Method for preparing bioconjugates

Abstract

The present invention relates to the use of surfactants in bioconjugation reactions in which biomolecules containing one or more azide moieties are connected to cyclic alkynes containing payloads. More particularly, the present invention relates to surfactants having the structure B-(ZLD) x A method for preparing a bioconjugate of (1), comprising: (i) reacting an alkyne or alkene compound of structure QLD(2), where Q is a click probe containing a cyclic alkyne or cyclic alkene moiety, L is a linker, and D is a payload; and (ii) reacting an alkyne or alkene compound of structure B-(F): x (3), wherein B is a biomolecule functionalized with x number of click probes F, F is a click probe capable of reacting with Q, and x is an integer ranging from 1 to 10, in the presence of a surfactant. The use of a surfactant increases the conversion rate of the bioconjugation reaction, increases the yield of the bioconjugation reaction, reduces the amount of organic cosolvent in the solvent system in which the bioconjugation reaction is carried out, provides flexibility in the concentration of the biomolecule during the bioconjugation reaction, reduces the excess amount of alkyne- or alkene-functionalized payload used during the bioconjugation reaction, reduces the degree of aggregate formation during the bioconjugation reaction, and simplifies downstream processing of the bioconjugate.

Description

Field of Invention

[0001]

[0001] The present invention relates to the field of bioconjugation, and more particularly to a method for preparing bioconjugates in the presence of a surfactant. Background of the Invention

[0002]

[0002] Antibody-drug conjugates (ADCs), considered highly effective therapeutic agents, consist of antibodies bound to a drug. Antibodies (also called ligands) can be in small protein formats (scFv, Fab fragments, DARPin, aphibodies, etc.), but are generally monoclonal antibodies (mAbs) selected based on high selectivity and affinity for a given antigen, a long circulating half-life, and little to no immunogenicity. Thus, mAbs as protein ligands for carefully selected biological receptors provide an ideal delivery platform for the selective targeting of pharmaceuticals. For example, monoclonal antibodies known to selectively bind to specific cancer-associated antigens can be used for the delivery of chemically conjugated cytotoxic substances to tumors via binding, internalization, intracellular processing, and finally the release of active catabolites. Cytotoxic substances can be in other formats such as small molecule toxins, protein toxins, or oligonucleotides. As a result, tumor cells can be selectively eradicated while preserving normal cells that were not targeted by the antibody. Similarly, while chemical conjugation of antibacterial drugs (antibiotics) to antibodies can be applied to the treatment of bacterial infections, the conjugation of anti-inflammatory drugs is under investigation for the treatment of autoimmune diseases, and, for example, the attachment of oligonucleotides to antibodies is a potentially promising technique for the treatment of neuromuscular diseases. Therefore, the concept of targeted delivery of active drugs to selected specific cellular sites is a powerful technique for the treatment of a wide range of diseases, offering many advantages over systemic delivery of the same drug.

[0003]

[0003] An alternative strategy for targeting and delivering specific protein substances using monoclonal antibodies is by gene fusion of the latter protein to one (or more) of the antibody's terminals, which may be the N-terminus or C-terminus of the light chain or heavy chain (or both). In this case, the bioactive protein of interest, such as a protein toxin like Pseudomonas exotoxin A (PE38) or an anti-CD3 single-chain variable fragment (scFv), is gene-encoded as a fusion to the antibody, though not necessarily, and possibly via a peptide spacer, and the antibody is expressed as a fusion protein. The peptide spacer may or may not contain a protease-sensitive cleavage site.

[0004]

[0004] In the field of ADCs, chemical linkers are typically employed to attach pharmaceuticals to antibodies. This linker needs to have several important attributes, including the need for plasma to be stable after long-term drug administration. A stable linker allows for the localization of the ADC to a planned site or cell in the body and prevents premature release of the payload in circulation. Its premature release indiscriminately induces all kinds of undesirable biological responses, thereby reducing the therapeutic index of the ADC. Once internalized, the ADC should be processed so that the payload is effectively released and therefore can bind to its target.

[0005]

[0005] Linkers belong to two families: non-cleavable linkers and cleavable linkers. Non-cleavable linkers consist of a chain of atoms between the antibody and the payload and are sufficiently stable under physiological conditions regardless of the organ or biological compartment in which the antibody-drug conjugate resides. As a result, the release of the payload from an ADC containing a non-cleavable linker depends on the complete (lysosomal) degradation of the antibody after the ADC has been moved into the cell. As a result of this degradation, the payload (still containing the linker), as well as peptide fragments and / or amino acids, are released from the antibody to which the linker was originally attached. Cleavable linkers utilize the intrinsic properties of the cell or cellular compartment for the selective release of the payload from the ADC, thereby generally leaving no trace of linker after metabolic processing. There are three commonly used mechanisms for cleavable linkers: 1) sensitivity to specific enzymes, 2) pH sensitivity, and 3) sensitivity to the cellular redox state (or its microenvironment). The cleavable linker may also include, for example, self-destructive units based on para-aminobenzyl alcohol groups and their derivatives. The linker may also include additional non-functional elements, often called spacer or stretcher units, for connecting the linker to reactive groups for reactions with biomolecules.

