH group) of the sugar or sugar derivative, pp is 0. If S (F ¹ ) _x is an azidoacetamide-sugar derivative and S (F ¹ ) _x is, for example, GalNAz or GlcNAz, pp is 1 and M is —N (H) C (O) CH ₂ —. is there. If the azide in S (F ¹ ) _x is present at the C6 position of the sugar or sugar derivative, pp is 0 and M is absent.

Linkers (L ² ), also referred to as linking units, are well known in the art. Linker as described herein - in conjugates, L is linked to the target molecule and the functional group Q ^1. L ² is, for example, a linear or branched C _{1 to} C ₂₀₀ alkylene group, a C _{2 to} C ₂₀₀ alkenylene group, a C _{2 to} C ₂₀₀ alkynylene group, a C _{3 to} C ₂₀₀ cycloalkylene group, or a C _{5 to} C _200. cycloalkenylene _groups, C _{8 -C 200} cycloalkynylene _groups, C _{7 -C 200} alkyl arylene _group, C _{7 -C 200} arylalkylene _group, C _{8 -C 200} aryl alkenylene _group, C _{9 -C 200} aryl alkynylene group Can be selected from the group consisting of Optionally, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group may be substituted. , Optionally, the group may be interrupted by one or more heteroatoms, preferably 1-100 heteroatoms, preferably from the group consisting of O, S and NR ³⁵ Wherein R ³⁵ is hydrogen, halogen, a C ₁ -C ₂₄ alkyl group, a C ₆ -C ₂₄ (hetero) aryl group, a C ₇ -C ₂₄ alkyl (hetero) aryl group and a C ₇ -C ₂₄ ( Independently selected from the group consisting of hetero) arylalkyl groups. Most preferably, the heteroatom is O. Examples of suitable linking units include (poly) ethylene glycol diamine chains (eg, 1,8-diamino-3,6-dioxaoctane, or equivalents containing longer ethylene glycol chains), polyethylene glycol chains or poly Examples include ethylene oxide chains, polypropylene glycol chains or polypropylene oxide chains, and 1, xx-diaminoalkanes where xx is the number of carbon atoms in the alkane.

  Another class of suitable linkers includes cleavable linkers. Cleaveable linkers are well known in the art. For example, Shabat et al., Soft Matter 2012, 6, 1073, incorporated herein by reference, discloses cleavable linkers that include self-sacrificial moieties that release upon biological triggers, such as enzymatic cleavage or oxidation events. Yes. Some examples of suitable cleavable linkers are peptide-linkers that are cleaved upon specific recognition by proteases such as cathepsin, plasmin or metalloproteases, or glycosidases such as glucuronidase, or oxygen-poor hypoxic regions. Glycoside based linkers that are cleaved with specific recognition by reduced nitroaromatics.

Preferred linkers L ² is, for the embodiment of "sulfamide linked", as well as the antibody according to the third aspect - the conjugate is further defined below. Preferred linker-conjugates are further defined below.

  Step (ii) is preferably in the range of about 20 ° C. to about 50 ° C., more preferably in the range of about 25 ° C. to about 45 ° C., more preferably in the range of about 30 ° C. to 40 ° C., and most preferably about 32 It is carried out at a temperature in the range from 0C to about 37C. Step (ii) is preferably carried out at a pH in the range of about 5 to about 9, preferably in the range of about 5.5 to about 8.5, more preferably in the range of about 6 to about 8. Most preferably, step (ii) is carried out at a pH in the range of about 7 to about 8. Step (ii) is preferably carried out in water. The water is more preferably purified water, more preferably ultrapure water, or type I water as defined according to ISO 3696. A suitable water is, for example, milliQ® water. The water is preferably buffered, for example with phosphate-buffered saline or Tris. Suitable buffers are known to those skilled in the art. In a preferred embodiment, step (ii) is performed in milliQ water buffered with phosphate-buffered saline or Tris.

（式中、環Ａは、７〜１０員の（ヘテロ）環状部分である）によって表される（１０ｅ）、（１０ｉ）又は（１０ｇ）によって、好ましくは（１０ｇ）によって表される接続部分Ｚ^３を形成するための（シクロ）アルキン−アジドコンジュゲーションである。 In one embodiment, the reaction of step (ii) comprises

(Wherein ring A is a 7-10 membered (hetero) cyclic moiety), represented by (10e), (10i) or (10g), preferably represented by (10g) (Cyclo) alkyne-azide conjugation to form ³ .

（式中：
ＡＢは抗体であり、Ｓは、糖又は糖誘導体であり、ＧｌｃＮＡｃはＮ−アセチルグルコサミンであり；
Ｒ^３１は、水素、ハロゲン、−ＯＲ^３５、−ＮＯ_２、−ＣＮ、−Ｓ（Ｏ）_２Ｒ^３５、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され、ここでアルキル基、（ヘテロ）アリール基、アルキル（ヘテロ）アリール基及び（ヘテロ）アリールアルキル基は、任意選択で置換されており、２個の置換基Ｒ^３１は一緒に連結されて縮環シクロアルキル又は縮環（ヘテロ）アレーン置換基を形成することができ、Ｒ^３５は、水素、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され；
Ｘは、Ｃ（Ｒ^３１）_２、Ｏ、Ｓ又はＮＲ^３２であり、ここでＲ^３２はＲ^３１又はＬ^２（Ｄ）_ｒであり、ここでＬ^２はリンカーであり、Ｄは請求項１に定義されている通りであり；
ｒは、１〜２０であり；
ｑは、０又は１であるが、ｑが０であるならばＸはＮＬ^２（Ｄ）_ｒであることを条件とし；
ａａは、０、１、２、３、４、５、６、７又は８であり；
ａａ’は、０、１、２、３、４、５、６、７又は８であり；
ａａ＋ａａ’＜１０であり；
ｂは、０又は１であり；
ｐｐは、０又は１であり；
Ｍは、−Ｎ（Ｈ）Ｃ（Ｏ）ＣＨ_２−、−Ｎ（Ｈ）Ｃ（Ｏ）ＣＦ_２−、−ＣＨ_２−、−ＣＦ_２−、又は０〜４個のフッ素置換基、好ましくは、フェニレンのＣ２及びＣ６に又はＣ３及びＣ５に好ましくは位置する２個のフッ素置換基を含有する１，４−フェニレンであり；
ｙは、１〜４であり；
Ｆｕｃは、フコースである）によって好ましくは表される。 A bioconjugate comprising or obtained by this mode of conjugation has the formula (40) or (40b):

(Where:
AB is an antibody, S is a sugar or sugar derivative, and GlcNAc is N-acetylglucosamine;
R ³¹ is hydrogen, _{^{halogen, -OR 35, -NO 2, -CN}} , -S (O) 2 R 35, C 1 ~C 24 alkyl _group, C 6 _{-C 24} (hetero) aryl _{group, C} 7 ~ C ₂₄ alkyl (hetero) aryl groups and C ₇ -C 24 independently from the group consisting of _(hetero) aryl group is selected, wherein the alkyl group, (hetero) aryl group, alkyl (hetero) aryl groups and (hetero ) An arylalkyl group is optionally substituted, and the two substituents R ³¹ can be joined together to form a fused cycloalkyl or fused (hetero) arene substituent, and R ³⁵ is , hydrogen, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl _groups, C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) A Independently selected from the group consisting of Ruarukiru group;
X is C (R ³¹ ) ₂ , O, S or NR ³² , wherein R ³² is R ³¹ or L ² (D) _r , where L ² is a linker and D is claim 1 As defined in
r is 1-20;
q is 0 or 1, provided that if q is 0, then X is NL ² (D) _r ;
aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa ′ is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa + aa ′ <10;
b is 0 or 1;
pp is 0 or 1;
M is —N (H) C (O) CH ₂ —, —N (H) C (O) CF ₂ —, —CH ₂ —, —CF ₂ —, or 0 to 4 fluorine substituents, preferably Is 1,4-phenylene containing two fluorine substituents preferably located at C2 and C6 of phenylene or at C3 and C5;
y is 1 to 4;
Fuc is preferably represented by fucose).

（式中、ＡＢ、Ｌ^２、Ｄ、Ｙ、Ｓ、Ｍ、ｘ、ｙ、ｂ、ｐｐ、Ｒ^３２、ＧｌｃＮＡｃ、Ｒ^３１、Ｒ^３３、Ｒ^３４、ｎｎ及びｒは、上で定義されている通りであり、上記Ｎ−アセチルグルコサミンは、任意選択でフコシル化されている（ｂは０又は１である））
の抗体−コンジュゲートである。 In a preferred embodiment, the antibody-conjugate according to the invention has the formula (41):

Wherein AB, L ² , D, Y, S, M, x, y, b, pp, R ³² , GlcNAc, R ³¹ , R ³³ , R ³⁴ , nn and r are defined above. And the N-acetylglucosamine is optionally fucosylated (b is 0 or 1))
Antibody-conjugate.

In a more preferred embodiment, R ³¹ , R ³³ and R ³⁴ are hydrogen, nn is 1 or 2, and in a more preferred embodiment x is 1.

（式中、ＡＢ、Ｌ^２、Ｄ、Ｘ、Ｓ、ｂ、ｐｐ、ｘ、ｙ、Ｍ及びＧｌｃＮＡｃは、上で定義されている通りであり、上記Ｎ−アセチルグルコサミンは任意選択でフコシル化されている（ｂは０又は１である））
の抗体−コンジュゲートであり；又は位置異性体（４２ｂ）：

（式中、ＡＢ、Ｌ^２、Ｄ、Ｘ、Ｓ、ｂ、ｐｐ、ｘ、ｙ、Ｍ及びＧｌｃＮＡｃは、上で定義されている通りであり、上記Ｎ−アセチルグルコサミンは任意選択でフコシル化されている）に従っている。 In another preferred embodiment, the antibody-conjugate has the formula (42):

^{(Wherein, AB, L 2, D,} X, S, b, pp, x, y, M and GlcNAc are as defined above, the N- acetylglucosamine fucosylated optionally (B is 0 or 1))
Or regioisomer (42b):

^{(Wherein, AB, L 2, D,} X, S, b, pp, x, y, M and GlcNAc are as defined above, the N- acetylglucosamine fucosylated optionally Follow).

（式中、ＡＢ、Ｌ^２、Ｄ、Ｘ、Ｓ、ｂ、ｐｐ、ｘ、ｙ、Ｍ及びＧｌｃＮＡｃは、上で定義されている通りであり、上記Ｎ−アセチルグルコサミンは任意選択でフコシル化されている）の抗体−コンジュゲートである。
スルファミド連結 In another preferred embodiment, the antibody-conjugate has the formula (35b):

^{(Wherein, AB, L 2, D,} X, S, b, pp, x, y, M and GlcNAc are as defined above, the N- acetylglucosamine fucosylated optionally Antibody-conjugate.
Sulfamide linkage

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択され、又はＲ^１は、さらなる標的分子Ｄであり、ここでＤは、スペーサー部分を介してＮに任意選択で接続されている）を含む。 In one embodiment, the mode of conjugation according to the present invention is referred to as “sulfamide linkage”, which refers to the presence of a specific linker L that links biomolecule B and target molecule D. All that is said about the linker L in the context of this embodiment, the linker according to an embodiment of the core -GlcNAc functionalized as modes of conjugation, preferably applies particularly to the linker L ^2. Linker L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ is independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is further target molecule D, where D is a spacer moiety Including connected optionally) the N and.

  When the group of formula (1) comprises a salt, the salt is preferably a pharmaceutically acceptable salt.

（式中、Ｒ^１は、上で定義されている通りである）を含む。 In a preferred embodiment, the linker L according to the invention comprises a group according to formula (1) wherein a is 0, or a salt thereof. In this embodiment, the linker L is thus a group according to formula (2) or a salt thereof:

In which R ¹ is as defined above.

（式中、Ｒ^１は、上で定義されている通りである）を含む。 In another preferred embodiment, the linker L according to the invention comprises a group according to formula (1) wherein a is 1 or a salt thereof. In this embodiment, the linker L is thus a group according to formula (3) or a salt thereof:

In which R ¹ is as defined above.

In the groups according to formulas (1), (2) and (3), R ¹ is hydrogen, C ₁ -C ₂₄ alkyl group, C ₃ -C ₂₄ cycloalkyl group, C ₂ -C ₂₄ (hetero) aryl. _groups, C 3 _{-C 24} alkyl is selected from the group consisting of (hetero) aryl group and _C 3 _{-C 24} (hetero) arylalkyl _groups, C 1 _{-C 24} alkyl _groups, C 3 _{-C 24} cycloalkyl groups, C _{The 2 to} C ₂₄ (hetero) aryl group, the C _{3 to} C ₂₄ alkyl (hetero) aryl group and the C _{3 to} C ₂₄ (hetero) arylalkyl group are optionally substituted and selected from O, S and NR ³ It is interrupted optionally by one or more heteroatoms that, where R ³ is independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is Salana A target molecule D, where D is connected optionally to N via a spacer moiety;

In a preferred embodiment, R ¹ is hydrogen or a C ₁ -C ₂₀ alkyl group, more preferably R ¹ is hydrogen or a C ₁ -C ₁₆ alkyl group, and R ¹ is hydrogen or a C ₁ -C ₁₀ alkyl group. More preferred is a group wherein the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , preferably O; Here, R ³ is independently selected from the group consisting of hydrogen and a C ₁ -C ₄ alkyl group. In a preferred embodiment, R ¹ is hydrogen. In another preferred embodiment, R ¹ is a C ₁ -C ₂₀ alkyl group, more preferably a C ₁ -C ₁₆ alkyl group, more preferably a C ₁ -C ₁₀ alkyl group, wherein the alkyl group is 1 Optionally interrupted by one or more O atoms, the alkyl group is optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment, R ¹ is more preferably a (poly) ethylene glycol chain comprising a terminal —OH group. In another preferred embodiment, R ¹ is from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, more preferably hydrogen, methyl, ethyl, It is selected from the group consisting of n-propyl and i-propyl, and more preferably from the group consisting of hydrogen, methyl and ethyl. Even more preferably, R ¹ is hydrogen or methyl, and most preferably R ¹ is hydrogen.

In another preferred embodiment, R ¹ is a further target molecule D. Optionally, D is connected to N via one or more spacer moieties. The spacer portion, if present, provides a distance between D and N, ie, a portion that provides a certain distance between D and N, and covalently connects D and N. Is defined.

  Target molecule D and preferred embodiments thereof are defined in more detail above.

To obtain a bioconjugate of formula (A), the group of formula (1) can be introduced in one of three options. First of all, a linker L comprising a group according to formula (1) or a salt thereof can be present in the linker-conjugate represented by Q ¹ -D, where L is Q ¹ and D It is a spacer between. Second, a linker L comprising a group according to formula (1) or a salt thereof can be present in the biomolecule represented by BF ¹ , where L is between B and F ¹ It is a spacer. Thirdly, a group according to formula (1) or a salt thereof can be formed in the conjugation reaction itself. In the latter option, Q ¹ and F ¹ are their reaction products, ie the connecting group Z ³ contains a group according to formula (1) or a salt thereof, or a group according to formula (1) Or it is selected to be its salt. The group according to formula (1) or a salt thereof is preferably introduced according to the first or second of the options described above, most preferably according to the first option. In the case where the group according to formula (1) or a salt thereof is already present as it is during the conjugation reaction, the positive effect on the solubility and absence of in-process aggregation is as described above of the conjugation reaction. Improve efficiency. In the case where a group according to formula (1) or a salt thereof is present in the linker-conjugate, even a hydrophobic drug can be readily subjected to the conjugation reaction.
Linker-conjugate

The linker-conjugate is represented by Q ¹ -D, preferably by Q ¹ -LD, where D is the target molecule and L is Q ¹ and D as further defined above. a linker connecting, Q ¹ is a reactive group capable of reacting with a functional group F ¹ of biomolecules, "-" each occurrence of is independently a bond or a spacer moiety. In one embodiment, “-” is a spacer moiety as defined herein. In one embodiment, “-” is a bond, typically a covalent bond. Linker - conjugate target molecules, preferably via a linker or spacer, and most preferably via a linker L, as defined above, is covalently coupled to a reactive group Q ¹ A compound. Linker - conjugate, linkers - can be obtained through reaction with a reactive group present on the reactive groups Q ² and the target molecule present in the construct.

The group according to formula (1) or a salt thereof is preferably located between Q ¹ and D. In other words, the reactive group Q ¹ is covalently bonded to the first end of the group according to formula (1) and the target molecule D is the second end of the group according to formula (1). Covalently bound to In this specification, “first end” and “second end” are both a carbonyl end or a carboxy end of a group according to formula (1), or a group according to formula (1). The sulfamide end of any of the above, but logically does not point to the same end.

As will be appreciated by those skilled in the art, a linker-conjugate according to the present invention can comprise more than one target molecule D, such as 2, 3, 4, 5, etc. Consequently, the linker-conjugate can thus comprise more than one “second end”. Likewise, linker - conjugates may include one than the reactive group Q ^1, i.e., the linker - conjugates may include a first end of one greater. When more than one reactive group Q ¹ is present, the Q ¹ groups may be the same or different, and when more than one target molecule D is present, the target molecules D may be the same or different.

The linker-conjugates according to the invention can therefore also be denoted as (Q ¹ ) _{y ′} Sp (D) _z , where y ′ is an integer in the range 1-10 and z is in the range 1-10. Is an integer. In this specification:
y ′ is an integer in the range of 1-10;
z is an integer in the range of 1-10;
Q ¹ is a reactive group that can react with the functional group F ¹ present in the biomolecule;
D is the target molecule;
Sp is a spacer moiety, where the spacer moiety spaces the reactive group Q ¹ and the target molecule D (ie provides a certain distance in between) and the reactive group Q ¹ and the target molecule D Wherein the spacer moiety is a linker L as defined above and therefore a group according to formula (1) or a salt thereof including.

y ′ is preferably 1, 2, 3, or 4, y ′ is more preferably 1 or 2, and y ′ is most preferably 1. z is preferably 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4, more preferably z is 1, 2 or 3, and z Is more preferably 1 or 2, and most preferably z is 1. y ′ is 1 or 2, preferably 1, z is more preferably 1, 2, 3 or 4, y ′ is 1 or 2, preferably 1, and z is 1, 2 or 3. Even more preferably, y ′ is 1 or 2, preferably 1, z is even more preferably 1 or 2, y ′ is 1 and z is most preferably 1. In preferred embodiments, the linker-conjugate is according to the formula Q ¹ Sp (D) ₄ , Q ¹ Sp (D) ₃ , Q ¹ Sp (D) ₂ or Q ¹ SpD.

  D is preferably an “active substance” or “pharmaceutically active substance” and is a pharmacological and / or biological substance, ie a biologically and / or pharmaceutically active substance, eg a drug , Prodrug, diagnostic agent. The active substance is preferably selected from the group consisting of drugs and prodrugs. More preferably, the active substance is a pharmaceutically active compound, in particular a low to medium molecular weight compound (eg about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In a further preferred embodiment, the active substance is selected from the group consisting of cytotoxins, antiviral agents, antibacterial agents, peptides and oligonucleotides. Examples of cytotoxins include colchicine, vinca alkaloid, anthracycline, camptothecin, doxorubicin, daunorubicin, taxane, calicheamicin, tubricin, irinotecan, enediyne, inhibitory peptide, amanitin, deBouganin, duocarmycin, maytansin, auristatin or Examples include pyrrolobenzodiazepine (PBD). Preferred active substances include vinca alkaloids, anthracyclines, camptothecins, taxanes, tubricin, amanitine, duocarmycin, maytansine, auristatin and pyrrolobenzodiazepines, especially vinca alkaloids, anthracyclines, camptothecins, taxanes, tubulicin, amanitin, maytansine and auris. Statins are mentioned.

The linker-conjugate comprises a reactive group Q ¹ that can react with a functional group F ¹ present in a biomolecule. Functional groups are known to those skilled in the art and can be defined as any molecular entity that confers particular properties to molecules with functional groups. For example, the functional group in the biomolecule can constitute an amino group, a thiol group, a carboxylic acid, an alcohol group, a carbonyl group, a phosphate group or an aromatic group. Functional groups in biomolecules can occur naturally or can be put into biomolecules by specific techniques, such as (bio) chemical or genetic techniques. The functional group incorporated into the biomolecule can be a naturally occurring functional group or can be a functional group prepared by chemical synthesis, such as an azide, terminal alkyne or phosphine moiety. As used herein, the term “reactive group” refers to a specific group that includes a functional group, but can also refer to the functional group itself. For example, a cyclooctynyl group is a reactive group containing a functional group, ie, a C—C triple bond. Similarly, the N-maleimidyl group is a reactive group containing a C—C double bond as a functional group. However, functional groups such as azide functional groups, thiol functional groups or amino functional groups can also be referred to herein as reactive groups.

Linker - conjugate can comprise of one than the reactive group Q ^1. Linker - If the conjugate comprises two or more reactive groups Q ^1, the reactive group Q ¹ may be different from each other. The linker-conjugate preferably comprises ^one reactive group Q1.

The reactive group Q ¹ present in the linker-conjugate can form a connecting group Z ³ by reacting with the functional group F ¹ present in the biomolecule. In other words, the reactive group Q ¹ needs to be complementary to the functional group F ¹ present in the biomolecule. As used herein, a reactive group is indicated as “complementary” to a functional group when it selectively reacts with the functional group to form a linking group Z ³ , optionally in the presence of another functional group. It is. Complementary reactive groups and functional groups are known to those skilled in the art and are described in more detail below.

In preferred embodiments, the reactive group Q ¹ is optionally substituted, an N-maleimidyl group, a halogenated N-alkylamide group, a sulfonyloxy N-alkylamide group, an ester group, a carbonate group, a sulfonyl halide. Group, thiol group or derivative thereof, alkenyl group, alkynyl group, (hetero) cycloalkynyl group, bicyclo [6.1.0] non-4-yn-9-yl] group, cycloalkenyl group, tetrazinyl group, azide group , Phosphine group, nitrile oxide group, nitrone group, nitrile imine group, diazo group, ketone group, (O-alkyl) hydroxylamino group, hydrazine group, halogenated N-maleimidyl group, 1,1-bis (sulfonylmethyl) methyl Carbonyl group or its leaving derivative, halogenated carbonyl group, allene It is selected from the group consisting of an amide group, a 1,2-quinone group, or a triazine group.

In a preferred embodiment, Q ¹ is an N-maleimidyl group. When Q ¹ is an N-maleimidyl group, Q ¹ is preferably unsubstituted. Q ¹ is therefore preferably according to formula (9a) as shown below. A preferred example of such a maleimidyl group is 2,3-diaminopropionic acid (DPR) maleimidyl, which can be connected to the remainder of the linker-conjugate via a carboxylic acid moiety.

In another preferred embodiment, Q ¹ is a halogenated N-alkylamide group. When Q ¹ is a halogenated N-alkylamide group, Q ¹ preferably conforms to formula (9b) as shown below, where k is an integer in the range of 1-10. , R ⁴ is selected from the group consisting of —Cl, —Br and —I. k is preferably 1, 2, 3, or 4, k is more preferably 1 or 2, and k is most preferably 1. R ⁴ is preferably —I or —Br. More preferably, k is 1 or 2, R ⁴ is —I or —Br, k is 1, and R ⁴ is —I or Br.

In another preferred embodiment, Q ¹ is a sulfonyloxy N-alkylamide group. When Q ¹ is a sulfonyloxy N-alkylamide group, Q ¹ preferably follows the formula (9b) as shown below, where k is an integer in the range of 1-10: , R ⁴ is selected from the group consisting of —O-mesyl, —O-phenylsulfonyl, and —O-tosyl. k is preferably 1, 2, 3 or 4, k is more preferably 1 or 2, and k is more preferably 1. Most preferably, k is 1 and R ⁴ is selected from the group consisting of —O-mesyl, —O-phenylsulfonyl, and —O-tosyl.

In another preferred embodiment, Q ¹ is an ester group. When Q ¹ is an ester group, the ester group is preferably an activated ester group. Activated ester groups are known to those skilled in the art. An activated ester group is defined herein as an ester group containing a good leaving group, wherein the ester carbonyl group is attached to the good leaving group. Good leaving groups are known to those skilled in the art. More preferably, the activated ester is according to formula (9c) as shown below, wherein R ⁵ is —N-succinimidyl (NHS), —N-sulfo-succinimidyl (sulfo-NHS), Selected from the group consisting of-(4-nitrophenyl), -pentafluorophenyl or -tetrafluorophenyl (TFP).

In another preferred embodiment, Q ¹ is a carbonate group. When Q ¹ is a carbonate group, the carbonate group is preferably an activated carbonate group. Activated carbonate groups are known to those skilled in the art. An activated carbonate group is defined herein as a carbonate group containing a good leaving group, wherein the carbonate carbonyl group is bonded to the good leaving group. More preferably, the carbonate group is according to formula (9d) as shown below, wherein R ⁷ is —N-succinimidyl, —N-sulfo-succinimidyl, — (4-nitrophenyl), — Selected from the group consisting of pentafluorophenyl or -tetrafluorophenyl.

In another preferred embodiment, Q ¹ is a sulfonyl halide group according to formula (9e) as shown below, wherein X is selected from the group consisting of F, Cl, Br and I Is done. X is preferably Cl or Br, more preferably Cl.