[0006]

[0006] Currently, payloads used in ADCs mainly include microtubule disruptors [e.g., auristatins such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), maytansinoids such as DM1 and DM4, tubulinin], DNA damaging agents [e.g., calicheamycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine (indolinobenzodiapine) dimers, duocalmycin, anthracyclines], topoisomerase inhibitors [e.g., DXd, SN-38, exatecan and its derivatives, simitecan], or RNA polymerase II inhibitors [e.g., amanitin]. Although ADCs have demonstrated clinical and preclinical activity, it remained unclear which factors, in addition to antigen expression in targeted tumor cells, determine such efficacy. For example, drug-to-antibody ratio (DAR), ADC binding affinity, payload potency, receptor expression level, internal transfer rate, transport, multidrug resistance (MDR) status, and other factors all played a role in influencing the outcomes of ADC treatment in vitro. In addition to the direct killing of antigen-positive tumor cells, ADCs also possess the ability to kill adjacent antigen-negative tumor cells, a so-called "bystander killing" effect, as reported by Sahin et al., Cancer Res., 1990, 50, 6944-6948 (incorporated by reference) and studied by Li et al., Cancer Res., 2016, 76, 2710-2719 (incorporated by reference). Generally, neutral cytotoxic payloads exhibit bystander killing, while ionic (charged) payloads do not exhibit bystander killing as a result of the fact that ionic species do not readily permeate the cell membrane by passive diffusion. For example, as disclosed by Ogitani et al., Cancer Sci., 2016, 107, 1039-1046, incorporated by reference, evaluation of various exatecan derivatives showed that acylation of primary amines with hydroxyacetic acid yielded substantially enhanced bystander-killer derivatives (DXd) compared to various aminoacylated exatecan derivatives.

[0007]

[0007] ADCs are prepared by a process called bioconjugation, which is the conjugation of a linker-drug and a protein. Many techniques for bioconjugation are known, as summarized in G. Thermanson, "Bioconjugate Techniques", Elsevier, 3rd edition, 2013, incorporated by reference. Two main techniques can be recognized for the preparation of ADCs by random conjugation: one based on acylation of the lysine side chain and the other on alkylation of the cysteine ​​side chain. Acylation of the ε-amino group in the lysine side chain is typically achieved by exposing the protein to a reagent based on an activated ester or activated carbonate derivative, e.g., SMCC, which is used in the production of Kadcyla®. The main chemistry for alkylation of the thiol group in the cysteine ​​side chain is based on the use of maleimide reagents, e.g., as applied in the production of Adcetris®. In addition to standard maleimide derivatives, various maleimide variants can also be applied to more stable cysteine ​​conjugations, as is evident from James Christie et al., J. Contr. Rel., 2015, 220, 660-670 and Lyon et al., Nat. Biotechnol., 2014, 32, 1059-1062, which are incorporated together by reference.Other methods for cysteine ​​alkylation include, for example, nucleophilic substitution of haloacetamides (typically bromoacetamide or iodoacetamide) (see, e.g., Alley et al., Bioconj. Chem., 2008, 19, 759-765, incorporated by reference), or various methods based on nucleophilic addition to unsaturated bonds, such as reactions with acrylate reagents (see, e.g., Bernardim et al., Nat. Commun., 2016, 7, DOI:10.1038 / ncomms13128 and Ariyasu et al., Bioconj. Chem., 2017, 28, 897-9, both incorporated by reference). This includes reactions with phosphoamides (see, for example, Kasper et al., Angew. Chem. Int. Ed., 2019, 58, 11625-11630, incorporated by reference), reactions with allenamides (see, for example, Abbas et al., Angew. Chem. Int. Ed., 2014, 53, 7491-7494, incorporated by reference), reactions with cyanoethynyl reagents (see, for example, Kolodych et al., Bioconj. Chem., 2015, 26, 197-200, incorporated by reference), reactions with vinylsulfones (see, for example, Gil de Montes et al., Chem. Sci., 2019, 10, 4515-4522, incorporated by reference), or reactions with vinylpyridines (see, for example, https: / / iksuda.com / science / permalink / (accessed January 7, 2020)).An alternative to antibody conjugation that does not involve antibody reengineering involves reducing interchain disulfide crosslinks and subsequently using bis-sulfone reagents (see, for example, Balan et al., Bioconj. Chem., 2007, 18, 61-76 and Bryant et al., Mol. Pharmaceuticals, 2015, 12, 1872-1879, both incorporated by reference), mono- or bis-bromomaleimides (see, for example, Smith et al., J.Am. Chem. Soc., 2010, 132, 1960-1965 and Schuma, both incorporated by reference). See cher et al., Org. Biomol. Chem., 2014, 37, 7261-7269), bis-maleimide reagent (see, e.g., International Publication No. 2014114207), bis(phenylthio)maleimide (see, e.g., both incorporated by reference, Schumacher et al., Org. Biomol. Chem., 2014, 37, 7261-7269 and Aubrey et al., Bioconj. Chem., 2018, 29, 3516-3521), bis-bromopyridazinedione (see, e.g., Robinson et al., incorporated by reference, RSC This involves the addition of a payload attached to a cysteine ​​crosslinking reagent, such as Advances, 2017, 7, 9073-9077), bis(halomethyl)benzene (see, e.g., Ramos-Tomillero et al., Bioconj. Chem., 2018, 29, 1199-1208, incorporated by reference), or other bis(halomethyl) aromatic compounds (see, e.g., International Publication No. 2013173391). Typically, ADCs prepared by cysteine ​​crosslinking have approximately 4 drug-antibody loadings (DAR4). Another technique useful for conjugation to cysteine ​​side chains is the technique using disulfide bonds, which are bioactivatable links that have been used to reversibly link protein toxins, chemotherapeutic drugs, and probes to carrier molecules (see, e.g., Pillow et al., Chem. Sci., 2017, 8, 366-370, incorporated by reference).

[0008]

[0008] In addition to conjugation to lysine or cysteine, various other conjugation techniques have been explored over the past decade. One method is based on the gene encoding of non-natural amino acids, such as p-acetophenylalanine suitable for oximligation, or p-azidomethylphenylalanine or p-azidophenylalanine suitable for click chemistry conjugation, as is evident, for example, from Axup et al., Proc. Nat. Acad. Sci., 2012, 109, 16101-16106, incorporated by reference. Similarly, Zimmerman et al., Bioconj. Chem., 2014, 25, 351-361, incorporated by reference, introduced azidomethylphenylalanine (AzPhe) into a monoclonal antibody for conversion to ADC by metal-free click chemistry using a cell-free protein synthesis method. Furthermore, Nairn et al., Bioconj. Chem., 2012, 23, 2087-2097, incorporated by reference, demonstrated that methionine analogs such as azidohomoalanine (Aha) can be introduced into proteins by nutrient-dependent bacteria and further converted into protein conjugates by (copper-catalyzed) click chemistry. Finally, pyrrolysyl-tRNA synthetase / tRNA CUA Gene encoding of aliphatic azides in recombinant proteins using pairs was shown by Nguyen et al., J.Am.Chem.Soc., 2009, 131, 8720-8721, via reference integration, and labeling was secured by click chemistry.

[0009]

[0009] Another method is based on the enzymatic introduction of non-natural functionalities. For example, Lhospice et al., Mol. Pharmaceut., 2015, 12, 1863-1871, incorporated by reference, employs the bacterial enzyme transglutaminase (BTG or TGase) for the introduction of the azide moiety into the antibody. A genetic method based on conversion in ADC using metal-free click chemistry following C-terminal TGase-mediated azide introduction was reported by Cheng et al., Mol. Cancer Therap., 2018, 17, 2665-2675, incorporated by reference.

[0010]

[0010] In International Publication No. 2014065661, all incorporated by reference, van Geel et al., Bioconj. Chem., 2015, 26, 2233-2242 and Verkade et al., Antibodies, 2018, 7, 12, it was shown that enzymatic remodeling of the natural antibody glycan at N297 enables the introduction of azide-modified sugars suitable for the attachment of cytotoxic payloads using click chemistry.

[0011]

[0011] Chemical methods for site-specific modification of antibodies without prior genetic modification have also been developed, as highlighted, for example, by Yamada and Ito, ChemBioChem., 2019, 20, 2729-2737.

[0012]

[0012] In addition to the reaction between azide and cyclooctin, bioconjugation of linker-drugs to proteins (and other biomolecules such as glycans and nucleic acids) can also be achieved by various other metal-free click chemistry. See, for example, Nguyen and Presser, Nature rev., 2020, doi:10.1038 / s41570-020-0205-0, incorporated by reference. For example, orthoquinones can be obtained by oxidation of specific tyrosines in proteins, which readily undergo cycloaddition with strained alkenes (e.g., TCO) or strained alkynes. See, for example, Bruins et al., Chem. Eur. J., 2017, 24, 4749-4756, incorporated by reference. In addition to cyclooctin, several cycloheptins are also suitable for metal-free click chemistry, as reported by Wetering et al., Chem. Sci., 2020, doi: 10.1039 / d0sc03477k, incorporated by reference. The tetrazine moiety can also be introduced into proteins or glycans by various means, such as gene coding or chemical acylation, and may undergo cycloaddition with cyclic alkenes and alkynes. A list of functional group F and Q pairs for metal-free click chemistry is shown in Figure 1.

[0013]

[0013] Based on the above, a general method for preparing the protein conjugate exemplified for the monoclonal antibody in Figure 2 requires the reaction of a protein containing x reactive moieties F with a linker-drug construct containing a single molecule Q. A schematic diagram showing how the reactive molecule F can be introduced into the monoclonal antibody is shown in Figure 3.

[0014]

[0014] A common method for linker-drug bioconjugation to azide-modified proteins is strain-enhanced alkyne-azide cycloaddition (SPAAC). In the SPAAC reaction, the linker-drug is functionalized with a cyclic alkyne, and the cycloaddition is driven by the release of ring strain. Various strained alkynes suitable for metal-free click chemistry are shown in Figure 4, as well as, for example, azides, quinones, or nitrile oxides.

[0015]

[0015] Conjugation of cytotoxic payloads to antibodies by any of the above methods is often challenging because the hydrophobicity of the payload, and possibly the hydrophobicity of the payload combined with the linker, interferes with its solubility in aqueous or buffer systems (preferred media for antibodies). As a result, conjugation of cytotoxic payloads is typically carried out in a medium consisting of water / buffer plus an organic cosolvent. Typical cosolvents for conjugation are DMSO, propylene glycol (PG), ethanol, DMF, DMA, and NMP, which promote linker-drug solubilization but are also well miscible with water. Typical amounts of cosolvent are 10-25% relative to the aqueous medium, but the cosolvent may be added up to 50% in some cases. Adding high amounts of cosolvent is particularly advantageous for conjugation processes where the payload is significantly hydrophobic (lipophilic), and where a large excess of linker-drug is required to achieve sufficient conversion to the desired product.