In another preferred embodiment, Q ¹ is a thiol group (9f) or a derivative or precursor of a thiol group. A thiol group may also be referred to as a mercapto group. When Q ¹ is a derivative or precursor of a thiol group, the thiol derivative preferably follows the formula (9g), (9h) or (9zb) as shown below, where R ⁸ is An optionally substituted C ₁ -C ₁₂ alkyl group or a C ₂ -C ₁₂ (hetero) aryl group, V is O or S, and R ¹⁶ is an optionally substituted C _1- C ₁₂ alkyl group. More preferably, R ⁸ is an optionally substituted C ₁ -C ₆ alkyl group or a C ₂ -C ₆ (hetero) aryl group, and R ⁸ is methyl, ethyl, n-propyl, i-propyl. More preferably, n-butyl, s-butyl, t-butyl or phenyl. More preferably, R ⁸ is methyl or phenyl, most preferably methyl. More preferably, R ¹⁶ is an optionally substituted C ₁ -C ₆ alkyl group, wherein R ¹⁶ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t -Butyl is more preferred and methyl is most preferred. Q ¹ is thiol according to formula (9 g) or (9zb) - a derivative, if Q ¹ is reacted with the reactive group F ¹ of the biomolecule, the thiol - derivative is converted to a thiol group in the process The When Q ¹ is according to formula (9h), Q ¹ is —SC (O) OR ⁸ or —SC (S) OR ⁸ , preferably SC (O) OR ⁸ , where R ⁸ and its preferred implementations. The form is as defined above.

In another preferred embodiment, Q ¹ is an alkenyl group, wherein the alkenyl group is straight or branched and the alkenyl group is optionally substituted. The alkenyl group can be a terminal or internal alkenyl group. An alkenyl group can contain more than one C—C double bond, and if so, preferably contains two C—C double bonds. More preferably, when the alkenyl group is a dienyl group, the two C—C double bonds are separated by one C—C single bond (ie, the dienyl group is a conjugated dienyl group). Preferably). The alkenyl group _is preferably a C 2 _{-C 24} alkenyl _group, more preferably a C 2 _{-C 12} alkenyl _group, and even more preferably C 2 -C ₆ alkenyl group. More preferably, the alkenyl group is a terminal alkenyl group. More preferably, the alkenyl group follows formula (9i) as shown below, where l is an integer in the range 0-10, preferably 0-6, and p is 0: It is an integer in the range of -10, preferably 0-6. l is more preferably 0, 1, 2, 3 or 4, l is more preferably 0, 1 or 2, and l is most preferably 0 or 1. More preferably, p is 0, 1, 2, 3 or 4, p is more preferably 0, 1 or 2, and p is most preferably 0 or 1. It is particularly preferred that p is 0 and l is 0 or 1, or p is 1 and l is 0 or 1.

In another preferred embodiment, Q ¹ is an alkynyl group, wherein the alkynyl group is straight or branched and the alkynyl group is optionally substituted. An alkynyl group can be a terminal or internal alkynyl group. Preferably the alkynyl group is _C 2 _{-C 24} alkynyl _group, more preferably a C 2 _{-C 12} alkynyl _group, and even more preferably C 2 -C ₆ alkynyl group. More preferably, the alkynyl group is a terminal alkynyl group. More preferably, the alkynyl group is according to formula (9j) as shown below, where l is an integer in the range of 0-10, preferably in the range of 0-6. l is more preferably 0, 1, 2, 3 or 4, l is more preferably 0, 1 or 2, and l is most preferably 0 or 1.

In another preferred embodiment, Q ¹ is a cycloalkenyl group. Cycloalkenyl groups are optionally substituted. The cycloalkenyl group is preferably a C _{3 to} C ₂₄ cycloalkenyl group, more preferably a C _{3 to} C ₁₂ cycloalkenyl group, and even more preferably a C _{3 to} C ₈ cycloalkenyl group. In a preferred embodiment, the cycloalkenyl group is a trans-cycloalkenyl group, more preferably a trans-cyclooctenyl group (also referred to as a TCO group), most preferably the formula (9zi) or ( 9zj) is a trans-cyclooctenyl group. In another preferred embodiment, the cycloalkenyl group is a cyclopropenyl group, wherein the cyclopropenyl group is optionally substituted. In another preferred embodiment, the cycloalkenyl group is a norbornenyl group, an oxanorbornenyl group, a norbornadienyl group or an oxanorbornadienyl group, wherein the norbornenyl group, oxanorbornenyl group, norbornadi group An enyl group or an oxanorbornadienyl group is optionally substituted. In a further preferred embodiment, the cycloalkenyl group is according to formula (9k), (9l), (9m) or (9zc) as shown below, wherein T is CH ₂ or O, R ⁹ is independently selected from the group consisting of hydrogen, a linear or branched C ₁ -C ₁₂ alkyl group or a C ₄ -C ₁₂ (hetero) aryl group, and R ¹⁹ is selected from hydrogen and fluorinated hydrocarbons. Selected from the group consisting of R ⁹ is preferably independently hydrogen or an aC ₁ -C ₆ alkyl group, and R ⁹ is more preferably independently hydrogen or C ₁ -C ₄ alkyl group. More preferably, R ⁹ is independently hydrogen or methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R ⁹ is independently hydrogen or methyl. In a further preferred ^{embodiment, R 19} is hydrogen and _{-CF 3, -C 2 F 5,} -C 3 F 7 and _-C 4 _{F 9,} and more preferably is selected from the group of hydrogen and -CF _3. In a further preferred embodiment, the cycloalkenyl group is according to formula (9k), wherein one R ⁹ is hydrogen and the other R ₉ is a methyl group. In another more preferred embodiment, the cycloalkenyl group is according to formula (91), wherein both R ⁹ are hydrogen. In these embodiments, it is more preferred that l is 0 or 1. In another more preferred embodiment, the cycloalkenyl group is a norbornenyl (T is CH ₂ ) or oxanorbornenyl (T is O) group according to formula (9m) or according to formula (9zc) Norbornadienyl (T is CH ₂ ) group or oxanorbornadienyl (T is O) group, wherein R ⁹ is hydrogen and R ¹⁹ is hydrogen or —CF ₃ , preferably is -CF _3.

In another preferred embodiment, Q ¹ is a (hetero) cycloalkynyl group. (Hetero) cycloalkynyl groups are optionally substituted. The (hetero) cycloalkynyl group is preferably a (hetero) cyclooctynyl group, ie a heterocyclooctynyl group or a cyclooctynyl group, wherein the (hetero) cyclooctynyl group is optionally substituted. In a further preferred embodiment, the (hetero) cyclooctynyl group is represented by formula (9n), also referred to as DIBO group, formula (9o), also referred to as DIBAC group (9p), or COMBO group. (9zk), all as indicated below, where U is O or NR ⁹ and preferred embodiments of R ⁹ are as defined above. The aromatic ring in (9n) is optionally O-sulfonylated at one or more positions, while the rings of (9o) and (9p) are halogenated at one or more positions. May be.

In a particularly preferred embodiment, the nitrogen atom bonded to R ¹ in compound (4b) is the nitrogen atom of the ring of the heterocycloalkyne group, for example the nitrogen atom in (9o). In other words, c, d and g are 0 in compound (4b) and R ¹ and Q ¹ together with the nitrogen atom to which they are attached, a heterocycloalkyne group, preferably a heterocyclooctyne group, Most preferably, a heterocyclooctyne group according to formula (9o) or (9p) is formed. Herein, the carbonyl moiety of (9o) is replaced by a sulfonyl group of the group according to formula (1). Alternatively, the nitrogen atom to which R ¹ is attached is the same atom as the atom designated U in formula (9n). In other words, when Q ¹ is according to formula (9n), U can be the nitrogen atom to the right of the group according to formula (1) or U = NR ⁹ and R ⁹ is equal to formula (1) The remainder of the group thus followed, R ¹ is the cyclooctyne moiety.

In another preferred embodiment, Q ¹ is an optionally substituted bicyclo [6.1.0] non-4-in-9-yl] group, also referred to as a BCN group. The bicyclo [6.1.0] non-4-yn-9-yl] group preferably follows the formula (9q) as shown below.

In another preferred embodiment, Q ¹ is a conjugated (hetero) diene group that can react in a Diels-Alder reaction. Preferred (hetero) diene groups include an optionally substituted tetrazinyl group, an optionally substituted 1,2-quinone group, and an optionally substituted triazine group. More preferably, the tetrazinyl group follows formula (9r) as shown below, wherein R ⁹ is hydrogen, a linear or branched C ₁ -C ₁₂ alkyl group or C ₄ -C ₁₂ selected from the group consisting of (hetero) aryl groups. R ⁹ is preferably hydrogen, a C ₁ -C ₆ alkyl group or a C ₄ -C ₁₀ (hetero) aryl group, and R ⁹ is hydrogen, a C ₁ -C ₄ alkyl group or C ₄ -C ₆ (hetero) More preferably, it is an aryl group. More preferably, R ⁹ is hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl or pyridyl. Even more preferably R ⁹ is hydrogen, methyl or pyridyl. More preferably, the 1,2-quinone group follows the formula (9zl) or (9zm). The triazine group can be any regioisomer. The triazine group is a 1,2,3-triazine group or a 1,2,4-triazine group that can be attached via any possible position, such as the position shown in formula (9zn). Is more preferable. 1,2,3-triazine is most preferred as the triazine group.

In another preferred embodiment, Q ¹ is an azide group according to formula (9s) as shown below.

In another preferred embodiment, Q ¹ is an optionally substituted triarylphosphine group that is suitable for undergoing a Staudinger ligation reaction. The phosphine group preferably follows the formula (9t) as shown below, where R ¹⁰ is a (thio) ester group. When R ¹⁰ is a (thio) ester group, R ¹⁰ is preferably —C (O) —V—R ¹¹ , where V is O or S and R ¹¹ is C ₁ -C _12. It is an alkyl group. R ¹¹ is preferably a C ₁ -C ₆ alkyl group, and more preferably a C ₁ -C ₄ alkyl group. Most preferably, R ¹¹ is a methyl group.

In another preferred embodiment, Q ¹ is a nitrile oxide group according to formula (9u) as shown below.

In another preferred embodiment, Q ¹ is a nitrone group. The nitrone group preferably follows the formula (9v) as shown below, wherein R ¹² is from a linear or branched C ₁ -C ₁₂ alkyl group and a C ₆ -C ₁₂ aryl group. Selected from the group consisting of R ¹² is preferably a C ₁ -C ₆ alkyl group, and R ¹² is more preferably a C ₁ -C ₄ alkyl group. More preferably, R ¹² is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R ¹² is methyl.

In another preferred embodiment, Q ¹ is a nitrile imine group. The nitrile imine group preferably conforms to formula (9w) or (9zd) as shown below, wherein R ¹³ is a linear or branched C ₁ -C ₁₂ alkyl group and C _6- It is selected from the group consisting of C ₁₂ aryl group. R ¹³ is preferably a C ₁ -C ₆ alkyl group, and R ¹³ is more preferably a C ₁ -C ₄ alkyl group. More preferably, R ¹³ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R ¹³ is methyl.

In another preferred embodiment, Q ¹ is a diazo group. The diazo group preferably conforms to formula (9x) as shown below, and R ¹⁴ is selected from the group consisting of hydrogen or a carbonyl derivative. More preferably, R ¹⁴ is hydrogen.

In another preferred embodiment, Q ¹ is a ketone group. More preferably, the ketone group is according to formula (9y) as shown below, wherein R ¹⁵ is a linear or branched C ₁ -C ₁₂ alkyl group and a C ₆ -C ₁₂ aryl group. Selected from the group consisting of R ¹⁵ is preferably a C ₁ -C ₆ alkyl group, and R ¹⁵ is more preferably a C ₁ -C ₄ alkyl group. More preferably R ¹⁵ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl. Even more preferably, R ¹⁵ is methyl.

In another preferred embodiment, Q ¹ is an (O-alkyl) hydroxylamino group. More preferably, the (O-alkyl) hydroxylamino group follows the formula (9z) as shown below.

In another preferred embodiment, Q ¹ is a hydrazine group. The hydrazine group preferably follows the formula (9za) as shown below.

In another preferred embodiment, Q ¹ is a halogenated N-malemidyl group or a sulfonylated N-malemidyl group. When Q ¹ is a halogenated or sulfonylated N-maleimidyl group, Q ¹ preferably follows the formula (9ze) as shown below, where R ⁶ is hydrogen F, Cl, Br, Independently selected from the group consisting of I-SR ^18a and -OS (O) ₂ R ^18b , wherein R ^18a is an optionally substituted C ₄ -C ₁₂ (hetero) aryl group, preferably phenyl Or R ^18b is selected from the group consisting of optionally substituted C ₁ -C ₁₂ alkyl groups and C ₄ -C ₁₂ (hetero) aryl groups, preferably tolyl or methyl, but at least The condition is that one R ⁶ is hydrogen. When R ⁶ is halogen (ie when R ⁶ is F, Cl, Br or I), R ⁶ is preferably Br. In one embodiment, the halogenated N-malemidyl group is a halogenated 2,3-diaminopropionic acid (DPR) maleimidyl that can be connected to the remainder of the linker-conjugate via a carboxylic acid moiety.

In another preferred embodiment, Q ¹ is a carbonyl halide group according to formula (9zf) as shown below, wherein X is selected from the group consisting of F, Cl, Br and I Is done. X is preferably Cl or Br, and most preferably X is Cl.

In another preferred embodiment, Q ¹ is an allenamide group according to formula (9zg).

（式中、ｋ、ｌ、Ｘ、Ｔ、Ｕ、Ｖ、Ｒ^４、Ｒ^５、Ｒ^６、Ｒ^７、Ｒ^８、Ｒ^９、Ｒ^１０、Ｒ^１１、Ｒ^１２、Ｒ^１３、Ｒ^１４、Ｒ^１５、Ｒ^１６、Ｒ^１８及びＲ^１９は、上で定義されている通りである。） In another preferred embodiment, Q ¹ is a 1,1-bis (sulfonylmethyl) methylcarbonyl group according to formula (9zh), or a leaving derivative thereof, wherein R ¹⁸ is optionally substituted. Selected from the group consisting of C ₁ -C ₁₂ alkyl groups and C ₄ -C ₁₂ (hetero) aryl groups. R ¹⁸ is more preferably an optionally substituted C ₁ -C ₆ alkyl group or C ₄ -C ₆ (hetero) aryl group, and most preferably a phenyl group.

(Wherein, k, l, X, T, U, V, R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁸ and R ¹⁹ are as defined above.)

In a preferred embodiment of the conjugation process according to the invention as described herein below, the conjugation is carried out by a Diels-Alder reaction or a 1,3-dipolar cycloaddition, preferably 1,3-bis. This is achieved via cycloaddition, such as polar cycloaddition. According to this embodiment, the reactive group Q ¹ (as well as F ^{1 of the} biomolecule) is selected from reactive groups in the cycloaddition reaction. In this specification, the reactive groups Q ¹ and F ¹ are complementary, ie, the reactive groups Q ¹ and F ¹ can react with each other in a cycloaddition reaction, and the resulting cyclic moiety is a linking group Z ³ . is there.

For the Diels-Alder reaction, ^one of F ¹ and Q ¹ is a diene and the other of F ¹ and Q ¹ is a dienophile. As understood by those skilled in the art, the term “diene” refers to a 1,3- (hetero) diene in the context of the Diels-Alder reaction, and is a conjugated diene (R ₂ C═CR—CR═ CR ₂ ), imine (eg R ₂ C═CR—N═CR ₂ or R ₂ C═CR—CR═NR, R ₂ C═N—N═CR ₂ ) and carbonyl (eg R ₂ C═CR—CR = O or O = CR-CR = O). Hetero-Diels-Alder reactions with N and O containing dienes are known to those skilled in the art. Any diene known in the art to be suitable for the Diels-Alder reaction can be used as the reactive group Q ¹ or F ¹ . Preferred dienes include tetrazine as described above, 1,2-quinone as described above, and triazine as described above. Any dienophile known in the art to be suitable for the Diels-Alder reaction can be used as the reactive group Q ¹ or F ¹ , but the dienophile is preferably described above. A straight alkene group or alkyne group, most preferably an alkyne group. For conjugation via the Diels-Alder reaction, it is preferred that F ¹ is a diene and Q ¹ is a dienophile. As used herein, when Q ¹ is a diene, F ¹ is a dienophile, and when Q ¹ is a dienophile, F ¹ is a diene. Q ¹ is most preferably a dienophile, Q ¹ is preferably an alkynyl group or contains an alkynyl group, and F ¹ is a diene, preferably a tetrazine group, a 1,2-quinone group or a triazine group.

For 1,3-dipolar cycloaddition, ^one of F ¹ and Q ¹ is a 1,3-dipole and the other of F ¹ and Q ¹ is a parent dipole. Any 1,3-dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q ¹ or F ¹ . Preferred 1,3-dipoles include an azide group, a nitrone group, a nitrile oxide group, a nitrile imine group, and a diazo group. Any parent dipole known in the art to be suitable for 1,3-dipolar cycloaddition can be used as the reactive group Q ¹ or F ¹ , An alkene group or an alkyne group is preferable, and an alkyne group is most preferable. For conjugation via 1,3-dipolar cycloaddition, it is preferred that F ¹ is a 1,3-dipole and Q ¹ is a parent dipole. As used herein, when Q ¹ is a 1,3-dipole, F ¹ is a parent dipole, Q ¹ is a case parent dipole, and F ¹ is a 1,3-dipole. Most preferably, Q ¹ is a parent dipole, Q ¹ is preferably an alkynyl group or includes an alkynyl group, and F ¹ is a 1,3-dipole, preferably an azide group.

Thus, in a preferred embodiment, Q ¹ is selected from a parent dipole and a dienophile. Q ¹ is preferably an alkene or alkyne group. In a particularly preferred embodiment, Q ¹ comprises an alkyne group, an alkynyl group as described above, a cycloalkenyl group as described above, a (hetero) cyclo as described above. Preferably, it is selected from an alkynyl group and a bicyclo [6.1.0] non-4-yn-9-yl] group, wherein Q ¹ is as defined above and illustrated below. More preferably selected from the formulas (9j), (9n), (9o), (9p), (9q) and (9zk), and the formulas (9n), (9o), (9p), (9q) and More preferably, it is selected from (9zk). Q ¹ is most preferably a bicyclo [6.1.0] non-4-yn-9-yl] group of formula (9q). These groups are known to be highly effective in conjugation with azide functionalized biomolecules as described herein, and the sulfamide linkers according to the present invention can be used in such linker-conjugates. If used, any agglomeration is beneficially reduced to a minimum.

In the linker-conjugate, as described above, Q ¹ can react with a reactive group F ¹ present on the biomolecule. Complementary reactive groups F ¹ to the reactive group Q ¹ are known to those skilled in the art and are described in more detail below. A representative example of the reaction between F ¹ and Q ¹ and part of its corresponding product containing the connecting group Z ³ is illustrated in FIG.

As described above, D and Q ¹ are covalently linked in the linker-conjugate according to the invention, preferably via a linker L as defined above. The covalent bond of D to the linker can occur, for example, via a reaction between a functional group F ² present on D and a reactive group Q ² present on the linker. Suitable organic reactions for the attachment of D to the linker are known to those skilled in the art as being a functional group F ² that is complementary to the reactive group Q ² . As a result, D can be bound to the linker via the connecting group Z.

The term “connecting group” as used herein refers to a structural element that connects one part of a compound and another part of the same compound. As will be appreciated by those skilled in the art, the nature of the connecting group depends on the type of organic reaction in which the connection between the parts of the compound is obtained. As an example, when the carboxyl group of R—C (O) —OH is reacted with the amino group of H ₂ N—R ′ to form R—C (O) —N (H) —R ′, R is connected Connected to R ′ via a group Z, Z can be represented by a —C (O) —N (H) — group.

The reactive group Q ¹ can be attached to the linker in a similar manner. As a result, Q ¹ can be bonded to the spacer moiety via the connecting group Z.

Numerous reactions are known in the art for binding of a target molecule to a linker and for binding of a reactive group Q ¹ to the linker. As a result, a wide variety of connecting groups Z can be present in the linker-conjugate.

In one embodiment, the linker-conjugate has the formula:
(Q ¹ ) _{y ′} (Z ^w ) Sp (Z ^x ) (D) _z
(Where:
y ′ is an integer in the range of 1-10;
z is an integer in the range of 1-10;
Q ¹ is a reactive group that can react with the functional group F ¹ present in the biomolecule;
D is the target molecule;
Sp is a spacer moiety that is defined as a moiety that is spaced apart (ie, provides a certain distance between) and covalently connects Q ¹ and D;
Z ^w is a connecting group that connects Q ¹ to the spacer portion;
Z ^x is a connecting group that connects D to the spacer portion, and;
The spacer moiety is a linker L and thus comprises a group according to formula (1) or a salt thereof, wherein the group according to formula (1) is as defined above) A compound.

  In a preferred embodiment, a in the group according to formula (1) is 0. In another preferred embodiment, a in the group according to formula (1) is 1.

Preferred embodiments for y ′ and z are as defined above for (Q ¹ ) _y Sp (D) _z . The compound has the formula Q ¹ (Z ^w ) Sp (Z ^x ) (D) ₄ , Q ¹ (Z ^w ) Sp (Z ^x ) (D) ₃ , Q ¹ (Z ^w ) Sp (Z ^x ) (D ) ₂ or Q ¹ (Z ^w ) Sp (Z ^x ) D, more preferably Q ¹ (Z ^w ) Sp (Z ^x ) (D) ₂ or Q ¹ (Z ^w ) Sp (Z ^x ) D and most preferably Is more preferably according to Q ¹ (Z ^w ) Sp (Z ^x ) D, where Z ^w and Z ^x are as defined above.

Z ^w and Z ^x are —O—, —S—, —NR ² —, —N═N—, —C (O) —, —C (O) NR ² —, —OC (O) —, — _{^{OC (O) O -, -}} OC (O) NR 2, -NR 2 C (O) -, - NR 2 C (O) O -, - NR 2 C (O) NR 2 -, - SC (O) ^{-, - SC (O) O} -, - SC (O) NR 2 -, - S (O) -, - S (O) 2 -, - OS (O) 2 -, - OS (O) 2 O- ^{_{, -OS (O) 2 NR 2}} -, - OS (O) -, - OS (O) O -, - OS (O) NR 2 -, - ONR 2 C (O) -, - ONR 2 C (O ) O—, —ONR ² C (O) NR ² —, —NR ² OC (O) —, —NR ² OC (O) O—, —NR ² OC (O) NR ² —, —ONR ² C ( S)-, -ONR ² C (S) O-, -ONR ² C (S) NR ^2- , —NR ² OC (S) —, —NR ² OC (S) O—, —NR ² OC (S) NR ² —, —OC (S) —, —OC (S) O—, —OC (S) NR ² —, —NR ² C (S) —, —NR ² C (S) O—, —NR ² C (S) NR ² —, —SS (O) ₂ —, —SS (O) ₂ O—, —SS (O) ₂ NR ² —, —NR ² OS (O) —, —NR ² OS (O) O—, —NR ² OS (O) NR ² —, —NR ² OS (O ) ₂ −, —NR ² OS (O) ₂ O—, —NR ² OS (O) ₂ NR ² —, —ONR ² S (O) —, —ONR ² S (O) O—, —ONR ² S (O) NR ² —, —ONR ² S (O) ₂ O—, —ONR ² S (O) ₂ NR ² —, —ONR ² S (O) ₂ —, —OP (O) (R ² ) ₂ -, -SP (O) (R ² ) _2- , -NR ² P (O) (R ² ) ₂ -and preferably selected independently from the group consisting of combinations of two or more thereof, wherein R ² is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl Alkyl groups are optionally substituted.

Preferred embodiments for D and Q ¹ are as defined above.

（式中：
ａは、独立して０又は１であり；
ｂは、独立して０又は１であり；
ｃは、０又は１であり；
ｄは、０又は１であり；
ｅは、０又は１であり；
ｆは、１〜１５０の範囲における整数であり；
ｇは、０又は１であり；
ｉは、０又は１であり；
Ｄは、標的分子であり；
Ｑ^１は、生体分子に存在する官能基Ｆ^１と反応できる反応性基であり；
Ｓｐ^１は、スペーサー部分であり；
Ｓｐ^２は、スペーサー部分であり；
Ｓｐ^３は、スペーサー部分であり；
Ｓｐ^４は、スペーサー部分であり；
Ｚ^１は、Ｑ^１又はＳｐ^３をＳｐ^２、Ｏ若しくはＣ（Ｏ）又はＮ（Ｒ^１）に接続する接続基であり；
Ｚ^２は、Ｄ若しくはＳｐ^４をＳｐ^１、Ｎ（Ｒ^１）、Ｏ若しくはＣ（Ｏ）に接続する接続基であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３−Ｃ_２４シクロアルキル基、Ｃ_１〜Ｃ_２４（ヘテロ）アリール基、Ｃ_１〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_１〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択されるか；又は
Ｒ^１は、Ｄ、−［（Ｓｐ^１）_ｂ（Ｚ^２）_ｅ（Ｓｐ^４）_ｉＤ］若しくは−［（Ｓｐ^２）_ｃ（Ｚ^１）_ｄ（Ｓｐ^３）_ｇＱ^１］であり、ここでＤは、さらなる標的分子であり、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２、Ｑ^１、ｂ、ｃ、ｄ、ｅ、ｇ及びｉは、上で定義されている通りである）に従った化合物、又はその塩である。 In one embodiment, the linker-conjugate is of formula (4a) or (4b):

(Where:
a is independently 0 or 1;
b is independently 0 or 1;
c is 0 or 1;
d is 0 or 1;
e is 0 or 1;
f is an integer in the range of 1 to 150;
g is 0 or 1;
i is 0 or 1;
D is the target molecule;
Q ¹ is a reactive group that can react with the functional group F ¹ present in the biomolecule;
Sp ¹ is a spacer moiety;
Sp ² is a spacer moiety;
Sp ³ is a spacer moiety;
Sp ⁴ is a spacer moiety;
Z ¹ is a connecting group connecting Q ¹ or Sp ³ to Sp ² , O or C (O) or N (R ¹ );
Z ² is a connecting group that connects D or Sp ⁴ to Sp ¹ , N (R ¹ ), O or C (O);
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₁ -C ₂₄ (hetero) aryl group, a C ₁ -C ₂₄ alkyl (hetero) aryl group, and a C ₁ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in ^{R 3} are either selected independently from the group consisting of hydrogen and _C 1 -C ₄ alkyl group; or R ^{1 _{is, D, - [(Sp 1}} ) b (Z 2) e (Sp 4) i ] Or ^- _[(Sp ²⁾ a ^{_{c (Z 1) d (Sp}} 3) g Q 1], where D is the additional target ^{molecules, Sp 1, Sp 2, Sp} 3, Sp 4, Z 1 , Z ² , Q ¹ , b, c, d, e, g and i are as defined above), or a salt thereof.

  In a preferred embodiment, a is 1 in the compound according to formula (4a) or (4b). In another preferred embodiment, a is 0 in the compounds according to formula (4a) or (4b).

As defined above, Z ¹ is a connecting group connecting Q ¹ or Sp ³ to Sp ² , O or C (O) or N (R ¹ ), Z ² is D or Sp ⁴ It is a connecting group connected to Sp ¹ , N (R ¹ ), O or C (O). As described in more detail above, the term “connecting group” refers to a structural element that connects one part of a compound and another part of the same compound.

In compounds according to formula (4a), the linking group Z ¹ , if present (ie when d is 1), Q ¹ (optionally via a spacer moiety Sp ³ ), in formula (4a) the O atom or a C (O) group followed compounds, connected via a spacer moiety Sp ² optionally. More particularly, when Z ¹ is present (ie, d is 1) and when Sp ³ and Sp ² are absent (ie, g is 0 and c is 0), Z ¹ is , Q ^{1 is connected} to the O atom (a is 1) or to the C (O) group (a is 0) of the linker-conjugate according to formula (4a). If Z ¹ is present (ie, if d is 1), Sp ³ is present (ie, g is 1), and Sp ² is not present (ie, c is 0), Z ¹ is The spacer moiety Sp ^{3 is connected} to the O atom (a is 1) or to the C (O) group (a is 0) of the linker-conjugate according to formula (4a). When Z ¹ is present (ie, d is 1) and when Sp ³ and Sp ² are present (ie, g is 1 and c is 1), Z ¹ is a spacer moiety Sp ³ the linker according to formula (4a) - connected to a spacer moiety Sp ² conjugates. If Z ¹ is present (ie, d is 1), Sp ³ is not present (ie, g is 0), and Sp ² is present (ie, c is 1), Z ¹ Connects Q ¹ to the spacer moiety Sp ² of the linker-conjugate according to formula (4a).

In a compound according to formula (4b), the linking group Z ¹ , if present (ie when d is 1), is Q ¹ (optionally via a spacer moiety Sp ³ ), in formula (4b) It is optionally connected to the N atom of the N (R ¹ ) group in the resulting linker-conjugate via a spacer moiety Sp ² . More particularly, when Z ¹ is present (ie, d is 1) and when Sp ³ and Sp ² are absent (ie, g is 0 and c is 0), Z ¹ is , Q ¹ is connected to the N atom of the N (R ¹ ) group of the linker-conjugate according to formula (4b). If Z ¹ is present (ie, if d is 1), Sp ³ is present (ie, g is 1), and Sp ² is not present (ie, c is 0), Z ¹ is The spacer moiety Sp ³ is connected to the N atom of the linker-conjugate N (R ¹ ) group according to formula (4b). When Z ¹ is present (ie, d is 1) and when Sp ³ and Sp ² are present (ie, g is 1 and c is 1), Z ¹ is a spacer moiety Sp ³ Is connected to the spacer moiety Sp ² of the linker-conjugate according to formula (4b). If Z ¹ is present (ie, d is 1), Sp ³ is not present (ie, g is 0), and Sp ² is present (ie, c is 1), Z ¹ Connects Q ¹ to the spacer moiety Sp ² of the linker-conjugate according to formula (4b).

In a compound according to formula (4a), when c, d and g are all 0, Q ¹ is at the O atom of the linker-conjugate according to formula (4a) (when a is 1) or It is directly attached to the C (O) group (when a is 0).

In compounds according to formula (4b), when c, d and g are all 0, Q ¹ is directly to the N atom of the N (R ¹ ) group of the linker-conjugate according to formula (4b) Is bound to.

In a compound according to formula (4a), the linking group Z ² is present (ie when e is 1), D is optionally (via the spacer moiety Sp ⁴ ) and according to formula (4a) The linker-conjugate is connected to the N atom of the N (R ¹ ) group, optionally via a spacer moiety Sp ¹ . More specifically, when Z ² is present (ie, e is 1) and when Sp ¹ and Sp ⁴ are absent (ie, b is 0 and i is 0), Z ² is , D is connected to the N atom of the N (R ¹ ) group of the linker-conjugate according to formula (4a). If Z ² is present (ie if e is 1), Sp ⁴ is present (ie i is 1) and Sp ¹ is absent (ie b is 0), Z ² is The spacer moiety Sp ⁴ is connected to the N atom of the N (R ¹ ) group of the linker-conjugate according to formula (4a). When Z ² is present (ie, e is 1), and when Sp ¹ and Sp ⁴ are present (ie, b is 1 and i is 1), Z ² is a spacer moiety Sp ¹ Is connected to the spacer moiety Sp ⁴ of the linker-conjugate according to formula (4a). If Z ² is present (ie if e is 1), Sp ⁴ is not present (ie i is 0) and Sp ¹ is present (ie b is 1), then Z ² connects D to the spacer moiety Sp ¹ of the linker-conjugate according to formula (4a).

In the compound according to formula (4a), when b, e and i are all 0, D is directly on the N atom of the N (R ¹ ) group of the linker-conjugate according to formula (4a). Are connected.

  In the compound according to formula (4b), when b, e and i are all 0, D is at the O atom of the linker-conjugate according to formula (4b) (when a is 1) or C It is directly bonded to the group (O) (when a is 0).

As will be appreciated by those skilled in the art, the nature of the linking group depends on the type of organic reaction in which the connection between the particular parts of the compound is obtained. Numerous organic reaction, to a reactive group Q ¹ is connected to a spacer moiety, and the target molecule is available to connect to the spacer moiety. As a result, there are a wide variety of connecting groups Z ¹ and Z ² .

In preferred embodiments of linker-conjugates according to formulas (4a) and (4b), Z ¹ and Z ² are —O—, —S—, —SS—, —NR ² —, —N═N—. ^{, -C (O) -, -} C (O) NR 2 -, - OC (O) -, - OC (O) O -, - OC (O) NR 2, -NR 2 C (O) -, - NR ² C (O) O—, —NR ² C (O) NR ² —, —SC (O) —, —SC (O) O—, —S—C (O) NR ² —, —S (O _{) -, - S (O)} 2 -, - OS (O) 2 -, - OS (O) 2 O -, - OS (O) 2 NR 2 -, - OS (O) -, - OS (O) ^{O -, - OS (O)} NR 2 -, - ONR 2 C (O) -, - ONR 2 C (O) O -, - ONR 2 C (O) NR 2 -, - NR 2 OC (O) - , —NR ² OC (O) O—, —NR ^{2 OC (O) NR 2 -,} - ONR 2 C (S) -, - ONR 2 C (S) O -, - ONR 2 C (S) NR 2 -, - NR 2 OC (S) -, - NR 2 OC (S) O—, —NR ² OC (S) NR ² —, —OC (S) —, —OC (S) O—, —OC (S) NR ² —, —NR ² C (S) — ^{, -NR 2 C (S) O} -, - NR 2 C (S) NR 2 -, - SS (O) 2 -, - SS (O) 2 O -, - SS (O) 2 NR 2 -, - NR ² OS (O) —, —NR ² OS (O) O—, —NR ² OS (O) NR ² —, —NR ² OS (O) ₂ —, —NR ² OS (O) ₂ O—, —NR ² OS (O) ₂ NR ² —, —ONR ² S (O) —, —ONR ² S (O) O—, —ONR ² S (O) NR ² —, —ONR ² S (O) ₂ O-, -ONR ² S (O) ₂ NR ^2- , -ONR ² S (O) _2- , -OP (O) (R ² ) _2- , -SP (O) (R ² ) _2- , -NR ² P (O) Independently selected from the group consisting of (R ² ) ₂ — and combinations of two or more thereof, wherein R ² is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₂₄ alkenyl group, C ₂ -C selected ₂₄ alkynyl and C ₃ -C ₂₄ independently from the group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group is optionally substituted.

As described above, in compounds according to formula (4a) or (4b), Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are spacer moieties. Sp ¹ , Sp ² , Sp ³ and Sp ⁴ may independently be absent or present (b, c, g and i are independently 0 or 1). Sp ¹ if present may be different from Sp ² if present and Sp ³ and / or Sp ⁴ if present.

  Spacer moieties are known to those skilled in the art. Examples of suitable spacer moieties include (poly) ethylene glycol diamine (eg, 1,8-diamino-3,6-dioxaoctane, or an equivalent containing a longer ethylene glycol chain), polyethylene glycol chain or polyethylene oxide Chains, polypropylene glycol chains or polypropylene oxide chains, and 1, xx-diaminoalkanes, where xx is the number of carbon atoms in the alkane.

  Another class of suitable spacer moieties includes cleavable spacer moieties, or cleavable linkers. Cleaveable linkers are well known in the art. For example, Shabat et al., Soft Matter 2012, 6, 1073, incorporated herein by reference, discloses cleavable linkers that contain self-sacrificial moieties that are released upon biological triggers, such as enzymatic cleavage or oxidation events. ing. Some examples of suitable cleavable linkers include disulfide-linkers that are cleaved upon reduction, peptide-linkers that are cleaved with specific recognition by proteases such as cathepsin, plasmin or metalloproteases, or glycosidases such as glucuronidase, or Glycoside-based linkers that are cleaved with specific recognition by nitroaromatics that are reduced in hypoxic regions that are poor in oxygen. As used herein, suitable cleavable spacer moieties also include spacer moieties that contain a specific cleavable sequence of amino acids. Examples include a spacer moiety that includes, for example, a Val-Ala (valine-alanine) or Val-Cit (valine-citrulline) moiety.

In a preferred embodiment of the linker-conjugate according to formula (4a) and (4b), the spacer moiety Sp ¹ , Sp ² , Sp ³ and / or Sp ⁴ comprises a sequence of amino acids, if present. Spacer moieties containing amino acid sequences are known in the art and can also be referred to as peptide linkers. Examples include spacer moieties containing a Val-Cit moiety, such as Val-Cit-PABC, Val-Cit-PABC, Fmoc-Val-Cit-PABC, and the like. It is preferred that the Val-Cit-PABC moiety is used in the linker-conjugate.

In preferred embodiments of linker-conjugates according to formulas (4a) and (4b), the spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are linear or branched C _1- C ₂₀₀ alkylene _groups, C _{2 -C 200} alkenylene _groups, C _{2 -C 200} alkynylene _groups, C _{3 -C 200} cycloalkylene _groups, C _{5 -C 200} cycloalkenylene _groups, C _{8 -C 200} cycloalkynylene groups, C ₇ -C ₂₀₀ alkyl arylene _group, C _{7 -C 200} arylalkylene group _is independently selected from C _{8 -C 200} aryl alkenylene group and _C 9 _{-C 200} group consisting of aryl alkynylene group, an alkylene group, an alkenylene group , Alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene , Alkylarylene groups, arylalkylene groups, aryl alkenylene and aryl alkynylene group is optionally substituted, O, interrupted optionally by one or more heteroatoms selected from S and NR ³ group Wherein R ³ is independently selected from the group consisting of hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₂₄ alkenyl group, a C ₂ -C ₂₄ alkynyl group, and a C ₃ -C ₂₄ cycloalkyl group. And the alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted. One alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group as defined above. Or when interrupted by a plurality of heteroatoms, the group is preferably interrupted by one or more O atoms and / or by one or more SS groups.

Spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are linear or branched C ₁ -C ₁₀₀ alkylene groups, C ₂ -C ₁₀₀ alkenylene groups, C ₂ -C ₁₀₀ alkynylene groups, C ₃ -C ₁₀₀ cycloalkylene _group, C _{5 -C 100} cycloalkenylene _group, C _{8 -C 100} cycloalkynylene _groups, C _{7 -C 100} alkyl arylene _group, C _{7 -C 100} arylalkylene _group, C 8 -C more preferably it is independently selected from the group consisting of ₁₀₀ aryl alkenylene group and C ₉ -C ₁₀₀ aryl alkynylene group, an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene groups , Alkylarylene group, arylalkylene group, arylalkene Emissions and aryl alkynylene group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen C ₁ -C ₂₄ alkyl group, C ₂ -C ₂₄ alkenyl group, C ₂ -C ₂₄ alkynyl group and C ₃ -C ₂₄ cycloalkyl group independently selected from the group consisting of alkyl group, alkenyl group, alkynyl Groups and cycloalkyl groups are optionally substituted.

Spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are linear or branched C ₁ -C ₅₀ alkylene groups, C ₂ -C ₅₀ alkenylene groups, C ₂ -C ₅₀ alkynylene groups, C ₃ -C ₅₀ cycloalkylene _group, C 5 _{-C 50} cycloalkenylene _groups, C 8 _{-C 50} cycloalkynylene _groups, C 7 _{-C 50} alkylarylene _group, C 7 _{-C 50} arylalkylene _groups, C 8 -C _More preferably it is independently selected from the group consisting of ₅₀ arylalkenylene groups and C ₉ -C ₅₀ arylalkynylene groups, alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups. Alkyl arylene group, aryl alkylene group, aryl alkenylene group and aryl Alkynylene group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group Are optionally substituted.

If present, the spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are linear or branched C ₁ -C ₂₀ alkylene groups, C ₂ -C ₂₀ alkenylene groups, C ₂ -C ₂₀ alkynylene groups, C ₃ -C ₂₀ cycloalkylene _group, C 5 _{-C 20} cycloalkenylene _groups, C 8 _{-C 20} cycloalkynylene _groups, C 7 _{-C 20} alkylarylene _group, C 7 _{-C 20} arylalkylene _groups, C 8 -C ₂₀ aryl alkenylene group and C ₉ -C ₂₀ aryl alkynylene is even more preferably selected independently from the group consisting of group, alkylene group, alkenylene group, alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene Group, alkylarylene group, arylalkylene group, arylalkenylene group and aryl group Ruarukiniren group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group Are optionally substituted.

In these preferred embodiments, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group are unsubstituted. And optionally interrupted by one or more heteroatoms selected from the group O, S and NR ³ , preferably O, wherein R ³ is hydrogen and a C ₁ -C ₄ alkyl group, More preferably it is independently selected from the group consisting of hydrogen or methyl.

Most preferably, the spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are independently selected from the group consisting of linear or branched C ₁ -C ₂₀ alkylene groups. Is optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR ³ , wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl groups, Optionally replaced. In this embodiment, the alkylene group is unsubstituted and optionally interrupted by one or more heteroatoms selected from the group O, S and NR ³ , preferably O and / or S; It is further preferred here that R ³ is independently selected from the group consisting of hydrogen and a C ₁ -C ₄ alkyl group, preferably hydrogen or methyl.

Preferred spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are therefore-(CH ₂ ) _n -,-(CH ₂ CH ₂ ) _n -,-(CH ₂ CH ₂ O) _n -,-( _{OCH 2 CH 2) n -,} - (CH 2 CH 2 O) n CH 2 CH 2 -, - CH 2 CH 2 (OCH 2 CH 2) n -, - (CH 2 CH 2 CH 2 O) n -, _{- (OCH 2 CH 2 CH 2} ) n -, - (CH 2 CH 2 CH 2 O) n CH 2 CH 2 CH 2 - and _{-CH 2 CH 2 CH 2 (OCH} 2 CH 2 CH 2) n - include Where n is an integer in the range 1-50, preferably in the range 1-40, more preferably in the range 1-30, more preferably in the range 1-20 and even more preferably in the range 1-15. It is. n is more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and more preferably 1, 2, 3, 4, 5, 6, 7 or 8. 1, 2, 3, 4, 5 or 6 is more preferred, and 1, 2, 3 or 4 is even more preferred.

Since Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are independently selected, Sp ¹ is different from Sp ² if present and Sp ³ and / or Sp ⁴ if present. It may be.

The reactive group Q ¹ is described in more detail above. In the linker-conjugate according to formulas (4a) and (4b), the reactive group Q ¹ is optionally substituted N-maleimidyl group, halogenated N-alkylamide group, sulfonyloxy N-alkylamide. Group, ester group, carbonate group, halogenated sulfonyl group, thiol group or derivatives thereof, alkenyl group, alkynyl group, (hetero) cycloalkynyl group, bicyclo [6.1.0] non-4-yn-9-yl] Group, cycloalkenyl group, tetrazinyl group, azide group, phosphine group, nitrile oxide group, nitrone group, nitrileimine group, diazo group, ketone group, (O-alkyl) hydroxylamino group, hydrazine group, halogenated N-maleimidyl group , Halogenated carbonyl group, allenamide group and 1,1-bis (sulfonylmethyl) It is preferably selected from the group consisting of methylcarbonyl groups or their leaving derivatives. In a more preferred embodiment, Q ¹ is represented by the formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j) , (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9v) 9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi) , (9zj) or (9zk), where (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9i), 9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9u) 9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh) , (9zi), (9zj), (9zk) and preferred embodiments thereof are as defined above. In a preferred embodiment, Q ¹ is represented by the formulas (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), According to (9t), (9zh) or (9r). In an even more preferred embodiment, Q ¹ is according to formula (9a), (9n), (9o), (9q), (9p), (9t), (9zh) or (9s), particularly preferred embodiments. Wherein Q ¹ is according to formula (9a), (9q), (9n), (9o), (9p), (9t) or (9zh), their preferred embodiments being defined above Street.

  Preferred embodiments for target molecule D and target molecule D in the linker-conjugate according to formulas (4a) and (4b) are as defined above.

As described above, R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero ) is selected from aryl and _C 3 _{-C 24} (hetero) the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl group, C ₃ -C ₂₄ alkyl (hetero) aryl group and _C 3 _{-C 24} (hetero) arylalkyl groups, optionally substituted, O, optionally by one or more hetero atoms selected from S and NR ³ Interrupted by selection, wherein R ³ is independently selected from the group consisting of hydrogen and a C ₁ -C ₄ alkyl group, or R ¹ is D, — [(Sp ¹ ) _b (Z ² ) _E (Sp ⁴ ) _i D] or — [(Sp ² ) _c (Z ¹ ) _d (Sp ³ ) _g Q ¹ ], where D is a further target molecule, Sp ¹ , Sp ² , Sp ³ , Sp ⁴ , Z ¹ , Z ² , Q ¹ , b, c, d, e, g and i are as defined above.

In a preferred embodiment, R ¹ is hydrogen or a C ₁ -C ₂₀ alkyl group, more preferably R ¹ is hydrogen or a C ₁ -C ₁₆ alkyl group, and R ¹ is hydrogen or a C ₁ -C ₁₀ alkyl group. More preferred is a group wherein the alkyl group is optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , preferably O; Here, R ³ is independently selected from the group consisting of hydrogen and a C ₁ -C ₄ alkyl group. In a further preferred embodiment, R ¹ is hydrogen. In another further preferred embodiment, R ¹ is a C ₁ -C ₂₀ alkyl group, more preferably a C ₁ -C ₁₆ alkyl group, more preferably a C ₁ -C ₁₀ alkyl group, wherein the alkyl group is Optionally interrupted by one or more O atoms, the alkyl group is optionally substituted with an —OH group, preferably a terminal —OH group. In this embodiment, R ¹ is more preferably a polyethylene glycol chain containing a terminal —OH group. In another further preferred embodiment, R ¹ is a C ₁ -C ₁₂ alkyl group, more preferably a C ₁ -C ₆ alkyl group, more preferably a C ₁ -C ₄ alkyl group, and R ¹ is methyl, Even more preferably, it is selected from the group consisting of ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl.

In another preferred embodiment, R ¹ is a further target molecule D,-[(Sp ¹ ) _b (Z ² ) _e (Sp ⁴ ) _i D] or-[(Sp ² ) _c (Z ¹ ) _d (Sp ³ ) _g Q ¹ ], where D is the target molecule, Sp ¹ , Sp ² , Sp ³ , Sp ⁴ , Z ¹ , Z ² , Q ¹ , b, c, d, e, g and i Is as defined above. It is further preferred that when R ¹ is D or-[(Sp ¹ ) _b (Z ² ) _e (Sp ⁴ ) _i D], the linker-conjugate is according to formula (4a). In this embodiment, the linker-conjugate (4a) comprises two target molecules D, which may be the same or different. If it is _{^{[(Sp 1) b (Z}} 2) e (Sp 4) i D], - - R 1 is _{^{[(Sp 1) b (Z}} 2) e (Sp 4) i D] Sp 1 in, b ^{, Z 2, e, Sp 4} , it and D is attached to the N atom of the _{^{N (R 1) [(Sp}} 1) b (Z 2) e (Sp 4) i D] Sp 1 in, b , Z ² , e, Sp ⁴ , i and D may be the same or different. In a preferred embodiment,-[(Sp ¹ ) _b (Z ² ) _e (Sp ⁴ ) _i D] and-[(Sp ¹ ) _b (Z ² ) _e (Sp ⁴ ) of the N atom of N (R ¹ ). _i D] is the same.

It is further preferred that when R ¹ is-[(Sp ² ) _c (Z ¹ ) _d (Sp ³ ) _g Q ¹ ], the linker-conjugate is according to formula (4b). In this embodiment, the linker - conjugate (4b) comprises the same or a target molecule Q ¹ also two which may of different. If it is _{^{[(Sp 2) c (Z}} 1) d (Sp 3) g Q 1], - - R 1 is _{^{[(Sp 1) b (Z}} 2) e (Sp 4) i D] in ^Sp 2, c, Z ¹ , d, Sp ³ , g and D are other — [(Sp ² ) _c (Z ¹ ) _d (Sp ³ ) _g Q ¹ ] bonded to the N atom of N (R ¹ ). Sp ¹ , b, Z ² , e, Sp ⁴ , i and Q ¹ may be the same as or different from each other. In a preferred embodiment, the — [(Sp ² ) _c (Z ¹ ) _d (Sp ³ ) _g Q ¹ ] group of the N atom of N (R ¹ ) is the same.

In the linker-conjugate according to formula (4a) and (4b), f is an integer in the range of 1-150. The linker-conjugate can thus comprise more than one group according to formula (1), wherein the group according to formula (1) is as defined above. When more than one group according to formula (1) is present, ie when f is 2 or more, a, b, Sp ¹ and R ¹ are independently selected. In other words, when f is 2 or more, each a is independently 0 or 1, each b is independently 0 or 1, each Sp ¹ may be the same or different, and each R ¹ May be the same or different. In preferred embodiments, f is an integer in the range 1-100, preferably in the range 1-50, more preferably in the range 1-25, and even more preferably in the range 1-15. More preferably, f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably f is 1, 2, 3, 4, 5, 6, 7 or 8. Preferably, f is even more preferably 1, 2, 3, 4, 5 or 6, f is even more preferably 1, 2, 3 or 4, and f is 1 in this embodiment. Is most preferred. In another preferred embodiment, f is in the range of 2-150, preferably in the range of 2-100, more preferably in the range of 2-50, more preferably in the range of 2-25, and even more preferably in the range of 2-15. An integer in the range. More preferably, f is 2, 3, 4, 5, 6, 7, 8, 9 or 10, more preferably f is 2, 3, 4, 5, 6, 7 or 8, and f is Even more preferably 2, 3, 4, 5 or 6, f is even more preferably 2, 3 or 4, and f is most preferably 2 in this embodiment.

（式中、ａ、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、ｉ、Ｄ、Ｑ^１、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２及びＲ^１、並びにそれらの好ましい実施形態は、（４ａ）及び（４ｂ）について上で定義されている通りである）でもあり得る。 As described above, in a preferred embodiment, a is 0 in the compound according to formula (4a) or (4b). The linker-conjugate is therefore a compound according to formula (6a) or (6b) or a salt thereof:

(Wherein a, b, c, d, e, f, g, i, D, Q ¹ , Sp ¹ , Sp ² , Sp ³ , Sp ⁴ , Z ¹ , Z ² and R ¹ , and preferred thereof) Embodiments may also be as defined above for (4a) and (4b).

（式中、ａ、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、ｉ、Ｄ、Ｑ^１、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２及びＲ^１、並びにそれらの好ましい実施形態は、（４ａ）及び（４ｂ）について上で定義されている通りである）でもあり得る。 As described above, in another preferred embodiment, a is 1 in the compound according to formula (4a) or (4b). The linker-conjugate is therefore a compound according to formula (7a) or (7b), or a salt thereof:

(Wherein a, b, c, d, e, f, g, i, D, Q ¹ , Sp ¹ , Sp ² , Sp ³ , Sp ⁴ , Z ¹ , Z ² and R ¹ , and preferred thereof) Embodiments may also be as defined above for (4a) and (4b).

（式中、ａ、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、Ｄ、Ｑ^１、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｚ^１、Ｚ^２及びＲ^１、並びにそれらの好ましい実施形態は、（４ａ）及び（４ｂ）について上で定義されている通りである）でもあり得る。 When Sp ⁴ is not present in the linker-conjugate according to formula (4a), ie i is 0, D is Z ² (when e is 1), Sp ¹ (e is 0) , B is 1) or N (R ¹ ) (when e is 0 and b is 0). When Sp ⁴ is not present in the linker-conjugate according to formula (4b), ie i is 0, D is Z ² (when e is 1), Sp ¹ (e is 0) , B is 1), O atom (when a is 1 and b and e are 0) or C (O) group (when a is 0 and b and e are 0) Connected. The linker-conjugate is therefore a compound according to formula (4c) or (4d), or a salt thereof:

Wherein a, b, c, d, e, f, g, D, Q ¹ , Sp ¹ , Sp ² , Sp ³ , Z ¹ , Z ² and R ¹ , and preferred embodiments thereof are ( 4a) and (4b) as defined above).

  In a preferred embodiment, in the linker-conjugate according to formula (4c) or (4d), a is 0. In another preferred embodiment, in the linker-conjugate according to formula (4c) or (4d), a is 1.