[0016]

[0016] Aside from the obvious benefits, a disadvantage of adding a substantial amount of organic co-solvent is that the antibody may be unstable in the solvent mixture and, as a result, may aggregate during the conjugation process. Typically, the level of aggregation is correlated with the amount of co-solvent, but this is also antibody-dependent. With particularly unstable antibodies, the level of aggregation can be significant, reaching 10% or even higher, resulting in a loss of process yield. Furthermore, these levels of aggregates may require additional processing steps (e.g., SEC or CHT) to remove the aggregates to an acceptable level.

[0017]

[0017] An additional disadvantage of high cosolvent levels during conjugation is that, in order to perform size exclusion purification (SEC), an additional process step is required to remove the excess cosolvent by, for example, dialysis, spin filtration, or TFF.

[0018]

[0018] Surfactants are well known in the art. Surfactants are a general class of organic compounds consisting of a single polar (or ionic) hydrophilic terminal group attached to a nonpolar hydrophobic hydrocarbon fragment. This amphiphilicity contributes to a unique phase behavior that reduces the surface tension between the two phases. Surfactants have a wide range of applications across various industries, including detergents, paints, plastics, cosmetics, agriculture, and pharmaceuticals. In the pharmaceutical industry, surfactants have been used primarily in formulations to improve drug solubility and stability in liquid form, for viral and bacterial inactivation, in upstream bioprocessing to enhance protein secretion, and in downstream bioprocesses to separate proteins from cells and tissues.

[0019]

[0019] Hu et al., Bioconj. Chem., 2018, 29, 3667-3676, incorporated by reference, reported that the surfactant sodium decanoate is used in the API process of the antibody-drug conjugate (ADC) Besponsa to promote bioconjugation via lysine acylation chemistry between an activated calicheamicin derivative (linker payload) and inotuzumab (monoclonal antibody). Micelle formation was shown to be critically important for an efficient conjugation reaction. Another screening study showed that sodium dodecyl sulfate, sodium deoxycholate, and dodecyltrimethylammonium bromide could also promote the conjugation reaction. However, it was also shown that the charge of the surfactant and the choice of linker payload affect the conjugate lysine site selectivity. Compared to the approximately 80 conjugate lysine sites typically observed in lysine conjugation in antibodies without surfactants, eight major conjugate lysine sites are observed in Besponsa.

[0020]

[0020] Anionic surfactants have also been shown to enhance protein conjugation processes using click chemistry. Specifically, Schneider et al., Bioorg. Med. Chem., 2016, 24, 995-1001, incorporated by reference, reported that anionic surfactants enhance conjugate formation using copper-catalyzed click chemistry (CuAAC) by up to 10-fold, and that high yields can be obtained even with low (i.e., micromolar) concentrations of reactants. However, it was not shown whether protein conjugation based on strain-enhanced (metal-free) click chemistry (SPAAC) is also improved. A study by Anderton et al., Bioconj. Chem., 2015, 26, 1687-1691, incorporated by reference, notes that strain-promoting azide-alkyne cycloaddition (SPAAC) can also be improved by using micellar catalysis with anionic and cationic surfactants, with a rate increase of up to 179 times for the reaction between benzyl azide and DIBAC cyclooctyne. A more moderate rate increase of 11 times was observed for the reaction between benzyl azide and DIBAC-functionalized DNA sequences using micellar catalysis, demonstrating that micellar catalysis can be successfully applied to nucleic acids. [Overview of the project]

[0021]

[0021] To our surprise, the inventors found that the bioconjugation reaction between biomolecules functionalized with click probe F and alkyne or alkene-functionalized payloads could be significantly improved by adding a surfactant to the reaction mixture. In the presence of the surfactant, higher conversion rates (and therefore higher yields and drug-antibody ratios closer to theoretical values) were obtained. Furthermore, the reaction could be carried out using fewer organic cosolvents and higher concentrations of biomolecules, resulting in less aggregation of conjugated biomolecules and easier purification. Moreover, the conjugation reaction could be carried out sufficiently using smaller excess amounts of alkyne or alkene-functionalized payloads, thus reducing the number of expensive and synthetically complex molecules required in the preparation of bioconjugates.

[0022]

[0022] The present invention may be defined by the following list of preferred embodiments. 1. Structure B-(ZLD) x (1) A method for preparing the bioconjugate, (i) Alkyne or alkene compounds of structure QLD(2) [In the structure, Q is a click probe that includes a cyclic alkyne portion or a cyclic alkene portion. L is a linker, D is the payload. (ii) Structure B-(F) x (3) Molecules [In the structure, B is a biomolecule functionalized with x click probes F, F is a click probe that can react to Q, x is an integer in the range of 1 to 10. A method comprising the step of reacting in the presence of a surfactant to form a bioconjugate in which the payload is covalently bonded to a biomolecule via a connecting group Z formed by a click reaction between Q and F. 2. The method according to Embodiment 1, wherein the surfactant includes a negatively charged portion, and preferably the surfactant is an anionic surfactant. 3. The method according to Embodiment 1 or 2, wherein the surfactant is selected from the group consisting of sodium decanoate, sodium dodecanoate, sodium lauryl sulfate (SDS), and sodium deoxycholate, and preferably the surfactant is sodium decanoate or sodium deoxycholate. 4. The method according to any one embodiment of Embodiments 1 to 3, wherein the reaction is carried out in a solvent system containing water and an organic solvent in a ratio of 50 / 50 to 100 / 0, preferably in the range of 75 / 25 to 95 / 5. 5. The method according to any one embodiment of Embodiments 1 to 4, wherein the concentration of the molecule of structure (3) is in the range of 1 to 100 mg / mL, preferably in the range of 5 to 50 mg / mL, and more preferably in the range of 10 to 20 mg / mL. 6. The method according to any one of embodiments 1 to 5, wherein the click probe Q contains a cyclic alkyne moiety, the click probe F is selected from the group consisting of azide, tetrazine, triazine, nitrone, nitrile oxide, nitrile imine, diazo compound, orthoquinone, dioxythiophene and sydnone, preferably the click probe F is an azide moiety. 7. The click probe Q is selected from the group consisting of (Q22) to (Q36),