Specific implementation of a linker-conjugate, in particular a linker-conjugate according to formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b) In forms, Sp ¹ , Sp ² Sp ³ and Sp ⁴ , if present, are independently selected from the group consisting of linear or branched C ₁ -C ₂₀ alkylene groups, wherein the alkylene groups are optionally Substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of O, S and NR ³ , wherein R ³ is a group consisting of hydrogen and a C ₁ -C ₄ alkyl group And Q ¹ is selected from equations (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j) ), (9k), (9l), (9m), (9n), (9o) , (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), ( 9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj) or (9zk), where (9a), (9b) , (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9 9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za) , (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi), (9zj), (9zk) and those Preferred embodiments are as defined above. In a preferred embodiment, Q ¹ is represented by the formulas (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s) ( 9t), (9zh) or (9r). In an even more preferred embodiment, Q ¹ is according to formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), particularly preferred embodiments. Wherein Q ¹ is according to formula (9a), (9q), (9n), (9p), (9t), (9zh) or (9o), and preferred embodiments thereof are as defined above. It is.

によって表すことができる。 Preferably a linker-conjugate according to formula (4a), (4b), (4c), (4d), (6a), (6b), (7a) or (7b) as defined above Linker L as included, linker as defined above is represented by formulas (8a) and (8b):

Can be represented by

As will be appreciated by those skilled in the art, preferred embodiments of spacer moieties (8a) and (8b) include, for example, the nature of the reactive groups Q ¹ and D in the linker-conjugate, the synthesis to prepare the linker-conjugate. Depending on the method (eg the nature of the complementary functional group F ² of the target molecule), the nature of the bioconjugate prepared using the linker-conjugate (eg the nature of the complementary functional group F ¹ of the biomolecule). obtain.

Sp ³ is present when Q ¹ is, for example, a cyclooctynyl group according to formula (9n), (9o), (9p), (9q) or (9zk) as defined above (g Is preferably 1).

For example, the linker-conjugate reacts the reactive group Q ² which is a cyclooctynyl group according to formula (9n), (9o), (9p), (9q) or (9zk) with the azide functional group F ^2. Preferably, Sp ⁴ is present (i is 1).

Furthermore, it is preferred that at least one of Sp ¹ , Sp ² , Sp ³ and Sp ⁴ is present, ie at least one of b, c, g and i is not zero. In another preferred embodiment, at least one of Sp ¹ and Sp ⁴ and at least one of Sp ² and Sp ³ are present.

When f is 2 or more, Sp ¹ is preferably present (b is 1).

  These preferred embodiments of the linker moieties (8a) and (8b) also hold for linker-conjugates when included in the bioconjugates according to the invention as described in more detail below.

Preferred embodiments of Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are as defined above.
Biomolecule

Biomolecules expressed by B-F ^1, where B is a biomolecule, F ¹ is a linker - a reactive group Q ¹ capable of reacting the functional group of a conjugate, "-" is a bond or a spacer moiety It is. Alternatively, the biomolecule is a modified antibody represented by formula (24), where F ¹ is a functional group capable of reacting with the reactive group Q ¹ of the linker-conjugate. The modified antibodies represented by formula (24) and their preferred embodiments are defined in detail above.

In one embodiment, “-” is a spacer moiety as defined herein. In one embodiment, “−” is a bond, typically a covalent bond. A biomolecule can also be referred to as a “target biomolecule” (BOI). The biomolecule may be a naturally occurring biomolecule, where the functional group F ¹ is already present in the biomolecule of interest, for example a thiol, amine, alcohol or hydroxyphenol unit. Conjugation with the linker-conjugate then occurs via the first approach as defined above. Alternatively, the biomolecule may be a modified biomolecule, wherein the functional group F ¹ is specifically incorporated into the biomolecule of interest, and conjugation with a linker-conjugate is the modified functional group, That is, it occurs through a two-stage approach to bioconjugation as defined above. Such modifications of biomolecules to incorporate specific functional groups are known, for example, from WO 2014/066561, which is hereby incorporated by reference in its entirety.

  In the bioconjugate according to the present invention, the biomolecule B is a protein (including glycoprotein and antibody), polypeptide, peptide, glycan, lipid, nucleic acid, oligonucleotide, polysaccharide, oligosaccharide, enzyme, hormone, amino acid and simple substance. It is preferably selected from the group consisting of sugars. The biomolecule B is more preferably selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes. The biomolecule B is more preferably selected from the group consisting of glycoproteins and proteins including antibodies, polypeptides, peptides and glycans. Biomolecule B is most preferably an antibody or an antigen-binding fragment thereof.

The functional group F ¹ can form a connecting group Z ³ by reacting with the reactive group Q ¹ of the linker-conjugate. Those skilled in the art will appreciate that the functional group F ¹ can react with the complementary reactive group Q ¹ . The functional group F ¹ as defined above and complementary to the reactive group Q ¹ as known to the person skilled in the art is described in more detail below. Representative examples of the reaction between F ¹ and Q ¹ and some of their corresponding products including the connecting group Z ³ are illustrated in FIG.

In the process for the preparation of a bioconjugate according to the invention, the reactive group Q ¹ present in the linker-conjugate is typically reacted with a functional group F ¹ . It can be a functional group F ¹ of the one than is present in the biomolecule. When two or more functional groups are present, the groups may be the same or different. In another preferred embodiment, the biomolecule comprises two or more functional groups F, which may be the same or different, and the two or more functional groups react with the complementary reactive group Q of the linker-conjugate. . For example, a biomolecule containing ^two functional groups F, ie F ¹ and F ² , reacts with two linker-conjugates containing a functional group Q ¹ that may be the same or different to form a bioconjugate. can do.

Examples of functional groups F ¹ in a biological molecule, comprising an amino group, a thiol group, a carboxylic acid, an alcohol group, a carbonyl group, a phosphate group, or an aromatic group. The functional groups in the biomolecule can be naturally occurring or can be introduced into the biomolecule by specific techniques such as (bio) chemical techniques or genetic techniques. The functional group incorporated in the biomolecule can be a naturally occurring functional group or a functional group prepared by chemical synthesis, such as an azide, terminal alkyne, cyclopropene moiety or phosphine moiety. In view of the preferred mode of conjugation by cycloaddition, F ¹ is a diene, a dienophile, such as 1,3-dipolar or dipolarophile is preferably a reactive group capable in cycloaddition, F ¹ is 1,3-dipole (typically an azide group, nitrone group, nitrile oxide group, nitrileimine group or diazo group) or a parent dipole (typically an alkenyl group or an alkynyl group). preferable. In this specification, ^{F 1} is a 1,3-dipolar When ^{Q 1} is a dipolarophile, ^{F 1} is an Shinso pole When ^{Q 1} is a 1,3-dipolar Or F ¹ is a diene when Q ¹ is a dienophile and F ¹ is a dienophile when Q ¹ is a diene. Most preferably, F ¹ is a 1,3-dipole, and F ¹ is preferably an azide group or contains an azide group.

Some examples of functional groups that can be incorporated into biomolecules are shown in FIG. FIG. 2 shows, for example, in the 2-position using a thiopropionyl group (11a), an azidoacetyl group (11b) or an azidodifluoroacetyl group (11c), or in the 6-position of N-acetylgalactosamine (11d) using an azide group. Figure 2 shows some structures of UDP sugar derivatives of galactosamine that can be modified. In one embodiment, the functional group F ¹ is a thiopropionyl group, an azidoacetyl group, or an azidodifluoroacetyl group.

  FIG. 3 shows how any of the UDP-saccharides 11a-d can be converted into a galactosyltransferase mutant or glycoprotein containing a GlcNAc moiety 12 (eg, a monoclonal antibody in which glycans are trimmed by endoglycosidase). Schematic representation of the ability to bind under the action of GalNAc-transferase, thereby producing 1-4 linkages of β-glycosides between GalNAc derivatives and GlcNAc (compounds 13a-d, respectively) .

Preferable examples of the naturally occurring functional group F ¹ include a thiol group and an amino group. Preferred examples of functional groups prepared by chemical synthesis for incorporation into biomolecules include ketone groups, terminal alkyne groups, azide groups, cyclo (hetero) alkyne groups, cyclopropene groups or tetrazine groups.

As described above, the complementary reactive group Q ¹ and functional group F ¹ are known to those skilled in the art, and some suitable combinations of Q ¹ and F ¹ are described above, and FIG. Is shown in A list of complementary groups Q ¹ and F ¹ can be found in G. T.A. Hermanson, “Bioconjugate Technologies” Elsevier, 3rd edition, 2013 (ISBN: 978-0-12-382239-0), Chapter 3, pages 230-232, Table 3.1. Which is expressly incorporated herein by reference.
Bioconjugate

A bioconjugate is defined herein as a compound in which a biomolecule is covalently connected to a target molecule D via a linker. The bioconjugate comprises one or more biomolecules and / or one or more target molecules. The linker can include one or more spacer moieties. The bioconjugate according to the present invention is conveniently prepared by a process for the preparation of a bioconjugate according to the present invention, wherein the linker-conjugate comprising the reactive group Q ¹ comprises a living body comprising the functional group F ^1. Conjugated to a molecule. In this conjugation reaction, the Q ¹ group and the F ¹ group react with each other to form a connecting group Z ³ that connects the target molecule D to the biomolecule B. All preferred embodiments described herein for linker-conjugates and biomolecules thus apply equally to the bioconjugates according to the invention except for all that is said for Q ¹ and F ¹ , Here, the bioconjugate according to the invention contains the reaction products of Q ¹ and F ¹ , ie the connecting group Z ³ . In one aspect, the present invention also relates to bioconjugates, preferably the antibody-conjugates described herein.

The bioconjugate according to the invention has the formula (A):
BLD
(A)
(Where:
B is a biomolecule, preferably antibody AB;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” independently has a bond or spacer moiety).

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基らなる群から独立して選択される；又はＲ^１は、標的分子Ｄであり、ここでＤは、スペーサー部分を介してＮに任意選択で接続されている）を含むリンカーＬを有する。 In a first preferred embodiment, the bioconjugate is according to the mode of conjugation defined as “core-GlcNAc functionalization”, ie by steps (i) and (ii) as defined above. Can be obtained. In a second preferred embodiment, the bioconjugate according to formula (A) is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ is independently selected from hydrogen and C ₁ -C ₄ alkyl group we made the group; or R ¹ is a target molecule D, where D is via a spacer moiety N A linker L comprising connected) optionally.

  In one embodiment, “-” is a spacer moiety as defined herein. In one embodiment, “−” is a bond, typically a covalent bond.

In a preferred embodiment, the bioconjugate is represented by BZ ³ -LD, where B, L, D and “-” are as defined above, and Z ³ is It is a connecting group that can be obtained by the reaction of Q ¹ and F ¹ . The moiety Z ³ is preferably obtainable by cycloaddition, preferably a 1,3-dipolar cycloaddition reaction, and Z ³ is a spacer moiety, preferably between B and L, most preferably B and Most preferred is a 1,2,3-triazole ring located in the spacer moiety between the carbonyl end or the carboxyl end of the group according to formula (1).

  When the bioconjugate according to the invention comprises a salt of a group according to formula (1), the salt is preferably a pharmaceutically acceptable salt.

  A bioconjugate according to the present invention can contain more than one target molecule. Similarly, a bioconjugate can include more than one biomolecule. Biomolecule B and target molecule D and their preferred embodiments are described in more detail above. Preferred embodiments for D in the bioconjugate according to the invention correspond to preferred embodiments for D in the linker-conjugate according to the invention as described in more detail above. Preferred embodiments for the linker (8a) or (8b) in the bioconjugate according to the invention correspond to the preferred embodiment of the linker in the linker-conjugate as described in more detail above. Preferred embodiments for B in a bioconjugate according to the invention correspond to preferred embodiments for B in a biomolecule according to the invention as described in more detail above.

  A bioconjugate according to the invention can also be defined as a bioconjugate in which a biomolecule is conjugated to a target molecule via a spacer moiety, wherein the spacer moiety is a group according to formula (1) or a group thereof The group comprising a salt, according to formula (1), is as defined above.

The bioconjugate according to the invention can also be denoted as (B) _{y ′} Sp (D) _z , where y ′ is an integer in the range 1-10 and z is an integer in the range 1-10. is there.

The present invention therefore has the formula:
(B) _y Sp (D) _z
(Where:
y ′ is an integer in the range of 1-10;
z is an integer in the range of 1-10;
B is a biomolecule;
D is the target molecule;
Sp is a spacer moiety, where a spacer moiety is defined as a moiety that covalently links biomolecule B and target molecule D with spacing (ie, providing a certain distance in between). The spacer moiety comprises a group according to formula (1) or a salt thereof, wherein the group according to formula (1) is as defined above) to the bioconjugate according to Also related.

  In a preferred embodiment, the spacer moiety is a cycloaddition, preferably a 1,3-dipolar cycloaddition reaction, most preferably 1,2,2, located between B and a group according to formula (1). Further included is a moiety obtainable by a 3-triazole ring.

y ′ is preferably 1, 2, 3 or 4, y ′ is more preferably 1 or 2, and y ′ is most preferably 1. z is preferably 1, 2, 3, 4, 5 or 6, more preferably z is 1, 2, 3 or 4, more preferably z is 1, 2 or 3, and z Even more preferred is 1 or 2, and most preferred is z. y ′ is 1 or 2, preferably 1, z is more preferably 1, 2, 3 or 4, y ′ is 1 or 2, preferably 1, and z is 1, 2 or 3. Even more preferably, y ′ is 1 or 2, preferably 1, z is even more preferably 1 or 2, y ′ is 1 and z is most preferably 1. In a preferred embodiment, the bioconjugate follows the formula BSp (D) ₄ , BSp (D) ₃ , BSp (D) ₂ or BSpD.

  As described above, the bioconjugate according to the invention comprises a group according to formula (1) as defined above or a salt thereof. In a preferred embodiment, the bioconjugate comprises a group according to formula (1) wherein a is 0 or a salt thereof. In this embodiment, the bioconjugate thus comprises a group according to formula (2) or a salt thereof, wherein (2) is as defined above.

  In another preferred embodiment, the bioconjugate comprises a group according to formula (1) wherein a is 1 or a salt thereof. In this embodiment, the bioconjugate thus comprises a group according to formula (3) or a salt thereof, wherein (3) is as defined above.

In the bioconjugate according to the invention, preferred embodiments of R ¹ , spacer moiety Sp, and R ¹ and Sp are as defined above for the linker-conjugate according to the invention.

（式中、ａ、ｂ、ｃ、ｄ、ｅ、ｆ、ｇ、ｈ、ｉ、Ｄ、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２、Ｚ^３及びＲ^１、並びにそれらの好ましい実施形態は、リンカー−コンジュゲート（４ａ）及び（４ｂ）について上で定義されている通りであり；
ｈは、０又は１であり；
Ｚ^３は、ＢをＳｐ^３、Ｚ^１、Ｓｐ^２、Ｏ又はＣ（Ｏ）に接続する接続基であり；
Ｂは、生体分子である）に従っている。 In a preferred embodiment, the bioconjugate has formula (5a) or (5b) or a salt thereof:

(Wherein a, b, c, d, e, f, g, h, i, D, Sp ¹ , Sp ² , Sp ³ , Sp ⁴ , Z ¹ , Z ² , Z ³ and R ¹ , and those Preferred embodiments of are as defined above for linker-conjugates (4a) and (4b);
h is 0 or 1;
Z ³ is a connecting group that connects B to Sp ³ , Z ¹ , Sp ² , O or C (O);
B is a biomolecule).

  h is preferably 1.

  Preferred embodiments of biomolecule B are as defined above.

  When the bioconjugate according to the invention is a salt of (5a) or (5b), the salt is preferably a pharmaceutically acceptable salt.

Z ³ is a connecting group. As described above, the term “connecting group” as used herein refers to a structural element that connects one part of a compound and another part of the same compound. Typically, the bioconjugate is prepared via reaction of the reactive group Q ¹ present in the linker-conjugate with the functional group F ¹ present in the biomolecule. As will be appreciated by those skilled in the art, the nature of the linking group Z ³ depends on the type of organic reaction used to establish the connection between the biomolecule and the linker-conjugate. In other words, the nature of Z ³ depends on the nature of the reactive group Q ¹ of the linker-conjugate and the nature of the functional group F ¹ in the biomolecule. As a result, there are many possibilities for Z ³ since there are many different chemical reactions available to establish a connection between the biomolecule and the linker-conjugate.

Some suitable combinations of F ¹ and Q ¹ present in the bioconjugate and some of the connecting groups Z ^{3 when} the linker-conjugate comprising Q ¹ is conjugated to a biomolecule comprising a complementary functional group F ¹ An example of this is shown in FIG.

When F ¹ is, for example, a thiol group, the complementary group Q ¹ includes an N-maleimidyl group and an alkenyl group, and the corresponding connecting group Z ³ is as shown in FIG. When F ¹ is a thiol group, the complementary group Q ¹ also includes an allenamide group.

When F ¹ is, for example, an amino group, the complementary group Q ¹ includes a ketone group and an activated ester group, and the corresponding connecting group Z ³ is as shown in FIG.

When F ¹ is, for example, a ketone group, complementary groups Q ¹ include (O-alkyl) hydroxylamino groups and hydrazine groups, and the corresponding connecting group Z ³ is as shown in FIG. is there.

When F ¹ is, for example, an alkynyl group, the complementary group Q ¹ includes an azide group, and the corresponding connecting group Z ³ is as shown in FIG.

When F ¹ is, for example, an azide group, the complementary group Q ¹ includes an alkynyl group, and the corresponding connecting group Z ³ is as shown in FIG.

When F ¹ is, for example, a cyclopropenyl group, a trans-cyclooctene group or a cyclooctyne group, the complementary group Q ¹ includes a tetrazinyl group, and the corresponding connecting group Z ³ is shown in FIG. Street. In these special cases, Z ³ is only an intermediate structure, which eliminates N ₂ , thereby yielding dihydropyridazine (from reaction with alkene) or pyridazine (from reaction with alkyne).

Additional suitable combinations of F ¹ and Q ^1, and the nature of the linking groups Z ³ resulting are known to those skilled in the art, for example, are incorporated by reference G. T.A. Hermanson, “Bioconjugate Techniques”, Elsevier, 3rd edition, 2013 (ISBN: 978-0-12-382239-0), especially Chapter 3, pages 229-258. A list of complementary reactive groups suitable for bioconjugation processes can be found in G. T.A. Hermanson, “Bioconjugate Technologies” Elsevier, 3rd edition, 2013 (ISBN: 978-0-12-382239-0), Chapter 3, pages 230-232, Table 3.1. Which is expressly incorporated herein by reference.

In the bioconjugate according to (5a) and (5b), at least one of Z ³ , Sp ³ , Z ¹ and Sp ² is present, ie at least of h, g, d and c Preferably one is not zero. It is also preferred that at least one of Sp ¹ , Z ² and Sp ⁴ is present, ie at least one of b, e and i is not zero. More preferably, at least one of Z ³ , Sp ³ , Z ¹ and Sp ² is present and at least one of Sp ¹ , Z ² and Sp ⁴ is present, ie b, e and i Preferably at least one of is not 0 and at least one of h, g, d and c is not 0.
Process for the preparation of bioconjugates

In various embodiments of the present invention, the bioconjugate according to the present invention is typically obtained by a process for the preparation of a bioconjugate as defined herein. Since the presence of a group according to formula (1) or a salt thereof in the linker L of the bioconjugate is key to the present invention, any method for preparing a bioconjugate is described herein where the resulting bioconjugate is Can be used as long as it contains a linker L as defined in. The group according to formula (1) can be present in the linker L between B and Z ³ , ie derived from a biomolecule, or between Z ³ and D, ie a linker-conjugate. Or Z ³ is a group according to formula (1) or comprises a group according to formula (1), ie a group according to formula (1) is formed by conjugation. The group according to formula (1) is preferably present in the linker L between B and Z ³ or between Z ³ and D, the group according to formula (1) being Z ³ and D and Most preferably it is present in the linker L between. Similarly, the exact mode of conjugation, including the nature of Q ¹ and F ¹ , has great flexibility in the context of the present invention. Many techniques for conjugating BOI to MOI are known to those skilled in the art and can be used in the context of the present invention. In one embodiment, the conjugation method comprises steps (i) and (ii) as defined above.

In one embodiment, the mode of conjugation is from any of the conjugation modes illustrated in FIG. 11, ie, a thiol to form a connecting moiety Z ³ that can be represented as (10a) or (10b). Alkene conjugation (preferably cysteine-alkene conjugation, preferably where the alkene is a pendant alkene (—C═CH ₂ ) or maleimide moiety, most preferably a maleimide moiety), can be represented as (10c) Amino- (activated) carboxylic acid conjugation to form connecting moiety Z ³ where (activated) carboxylic acid is represented by —C (O) X, where X is a leaving group and forming a connecting portion ^{Z 3,} which may be expressed as Y = a NH (10d) Ketone - hydrazino conjugation (preferably acetyl - hydrazino conjugation), Y = a O (10d) ketone to form a connecting portion Z ³ which may be represented as - Oxyamino conjugation (preferably acetyl - oxyamino conjugation), (10e), (10f ), (10i) or (alkyne to form a connecting portion ^{Z 3,} which may be expressed as 10 g) - azido conjugation (preferably where alkyne pendant Alkene (—C≡CH) or a cyclooctyne moiety, most preferably a cyclooctyne moiety), an alkene-1,2 to form a connecting moiety Z ³ that can be represented as N ₂ removed (10 h) , 4,5-tetrazine conjugation or alkyne- It is selected from 2,4,5 tetrazine conjugation. Particularly preferred conjugation modes are cysteine-alkene conjugation and alkyne-azide conjugation, more preferably cysteine-maleimide conjugation and cyclooctyne-azide conjugation.

The bioconjugate according to the invention comprises a process comprising reacting a reactive group Q ¹ of a linker-conjugate as defined herein with a functional group F ¹ of a biomolecule, also referred to as a biomolecule. Typically. Linker-conjugates and biomolecules, and preferred embodiments thereof, are described in more detail above. Such a process is known to those skilled in the art as conjugation or bioconjugation. FIG. 1 illustrates the general concept of biomolecule conjugation: a biomolecule of interest (BOI) containing one or more functional groups F ¹ is shared with a reactive group Q ¹ via a specific linker. Incubated with bound (excess) target molecule D (also referred to as molecule of interest or MOI). In the above process of bioconjugation, a chemical reaction between F ¹ and Q ¹ takes place, thereby forming a bioconjugate that includes a covalent bond between BOI and MOI. The BOI can be, for example, a peptide / protein, glycan or nucleic acid.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、スペーサー部分を介してＮに任意選択で接続されているさらなる標的分子Ｄである）を含む。 The bioconjugation reaction typically includes reacting a linker-conjugate reactive group Q ¹ with a biomolecule functional group F ¹ , where a bioconjugate of formula (A) is formed, wherein And the linker L is a group according to the formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ is independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is connected optionally to N via a spacer moiety It made containing the target molecule is a D).

In preferred embodiments, the bioconjugate undergoes cycloaddition, such as (4 + 2) -cycloaddition (eg, Diels-Alder reaction) or (3 + 2) -cycloaddition (eg, 1,3-bipolar cycloaddition). Prepared. The conjugation is preferably a Diels-Alder reaction or a 1,3-dipolar cycloaddition. A preferred Diels-Alder reaction is the reverse electron demand Diels-Alder cycloaddition. In another preferred embodiment, 1,3-dipolar cycloaddition, more preferably alkyne-azide cycloaddition is used, most preferably where Q ¹ is an alkyne group or comprises an alkyne group, F ¹ is an azide group. Cycloadditions such as the Diels-Alder reaction and 1,3-dipolar cycloaddition are known in the art and the skilled person knows how to perform the cycloaddition.

When Q ¹ reacts with F ¹ , a covalent bond is formed between the biomolecule derived from the linker-conjugate and the target molecule. The complementary reactive group Q ¹ and the functional group F ¹ are described in more detail above and below.

  In a preferred embodiment of the process for preparing the bioconjugate, a is 0 in the group according to formula (1). In this embodiment, the linker-conjugate thus comprises a group according to formula (2) as defined above. In another preferred embodiment of the process for preparing the bioconjugate, a is 1 in the group according to formula (1). In this embodiment, the linker-conjugate thus comprises a group according to formula (3) as defined above.

  Biomolecules are described in more detail above. In the process according to the invention, biomolecules consist of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids and monosaccharides. Preferably it is selected from the group. The biomolecule B is more preferably selected from the group consisting of proteins (including glycoproteins and antibodies), polypeptides, peptides, glycans, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides and enzymes. The biomolecule B is more preferably selected from the group consisting of glycoproteins and proteins including antibodies, polypeptides, peptides and glycans. Most preferably, B is an antibody or antigen-binding fragment thereof.

In the process for preparing the bioconjugate, the reactive group Q ¹ is optionally substituted N-maleimidyl group, halogenated N-alkylamide group, sulfonyloxy N-alkylamide group, ester group, carbonate Group, sulfonyl halide group, thiol group or derivative thereof, alkenyl group, alkynyl group, (hetero) cycloalkynyl group, bicyclo [6.1.0] non-4-in-9-yl] group, cycloalkenyl group, Tetrazinyl group, azide group, phosphine group, nitrile oxide group, nitrone group, nitrileimine group, diazo group, ketone group, (O-alkyl) hydroxylamino group, hydrazine group, halogenated N-malemidyl group, 1,1-bis (Sulfonylmethyl) methylcarbonyl group or its leaving derivative, halogenated potassium It is preferably selected from the group consisting of a rubonyl group and an allenamide group.

In a more preferred embodiment, Q ¹ is represented by the formulas (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j) , (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9v) 9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh), (9zi) , (9zj) or (9zk), where (9a), (9b), (9c), (9d), (9e), (9f), (9g), (9h), (9i), (9j), (9k), (9l), (9m), (9n), (9o), (9p), (9q), (9r), (9s), (9t), (9u), (9v), (9w), (9x), (9y), (9z), (9za), (9zb), (9zc), (9zd), (9ze), (9zf), (9zg), (9zh) ), (9zi), (9zj), (9zk) and their preferred embodiments are as defined above for the linker-conjugates according to the invention. Q ¹ is defined by the equations (9a), (9b), (9c), (9f), (9j), (9n), (9o), (9p), (9q), (9s), (9t), (9t), 9zh) or (9r) is more preferred. Q ¹ is more preferably according to formula (9a), (9n), (9o), (9p), (9q), (9t), (9zh) or (9s), and Q ¹ is represented by formula (9a) ), (9p), (9q), (9n), (9t), (9zh) or (9o) are most preferred and their preferred embodiments are as defined above.

In a particularly preferred embodiment, Q ¹ is an alkynyl group as described above, a cycloalkenyl group as described above, a (hetero) cycloalkynyl group as described above and a bicyclo [ 6.1.0] Non-4-in-9-yl] groups, preferably selected from alkyne groups, wherein Q ¹ is defined by the formulas (9j), (9n), (9 More preferably, it is selected from 9o), (9p), (9q) and (9zk). Q ¹ is most preferably a bicyclo [6.1.0] non-4-yn-9-yl] group of formula (9q).

（式中：
ａは、独立して０又は１であり；
ｂは、独立して０又は１であり；
ｃは、０又は１であり；
ｄは、０又は１であり；
ｅは、０又は１であり；
ｆは、１〜１５０の範囲における整数であり；
ｇは、０又は１であり；
ｉは、０又は１であり；
Ｄは、標的分子であり；
Ｑ^１は、生体分子に存在する官能基Ｆ^１と反応できる反応性基であり；
Ｓｐ^１は、スペーサー部分であり；
Ｓｐ^２は、スペーサー部分であり；
Ｓｐ^３は、スペーサー部分であり；
Ｓｐ^４は、スペーサー部分であり；
Ｚ^１は、Ｑ^１又はＳｐ^３を、Ｓｐ^２、Ｏ若しくはＣ（Ｏ）又はＮ（Ｒ^１）に接続する接続基であり；
Ｚ^２は、Ｄ又はＳｐ^４を、Ｓｐ^１、Ｎ（Ｒ^１）、Ｏ又はＣ（Ｏ）に接続する接続基であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される；又はＲ^１は、Ｄ、−［（Ｓｐ^１）_ｂ（Ｚ^２）_ｅ（Ｓｐ^４）_ｉＤ］若しくは−［（Ｓｐ^２）_ｃ（Ｚ^１）_ｄ（Ｓｐ^３）_ｇＱ^１］であり、ここでＤは標的分子であり、Ｓｐ^１、Ｓｐ^２、Ｓｐ^３、Ｓｐ^４、Ｚ^１、Ｚ^２、Ｑ^１、ｂ、ｃ、ｄ、ｅ、ｇ及びｉは、上で定義されている通りである）に従っている。 In a further preferred embodiment of the process for preparing the bioconjugate, the linker-conjugate is of formula (4a) or (4b), or a salt thereof:

(Where:
a is independently 0 or 1;
b is independently 0 or 1;
c is 0 or 1;
d is 0 or 1;
e is 0 or 1;
f is an integer in the range of 1 to 150;
g is 0 or 1;
i is 0 or 1;
D is the target molecule;
Q ¹ is a reactive group that can react with the functional group F ¹ present in the biomolecule;
Sp ¹ is a spacer moiety;
Sp ² is a spacer moiety;
Sp ³ is a spacer moiety;
Sp ⁴ is a spacer moiety;
Z ¹ is a connecting group that connects Q ¹ or Sp ³ to Sp ² , O or C (O) or N (R ¹ );
Z ² is a connecting group that connects D or Sp ⁴ to Sp ¹ , N (R ¹ ), O or C (O);
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in ^{R 3} are independently selected from the group consisting of hydrogen and _C 1 -C ₄ alkyl group; or ^{R 1 _{is, D, - [(Sp 1}} ) b (Z 2) e (Sp 4) i D Or ^- a _{^{[(Sp 2) c (Z}} 1) d (Sp 3) g Q 1], where D is the target ^{molecule, Sp 1, Sp 2, Sp} 3, Sp 4, Z 1, Z 2 , Q ¹ , b, c, d, e, g and i are as defined above).

Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are independently spacer portions, in other words, Sp ¹ , Sp ² , Sp ³ and Sp ⁴ may be different from each other. Sp ¹ , Sp ² , Sp ³ and Sp ⁴ may be present or absent (b, c, g and i are independently 0 or 1). However, it is preferred that at least one of Sp ¹ , Sp ² , Sp ³ and Sp ⁴ is present, ie at least one of b, c, g and i is not zero.

If present, Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are linear or branched C ₁ -C ₂₀₀ alkylene groups, C ₂ -C ₂₀₀ alkenylene groups, C ₂ -C ₂₀₀ alkynylene groups, C ₃ -C ₂₀₀ cycloalkylene _group, C _{5 -C 200} cycloalkenylene _group, C _{8 -C 200} cycloalkynylene _groups, C _{7 -C 200} alkyl arylene _group, C _{7 -C 200} arylalkylene _group, C _{8 -C 200} aryl it is preferably independently selected from the group consisting of alkenylene and C ₉ -C ₂₀₀ aryl alkynylene group, an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene groups, alkylarylene Group, arylalkylene group, arylalkenylene group and aryl Alkynylene group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group Are optionally substituted. One alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group as defined above. Or when interrupted by a plurality of heteroatoms, the group is preferably interrupted by one or more O atoms and / or by one or more SS groups.

Spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are linear or branched C ₁ -C ₁₀₀ alkylene groups, C ₂ -C ₁₀₀ alkenylene groups, C ₂ -C ₁₀₀ alkynylene groups, C ₃ -C ₁₀₀ cycloalkylene _group, C _{5 -C 100} cycloalkenylene _group, C _{8 -C 100} cycloalkynylene _groups, C _{7 -C 100} alkyl arylene _group, C _{7 -C 100} arylalkylene _group, C 8 -C more preferably it is independently selected from the group consisting of ₁₀₀ aryl alkenylene group and C ₉ -C ₁₀₀ aryl alkynylene group, an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene groups , Alkylarylene group, arylalkylene group, arylalkene Emissions and aryl alkynylene group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen C ₁ -C ₂₄ alkyl group, C ₂ -C ₂₄ alkenyl group, C ₂ -C ₂₄ alkynyl group and C ₃ -C ₂₄ cycloalkyl group independently selected from the group consisting of alkyl group, alkenyl group, alkynyl Groups and cycloalkyl groups are optionally substituted.

Spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are linear or branched C ₁ -C ₅₀ alkylene groups, C ₂ -C ₅₀ alkenylene groups, C ₂ -C ₅₀ alkynylene groups, C ₃ -C ₅₀ cycloalkylene _group, C 5 _{-C 50} cycloalkenylene _groups, C 8 _{-C 50} cycloalkynylene _groups, C 7 _{-C 50} alkylarylene _group, C 7 _{-C 50} arylalkylene _groups, C 8 -C _More preferably it is independently selected from the group consisting of ₅₀ arylalkenylene groups and C ₉ -C ₅₀ arylalkynylene groups, alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups. Alkyl arylene group, aryl alkylene group, aryl alkenylene group and aryl Alkynylene group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group Are optionally substituted.

If present, the spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ are linear or branched C ₁ -C ₂₀ alkylene groups, C ₂ -C ₂₀ alkenylene groups, C ₂ -C ₂₀ alkynylene groups, C ₃ -C ₂₀ cycloalkylene _group, C 5 _{-C 20} cycloalkenylene _groups, C 8 _{-C 20} cycloalkynylene _groups, C 7 _{-C 20} alkylarylene _group, C 7 _{-C 20} arylalkylene _groups, C 8 -C ₂₀ aryl alkenylene group and C ₉ -C ₂₀ aryl alkynylene is even more preferably selected independently from the group consisting of group, alkylene group, alkenylene group, alkynylene group, a cycloalkylene group, cycloalkenylene group, cycloalkynylene Group, alkylarylene group, arylalkylene group, arylalkenylene group and aryl group Ruarukiniren group is optionally substituted, O, is interrupted optionally by one or more heteroatoms selected from the group of S and NR ^3, wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl group Are optionally substituted.

In these preferred embodiments, the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkynylene group, alkylarylene group, arylalkylene group, arylalkenylene group and arylalkynylene group are unsubstituted. More preferably, it is further optionally interrupted by one or more heteroatoms selected from the group O, S and NR ³ , preferably O, wherein R ³ is hydrogen and C ₁ -C It is independently selected from the group consisting of ₄ alkyl groups, preferably hydrogen or methyl.

Most preferably, the spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are independently selected from the group consisting of linear or branched C ₁ -C ₂₀ alkylene groups. Is optionally substituted and optionally interrupted by one or more heteroatoms selected from the group of O, S and NR ³ , wherein R ³ is hydrogen, C ₁ -C ₂₄ alkyl _group, C 2 _{-C 24} alkenyl group _is independently selected from C 2 _{-C 24} alkynyl and _C 3 _{-C 24} group consisting of cycloalkyl, alkyl group, alkenyl group, alkynyl group and cycloalkyl groups, Optionally replaced. In this embodiment, the alkylene group is unsubstituted and optionally interrupted by one or more heteroatoms selected from the group O, S and NR ³ , preferably O and / or S—S. It is further preferred that R ³ is independently selected from the group consisting of hydrogen and a C ₁ -C ₄ alkyl group, preferably hydrogen or methyl.

Particularly preferred spacer moieties Sp ¹ , Sp ² , Sp ³ and Sp ⁴ include — (CH ₂ ) _n —, — (CH ₂ CH ₂ ) _n —, — (CH ₂ CH ₂ O) _n —, — (OCH _{2 CH 2) n -, -} (CH 2 CH 2 O) n CH 2 CH 2 -, - CH 2 CH 2 (OCH 2 CH 2) n -, - (CH 2 CH 2 CH 2 O) n -, - _{(OCH 2 CH 2 CH 2)} n -, - (CH 2 CH 2 CH 2 O) n CH 2 CH 2 CH 2 - and _{-CH 2 CH 2 CH 2 (OCH} 2 CH 2 CH 2) n - can be mentioned Where n is an integer in the range 1-50, preferably in the range 1-40, more preferably in the range 1-30, more preferably in the range 1-20 and even more preferably in the range 1-15. is there. n is more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and more preferably 1, 2, 3, 4, 5, 6, 7 or 8. 1, 2, 3, 4, 5 or 6 is more preferred, and 1, 2, 3 or 4 is even more preferred.

In another preferred embodiment of the process according to the invention, in the linker-conjugates according to formulas (4a) and (4b), the spacer moieties Sp ¹ , Sp ² , Sp ³ and / or Sp ⁴ are present A sequence of amino acids. Spacer moieties containing amino acid sequences are known in the art and can also be referred to as peptide linkers. Examples include spacer moieties containing a Val-Ala moiety or a Val-Cit moiety, such as Val-Cit-PABC, Val-Cit-PABC, Fmoc-Val-Cit-PABC, and the like.

As described above, Z ¹ and Z ² are connecting groups. In a preferred embodiment of the process according to the invention, Z ¹ and Z ² are —O—, —S—, —NR ² —, —N═N—, —C (O) —, —C (O) NR ^2. -, - OC (O) - , - OC (O) O -, - OC (O) NR 2, -NR 2 C (O) -, - NR 2 C (O) O -, - NR 2 C (O ^{) NR 2 -, - SC (} O) -, - SC (O) O -, - SC (O) NR 2 -, - S (O) -, - S (O) 2 -, - OS (O) 2 ^{_{-, - OS (O) 2}} O -, - OS (O) 2 NR 2 -, - OS (O) -, - OS (O) O -, - OS (O) NR 2 -, - ONR 2 C ( ^{O) -, - ONR 2 C} (O) O -, - ONR 2 C (O) NR 2 -, - NR 2 OC (O) -, - NR 2 OC (O) O -, - NR 2 OC (O ^{) NR 2 -, - ONR 2} C (S) -, ^{ONR 2 C (S) O -} , - ONR 2 C (S) NR 2 -, - NR 2 OC (S) -, - NR 2 OC (S) O -, - NR 2 OC (S) NR 2 -, ^{-OC (S) -, - OC} (S) O -, - OC (S) NR 2 -, - NR 2 C (S) -, - NR 2 C (S) O -, - NR 2 C (S) _{^{NR 2 -, - SS (O}} ) 2 -, - SS (O) 2 O -, - SS (O) 2 NR 2 -, - NR 2 OS (O) -, - NR 2 OS (O) O-, —NR ² OS (O) NR ² —, —NR ² OS (O) ₂ —, —NR ² OS (O) ₂ O—, —NR ² OS (O) ₂ NR ² —, —ONR ² S (O )-, -ONR ² S (O) O-, -ONR ² S (O) NR ^2- , -ONR ² S (O) ₂ O-, -ONR ² S (O) ₂ NR ^2- , -ONR ² S _{(O) 2 ^{, -OP (O) (R 2}} ) 2 -, - SP (O) (R 2) 2 -, - NR 2 P (O) (R 2) 2 - and from the group consisting of combinations of two or more thereof Independently selected, wherein R ² is selected from the group consisting of hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₂ -C ₂₄ alkenyl group, a C ₂ -C ₂₄ alkynyl group, and a C ₃ -C ₂₄ cycloalkyl group. Independently selected, alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

（式中：
Ｒ^１０は、（チオ）エステル基であり；
Ｒ^１８は、任意選択で置換されている、Ｃ_１〜Ｃ_１２アルキル基及びＣ_４〜Ｃ_１２（ヘテロ）アリール基からなる群から選択される）に従っている。 In a particularly preferred process according to the invention, Sp ¹ , Sp ² , Sp ³ and Sp ⁴ , if present, are independently selected from the group consisting of linear or branched C ₁ -C ₂₀ alkylene groups The group is optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of O, S and NR ³ , wherein R ³ is hydrogen and C ₁ -C Independently selected from the group consisting of ₄ alkyl groups, Q ¹ is of the formula (9a), (9p), (9q), (9n), (9t), (9zh) or (9o):

(Where:
R ¹⁰ is a (thio) ester group;
R ¹⁸ is optionally selected from the group consisting of a C ₁ -C ₁₂ alkyl group and a C ₄ -C ₁₂ (hetero) aryl group).

  An embodiment of a process for preparing a bioconjugate is illustrated in FIG. FIG. 4 shows that modified antibodies 13a-d are directed to modified cysteine residues related to 3-mercaptopropionyl-galactosamine modified 13a leading to thioether conjugate 14 or to thioether conjugate 17 by means of nucleophilic addition with maleimide. It shows how the bioconjugation process is subjected, with respect to conjugation), or with strain-promoted cycloaddition with a cyclooctyne reagent (for 13b, 13c or 13d leading to triazole 15a, 15b or 16, respectively).

In addition to increasing the therapeutic index of the bioconjugates according to the present invention, further advantages of the process for the preparation of bioconjugates as described herein and the linker-conjugates and sulfamide linkers according to the present invention are The conjugation efficiency is increased when a sulfamide linker is used instead of a typical polyethylene glycol (PEG) spacer. The additional advantages of sulfamide groups, especially acylsulfamide groups or carbamoylsulfamide groups, are relative to the solubility of linkers containing such groups and to the overall construct before, during and after conjugation. High polarity of sulfamide group that imparts a positive effect. In view of this increased polarity, conjugation using linker-conjugates containing sulfamide linkers according to the present invention is particularly suitable for conjugating hydrophobic target compounds to biomolecules. The high polarity of sulfamides allows the hydrophobic moiety to be conjugated to a biomolecule of interest known to require a large amount of organic co-solvent during conjugation and / or induce aggregation of the bioconjugate. Have a positive effect. High levels of co-solvents (up to 25% of DMF or indeed 50% of DMA, propylene glycol or DMSO) can induce protein denaturation during the conjugation process and / or require special equipment in the manufacturing process There are things to do. Therefore, aggregation of the problems associated with the connection portions of the hydrophobic in bioconjugates, in the formation of bioconjugates, linker - a spacer between the target molecule in the conjugate with the reactive group Q ^1, according to the invention It is effectively solved by using a sulfamide linker. An additional advantage of the use of the sulfamide linker and sulfamide linker in the bioconjugation process according to the present invention is the ease of synthesis and high yield.

For evidence of these beneficial effects of the use of sulfamide linkers according to the present invention, see international application PCT / NL2015 / 050697, in particular its Tables 1-3, FIGS. 11-14, 23 and 24, and Examples 57, 58, 60. And 61 are referred to. These tables, figures and examples of International Application PCT / NL2015 / 050506 are incorporated herein.
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（式中、Ｓ（Ｆ^１）_ｘ及びｘは、上で定義されている通りであり；ＡＢは抗体を表し；ＧｌｃＮＡｃはＮ−アセチルグルコサミンであり；Ｆｕｃはフコースであり；ｂは、０又は１であり；ｙは１、２、３又は４である）に従った修飾糖タンパク質を得ることができる、ステップ；並びに
（ｉｉ）修飾糖タンパク質を、官能基Ｆ^１と反応できる官能基Ｑ^１及びリンカーＬ^２を介してＱ^１に接続されている標的分子Ｄを含むリンカー−コンジュゲートと反応させることで抗体−コンジュゲートを得るステップであって、リンカーＬはＳ−Ｚ^３−Ｌ^２を含み、Ｚ^３は、Ｑ^１とＦ^１との間の反応から生じる接続基である、ステップ。 The present invention thus provides, in a first aspect, at least one of “core-GlcNAc functionalization” and “sulfamide linkage” as defined above for increasing the therapeutic index of a bioconjugate. To the use of a mode of conjugation comprising The invention according to this aspect can also be expressed as a method for increasing the therapeutic index of a bioconjugate. In a preferred embodiment, the mode of conjugation is used to connect biomolecule B to target molecule D via linker L, where the mode of conjugation includes:
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is , A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) By transferring the _x moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or be 1; y can be obtained a modified glycoprotein according to 1, 2, 3 or 4), the step; and (ii) a modified glycoprotein, the functional group ^{Q 1} capable of reacting with functional groups ^{F 1} And a linker-conjugate comprising a target molecule D connected to Q ¹ via the linker L ² to obtain an antibody-conjugate, wherein the linker L converts SZ ³ -L ² And Z ³ is a connecting group resulting from the reaction between Q ¹ and F ¹ .

  Herein, the biomolecule is preferably an antibody and the bioconjugate is preferably an antibody-conjugate.

  Herein, the therapeutic index is increased compared to a bioconjugate that does not include a mode of conjugation according to the present invention, or can be obtained by a mode of conjugation according to the present invention. Thus, in a first embodiment, the therapeutic index is a bioconjugate that cannot be obtained by steps (i) and (ii) as defined above, or in other words, a direct consequence of the conjugation process. Increased compared to bioconjugates that do not contain the structural features of linker L that result from linking an antibody to a target molecule. Thus, in a second embodiment, the therapeutic index is increased compared to a bioconjugate of formula (A) in which the linker L does not comprise a group according to formula (1) or a salt thereof.

  The inventors surprisingly found that when the mode of conjugation according to the invention was used, all other factors, in particular the type and type of biomolecule and the biomolecule-target molecule-ratio were kept constant. Even so, it was found that the therapeutic index of the bioconjugate according to the invention was significantly increased. The increase in therapeutic index is preferably an increase in therapeutic index in the treatment of cancer or alternatively in targeting CD30 expressing tumors.

  The method according to the first aspect of the invention can also be expressed as a method for increasing the therapeutic index of a bioconjugate comprising providing a bioconjugate having a mode of conjugation according to the invention.

  We have for both aspects of the therapeutic index when the mode of conjugation according to the invention is included in a bioconjugate according to the invention: (a) for therapeutic efficacy and (b ) It has been found to have an effect on tolerance. Thus, the present use or method for increasing the therapeutic index preferably (a) increases therapeutic efficacy and / or (b) increases the tolerability of the bioconjugate of formula (A). Because. The bioconjugate is preferably an antibody-conjugate, and the use or method preferably increases the therapeutic index of the antibody-conjugate, preferably (a) increases the therapeutic effectiveness of the antibody-conjugate. And / or (b) to increase the tolerability of the antibody-conjugate. In one embodiment, the method or use is for increasing the therapeutic effectiveness of a bioconjugate, preferably an antibody-conjugate. In one embodiment, the method or use is for increasing the tolerability of a bioconjugate, preferably an antibody-conjugate.

Accordingly, in one embodiment, the use or method according to the first aspect is for increasing the therapeutic effectiveness of a bioconjugate of formula (A). As used herein, “increasing therapeutic efficacy” can also be expressed as “decreasing the effective amount”, “decreasing the ED ₅₀ value” or “increasing the protective index”. . Similarly, in one embodiment, the method according to the first aspect is for increasing the tolerability of the bioconjugate of formula (A). As used herein, “increasing tolerability” means “increasing maximum tolerated dose (MTD)”, “increasing TD ₅₀ value”, “increasing safety” or “reducing toxicity” It can also be expressed as “to do”. In a particularly preferred embodiment, the method according to the first aspect is for (a) increasing the therapeutic effectiveness and (b) increasing the tolerability of the bioconjugate of formula (A).

  The method according to the first aspect is very non-medical. In one embodiment, the method is a non-medical method for increasing the therapeutic index of a bioconjugate.

  The first aspect of the invention can also be expressed as a mode of conjugation for use in improving the therapeutic index (therapeutic efficacy and / or tolerability) of a bioconjugate, wherein the conjugate The mode of gation is as defined above. In one embodiment, this aspect is according to “core-GlcNAc functionalization” as defined above for use in improving the therapeutic efficacy of a bioconjugate of formula (A). It is also expressed as a mode of conjugation, where L and (A) are as defined above. In one embodiment, this aspect is also expressed as a mode of conjugation for use in improving the therapeutic index (therapeutic efficacy and / or tolerability) of a bioconjugate, preferably an antibody-conjugate. The In other words, the first aspect is the conjugation for the preparation of a bioconjugate, preferably an antibody-conjugate, to improve the therapeutic index (therapeutic efficacy and / or tolerability) of the bioconjugate. Regarding the use of modes. The invention according to the first aspect is a bioconjugate, preferably in an antibody-conjugate, or in a bioconjugate, preferably for increasing the therapeutic index (therapeutic efficacy and / or tolerability) of the bioconjugate. Can also be expressed as the use of a mode of conjugation in the preparation of antibody-conjugates.

  In one embodiment, the method, use or mode of conjugation for use according to the first aspect of the invention is directed to a subject in need thereof, suitably a lymphoma such as Hodgkin lymphoma (HL), non-Hodgkin Lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B cell lymphoma, childhood lymphoma, T cell lymphoma and enteropathy-associated T cell lymphoma (EATL), leukemia such as acute myeloid leukemia (AML), acute lymphoblast For patients suffering from disorders associated with CD30 expression selected from spheroidal leukemia (ALL) and mast cell leukemia, germ cell cancer, transplant versus host disease (GvHD) and lupus, especially systemic lupus lupus erythematosus (SLE), Further comprising administration of a bioconjugate according to the invention. In one embodiment, the subject is a cancer patient, more suitably a patient suffering from a CD30 expressing tumor. The use of bioconjugates such as antibody-drug-conjugates is well known in the field of cancer treatment, and the bioconjugates according to the invention are particularly suitable in this regard.

Typically, the bioconjugate is administered in a therapeutically effective amount. Administration can be a single dose or can occur, for example, 1 to 4 times a month, preferably 1 to 2 times a month. In a preferred embodiment, administration occurs every 3 or 4 weeks, most preferably once every 4 weeks. In view of increased therapeutic efficacy, administration may not occur more frequently than during treatment with conventional bioconjugates. As will be appreciated by those skilled in the art, the dose of the bioconjugate according to the present invention may depend on many factors, and the optimal dosing schedule can be determined by one skilled in the art through routine experimentation. . The bioconjugate is 0.01-50 mg / kg subject body weight, more precisely 0.03-25 mg / kg or most precisely 0.05-10 mg / kg, or alternatively 0.1-25 mg / kg or Typically administered at a dose of 0.5-10 mg / kg. In one embodiment, administration is via intravenous injection.
Methods for targeting CD30 expressing cells

  The invention relates in a second aspect to a method for targeting CD30 expressing cells comprising the administration of a bioconjugate according to the invention. The subject in need thereof is most preferably a cancer patient. The use of bioconjugates such as antibody-drug-conjugates is well known in the field of cancer treatment, and the bioconjugates according to the invention are particularly suitable in this regard. The method as described is typically suitable for the treatment of cancer. The bioconjugates according to the invention have been described in great detail above, and the above description applies equally to the bioconjugates used in the second aspect of the invention. The second aspect of the invention can also be expressed as a bioconjugate according to the invention for use in targeting CD30 expressing cells in a subject in need thereof. In other words, the second aspect relates to the use of a bioconjugate according to the invention for the preparation of a medicament for use in targeting CD30 expressing cells in a subject in need thereof.

  In the context of this embodiment, targeting CD30 expressing cells includes treating, imaging, diagnosing, preventing their growth, containment and reducing CD30 expressing cells, particularly CD30 expressing tumors. One or more of these. Most preferably, this embodiment is for the treatment of CD30 expressing tumors.

  In one embodiment, this aspect relates to a method for the treatment of a subject in need thereof. The second aspect of the invention can also be expressed as a bioconjugate according to the invention for use in the treatment of a subject in need thereof, preferably for the treatment of cancer. In other words, the second aspect relates to the use of a bioconjugate according to the invention for the preparation of a medicament for use in the treatment of a subject in need thereof, preferably for use in the treatment of cancer.