Chemical formula

Chemical formula

[0023] [Figure 1] This figure shows a representative (but not exhaustive) group of functional groups (F) that can be introduced into biomolecules by engineering, chemical modification, or enzymatic means, yielding a conjugate group Z after a metal-free click reaction with a complementary reactive group Q. Functional groups F can be artificially introduced (engineered) at any position of choice in the biomolecule. Some functional groups F (e.g., nitrile oxides, quinones) can react with strained alkenes as well as strained alkynes, for example, triazine or tetrazine are shown (bottom row). Pyridine or pyridazine conjugate groups are rearrangement products of the tetraazabicyclo[2.2.2]octane conjugate group, formed by the loss of N2 after the reaction of triazine or tetrazine with an alkyne (not an alkene), respectively. Conjugate group Z is a preferred conjugate group for use in the present invention. [Figure 2] This figure shows a general scheme for preparing antibody-drug conjugates by the reaction of a monoclonal antibody (usually a symmetric dimer) containing x functional groups F. The conjugate is obtained by incubation of antibody-(F)x with an excess of the linker-drug construct (Q-spacer-linker-payload), where F and Q react to form conjugate Z. [Figure 3]This figure shows the general process of non-genetic conversion of a monoclonal antibody to an antibody containing a probe (F) for click conjugation. The click probe can be located at various positions on the antibody, depending on the technique employed. For example, the antibody may be converted to an antibody containing two click probes (left structure), four click probes (bottom structure), or eight probes (right structure) for click conjugation. [Figure 4] This figure shows preferred embodiments of cyclic alkynes and reactive moieties Q suitable for metal-free click chemistry. This list is not exhaustive, and alkynes may be further activated by fluorination, substitution of aromatic rings, or introduction of heteroatoms in aromatic rings, for example. [Figure 5] This figure illustrates a specific example of site-specific payload conjugation based on azido-cyclooctin click chemistry following glycan remodeling of full-length IgG. First, IgG is enzymatically remodeled by endoglycosidase-mediated trimming of all different glycoforms, followed by glycosyltransferase-mediated transfer of azido sugars to the endoglycosidase-released core GlcNAc. In the next step, the azido-remodeled IgG is exposed to polypeptides and modified with single cyclooctin (SPAAC) for metal-free click chemistry, yielding a bispecific antibody in a 2:2 molecular format. It has also been shown that cyclooctin-polypeptide constructs may have specific spacers that allow for adjustment of the IgG-polypeptide distance between cyclooctin and polypeptide, or confer other properties to the resulting bispecific antibody. [Figure 6] Figure 5 shows plots illustrating the effect of various surfactants on the conjugation efficiency of linker-drugs X1 and X2 to azide-remodeling antibodies obtained by the process shown. X1 or X2 reacts with the cyclooctin moiety containing the azide-remodeling antibody as a result of metal-free click chemistry. Clearly, the addition of surfactants improves the drug-antibody ratio (DAR). [Figures 7A-7F] This diagram shows the structure of BCN-containing linkers - drugs X1 to X12. Detailed description of the invention

[0024] definition

[0030] As used herein and in the claims, the verb “to comprise” and its conjugations are used in their non-restrictive sense to mean that they include the matter following the word, but do not exclude matters that are not specifically stated. Furthermore, references to “elements” with the indefinite article “a” or “an” do not rule out the possibility that there may be more than one element unless the context explicitly requires that there be only one element; therefore, the indefinite article “a” or “an” usually means “at least one.”

[0025]

[0031] The compounds disclosed herein and in the claims may contain one or more chiral centers, and various diastereomers and / or enantiomers of the compounds may exist. Unless otherwise stated, any description of a compound herein and in the claims includes all diastereomers and mixtures thereof. Furthermore, unless otherwise stated, any description of a compound herein and in the claims includes individual enantiomers, any mixture of enantiomers, racemic or non-racemic. When the structure of a compound is shown as a specific enantiomer, it should be understood that the invention of this application is not limited to that specific enantiomer.

[0026]

[0032] Compounds may appear in various tautomer forms. Unless otherwise stated, the compounds according to this invention are intended to include all tautomer forms. When the structure of a compound is shown as a specific tautomer, it should be understood that the invention of this application is not limited to that specific tautomer.

[0027]

[0033] The compounds disclosed herein and in the claims may further exist as exo and endodiastereomers. Unless otherwise stated, any description of a compound herein and in the claims includes both the individual exodiastereomers and individual endodiastereomers of the compound, as well as mixtures thereof. When the structure of a compound is shown as an endo or exodiastereomer, it should be understood that the invention of this application is not limited to that particular endo or exodiastereomer.