  In the context of this embodiment, the subject is a lymphoma, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B cell lymphoma, childhood lymphoma, T cell lymphoma and enteropathy-related T Cellular lymphoma (EATL), leukemias such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell cancer, transplant versus host disease (GvHD), and lupus, especially systemic lupus Suitably suffers from a disorder selected from lupus erythematosus (SLE). The disorder is more suitably a cancer, most suitably a lymphoma such as Hodgkin lymphoma (HL).

  In the context of this embodiment, the target molecule D is preferably an anticancer agent, preferably a cytotoxin.

  In the method according to the second aspect, the bioconjugate is typically administered in a therapeutically effective amount. Administration can be a single dose or can occur, for example, 1 to 4 times a month, preferably 1 to 2 times a month. In a preferred embodiment, administration occurs every 3 or 4 weeks, most preferably once every 4 weeks. As will be appreciated by those skilled in the art, the dose of the bioconjugate according to the present invention may depend on many factors, and the optimal dosing schedule can be determined by one skilled in the art through routine experimentation. . The bioconjugate is 0.01-50 mg / kg subject body weight, more precisely 0.03-25 mg / kg or most precisely 0.05-10 mg / kg, or alternatively 0.1-25 mg / kg or Typically administered at a dose of 0.5-10 mg / kg. In one embodiment, administration is via intravenous injection.

In view of increased therapeutic efficacy, administration may occur less frequently and / or at lower doses than in treatments using conventional bioconjugates. In one embodiment, the administration of the bioconjugate according to the present invention is a lower dose than the TD _{50 of a} bioconjugate that is the same bioconjugate but does not include a mode of conjugation according to the present invention, preferably the dose is At most 99-90%, more preferably at most 89-60%, more preferably at most 59-30, of the TD ₅₀ of the same bioconjugate but without the mode of conjugation according to the invention. %, Most preferably at most 29-10%. In one embodiment, the administration of the bioconjugate according to the invention does not occur more frequently than the administration performed for a bioconjugate that is the same bioconjugate but does not comprise a mode of conjugation according to the invention, preferably The number of administration events is at most 75%, more preferably at most 50% of the number of administration events of a bioconjugate that is the same bioconjugate but does not include a mode of conjugation according to the present invention. Alternatively, taking into account increased tolerability, administration may occur at higher doses than treatment with conventional bioconjugates. In one embodiment, the administration of the bioconjugate according to the present invention is a higher dose than the TD _{50 of a} bioconjugate that is the same bioconjugate but does not include a mode of conjugation according to the present invention, preferably the dose is At most 25-50%, more preferably at most 50-75%, most preferably at most 75-100 of the TD ₅₀ of the same bioconjugate but without the mode of conjugation according to the invention. %.

In view of increased therapeutic efficacy, administration may occur less frequently and / or at lower doses than treatment with conventional bioconjugates. In one embodiment, the administration of the bioconjugate according to the invention is a lower dose than the ED _{50 of a} bioconjugate that is the same bioconjugate but does not contain a mode of conjugation according to the invention, preferably the dose is At most 99-90%, more preferably at most 89-60%, more preferably at most 59-30 of the ED ₅₀ of a bioconjugate which is the same bioconjugate but does not include the mode of conjugation according to the invention. %, Most preferably at most 29-10%. In one embodiment, the administration of the bioconjugate according to the invention does not occur more frequently than the administration performed for a bioconjugate that is the same bioconjugate but does not comprise a mode of conjugation according to the invention, preferably The number of administration events is at most 75%, more preferably at most 50% of the number of administration events of a bioconjugate that is the same bioconjugate but does not include a mode of conjugation according to the present invention. Alternatively, taking into account increased tolerability, administration may occur at higher doses than in treatments using conventional bioconjugates. In one embodiment, the administration of the bioconjugate according to the present invention is a higher dose than the TD _{50 of a} bioconjugate that is the same bioconjugate but does not include a mode of conjugation according to the present invention, preferably the dose is At most 1.1 to 1.49 times, more preferably at most 1.5 to 1.99 times the TD ₅₀ of the same bioconjugate but not including the mode of conjugation according to the invention Higher, more preferably 2 to 4.99 times higher, most preferably 5 to 10 times higher.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含む］によって表される。 In one embodiment, a use or method according to this aspect or a conjugation mode for use is a bioconjugate for use in the treatment of a subject in need thereof, wherein the bioconjugate is of formula (A ):
BLD
(A)
[Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or a spacer moiety;
Where L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I wherein the target molecule is a spacer Min represented by including that) is connected optionally] the N through.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含む］によって表される。
本発明による抗体−コンジュゲート In one embodiment, the use or method according to this aspect or the conjugation mode for use is not a bioconjugate for use in the treatment of a subject in need thereof, wherein the bioconjugate is of formula (A ):
BLD
(A)
[Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or a spacer moiety;
Where L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I wherein the target molecule is a spacer Min represented by including that) is connected optionally] the N through.
Antibody-conjugates according to the invention

（式中、Ｓ（Ｆ^１）_ｘ及びｘは、上で定義されている通りであり；ＡＢは抗体を表し；ＧｌｃＮＡｃはＮ−アセチルグルコサミンであり；Ｆｕｃはフコースであり；ｂは０又は１であり；ｙは１、２、３又は４である）に従った修飾抗体を得ることができる、ステップ；並びに
（ｉｉ）修飾抗体を、官能基Ｆ^１と反応できる官能基Ｑ^１及びリンカーＬ^２を介してＱ^１に接続されている標的分子Ｄを含むリンカー−コンジュゲートと反応させることで抗体−コンジュゲートを得るステップであって、リンカーＬは、Ｓ−Ｚ^３−Ｌ^２を含み、Ｚ^３は、Ｑ^１とＦ^１との間の反応から生じる接続基である、ステップ。 In a third aspect, the present invention relates to antibody-conjugates that are particularly suitable for targeting CD30 expressing tumors. The antibody-conjugate according to the invention comprises an antibody AB connected to the target molecule D via a linker L, wherein the antibody-conjugate comprises a mode of conjugation according to the invention or according to the invention It can be obtained by the mode of conjugation. In particular, antibody-conjugates according to the invention can be obtained by:
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is , A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) By transferring the _x moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 And y is 1, 2, 3 or 4), and (ii) a functional group Q ¹ and a linker L that can react the modified antibody with the functional group F ^1. Obtaining an antibody-conjugate by reacting with a linker-conjugate comprising a target molecule D connected to Q ¹ via ^{2 wherein} the linker L comprises SZ ³ -L ² ; Z ³ is a connecting group resulting from the reaction between Q ¹ and F ¹ , step.

  As used herein, antibody AB can target CD30 expressing tumors and target molecule D is taxane, anthracycline, camptothecin, epothilone, mitomycin, combretastatin, vinca alkaloid, maytansinoid, calicheamicin and enediyne, duoin Carmycin, tubricin, amatoxin, dolastatin and auristatin, pyrrolobenzodiazepine dimer, indoline o-benzodiazepine dimer, radioisotope, therapeutic protein and peptide (or fragments thereof), kinase inhibitor, MEK inhibitor, Selected from the group consisting of KSP inhibitors, as well as their analogs or prodrugs. Alternatively, the target molecule D is a cytotoxin. In one embodiment according to this aspect, the target molecule D is an anthracycline, maytansinoid, calicheamicin and enediyne, duocarmycin, tubricin, dolastatin and auristatin, pyrrolobenzodiazepine dimer, indoline o-benzodiazepine dimer. And more preferably selected from the group consisting of anthracyclines, maytansinoids, dolastatins and auristatins, and pyrrolobenzodiazepine dimers. In a preferred embodiment according to this aspect, the target molecule D is auristatin, more preferably auristatin selected from the group of MMAD, MMAE and MMAF, most preferably D = MMAD or MMAE.

  Preferred embodiments of steps (i) and (ii) apply equally to the antibody-conjugates according to the invention as defined above. The skilled artisan knows how to translate these preferred features into the structural features of the antibody-conjugate. In a preferred embodiment, the antibody-conjugate according to this aspect is according to formula (A), preferably the linker L contains a group according to formula (1) or a salt thereof, wherein (A) and ( 1) is as defined above.

Preferred options for S (F ¹ ) _x are described above. In one preferred embodiment, S (F ¹ ) _x is 6-azido-6-deoxy-N-acetylgalactosamine.

  In a preferred embodiment, an antibody AB capable of targeting a CD30 expressing tumor is Ki-2, Ki-2, Ki-4, Ki-6, Ki-7, HRS-1, HRS-4, Ber-H8, Ber- From the group consisting of H2, 5F11 (MDX-060, Iratumumab), Ki-1, Ki-5, M67, Ki-3, M44, HeFi-1, AC10, cAC10 (brentuximab) and their functional analogues Selected. The antibody AB capable of targeting a CD30-expressing tumor is more preferably iratumumab or brentuximab, and most preferably brentuximab.

  In a particularly preferred embodiment, the antibody AB is iritumumab or brentuximab, most preferably brentuximab and the target molecule D is an auristatin selected from the group consisting of MMAD, MMAE and MMAF, most preferably D = MMAD or MMAE.

（式中：
Ｒ^３１は、水素、ハロゲン、−ＯＲ^３５、−ＮＯ_２、−ＣＮ、−Ｓ（Ｏ）_２Ｒ^３５、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され、アルキル基、（ヘテロ）アリール基、アルキル（ヘテロ）アリール基及び（ヘテロ）アリールアルキル基は、任意選択で置換されており、ここで２個の置換基Ｒ^３１は、一緒に連結されて縮環シクロアルキル又は縮環（ヘテロ）アレーン置換基を形成することができ、Ｒ^３５は、水素、ハロゲン、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_６〜Ｃ_２４（ヘテロ）アリール基、Ｃ_７〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_７〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から独立して選択され；
Ｘは、Ｃ（Ｒ^３１）_２、Ｏ、Ｓ又はＮＲ^３２であり、ここでＲ^３２は、Ｒ^３１又はＬ^３（Ｄ）_ｒであり、ここでＬ^３はリンカーであり、Ｄは、請求項１に定義されている通りであり；
ｒは、１〜２０であり；
ｑは、０又は１であるが、ｑが０であるならば、ＸはＮ−Ｌ^２（Ｄ）_ｒであることを条件とし；
ａａは、０、１、２、３、４、５、６、７又は８であり；
ａａ’は、０、１、２、３、４、５、６、７又は８であり；
ａａ＋ａａ’＜１０であり；
ｂは、０又は１であり；
ｐｐは、０又は１であり；
Ｍは、−Ｎ（Ｈ）Ｃ（Ｏ）ＣＨ_２−、−Ｎ（Ｈ）Ｃ（Ｏ）ＣＦ_２−、−ＣＨ_２−、−ＣＦ_２−、又は０〜４個のフッ素置換基、好ましくは、フェニレンのＣ２及びＣ６又はＣ３及びＣ５に好ましくは位置する２個のフッ素置換基を含有する１，４−フェニレンであり；
ｙは、１〜４であり；
Ｆｕｃは、フコースである）によって表される。 In a preferred embodiment, the antibody-conjugate is of formula (40) or (40b):

(Where:
R ³¹ is hydrogen, _{^{halogen, -OR 35, -NO 2, -CN}} , -S (O) 2 R 35, C 1 ~C 24 alkyl _group, C 6 _{-C 24} (hetero) aryl _{group, C} 7 ~ C ₂₄ alkyl is independently selected from the group consisting of (hetero) aryl groups and _C 7 _{-C 24} (hetero) aryl group, an alkyl group, (hetero) aryl group, alkyl (hetero) aryl groups and (hetero) aryl The alkyl group is optionally substituted, wherein the two substituents R ³¹ can be joined together to form a fused cycloalkyl or fused (hetero) arene substituent, R ³⁵ is hydrogen, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl _groups, C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) Independently selected from the group consisting of reel alkyl group;
X is C (R ³¹ ) ₂ , O, S or NR ³² where R ³² is R ³¹ or L ³ (D) _r where L ³ is a linker and D is claimed As defined in paragraph 1;
r is 1-20;
q is 0 or 1, but if q is 0, provided that X is NL ² (D) _r ;
aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa ′ is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa + aa ′ <10;
b is 0 or 1;
pp is 0 or 1;
M is —N (H) C (O) CH ₂ —, —N (H) C (O) CF ₂ —, —CH ₂ —, —CF ₂ —, or 0 to 4 fluorine substituents, preferably Is 1,4-phenylene containing two fluorine substituents preferably located at C2 and C6 or C3 and C5 of phenylene;
y is 1 to 4;
Fuc is fucose).

Preferably aa = 2, aa ′ = 3, X═C (R ³¹ ) ₂ (ie, there is a fused cyclooctene ring), where one R ³¹ = H and the other R ³¹ Are linked together with further R ³¹ substituents present in the structure according to formula (20b), and carbon atoms 5 and 6 of the cyclooctene moiety (numbered carbon atoms shared with the triazole ring are numbered 1 and 2). To form a condensed cyclopropyl ring.

  Preferred features of the antibody-conjugates according to the invention are described above in particular in the description of step (ii) of “core-GlcNAc functionalization” as a mode of conjugation and its products.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含む］によって表されるバイオコンジュゲートである。 In one embodiment, the antibody-conjugate according to this aspect has the formula (A):
BLD
(A)
[Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or a spacer moiety;
Where L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I wherein the target molecule is a spacer Min is a bioconjugate represented by including connected) optionally] the N through.

（式中：
ａは、０又は１であり；
Ｒ^１は、水素、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基からなる群から選択され、Ｃ_１〜Ｃ_２４アルキル基、Ｃ_３〜Ｃ_２４シクロアルキル基、Ｃ_２〜Ｃ_２４（ヘテロ）アリール基、Ｃ_３〜Ｃ_２４アルキル（ヘテロ）アリール基及びＣ_３〜Ｃ_２４（ヘテロ）アリールアルキル基は、任意選択で置換され、Ｏ、Ｓ及びＮＲ^３から選択される１個若しくは複数のヘテロ原子によって任意選択で中断されており、ここでＲ^３は、水素及びＣ_１〜Ｃ_４アルキル基からなる群から独立して選択される、又はＲ^１は、追加の標的分子Ｄであり、ここで標的分子は、スペーサー部分を介してＮに任意選択で接続されている）を含む］によって表されるバイオコンジュゲートではない。 In one embodiment, the antibody-conjugate according to this aspect has the formula (A):
BLD
(A)
[Where:
B is a biomolecule;
L is a linker connecting B and D;
D is the target molecule;
Each occurrence of “-” is independently a bond or a spacer moiety;
Where L is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S and NR ³ , wherein in R ³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ¹ is an additional target particle D, I wherein the target molecule is a spacer Not a bioconjugate represented by] comprising the connections are) optionally the N through a minute.

Preferred antibody-conjugates according to this aspect are listed below as conjugates (I)-(VII) below. In one embodiment, the antibody-conjugate according to this aspect is selected from the conjugates defined herein below as (I)-(VII), more preferably herein below (IV)-(VII). Selected from the conjugates defined as In one embodiment, an antibody-conjugate according to this aspect is not a conjugate as defined herein below as (I)-(III), preferably herein as defined below as (I)-(VII). It is not a conjugate.
(I) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) _{2 -NH- (CH 2 -CH 2 -O)} 2 -CO-Val-Cit-PABC-, is D = MMAE;
(II) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ ) ₃ —CO _{-NH-S (O) 2 -NH-} (CH 2 -CH 2 -O) 2 -CO-Val-Cit-PABC- a and is the D = MMAE;
(III) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH _2- O) _4- CO-Val-Cit-PABC-, D = MMAE;
(IV) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH _{2 -O)} a _{4 -CO-N (CH 2 -CH} 2 -O-CO-Val-Cit-PABC-D) 2, is each occurrence = MMAE of D;
(V) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) ₂ -NH- _{(CH 2} -CH _{2 -O)} a _{2 -CO-N (CH 2 -CH} 2 -O-CO-Val-Cit-PABC-D) 2, is each occurrence = MMAE of D;
(VI) AB = Iratumumab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N292, and S (F ¹ ) _x = 6-azido-6-deoxy- N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH ₂ — O) _4- CO-Val-Cit-PABC-, D = MMAE;
(VII) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine (ie F ¹ = N ₃ and x = 1), Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) ₂ -NH- _{(CH 2} -CH ₂ -O) a 2 -CO-Val-Cit-PABC- , a D = MMAD.

によって表される。 In this specification, (9q) is

Represented by

^One skilled in the art will recognize that the antibody-conjugates defined herein as (I)-(X) contain neither F ¹ = N ₃ nor Q ¹ = (9q), but between F ¹ and Q ¹ It is understood that it contains the connecting group Z ³ resulting from the reaction. More specifically, an antibody-conjugate as defined above follows formula (40b), where S = GalNAc, y = 2, x = 1, b = 0 or 1, pp = 0. (Ie, M does not exist), aa = 2, aa ′ = 3, X = C (R ³¹ ) ₂ , where one R ³¹ = H and the other R ³¹ is represented by the formula (40b ) Together with further R ³¹ substituents present in the structure according to), when the carbon atoms 5 and 6 of the cyclooctene moiety are numbered 1 and 2 (carbon atoms shared with the triazole ring) , See structure (9q) above), q = 1, r = 1 or 2 and D and L ² are defined above.

  Antibody-conjugates according to this aspect have an improved therapeutic index compared to known antibody-conjugates, wherein the therapeutic index is preferably for the treatment of CD30 expressing tumors. Improvement of the therapeutic index may take the form of improved therapeutic efficacy and / or improved tolerability. In one embodiment, an antibody-conjugate according to this aspect has improved therapeutic efficacy compared to known antibody-conjugates for the treatment of CD30 expressing tumors. In one embodiment, antibody-conjugates according to this aspect have improved tolerability compared to known antibody-conjugates for the treatment of CD30 expressing tumors.

The antibody-conjugates according to the present invention outperform known antibody-conjugates in other embodiments. The inventors have found that the antibody-conjugate exhibits increased stability (ie, the antibody-conjugate exhibits less degradation over time). The inventors have also found that the antibody-conjugate exhibits a reduced aggregation challenge (ie, the antibody-conjugate exhibits less aggregation over time). Given the improved therapeutic index, increased stability and decreased aggregation of the antibody-conjugate, the antibody-conjugate is a significant improvement over prior art antibody-conjugates. The present invention therefore also relates to the use of a mode of conjugation as defined herein to improve the stability of bioconjugates, typically antibody-conjugates. The invention therefore also relates to the use of a mode of conjugation as defined herein to reduce bioconjugate, typically antibody-conjugate aggregation.
Endoglycosidase fusion enzyme

In a fourth aspect, the present invention relates to a fusion enzyme comprising two endoglycosidases. In a particular example, two endoglycosidases EndoS and EndoH, the linker, preferably _{- (Gly 4 Ser) 3 -} (His) 6 - (Gly 4 Ser) 3 - are connected via a linker. The fusion enzyme according to the present invention is also referred to as EndoSH. The enzyme according to the present invention has at least 50% sequence identity with SEQ ID NO: 1, preferably at least 70% sequence identity with SEQ ID NO: 1, more preferably at least 80% sequence identity, eg at least 81% with SEQ ID NO: 1, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Identity is known and / or computer program methods known in the art, such as BLASTP and other sources publicly available from NCBI (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda , MD 20894; Altschul, S. et al., J. Mol.Biol.215: 403-410 (1990), having the sequence identity shown above for SEQ ID NO: 1. The enzyme of the present invention preferably has EndoS and EndoH activity, and most preferably the enzyme according to the present invention has 100% sequence identity with SEQ ID NO: 1.

  Also included are EndoS and EndoH fusion enzymes, wherein the linker has been replaced by another suitable linker known in the art, wherein the linker can be rigid or flexible. The linker is preferably a flexible linker that allows adjacent protein domains to relatively freely transfer to each other. The flexible linker is composed of amino residues such as glycine, serine, histidine and / or alanine and 3 to 59 amino acid residues, preferably 10 to 45 or 15 to 40 amino acid residues, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or It preferably has a length of 40 amino acid residues, or 20-38, 25-37 or 30-36 amino acid residues. Optionally, the fusion enzyme is easily purified and / or detected as known in the art, such as Fc-tag, FLAG-tag, poly (His) -tag, HA-tag and Myc-tag. Is covalently linked to or includes a tag.

  Glycoprotein trimming is known in the art, for example from WO 2007/133855 or WO 2014/066561. The enzyme according to the present invention exhibits both EndoS and EndoH activity and can trim glycans of glycoproteins (such as antibodies) in the core-GlcNAc unit, leaving only the core-GlcNAc residue of the glycoprotein (EndoS activity). Also, high mannose glycans (EndoH activity) can be removed. Surprisingly, both activities of the fusion enzyme function smoothly at a pH of about 7-8, while monomeric EndoH requires a pH of 6 to operate optimally.

The fusion enzyme according to the present invention is routine in the art, such as an expression vector (eg, plasmid) containing an enzyme coding sequence can be introduced into a host cell (eg, E. coli) and expressed, and the enzyme can be isolated from the host cell. Can be prepared by techniques. Possible approaches for the preparation and purification of the fusion enzyme according to the invention are given in Examples 4-6, the function of the fusion enzyme according to the invention is demonstrated in Examples 7 and 9, where Brentuxima Bulb and Iratumumab are effectively trimmed in a single step.
Sequence identification of the fusion protein EndoSH (SEQ ID NO: 1):
1 MPSIDSLHYL SENSKKEFKEELSKAGQESQ KVKEILAKAQ QADKQAQELA
51 KMKIPEKIPM KPLHGPLYGG YFRTWHDKTSDPTEKDKVNS MGELPKEVDL
101 AFIFHDWTKD YSLFWKELAT KHVPKLNKQGTRVIRTIPWR FLAGGDNSGI
151 AEDTSKYPNT PEGNKALAKA IVDEYVYKYNLDGLDVDVEH DSIPKVDKKE
201 DTAGVERSIQ VFEEIGKLIG PKGVDKSRLFIMDSTYMADK NPLIERGAPY
251 INLLLVQVYG SQGEKGGWEP VSNRPEKTMEERWQGYSKYI RPEQYMIGFS
301 FYEENAQEGN LWYDINSRKD EDKANGINTDITGTRAERYA RWQPKTGGVK
351 GGIFSYAIDR DGVAHQPKKY AKQKEFKDATDNIFHSDYSV SKALKTVMLK
401 DKSYDLIDEK DFPDKALREA VMAQVGTRKGDLERFNGTLR LDNPAIQSLE
451 GLNKFKKLAQ LDLIGLSRIT KLDRSVLPANMKPGKDTLET VLETYKKDNK
501 EEPATIPPVS LKVSGLTGLK ELDLSGFDRETLAGLDAATL TSLEKVDISG
551 NKLDLAPGTE NRQIFDTMLS TISNHVGSNEQTVKFDKQKP TGHYPDTYGK
601 TSLRLPVANE KVDLQSQLLF GTVTNQGTLINSEADYKAYQ NHKIAGRSFV
651 DSNYHYNNFK VSYENYTVKV TDSTLGTTTDKTLATDKEET YKVDFFSPAD
701 KTKAVHTAKV IVGDEKTMMV NLAEGATVIGGSADPVNARK VFDGQLGSET
751 DNISLGWDSK QSIIFKLKED GLIKHWRFFNDSARNPETTN KPIQEASLQI
801 FNIKDYNLDN LLENPNKFDD EKYWITVDTYSAQGERATAF SNTLNNITSK
851 YWRVVFDTKG DRYSSPVVPE LQILGYPLPNADTIMKTVTT AKELSQQKDK
901 FSQKMLDELK IKEMALETSL NSKIFDVTAINANAGVLKDC IEKRQLLKK G
951 GGGSGGGGSG GGGSHHHHHHGGGGSGGGGS GGGGS APAPV KQGPTSVAYV
1001 EVNNNSMLNV GKYTLADGGG NAFDVAVIFAANINYDTGTK TAYLHFNENV
1051 QRVLDNAVTQ IRPLQQQGIK VLLSVLGNHQGAGFANFPSQ QAASAFAKQL
1101 SDAVAKYGLD GVDFDDEYAE YGNNGTAQPNDSSFVHLVTA LRANMPDKII
1151 SLYNIGPAAS RLSYGGVDVS DKFDYAWNPYYGTWQVPGIA LPKAQLSPAA
1201 VEIGRTSRST VADLARRTVD EGYGVYLTYNLDGGDRTADV SAFTRELYGS
1251 EAVRTP
(The linker is underlined and the EndoH sequence is shown in italics)

RP-HPLC analysis of reduced monoclonal antibodies : Prior to RP-HPLC analysis, a solution of 10 μg (modified) IgG is incubated for 15 minutes at 37 ° C. with 10 mM DTT and 100 mM Tris pH 8.0 in a total volume of 50 μL. To reduce the sample. A solution of 49% ACN, 49% MQ and 2% formic acid (50 μL) was added to the reduced sample. Using a ZORBAX Pheroshell 300SB-C8 1 × 75 5 μm (Agilent Technologies) column running at 70 ° C. at 1 ml / min on an Agilent 1100 HPLC, a 16.9 minute linear gradient (buffer) of 25-50% Buffer B Solution A = 90% MQ, 10% ACN, 0.1% TFA and buffer B = 90% ACN, 10% MQ, 0.1% TFA) HPLC was performed.

Mass spectral analysis of monoclonal antibodies : Prior to mass spectral analysis, IgG was treated with DTT allowing analysis of both light and heavy chains, or a fabricator that allows analysis of Fc / 2 fragments ( Fabricator ™ (commercially available from Genovis, Lund, Sweden). For analysis of both light and heavy chains, a solution of 20 μg (modified) IgG was incubated for 5 minutes at 37 ° C. with 100 mM DTT in a total volume of 4 μL. If present, the azide functional group is reduced to an amine under these conditions. For analysis of Fc / 2 fragments, a solution of 20 μg (modified) IgG was used with phosphate-buffered saline (PBS) with Fabricator ™ (1.25 U / μL) at 37 ° C. for 1 hour. ) Incubated with pH 6.6 in a total volume of 10 μL. After reduction or Fabricator digestion, the sample was washed twice using Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore) with milliQ, resulting in a final sample volume of approximately 40 μL. The samples were then analyzed on a JEOL AccuTOF by electrospray ionization time of flight (ESI-TOF). Deconvoluted spectra were obtained using Magtran software.