[0028]

[0034] Compounds according to the present invention may exist in the form of salts, which are also encompassed by the present invention. A salt is a pharmaceutically acceptable salt that typically contains a pharmaceutically acceptable anion. The term “its salt” means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation. Where applicable, a 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 by an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid is the anionic component of the salt.

[0029]

[0035] The term “pharmaceutically acceptable” salt means a salt that is acceptable for administration to patients, such as mammals (a salt containing a counterion that has mammalian safety acceptable for a given administration regimen). Such salts can be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. “pharmaceutically acceptable salt” refers to a pharmaceutically acceptable salt of a compound. Such salts are derived from a variety of organic and inorganic counterions known in the art, and include, for example, sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, tetraalkylammonium salts, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochlorides, hydrobromides, formates, tartrates, besylates, mesylates, acetates, maleates, and oxalates.

[0030]

[0036] In this specification, the term "protein" is used in its ordinary scientific sense. In this specification, polypeptides containing approximately 10 or more amino acids are considered proteins. Proteins may contain natural amino acids, but they may also contain non-natural amino acids.

[0031]

[0037] In this specification, the term “antibody” is used in its ordinary scientific sense. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Antibodies are an example of glycoproteins. In this specification, the term “antibody” is used in its broadest sense, specifically including monoclonal antibodies, polyclonal antibodies, dimers, multimers, polyspecific antibodies (e.g., bispecific antibodies), antibody fragments, and double-chain and single-chain antibodies. In this specification, the term “antibody” is also intended to include human antibodies, humanized antibodies, chimeric antibodies, and antibodies that specifically bind to cancer antigens. The term “antibody” includes all immunoglobulins, but is also intended to include antigen-binding fragments of antibodies. Furthermore, the term includes genetically engineered antibodies and antibody derivatives. Antibodies, antibody fragments, and genetically engineered antibodies can be obtained by methods known in the art. Typical examples of antibodies include, in particular, absiximab, rituximab, basiliximab, palivizumab, infliximab, trastuzumab, efalizumab, alemtuzumab, adalimumab, tocitumomab, cetuximab, ibrituximab, omalizumab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, certolizumab pegol, golimumab, canakinumab, catumaxomab, ustekinumab, tocilizumab, ofatumumab, denosumab, belimumab, ipilimumab, and brentuximab.

[0032]

[0038] In this specification, “antibody fragment” is defined as a portion of an intact antibody that includes its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, minibodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments(s), fragments(s) produced by Fab expression libraries, or any of the above epitope-binding fragments that bind immunospecifically to target antigens (e.g., cancer cell antigens, viral antigens, or microbial antigens).

[0033]

[0039] In this specification, "antigen" is defined as the unit to which an antibody specifically binds.

[0034]

[0040] In this specification, the terms “specific binding” and “specifically binding” are defined as a highly selective manner in which an antibody or antibody binds to its corresponding target antigen epitope and not to a large number of other antigens. Typically, an antibody or antibody derivative binds to at least about 1 × 10⁻¹⁶ antigens. -7 M, preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 It binds with affinity M, and binds to a predetermined antigen with an affinity at least twice as high as the affinity it has for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein).

[0035]

[0041] In this specification, the terms “substantial” or “substantially” are defined as the majority of a group of mixtures or samples, i.e., >50%, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the group.

[0036]

[0042] In this specification, “linker” is defined as a portion that connects two or more elements of a compound. For example, in an antibody conjugate, the antibody and payload are covalently bonded to each other via a linker. A linker may include one or more linkers and spacer portions that connect various parts within the linker.

[0037]

[0043] In this specification, a “spacer” or spacer portion is defined as a portion of a linker that provides a certain distance between two or more parts of the linker and covalently connects those parts. The linker may be, for example, a linker-structure portion, a linker conjugate, or a bioconjugate, as defined below.

[0038]

[0044] In this specification, “self-destructive group” is defined as a portion of a linker in an antibody-drug conjugate that has the function of conditionally releasing a free drug at a site targeted by a ligand unit. An activatable self-destructive moiety comprises an activatable group (AG) and a self-destructive spacer unit. Activation of the activatable group, for example, by enzymatic conversion of an amide group to an amino group or reduction of a disulfide to a free thiol group, initiates a self-destructive reaction sequence and causes the release of a free drug by one or more of various mechanisms, which may include a (transient) 1,6-elimination reaction of a p-aminobenzyl group to a p-quinone methide, and / or a subsequent second cyclization release mechanism, optionally accompanied by the release of carbon dioxide. The self-destructive assembly unit may be a portion of a chemical spacer connecting the antibody and payload (via a functional group). Alternatively, the self-destructive group may branch off from the chemical spacer connecting the antibody and payload, rather than being an inherent part of the chemical spacer.

[0039]

[0045] In this specification, “bioconjugate” is defined as a compound in which a biomolecule is covalently bonded to a payload via a linker. A bioconjugate comprises one or more biomolecules and / or one or more payloads. Antibody conjugates, such as antibody-payload conjugates and antibody-drug conjugates, are bioconjugates in which the biomolecule is an antibody.

[0040]

[0046] In this specification, “biomolecules” are defined as any molecules that can be isolated from nature, or any molecules composed of smaller building blocks, particularly nucleic acids, proteins, glycans, and lipids, which are components of macromolecular structures of natural origin. Examples of biomolecules include enzymes, (non-catalytic) proteins, polypeptides, peptides, amino acids, oligonucleotides, monosaccharides, oligosaccharides, polysaccharides, glycans, lipids, and hormones.

[0041]

[0047] The term "payload" refers to a portion that is covalently bound to the targeting portion of an antibody or similar molecule, but is also covalently bound to molecules released from the conjugate after the protein conjugate is taken up and / or the linker is cleaved. Therefore, in the context of this invention, the payload is referred to as D and also refers to the monovalent portion having one open end that is covalently bound to the targeting portion via the linker, and the molecules released therefrom.

[0042]

[0048] The term “drug-antibody ratio” or “DAR” refers to the number of payloads attached to a biomolecule. In the context of the term DAR, “drug” should not be interpreted narrowly and refers to any payload suitable in the context of the present invention, but preferably the payload is a drug. Similarly, in the context of the term DAR, “antibody” should not be interpreted narrowly and refers to any biomolecule suitable in the context of the present invention, but preferably the biomolecule is an antibody. Therefore, the term DAR is sometimes called the “payload biomolecule ratio.” Any bioconjugation reaction and the resulting bioconjugate have a theoretical DAR determined by the amount of biomolecules attached to each conjugation site and the amount of conjugation sites in the payload portion. For example, the conjugation reaction shown in Figure 5 involves an antibody having two conjugation sites (azide portions) and the attachment of one payload (polypeptide) to each conjugation site, resulting in a theoretical DAR of 2. In reality, bioconjugation reactions can have conversion rates of less than 100%, resulting in a DAR lower than the theoretical DAR for the resulting conjugate.

[0043]

[0049] The term "surfactant" or "surface-active agent" refers to a class of compounds that reduce the surface tension between two liquids. Surfactants are amphiphilic organic molecules that contain both a hydrophobic group (usually called the "tail") and a hydrophilic group (usually called the "head"). Surfactants can be nonionic, anionic, cationic, or zwitterionic. When a surfactant is part of a salt, such as sodium dodecyl sulfate (SDS), only amphiphilic ions are called surfactants; in this case, the dodecyl sulfate anion, where the dodecyl portion is lipophilic and the sulfate portion is hydrophilic, is called a surfactant. The presence of cationic sodium is further irrelevant to the surface activity of SDS, and therefore SDS is an anionic surfactant.

[0044] This invention

[0050] To our surprise, the inventors discovered that the bioconjugation reaction between a biomolecule functionalized with a click probe F, which can react with a payload functionalized with a cyclic alkyne or alkene portion Q, and that payload, can be significantly improved by adding a surfactant to the reaction mixture. In the presence of the surfactant, a higher conversion rate (and therefore yield) was obtained. Furthermore, the reaction could be carried out using less organic cosolvent and a higher concentration of biomolecules, and purification became easier. Moreover, the conjugation reaction could be carried out sufficiently with a smaller excess amount of the cyclic alkyne or alkene functionalized payload, thus reducing the number of expensive and synthetically complex molecules required in the preparation of the bioconjugate. Therefore, the present invention relates to the bioconjugation reaction in the presence of a surfactant and to the use of a surfactant in bioconjugation.

[0045]

[0051] The use of surfactants to improve the bioconjugation reaction between a cyclic alkyne or alkene compound and a hydrophilic azide moiety, as the subject of this invention, is unprecedented in the art, particularly in the context of strain-enhanced alkyne-azide rigation. The rare use of surfactants to enhance metal-free click reactions involves a hydrophobic azide moiety whose solubility is improved by the use of a surfactant. In this invention, the solubility of the azide moiety is not critical. However, the inventors have found that the improvement in conjugation reaction efficiency is remarkable for azides that are already highly soluble in aqueous systems.

[0046]

[0052] More specifically, in a first embodiment, the present invention relates to structure B-(ZLD) x A method for preparing the bioconjugate of (1), comprising (i) a cyclic alkyne or cyclic alkene compound of structure QLD(2) [wherein Q is the cyclic alkyne or cyclic alkene moiety, L is the linker, and D is the payload] and (ii) structure B-(F) xThe present invention provides a method comprising the step of reacting the molecule of (3) [in the structure, B is a biomolecule functionalized with x click probes F, where x is an integer in the range of 1 to 10] in the presence of a surfactant to form a bioconjugate in which the payload is covalently bonded to the biomolecule via a connecting group Z formed by a click reaction between click probe Q and click probe F, typically 1,3-dipolar cycloaddition or (4+2) cycloaddition.

[0047]

[0053] In a second embodiment, the present invention relates to structure B-(ZLD) x (1) Use of a surfactant in a bioconjugation reaction to prepare the bioconjugate, wherein x payloads D are covalently bonded to biomolecule B via a connecting group Z containing a cyclic alkyne or cyclic alkene moiety and a moiety formed by 1,3-dipolar addition cyclization or (4+2) cycloaddition of a click probe F, and the reaction involves (i) an alkyne compound of structure QLD(2) [wherein Q is a cyclic alkyne or cyclic alkene moiety, L is a linker, and D is a payload] and (ii) structure B-(F) x (3) Provides use between molecules [in the structure, B is a biomolecule functionalized with x click probes F, where x is an integer in the range of 1 to 10].

[0048]

[0054] As will be clear from the context of the present invention, everything defined herein for the method according to the first embodiment applies equally to the use according to the second embodiment, and vice versa.