Preparation of protein constituents: Examples 1-3:
Example 1: Transient expression and purification of cAC10 cAC10 was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) on a 5L scale. The supernatant was purified using an XK 26/20 column packed with 50 mL protein A sepharose. In a single run, 5 L of supernatant was loaded onto the column followed by washing with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl. The retained protein was eluted with 0.1 M glycine pH 2.7. The eluted cAC10 was immediately neutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against 25 mM Tris pH 8.0. The IgG was then concentrated to approximately 20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at −80 ° C. before further use.

Example 2: Transient expression and purification of iratumumab Iratumumab was transiently expressed in CHO K1 cells by Evitria (Zurich, Switzerland) on a 125 mL scale. The supernatant was purified using a HiTrap mAbSelect SuRe 5 mL column (GE Healthcare). The supernatant was loaded onto the column and subsequently washed with at least 10 column volumes of 25 mM Tris pH 7.5, 150 mM NaCl. The retained protein was eluted with 0.1 M glycine pH 2.7. The eluted product was immediately neutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against 20 mM Tris pH 7.5. The product was then concentrated to 14.4 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius) and stored at −80 ° C. before further use.

Example 3: Transient expression and purification of His-TnGalNAcT (33-421) His-TnGalNAcT (33-421) (identified by SEQ ID NO: 2) was purified by 5 L by Evitria (Zurich, Switzerland) in CHO K1 cells. It was expressed transiently on a scale. The supernatant was purified using an XK 16/20 column packed with 25 mL of Ni Sepharose Excel (GE Healthcare). In each run, approximately 1.5 L of supernatant was loaded onto the column, followed by washing with at least 10 column volumes of buffer A (20 mM Tris buffer, 5 mM imidazole, 500 mM NaCl, pH 7.5). The retained protein was eluted with buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5). Using a HiPrep H26 / 10 desalting column (GE Healthcare), the buffer of the eluted fraction was exchanged with 25 mM Tris pH 8.0. The purified protein was concentrated to at least 3 mg / mL using a Vivaspin Turbo 4 ultrafiltration unit (Sartorius) and stored at −80 ° C. before further use.
His-TnGalNAcT (33-421) sequence (SEQ ID NO: 2):
1 HHHHHHSPLR TYLYTPLYNATQPTLRNVER LAANWPKKIP SNYIEDSEEY
51 SIKNISLSNH TTRASVVHPP SSITETASKLDKNMTIQDGA FAMISPTPLL
101 ITKLMDSIKS YVTTEDGVKK AEAVVTLPLCDSMPPDLGPI TLNKTELELE
151 WVEKKFPEVE WGGRYSPPNC TARHRVAIIVPYRDRQQHLA IFLNHMHPFL
201 MKQQIEYGIF IVEQEGNKDF NRAKLMNVGFVESQKLVAEG WQCFVFHDID
251 LLPLDTRNLY SCPRQPRHMS ASIDKLHFKLPYEDIFGGVS AMTLEQFTRV
301 NGFSNKYWGW GGEDDDMSYR LKKINYHIARYKMSIARYAM LDHKKSTPNP
351 KRYQLLSQTS KTFQKDGLST LEYELVQVVQYHLYTHILVN IDERS

Examples 4-6: endoglycosidase EndoSH product <br/> Example 4: between cloning NdeI-HindIII site of the fusion protein EndoSH into pET22B expression vector _{EndoS- (G 4 S) 3 -} (His) 6 - ( The pET22B-vector containing the G ₄ S) ₃ -EndoH (EndoSH) coding sequence (EndoSH is identified by SEQ ID NO: 1) was obtained from Genscript. The DNA sequence for the EndoSH fusion protein consists of EndoS encoding residues 48-995 fused to EndoH via an N-terminally linked glycine-serine (GS) linker. Glycine - serine (GS) _{linker, - (G 4 S) 3} - (His) 6 - (G 4 S) 3 - comprises a format, to allow the spacing of the two enzymes, simultaneously IMAC- introduced purification tag To do.

Example 5: E. coli Expression of Fusion Protein EndoSH Expression of EndoSH fusion protein (identified by SEQ ID NO: 1) begins with transformation of plasmid (pET22b-EndoSH) into BL21 cells. The next step is inoculation of 500 mL cultures with BL21 cells (LB medium + Americin). When OD600 reached 0.7, the culture was induced with 1 mM IPTG (500 μL of 1M stock solution).

Example 6: Purification of the fusion protein EndoSH from E. coli After induction overnight at 37 ° C, the culture was pelleted by centrifugation. The pellet was resuspended in 40 mL PBS and incubated with 5 ml lysozyme (10 mg / mL) on ice for 30 minutes. Half an hour later, 5 ml of 10% Triton-X-100 was added and sonicated on ice (10 minutes). After sonication, cell debris was removed by centrifugation (10 min 8000 × g) followed by filtration through a 0.22 μM pore size filter. The soluble extract was loaded onto a HisTrap HP 5 mL column (GE Healthcare). The column was first washed with buffer A (20 mM Tris buffer, 20 mM imidazole, 500 mM NaCl, pH 7.5). The retained protein was eluted with buffer B (20 mM Tris, 500 mM NaCl, 250 mM imidazole, pH 7.5, 10 mL). Fractions were analyzed on polyacrylamide gels (12%) by SDS-PAGE. Fractions containing purified target protein were combined and the buffer was exchanged against 20 mM Tris pH 7.5 and 150 mM NaCl by dialysis performed overnight at 4 ° C. The purified protein was concentrated to at least 2 mg / mL using Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore). The product is stored at −80 ° C. before further use.

cAC10 Remodeling: Examples 7-8:
Example 7: Preparation of trimming cAC10 by means of the fusion protein EndoSH Glycan trimming of cAC10 (obtained via transient expression in CHO K1 cells performed by Evitria, Zurich, Switzerland) was performed with the fusion protein EndoSH Therefore, cAC10 (14.5 mg / mL) was incubated with EndoSH (1 w / w%) in 150 mM NaCl in 25 mM Tris pH 7.5 for approximately 16 hours at 37 ° C. Trimmed. IgG was dialyzed against 3 × 1 L of 25 mM Tris-HCl pH 8.0 Mass spectral analysis of fabricator digested samples showed one major product corresponding to core-GlcNAc (Fuc) substituted cAC10 ( And approximately 2% of the secondary products corresponding to core-GlcNAc-substituted cAC10 and core-GlcNAc (Fuc) -substituted cAC10 with C-terminal lysine (23959 Da) Three peaks of Fc / 2-fragment belonging to the observed mass of 24233 Da, approximately 5% and 15% of the total Fc / 2 fragment) were shown.

Example 8: Glycotransfer from 6-N ₃ -GalNAc-UDP to trimming cAC10 under the action of TnGalNAcT The substrate 6-N ₃ -GalNAc-UDP (11d) is a suitable modified biomolecule in the context of the present invention. Used for the preparation of the molecule cAC10- (6-N ₃ -GalNAc) ₂ 13d.

Trimmed cAC10 (10 mg / mL) obtained by cAC10EndoSH treatment as described above in Example 7 was used for the substrate 6-N ₃ -GalNAc- in 10 mM MnCl ₂ and 25 mM Tris-HCl pH 8.0. Incubated at 30 ° C. with UDP (2.5 mM, commercially available from GlycoHub) and 0.5 mg / mL His-TnGalNAcT (33-421) (5 w / w%). After 3 hours, the amount of His-TnGalNAcT (33-421) was increased to a final concentration of 1 mg / mL (10 w / w%) and the reaction was incubated at 30 ° C. overnight. Biomolecule 13d was purified from the reaction mixture on a HiTrap MabSelect SuRe 5 ml column (GE Healthcare) using AKTA purifier 10 (GE Healthcare). The eluted IgG was immediately neutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against PBS pH 7.4. Next, IgG was concentrated to a concentration of 23.4 mg / mL using Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 24333 Da, approximately 80% of total Fc / 2 fragments) corresponding to the core 6-N ₃ -GalNAc-GlcNAc (Fuc) substituted cAC10, And two secondary products (24187 Da and 24461 Da) corresponding to core 6-N ₃ -GalNAc-GlcNAc substituted cAC10 and C 6-N ₃ -GalNAc-GlcNAc (Fuc) substituted cAC10 with C-terminal lysine 3 peaks of Fc / 2− fragments belonging to approximately 5% and 15% of the total Fc / 2 fragments).

Iratumumab Remodeling: Examples 9-10
Example 9: Preparation of trimming iratumumab by means of the fusion protein EndoSH Glycan trimming of iratumumab (obtained via transient expression in CHO K1 cells performed by Evitria, Zurich, Switzerland) was performed on the fusion protein EndoSH. Therefore, Iratumumab (14.4 mg / mL) was incubated with EndoSH (1 w / w%) in 20 mM Tris pH 7.5 for approximately 16 hours at 37 ° C. Trimming IgG was 3 × 1 L of 25 mM. The mass spectrometric analysis of the fabricator digested sample showed that one major product corresponding to the core-GlcNAc (Fuc) substituted iratumumab (observed mass 24104 Da, total Fc / Approximately 85% of the fragments), and two secondary products corresponding to core-GlcNAc-substituted Iratumumab with C-terminal lysine and core-GlcNAc (Fuc) -substituted Iratumumab (23957 Da and 24232 Da, total Fc / 2 Three peaks of Fc / 2-fragment belonging to approximately 5% and 10% of the fragment) were shown.

Example _{10: 6-N 3 -GalNAc UDP} - glycosyltransferase substrate to trim Ira zum Mab from _{6-N 3 -GalNAc-UDP (} 11d) under the action of TnGalNAcT is suitable as a biological molecule in the context of the present invention modified biomolecule Iratsumumabu _- are used for the (6-N 3 _{-GalNAc) 2} preparation.

Trimmed irtumumab (10 mg / mL) obtained by EndoSH treatment of iratumumab as described above in Example 9 was converted to substrate 6 in 10 mM MnCl ₂ and 20 mM Tris-HCl pH 7.5 overnight. and incubated at _{-N 3 -GalNAc-UDP His-TnGalNAcT} (33-421) and of 0.5mg / mL (5mM, commercially available and from GlycoHub) (5w / w%) with 30 ° C.. Azide modified iratumumab was purified from the reaction mixture on a HiTrap MabSelect SuRe 5 ml column (GE Healthcare) using an AKTA purifier 10 (GE Healthcare). The eluted IgG was immediately neutralized with 1.5 M Tris-HCl pH 8.8 and dialyzed against PBS pH 7.4. Next, IgG was concentrated to a concentration of 25.6 mg / mL using Amicon Ultra-0.5, Ultracel-10 Membrane (Millipore). Mass spectral analysis of the fabricator digested sample shows one major product (observed mass 24332 Da, approximately 85% of total Fc / 2 fragments) corresponding to the core 6-N ₃ -GalNAc-GlcNAc (Fuc) substituted iratumab. and the core _6-N 3 -GalNAc-GlcNAc substituted Iratsumumabu and core _{6-N 3 -GalNAc-GlcNAc (} Fuc) 2 kinds of secondary product corresponding to the replacement Iratsumumabu with a C-terminal lysine (24187Da and 24461Da observations mass 3 peaks of Fc / 2-fragment belonging to approximately 5% and 10% of the total Fc / 2 fragment).

Linker-conjugate synthesis: Examples 11-29:

Example 11: Preparation of Compound 100 Compound 99 (prepared via activation of Compound 58 as disclosed in Example 50 of International Application PCT / NL2015 / 050697 in DMF (200 μL) NL2015 / 050697 prepared according to Example 50; 4.7 mg, 9.0 μmol) was added to solid Val-Cit-PABC-MMAE (vc-PABC-MMAE, 10 mg, 8.1 μmol) followed by Et ₃ N (3.7 μL, 2.7 mg, 27 μmol) was added. After 23 hours, 2 ′-(ethylenedioxy) bis (ethylamine) (1.3 μL, 1.3 mg, 8.9 μmol) in DMF was added (13 μL of 10% solution in DMF). The mixture was left for 4 hours and purified via reverse phase (C18) HPLC chromatography (30 → 90% MeCN (1% AcOH) in H ₂ O (1% AcOH)). LCMS (ESI ⁺ ) Calculated for C ₇₄ H ₁₁₇ N ₁₂ O ₁₉ S ⁺ (M + H ⁺ ) as a colorless film (10.7 mg, 7.1 μmol, 87%) 1509.83 Found 1510.59

Example 12: Preparation of compound 101 To a solution of BCN-OSu (1.00 g, 3.43 mmol) in a mixture of THF and water (80 mL / 80 mL) was added γ-aminobutyric acid (0.60 g, 5.12 mmol) and Et ₃ N (1.43 mL, 1.04 g, 10.2 mmol) was added. The mixture was stirred for 4 h, followed by addition of DCM (200 mL) and a saturated aqueous solution of NH ₄ Cl (80 mL). After separation, the aqueous layer was extracted with DCM (2 × 200 mL). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by column chromatography (0 → 10% MeOH in DCM). The product BCN-GABA was obtained as a colorless thick oil (730 mg, 2.61 mmol, 76%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 4.81 (bs, 1H), 4.15 (d, J = 8.4 Hz, 2H), 3.30 to 3.21 (m, 2H), 2.42 (t, J = 7.2 Hz, 2H), 2.35 to 2.16 (m, 6H), 1.85 (quintage, J = 6.9 Hz, 2H), 1.64 to 1 .51 (m, 2H), 1.35 (quintage, J = 8.4 Hz, 1H), 1.00-0.90 (m, 2H)

Example 13: Compound 102 Preparation chlorosulfonyl isocyanate of (CSI; 0.91mL, 1.48g, 10mmol ) _and, Et 2 O (50mL) in tert- butanol (5.0mL, 3.88g, 52mmol) of Added to the cooled (−78 ° C.) solution. The reaction mixture was warmed to room temperature and concentrated. The residue was suspended in DCM (200 mL) followed by Et ₃ N (4.2 mL, 3.0 g, 30 mmol) and 2- (2-aminoethoxy) ethanol (1.0 mL, 1.05 g; 10 mmol). Was added. The resulting mixture was stirred for 10 minutes and concentrated. The residue was purified twice by column chromatography (0 → 10% MeOH in DCM). The product was obtained as a colorless thick oil (2.9 g, 10 mmol, 100%) ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.75 (bs, 1H), 3.79-3.74. (M, 2H), 3.67 to 3.62 (m, 2H), 3.61 to 3.57 (m, 2H), 3.35 to 3.28 (m, 2H), 1.50 (s , 9H).

Example 14: To a solution of 102 in Preparation DCM (40 mL) of compound _{103 (2.9g, 10mmol), Ac} 2 O (2.9mL, 3.11g, 30.5mmol) and _Et 3 N (12.8 mL , 9.29 g, 91.8 mmol). The reaction mixture was stirred for 2 h, washed with a saturated aqueous solution of NaHCO ₃ (50 mL) and dried (Na ₂ SO ₄ ). The residue was purified twice by column chromatography (20% → 100% EtOAc in heptane). The product was obtained as a colorless oil (2.5 g, 7.7 mmol, 77%) ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.48 (bs, 1H), 4.25 to 4.20. (M, 2H), 3.70 to 3.60 (m, 4H), 3.33 to 3.23 (m, 2H), 2.10 (s, 3H), 1.50 (s, 9H)

Example 15: Preparation of compound 104 To a solution of 103 (80 mg, 0.25 mmol) in DCM (8 mL) was added TFA (2 mL). After 40 minutes, the reaction mixture was concentrated. The residue was dissolved in toluene (30 mL) and the mixture was concentrated. The product was obtained as a colorless oil (54 mg, 0.24 mmol, 95%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.15 (bs, 2H), 4.26 to 4.18 (m, 2H), 3.71 to 3.60 (m, 4H), 3. 35-3.27 (m, 2H), 2.08 (s, 3H).

Example 16: Preparation of compound 105 To a mixture of BCN-GABA (101) (67 mg, 0.24 mmol) and 104 (54 mg, 0.24 mmol) in DCM (20 mL) was added N- (3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride (EDCI.HCl; 55 mg, 0.29 mmol) and DMAP (2.8 mg, 23 μmol) were added. The mixture was stirred for 16 and washed with a saturated aqueous solution of NH ₄ Cl (20 mL). After separation, the aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by column chromatography (0 → 10% MeOH in DCM). The product was obtained as a colorless thick oil (50 mg, 0.10 mmol, 42%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.83 to 5.72 (m, 1H), 5.14 to 5.04 (m, 1H), 4.23 to 4.19 (m, 2H) ), 4.15 (d, J = 8.1 Hz, 2H), 3.67 to 3.57 (m, 4H), 3.29 to 3.18 (m, 4H), 2.41 to 2.32 (M, 2H), 2.31 to 2.15 (m, 6H), 2.10 (s, 3H), 1.85 (quintage, J = 6.6 Hz, 2H), 1.65 to 1 .49 (m, 2H), 1.38 to 1.28
(M, 1H), 1.00 to 0.89 (m, 2H)

Example 17: To a solution of the preparation of compounds 106 MeOH (10 mL) in 105 _(50mg, 0.10mmol), was added _{K 2 CO 3 (43mg, 0.31mmol} ). The mixture was stirred for 3 hours and diluted with a saturated aqueous solution of NH ₄ Cl (20 mL). The mixture was extracted with DCM (3 × 20 mL). The combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The product was obtained as a colorless film (39 mg, 0.088 mmol, 88%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 6.25 (bs, 1H), 5.26-5.18 (m, 1H), 4.15 (d, J = 8.0 Hz, 2H), 3.77 to 3.71 (m, 2H), 3.63 to 3.53 (m, 4H), 3.33 to 3.27 (m, 2H), 3.27 to 3.17 (m, 2H) ), 2.45 to 2.34 (m, 2H), 2.34 to 2.14 (m, 6H), 1.85 (quintet, J = 6.7 hz, 2H), 1.65 to 1 .48 (m, 2H), 1.41-1.28 (m, 1H), 1.01-0.88 (m, 2H).

Example 18: Preparation of compound 107 To a solution of 106 (152 mg, 0.34 mmol) in DCM (20 mL) was added p-nitrophenyl chloroformate (PNP-COCl; 69 mg, 0.34 mmol) and pyridine (28 [mu] L, 27 mg). 0.34 mmol) was added. The mixture was stirred for 1.5 hours and concentrated. The residue was purified by column chromatography (50% → 100% EtOAc in heptane). The product was obtained as a colorless thick oil (98 mg, 0.16 mmol, 47%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 8.31-8.26 (m, 2H), 7.46-7.40 (m, 2H), 5.69-5.59 (m, 1H) ) 4.98-4.91 (m, 1H), 4.46-4.42 (m, 2H), 4.18 (d, J = 8.1 Hz, 2H), 3.79-3.75 (M, 2H), 3.69 to 3.64 (m, 2H), 3.33 to 3.24 (m, 4H), 2.39 to 2.31 (m, 2H), 2.32 to 2 .18 (m, 6H), 1.84 (quintet, J = 6.3 Hz 2H), 1.65 to 1.50 (m, 2H), 1.35 (quintet, J = 8.5 Hz) , 1H), 1.01 to 0.91 (m, 2H).

Example 19: Preparation of linker-conjugate 108 To a solution of Val-Cit-PABC-MMAE (16.4 mg, 13.2 μmol) in DMF (400 μL), Et ₃ N (3.4 μL, 2.5 mg, 24 μmol). ) Was added. The resulting solution was added to a solution of 107 (6.7 mg, 11 μmol) in DMF (300 μL). DMF (50 μL) was added. After 21.5 hours, 2 ′-(ethylenedioxy) bis (ethylamine) (1.2 μL, 1.2 mg, 8.2 μmol) in DMF was added (12 μL of a 10% solution in DMF). The mixture was purified via reverse phase (C18) HPLC chromatography (30 → 90% MeCN (1% AcOH) in H ₂ O (1% AcOH). The product was obtained as a colorless membrane ( 4.3 mg, 2.7 μmol, 25%) LCMS (ESI ⁺ ) Calculated for C ₇₈ H ₁₂₄ N ₁₃ O ₂₀ S ⁺ (M + H ⁺ ) 1594.88 Found 1594.97

Example 20: Preparation of compound 109 To a solution of 2- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethanol (539 mg, 2.79 mmol) in DCM (100 mL) was added BCN-OSu (0. 74 g, 2.54 mmol) and Et ₃ N (1.06 mL, 771 mg, 7.62 mmol) were added. The resulting solution was stirred for 2.5 hours and washed with a saturated aqueous solution of NH ₄ Cl (100 mL). After separation, the aqueous phase was extracted with DCM (100 mL). The combined organic phases were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by column chromatography (MeOH in DCM 0 → 10%). The product was obtained as a colorless oil (965 mg, 2.61 mmol amount). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.93 (bs, 1H), 4.14 (d, J = 8.0 Hz, 2H), 3.77 to 3.69 (m, 4H), 3.68 to 3.59 (m, 8H), 3.58 to 3.52 (m, 2H), 3.42 to 3.32 (m, 2H), 2.35 to 2.16 (m, 6H) ), 1.66 to 1.51 (m, 2H), 1.36 (quintet, J = 8.7 Hz, 1H), 0.99 to 0.87 (m, 2H).

Example 21: 109 in the preparation of compound 110 DCM (50mL) (0.96g, 2.59mmol) to a solution of, p- nitrophenyl chloroformate (680 mg, 3.37 mmol) and _Et 3 N (1.08 mL 784 mg, 7.75 mmol). The mixture was stirred for 16 hours and concentrated. The residue was purified twice by column chromatography (20% → 70% EtOAc in heptane (column 1) and 20% → 100% EtOAc in heptane (column 2)). The product was obtained as a slightly yellow thick oil (0.91 g, 1.70 mmol, 66%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 8.31-8.26 (m, 2H), 7.42-7.37 (m, 2H), 5.19 (bs, 1H), 4. 47 to 4.43 (m, 2H), 4.15 (d, J = 8.0 Hz, 2H), 3.84 to 3.80 (m, 2H), 3.74 to 3.61 (m, 8H) ), 3.59 to 3.53 (m, 2H), 3.42 to 3.32 (m, 2H), 2.35 to 2.16 (m, 6H), 1.66 to 1.50 (m) , 2H), 1.40 to 1.30 (m, 1H), 1.00 to 0.85 (m, 2H).

Example 22: Linker - during the preparation of the conjugate 111 DMF (400μL) Val-Cit -PABC-MMAE (vc-PABC-MMAE; 13.9mg; To a solution of _0.011mmol, Et 3 N _(3.4μL, 2.5 mg, 24.3 μmol) and a solution of BCN-PEG4-OPNP (110, 3.0 mg, 5.6 μmol) in DMF (200 μL) were added, and after 25 minutes additional Et ₃ N (1. 1 μL, 0.80 mg, 7.9 μmol) and BCN-PEG ₄ -OPNP (110, 2.2 mg in DMF (33 μL), 4.1 μmol) were added 17.5 hours later, 2 ′-( Ethylenedioxy) bis (ethylamine) (1.2 μL, 1.2 mg, 8.1 μmol) was added (12 μL of a 10% solution in DMF) and mixed. Was left overnight in a freezer, reverse phase (C18) HPLC chromatography _(H 2 O _(1% of AcOH) in 30 → 90% of MeCN (1% of AcOH) and purified via. The product of colorless LCMS (ESI ⁺ ) C ₇₈ H ₁₂₄ N ₁₁ O ₁₉ ⁺ (M + H ⁺ ) calculated as a film (10.9 mg, 7.2 μmol. 74%) 1518.91 found 1519.09

To a solution of; (0.734 mmol 0.39 g), diethanolamine in DMF (2mL) (DEA, 107mg ; 1.02mmol) compound in the preparation of DCM Example 23:61 (30mL) 99 solution, and Et ₃ N (305 μL; 221 mg; 2.19 mmol) was added. The resulting mixture was stirred at room temperature for 17 hours and washed with a saturated aqueous solution of NH ₄ Cl (30 mL). The aqueous phase was extracted with DCM (30 mL) and the combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (DCM → MeOH / DCM 1/9). The product was obtained as a colorless film (163 mg; 0.33 mmol; 45%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 6.29 (bs, 1H), 4.33 to 4.29 (m, 2H), 4.28 (d, J = 8.2 Hz, 2H), 3.90 to 3.80 (m, 4H), 3.69 to 3.64 (m, 2H), 3.61 (t, J = 4.8 Hz, 2H), 3.52 (t, J = 5) .0Hz, 4H), 3.32 (t, J = 5.1 Hz, 2H), 2.37 to 2.18 (m, 6H), 1.60 to 1.55 (m, 2H), 1.39 (Quintage, J = 8.7 Hz, 1H), 1.05-0.94 (m, 2H).

To a solution of 61 in the preparation of DCM Example 24:62 (10mL) (163mg, 0.33mmol ) and 4-nitrophenyl chloroformate _{(134mg, 0.66mmol), Et 3} N (230μL; 167mg; 1. 65 mmol) was added. The reaction mixture was stirred for 17 hours and concentrated. The residue was purified by flash column chromatography (50% EtOAc in heptane → 100% EtOAc). The product was obtained as a colorless oil (69 mg; 0.084 mmol; 25%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 8.29 to 8.23 (m, 4H), 7.42 to 7.35 (m, 4H), 5.81 to 5.71 (m, 1H) ), 4.53 to 4.43 (m, 4H), 4.36 to 4.30 (m, 2H), 4.25 (d, J = 8.2 Hz, 2H), 3.81 to 3.70. (M, 4H), 3.70 to 3.65 (m, 2H), 3.62 to 3.56 (m, 2H), 3.32 to 3.24 (m, 2H), 2.34 to 2 .14 (m, 6H), 1.60 to 1.45 (m, 2H), 1.35 (quintage, J = 8.7 Hz, 1H), 1.02 to 0.91 (m, 2H) .

Example 25: Preparation of linker-conjugate 63 To a solution of 62 (27 mg, 33 μmol) in DMF (400 μL), triethylamine (22 μl; 16 mg; 158 μmol) and vc-PABC-MMAE in DMF (1.0 mL). . A solution of TFA (96 mg; 78 μmol) was added. The mixture was allowed to stand for 19 hours and 2,2 ′-(ethylenedioxy) bis (ethylamine) (37 μL, 38 mg, 253 μmol) was added. After 2 hours, the reaction mixture was diluted with DMF (100 μL) and purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH). Obtained as a colorless film (41 mg, 14.7 μmol, 45%) LCMS (ESI ⁺ ) calcd for C ₁₃₈ H ₂₁₉ N ₂₃ O ₃₅ S ²⁺ (M + 2H ⁺ ) 139.79 found 1396.31.

In; (1.69mmol 0.90g), diethanolamine in DMF (7mL) (DEA, 231mg ; 2.20mmol) solution 110 in the preparation of DCM Example 26:64 (50mL) solution and _Et 3 N _(707μL 513 mg; 5.07 mmol) was added. The resulting mixture was stirred at room temperature for 43 hours and washed with a saturated aqueous solution of NH ₄ Cl (50 mL). The aqueous phase was extracted with DCM (50 mL) and the combined organic layers were dried (Na ₂ SO ₄ ) and concentrated. The residue was purified by flash column chromatography (DCM → MeOH / DCM 1/9). The product was obtained as a colorless film (784 mg; 1.57 mmol; 93%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 5.67 to 5.60 (m, 1H), 4.32 to 4.27 (m, 2H), 4.14 (d, J = 8.4 Hz) , 2H), 3.89 to 3.79 (m, 4H), 3.75 to 3.60 (m, 10H, 3.58 to 3.53 (m, 2H), 3.53 to 3.44 ( m, 4H), 3.40 to 3.33 (m, 2H), 2.35 to 2.18 (m, 6H), 1.62 to 1.56 (m, 2H), 1.42 to 1.2. 30 (m, 1H), 1.00 to 0.88 (m, 2H).

64 in the preparation of DCM Example 27:65 (20mL); To a solution of (0.78g 1.55mmol), 4- nitrophenyl chloroformate (938 mg; 4.65 mmol) and _Et 3 N _(1.08mL; 784 mg; 7.75 mmol) was added. The resulting mixture was stirred at room temperature for 17 hours and concentrated. The residue was purified twice by flash column chromatography (DCM → MeOH / DCM 1/9 (column 1), 50% EtOAc in heptane → EtOAc (column 2)). The product was obtained as a slightly yellow oil (423 mg; 0.51 mmol; 33%). ¹ H NMR (400 MHz, CDCl ₃ ) δ (ppm) 8.31-8.25 (m, 4H), 7.42-7.35 (m, 4H), 5.22-5.14 (m, 1H) ), 4.48 to 4.43 (m, 4H), 4.33 to 4.28 (m, 2H), 4.14 (d, J = 8.4 Hz, 2H), 3.78 to 3.68 (M, 6H), 3.67 to 3.59 (m, 8H), 3.57 to 3.51 (m, 2H), 3.39 to 3.32 (m, 2H), 2.34 to 2 .16 (m, 6H), 1.60 to 1.55 (m, 2H), 1.40 to 1.30 (m, 1H), 0.99 to 0.88 (m, 2H)

Example 28: Preparation of linker-conjugate 66 To a solution of 65 (34 mg, 41 μmol) in DMF (400 μL), triethylamine (28 μl; 20 mg; 201 μmol) and vc-PABC-MMAE in DMF (1.0 mL). . A solution of TFA (83 mg; 67 μmol) was added. The mixture was diluted with DMF (1200 μL), allowed to stand for 41 hours, and 2,2 ′-(ethylenedioxy) bis (ethylamine) (47 μL, 48 mg, 322 μmol) was added. After 80 minutes, the reaction mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH) to give the desired product as a colorless oil (66 mg). , 24 μmol, 58% (based on 65) LCMS (ESI ⁺ ) Calculated for C ₁₄₂ H ₂₂₆ N ₂₂ O ₃₅ ²⁺ (M + 2H ⁺ ) 1400.33 Found 1401.08.

Example 29: Preparation of linker-conjugate 67 vc-PABC-MMAD.D in DMF (0.42 mL) ^. To a solution of TFA (5.0 mg; 3.87 μmol) was added triethylamine (1.6 μl; 1.2 mg; 11 μmol) and a solution of 99 (2.5 mg, 4.8 μmol) in DMF (135 μL). The mixture was left to stand for 23 hours and 2,2 ′-(ethylenedioxy) bis (ethylamine) (3.4 μL, 3.5 mg, 23 μmol) was added. After 2 h, the reaction mixture was purified by RP HPLC (C18, 30% → 90% MeCN (1% AcOH) in water (1% AcOH) to give the desired product (3.1 mg, 2 LCMS (ESI ⁺ ) Calculated for C ₇₆ H ₁₁₆ N ₁₃ O ₁₈ S ₂ ⁺ (M + H ⁺ ) 1562.80 Found 1562.84

Antibody-drug-conjugate generation: Examples 30-36:

Example 30: Conjugation of cAC10 with 100 to obtain cAC10-MMAE conjugate 53 Conjugation of compound 100 as a linker-conjugate with a bioconjugate according to the present invention to azide modified cAC10 as a biomolecule It was prepared by. cAC10- (6-N ₃ -GalNAc) ₂ (13d) (287 μL, 6.7 mg, 23.38 mg / ml in PBS pH 7.4) was added to PBS pH 7.4 (133 μL) and Compound 100 (27 μL, DMF). Medium 10 mM solution) was added. The reaction was incubated overnight at room temperature and subsequently purified on a Superdex 200 10/300 GL (GE Healthcare) on an AKTA purifier 10 (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 25844 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 1.88.

Example 31: Conjugation of cAC10 with 108 to obtain cAC10-MMAE conjugate 54 Conjugation of compound 108 as a linker-conjugate to azide modified cAC10 as biomolecule with a bioconjugate according to the present invention It was prepared by. _{cAC10- (6-N 3 -GalNAc)} 2 (13d) (287μL, 6.7mg, in PBS pH7.4 23.38mg / ml) to a solution of, PBS pH7.4 (133μL) and compound 108 (27 [mu] L, DMF Medium 10 mM solution) was added. The reaction was incubated overnight at room temperature, followed by purification with AKTA purifier 10 (GE Healthcare) on Superdex 200 10/300 GL (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 25928 Da, approximately 70% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 1.85.

Example 32: Conjugation of cAC10 with 111 to obtain conjugate cAC10-MMAE52 A bioconjugate according to the present invention is obtained by conjugation of compound 111 as a linker-conjugate to azide modified cAC10 as a biomolecule. Prepared. In a solution of cAC10- (6-N ₃ -GalNAc) ₂ (13d) (287 μL, 6.7 mg, 23.38 mg / ml in PBS pH 7.4), PBS pH 7.4 (48.2 μL) Compound 111 (111. 8 μL, 4 mM solution in DMF) was added. The reaction was incubated overnight at room temperature, followed by purification with AKTA purifier 10 (GE Healthcare) on Superdex 200 10/300 GL (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 25853 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 1.88.

Example 33: Conjugation of cAC10 with 67 to obtain conjugate cAC10-MMAD55 A bioconjugate according to the present invention is obtained by conjugation of compound 67 as a linker-conjugate to azide modified cAC10 as a biomolecule. Prepared. In a solution of cAC10- (6-N ₃ -GalNAc) ₂ (13d) (243 μL, 5.0 mg, 20.56 mg / ml in PBS pH 7.4), PBS pH 7.4 (57 μL) and compound 67 (33 μL, DMF Medium 10 mM solution) was added. The reaction was incubated overnight at room temperature, followed by purification with AKTA purifier 10 (GE Healthcare) on Superdex 200 10/300 GL (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 25896 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 1.88.

Example 34: Conjugation of cAC10 with 66 to give cAC10- (MMAE) ₂ conjugate 56 Compound 66 as a linker-conjugate to azide modified cAC10 as a biomolecule with a bioconjugate according to the invention Prepared by conjugation. To a solution of cAC10- (6-N ₃ -GalNAc) ₂ (13d) (8.408 mL, 246.0 mg, 29.3 mg / ml in PBS pH 7.4) was added propylene glycol (11.909 mL) and compound 66 (410 .6 μL, 40 mM solution in DMF). The reaction was incubated at room temperature for approximately 40 hours. The reaction mixture was dialyzed against PBS pH 7.4 and purified with HiLoad 26/600 Superdex 200 PG (GE Healthcare) with AKTA purifier 10 (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 27132 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 3.81.

Example 35: Conjugation of cAC10 with 63 to obtain cAC10- (MMAE) ₂ conjugate 57 Compound 63 as a linker-conjugate to azide modified cAC10 as biomolecule with a bioconjugate according to the invention Prepared by conjugation. To a solution of cAC10- (6-N ₃ -GalNAc) ₂ (13d) (9.95 mL, 205 mg, 20.7 mg / ml in PBS pH 7.4), PBS pH 7.4 (1.0 mL), DMF (2. 568 mL) and compound 63 (171.7 μL, 40 mM solution in DMF) were added. The reaction was incubated overnight at room temperature, followed by dialysis and purification on a HiLoad 26/600 Superdex 200 PG (GE Healthcare) with an AKTA purifier 10 (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 27124 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 3.79.

Example 36: Conjugation of Iratumumab with 100 to obtain Iratumumab-MMAE conjugate 59

A bioconjugate according to the present invention was prepared by conjugation of compound 100 as a linker-conjugate to an azide modified iratumumab as a biomolecule. A solution of iratumumab (6-N ₃ -GalNAc) ₂ (189 μL, 4.8 mg, 25.6 mg / ml in PBS pH 7.4), PBS pH 7.4 (51 μL) and compound 100 (80 μL, 4 mM solution in DMF) ) Was added. The reaction was incubated overnight at room temperature, followed by purification with AKTA purifier 10 (GE Healthcare) on Superdex 200 10/300 GL (GE Healthcare). Mass spectral analysis of the fabricator digested sample showed one major product (observed mass 25853 Da, approximately 80% of total Fc / 2 fragments) corresponding to the conjugated Fc / 2 fragment. RP-HPLC analysis of the reduced sample showed an average DAR of 1.89.

Examples 37-39: Efficacy, tolerability and stability studies Example 37a: CD30 efficacy study 8-12 weeks old CR female CB. 17 SCID mice (obtained from Charles River Laboratories, USA) were injected subcutaneously in the flank with 1 × 10 ⁷ KARPAS-299 tumor cells in 50% Matrigel (Karpas-299 cell xenograft model). When tumor volumes ranged from 100-150 mm ³ , groups of 8 mice were given a single dose of either vehicle (control), Adcetris (A, 1 mg / kg) and 56 (at 1 mg / kg). The dose was injected intravenously on day 1. Tumors were measured twice weekly for a period of 60 days. Results for tumor volume (mean) are illustrated in FIG. 8A.

Example 37b: CD30 efficacy study 8-12 weeks old CR female CB. 17 SCID mice (obtained from Charles River Laboratories, USA) were injected subcutaneously in the flank with 1 × 10 ⁷ KARPAS-299 tumor cells in 50% Matrigel (Karpas-299 cell xenograft model). . When tumor volumes ranged from 100 to 150 mm ³ , groups of 8 mice were assigned to vehicle (control), Adcetris (A, 1 mg / kg), 53 (4 mg / kg), 55 (2 mg / kg). A single dose of 55 (at 4 mg / kg), 57 (at 1 mg / kg) and 57 (at 2 mg / kg) were injected intravenously on day 1. Tumors were measured twice weekly for a 30 day period. Results for tumor volume (median) are illustrated in FIG. 8B.

Example 38: CD30 Tolerance Study 56 or 57 (40 mg / kg) CR female Wistar rats (2 females per group) 5-6 weeks old at the start of the experimental phase, obtained from Charles River Laboratories, USA , 60 mg / kg, 70 mg / kg and 80 mg / kg), or 52, 53 or 54 (80 mg / kg, 120 mg / kg, 140 mg / kg and 160 mg / kg) and Adcetris (15 mg / kg, 20 mg / Kg and 40 mg / kg). Test items were administered via intravenous (bolus) injection using a microflex infusion set introduced into the tail vein (2 mL / kg at 1 mL / min). One group of animals was treated with vehicle (control). After dosing, all animals were maintained for a 12 day observation period. Surviving animals were euthanized on day 12. Each animal was weighed at the time of randomization / selection before dosing (day 0) and on all subsequent days up to day 12. Any individual animal with a single observation of weight loss greater than 30% or three consecutive measurements of weight loss greater than 25% was euthanized. All animals (including those found dead or moribund killed) were subjected to a complete autopsy procedure. Histopathological examination of liver, spleen and sciatic nerve was performed on all animals. Blood samples (including animals moribund and dead) were collected and subjected to determination of both hematological as well as serum clinical chemistry parameters.

  Results for percent body weight loss for rats for different dose plans per ADC are illustrated in FIG. It is clear from these results that the maximum tolerated dose (MTD) for Adcetris is between 15 and 20 mg / kg, while the MTD for ADC56 and 57 (both DAR = 4) is 60-70 mg / Kg was found to be in the range. For ADCs 52, 53 and 54 (all DAR = 2), the MTD was found to be between 120-140 mg / kg.

Example 39: In vitro serum stability assay Incubating human serum (Sigma, H4522-100 mL) with protein A sepharose (1 mL Sepharose / mL serum, commercially available from Repligen) for 1 hour at 4 ° C. IgG was depleted. The depleted serum was filter sterilized using a 0.22 μm filter (Millipore), divided into aliquots, snap frozen and stored at −20 ° C. until further use (avoid multiple freeze-thaw cycles). ADC56, 57 and Adcetris were added to a final concentration of 0.1 mg / mL and incubated at 37 ° C. At the preset time point, a sample (0.5 mL) was taken and stored at −20 ° C. until further analysis. For analysis, samples were incubated with protein A sepharose (20 μL Sepharose, commercially available from Repligen) for 1 hour at room temperature. The beads were then washed with PBS (3 × 1 mL) and subsequently eluted with 0.1 M glycine-HCl pH 2.7 (0.4 mL). After elution, the samples were immediately neutralized with 1.5 M Tris pH 8.8 (0.1 mL) and spin filtered to a final volume of approximately 40 μL in PBS pH 7.4. Samples were analyzed by RP-HPLC and MS according to standard procedures.

  The results of stability studies of different ADCs in human serum are illustrated in FIG. 9, which shows the superior stability of ADCs 56 and 57 compared to Adcetris.

Claims (18)

An antibody-conjugate comprising an antibody AB connected to a target molecule D via a linker L, comprising
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) _{By transferring the x} moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 And y is 1, 2, 3 or 4), and (ii) a functional group Q ¹ and a linker L that can react the modified antibody with the functional group F ^1. To obtain an antibody-conjugate by reacting with a linker-conjugate comprising a target molecule D connected to Q ¹ via ² , wherein the linker L comprises SZ ³ -L ² and Z ³ Can be obtained by the step, which is the connecting group resulting from the reaction between Q ¹ and F ¹ ,
Antibody AB can target CD30-expressing tumors and target molecule D is taxane, anthracycline, camptothecin, epothilone, mitomycin, combretastatin, vinca alkaloid, maytansinoid, calicheamicin and enediyne, duocarmycin, tubricin , Amatoxin, dolastatin and auristatin, pyrrolobenzodiazepine dimer, indoline o-benzodiazepine dimer, radioisotope, therapeutic protein and peptide (or fragments thereof), kinase inhibitor, MEK inhibitor, KSP inhibitor, As well as antibody-conjugates selected from the group consisting of analogs or prodrugs thereof.   The antibody can target CD30 expressing tumors, Ki-2, Ki-4, Ki-6, Ki-7, HRS-1, HRS-4, Ber-H8, Ber-H2, 5F11, Ki-1, Ki- 5. The antibody-conjugate of claim 1 selected from 5, M67, Ki-3, M44, HeFi-1, AC10, cAC10, and functional analogs thereof. The linker L ² is a group according to formula (1) or a salt thereof:

(Where:
a is 0 or 1;
R ³¹ is hydrogen, a C ₁ -C ₂₄ alkyl group, a C ₃ -C ₂₄ cycloalkyl group, a C ₂ -C ₂₄ (hetero) aryl group, a C ₃ -C ₂₄ alkyl (hetero) aryl group, and a C ₃ -C. ₂₄ (hetero) is selected from the group consisting of arylalkyl _group, C 1 _{-C 24} alkyl _group, C 3 _{-C 24} cycloalkyl _group, C 2 _{-C 24} (hetero) aryl _group, C 3 _{-C 24} alkyl (hetero The aryl group and the C ₃ -C ₂₄ (hetero) arylalkyl group are optionally substituted and optionally interrupted by one or more heteroatoms selected from O, S or NR ³³ , wherein in R ³³ are independently selected from the group consisting of hydrogen and C ₁ -C ₄ alkyl group, or R ³¹ is an additional target particle D, I wherein the target molecule Via a spacer moiety comprising a connected optionally) in N, according to claim 1 or 2 antibody - conjugate.   The antibody AB is brentuximab and the target molecule D is selected from the group of auristatins consisting of MMAD, MMAE and MMAF, preferably D = MMAD or MMAE. The antibody-conjugate described. (I) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) ₂ —NH— (CH ₂ —CH ₂ —O) ₂ -CO-Val-Cit-PABC-, D = MMAE;
(II) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ ) ₃ —CO—NH—S (O) ₂ —NH— _(CH 2 _-CH 2 _-O) a 2 -CO-Val-Cit-PABC- , is D = MMAE;
(III) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH ₂ —O) ₄ —CO—Val-Cit-PABC − And D = MMAE;
(IV) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH ₂ —O) ₄ —CO—N (CH ₂ — a _{CH 2 -O-CO-Val-} Cit-PABC-D) 2, is each occurrence = MMAE of D;
(V) AB = brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297, and S (F ¹ ) _x = 6-azido-6 Deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) ₂ —NH— (CH ₂ —CH ₂ —O) ₂ a _{-CO-N (CH 2 -CH 2} -O-CO-Val-Cit-PABC-D) 2, is each occurrence = MMAE of D;
(VI) AB = Iratumumab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N292, and S (F ¹ ) _x = 6-azido-6-deoxy- N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH— (CH ₂ —CH ₂ —O) ₄ —CO—Val-Cit-PABC— Yes, D = MMAE; or (VII) AB = Brentuximab, where S (F ¹ ) _x is connected to the core-GlcNAc linked to amino acid N297 and S (F ¹ ) _X = 6-azido-6-deoxy-N-acetylgalactosamine, Q ¹ is according to formula (9q), L ² = —O—C (O) —NH—S (O) ₂ —NH— ( CH ₂ -CH ₂ - ) And _{2 -CO-Val-Cit-PABC-} , a D = MMAD,
The antibody-conjugate according to any one of claims 1 to 4. Use of a conjugation mode to increase the therapeutic index of a bioconjugate, wherein the conjugation mode is used to connect biomolecule B via target molecule D and linker L ,
(Ii) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) _{By transferring the x} moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 in it; y can be obtained a modified glycoprotein according to 1, 2, 3 or 4), the step; and (ii) a modified glycoprotein, functional groups ^{Q 1} and can react with functional groups ^{F 1} Reacting with a linker-conjugate comprising a target molecule D connected to Q ¹ via a linker L ² to obtain an antibody-conjugate, wherein the linker L comprises SZ ³ -L ² ; Use, wherein Z ³ is a connecting group resulting from a reaction between Q ¹ and F ¹ .   Antibody AB is 5T4 (TPBG), αv-integrin / ITGAV, BCMA, C4.4a, CA-IX, CD19, CD19b, CD22, CD25, CD30, CD33, CD37, CD40, CD56, CD70, CD74, CD79b, c -KIT (CD117), CD138 / SDC1, CEACAM5 (CD66e), crypto, CS1, DLL3, EFNA4, EGFR, EGFRvIII, endothelin B receptor (ETBR), ENPP3 (AGS-16), EpCAM, EphA2, FGFR2, FGFR3, FOLR1 (folate receptor a), gpNMB, guanyl cyclase C (GCC), HER2, Erb-B2, Lamp-1, Lewis Y antigen, LIV-1 (SLC39A6, ZIP6), mesothelin (MSLN), UC1 (CA6, huDS6), MUC16 / EA-125, NaPi2b, Nectin-4, Notch3, P-cadherin, PSMA / FOLH1, PTK7, SLITRK6 (SLC44A4), STEAP1, TF (CD142), Trop-1, Trop-2 Use according to claim 6, which can target tumors expressing antigens selected from / EGP-1, Trop-3, Trop-4, preferably CD30 expressing tumors. Increasing the therapeutic index of the antibody-conjugate
8. Use according to claim 6 or 7, selected from (a) increasing the therapeutic efficacy of the antibody-conjugate; and / or (b) increasing the tolerability of the antibody-conjugate. The reaction of step (ii) is

(Wherein cycle A is a 7-10 membered (hetero) cyclic moiety), a connecting moiety Z represented by (10e), (10i) or (10g), preferably represented by (10g) 9. Use according to any one of claims 6 to 8, which is a (cyclo) alkyne-azide conjugation reaction to form ³ . One of the F ¹ is an azido moiety, a Q ¹ is (cyclo) alkyne moiety, Z ³ is triazole moiety Use according to any one of claims 6-9.   11. Use according to any one of claims 6 to 10, wherein x is 1 or 2, preferably x is 1. S ^(F _{1) x} is 6-azido-6-deoxy -N- acetylgalactosamine Use according to any one of claims 6-11. The antibody-conjugate is of formula (40) or (40b):

(Where:
R ³¹ is hydrogen, _{^{halogen, -OR 35, -NO 2, -CN}} , -S (O) 2 R 35, C 1 ~C 24 alkyl _group, C 6 _{-C 24} (hetero) aryl _{group, C} 7 ~ C ₂₄ alkyl is independently selected from the group consisting of (hetero) aryl groups and _C 7 _{-C 24} (hetero) aryl group, an alkyl group, (hetero) aryl group, alkyl (hetero) aryl groups and (hetero) aryl The alkyl group is optionally substituted, wherein the two substituents R ³¹ can be joined together to form a fused cycloalkyl or fused (hetero) arene substituent, R ³⁵ is hydrogen, _halogen, C 1 _{-C 24} alkyl _group, C 6 _{-C 24} (hetero) aryl _groups, C 7 _{-C 24} alkyl (hetero) aryl groups and _C 7 _{-C 24} (hetero) Independently selected from the group consisting of reel alkyl group;
X is C (R ³¹ ) ₂ , O, S or NR ³² , wherein R ³² is R ³¹ or L ³ (D) _r , where L ³ is a linker, and D is claimed As defined in 1;
r is 1-20;
q is 0 or 1, but if q is 0, provided that X is NL ² (D) _r ;
aa is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa ′ is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
aa + aa ′ <10;
b is 0 or 1;
pp is 0 or 1;
M is —N (H) C (O) CH ₂ —, —N (H) C (O) CF ₂ —, —CH ₂ —, —CF ₂ —, or C2 and C6 or C3 and C5 of phenylene. Preferably 1,4-phenylene containing 0 to 4 fluorine substituents, preferably 2 fluorine substituents located;
y is 1 to 4;
Use according to any one of claims 6 to 12, wherein Fuc is fucose).   14. Use according to any one of claims 6 to 13, wherein D is an active substance, preferably an anticancer agent. A method for targeting a CD30-expressing cell comprising administering an antibody-conjugate comprising an antibody AB connected to a target molecule D via a linker L to a subject in need thereof, comprising: The antibody-conjugate is
(I) contacting a glycoprotein comprising 1-4 core N-acetylglucosamine moieties with a compound of formula S (F ¹ ) _x -P in the presence of a catalyst, wherein S (F ¹ ) _x is A sugar derivative containing x functional groups F ¹ capable of reacting with the functional group Q ¹ , x is 1 or 2, P is nucleoside monophosphate or diphosphate, and the catalyst is S (F ¹ ) _{By transferring the x} moiety to the core-GlcNAc moiety, formula (24):

Wherein S (F ¹ ) _x and x are as defined above; AB represents an antibody; GlcNAc is N-acetylglucosamine; Fuc is fucose; b is 0 or 1 Wherein y is 1, 2, 3 or 4), and (ii) a linker comprising a functional group Q ¹ capable of reacting the modified antibody with a functional group F ¹ A step of obtaining an antibody-conjugate by reacting with the target molecule D connected to Q ¹ via the conjugate and linker L ² , wherein the linker L comprises SZ ³ -L ² and Z ³ Can be obtained by the step, which is the connecting group resulting from the reaction between Q ¹ and F ¹ ,
A method wherein antibody AB can target a CD30 expressing tumor.   Targeting CD30 expressing cells is one of treating, imaging, diagnosing, preventing their growth, containment and reducing CD30 expressing cells, particularly CD30 expressing tumors 16. The method of claim 15, comprising a plurality.   The subject is a lymphoma, such as Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), anaplastic large cell lymphoma (ALCL), large B cell lymphoma, childhood lymphoma, T cell lymphoma and enteropathy-related T cell lymphoma (EATL), Selected from leukemias such as acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and mast cell leukemia, germ cell cancer, transplant versus host disease (GvHD), and lupus, especially systemic lupus lupus erythematosus (SLE) 17. The method of claim 15 or 16, wherein the method suffers from a disorder that is caused.   The method according to any one of claims 15 to 17, wherein the target molecule D is an anticancer agent, preferably a cytotoxin.

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