[0049] Bioconjugation reaction

[0055] The present invention revolves around bioconjugation reactions. Bioconjugation reactions are well known in the art and relate to the covalent bonding of one or more payloads to biomolecules. In the context of the present invention, click probe Q forms a covalent bond to click probe F on a biomolecule. Such conjugation reactions with alkynes and alkene moieties Q are well known in the art as click reactions (see, for example, International Publication No. 2014 / 065661, both incorporated by reference, and Nguyen and Presser, Nature rev., 2020, doi:10.1038 / s41570-020-0205-0) and can typically take the form of 1,3-dipolar cycloaddition or (4+2) cycloaddition. Alkyne-azide cycloaddition can be strain-accelerated (e.g., strain-accelerated alkyne-azide cycloaddition, SPAAC) or catalyzed (e.g., by copper), both of which are well known. In a preferred embodiment, the bioconjugation reaction is a metal-free strain-promoting cycloaddition, most preferably a metal-free strain-promoting alkyne-azide cycloaddition.

[0050]

[0056] The bioconjugation reaction according to the present invention involves structure B-(ZLD) x (1) To form the bioconjugate [in the structure, payload D is covalently bonded to biomolecule B via a connecting group Z formed by a click reaction between Q and F], (i) a cyclic alkyne or alkene compound of structure QLD(2) [in the structure, Q contains the cyclic alkyne or alkene moiety, L is the linker, and D is the payload] and (ii) structure B-(F) x The reaction involves the molecule of (3) [in the structure, B is a biomolecule functionalized with x click probes F, where x is an integer in the range of 1 to 10]. In this specification, one structure B-(F) x The molecule in (3) reacts with x molecules of structure QLD(2). The bioconjugation reaction takes place in the presence of a surfactant.

[0051]

[0057] Therefore, biomolecules are functionalized with x reactive groups F that react with alkynes or alkenes in cycloaddition, or in other words, can form covalent bonds with alkyne or alkene moieties. Those skilled in the art recognize that such reactive groups may be selected from azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and cydonones. Preferred structures for the reactive groups are shown below, structures (F1) to (F10). [ka]

[0052]

[0058] In this specification, a waveform bond represents a connection to a biomolecule. For (F3), (F4), (F8), and (F9), a biomolecule can be connected to any one of the waveform bonds. Then, the other waveform bonds can be connected to hydrogen, C1-C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl group, C3-C 24 (hetero)arylalkyl groups and C1-C 24 It can be attached to an R group selected from sulfonyl groups, and each of these (except hydrogen) may be optionally substituted with O, S, and NR. 32 [Here, R 32The R group may be optionally interrupted by one or more heteroatoms independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Those skilled in the art will understand which R groups can be applied to each of the click probes F. For example, the R group connected to the nitrogen atom of (F3) can be selected from alkyl and aryl groups, and the R group connected to the carbon atom of (F3) can be selected from hydrogen, alkyl, aryl, acyl and sulfonyl groups. Preferably, the click probe F is selected from azide or tetrazine. Most preferably, the click probe F is azide.

[0053]

[0059] The properties of biomolecules are not limited in the context of this reaction. In preferred embodiments, biomolecules are selected from the group consisting of proteins (including glycoproteins such as antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides. More preferably, they are selected from the group consisting of proteins, polypeptides, peptides, and glycans. Preferably, the biomolecule include a polypeptide portion. Particularly preferred classes of biomolecules are glycoproteins such as antibodies, which combine hydrophilic peptide chains with hydrophilic sugar chains (glycans). In preferred embodiments, biomolecules are selected from the group consisting of antibodies, Fab, VHH, scFv, diabody, minibody, aphibody, affin, affimer, atrimer, finomer, Cys-knot, DARPin, adnectin / sentriin, notchin, anticarin, FN3, Knitz domain, OBody, bicyclic peptides, and tricyclic peptides.

[0054]

[0060] Biomolecules are preferably characterized as hydrophilic and / or water-soluble. The hydrophilicity of azides is particularly apparent when the azide moiety is attached to the monosaccharide moiety of the glycoprotein glycan. Most conveniently, the azide moiety is attached to the monosaccharide moiety of the glycan, preferably the terminal monosaccharide moiety of the glycoprotein glycan, most preferably the terminal monosaccharide moiety of the antibody glycan.

[0055]

[0061] x represents the amount of click probe F present on the biomolecule of structure (3) and is an integer in the range of 1 to 10. Preferably, x is an integer in the range of 1 to 8, more preferably x=1, 2, 3 or 4, even more preferably x=1 or 2, and most preferably x=2. The bioconjugate of structure (1) usually has the same amount of part ZLD attached to the biomolecule, although the bioconjugation reaction may sometimes be slightly incomplete. Notably, each linker L can contain more than one payload D, such as one or two payload molecules per linker L.

[0056]

[0062] Z is a linking group. The term “linking group” refers to a structural element that connects the bioconjugate of one part to the same bioconjugate of another part. In (1), Z connects biomolecule B to payload D via linker L. As those skilled in the art will understand, the exact properties of Z depend on the properties of F and Q. Preferred embodiments of Q, which are further defined below, include at least a cyclic alkyne or alkene moiety. Preferably, Q includes a cyclic alkyne moiety. The conjugation group Z can include a triazole moiety, an isoxazole moiety, a dihydroisoxazole moiety, a bicyclo[2.2.2]octa-5,7-diene-2,3-dione moiety, a bicyclo[2.2.2]octa-5-ene-2,3-dione moiety, a 7-thiabicyclo[2.2.1]hepta-2,5-diene-7,7-dioxide moiety, a 7-thiabicyclo[2.2.1]hepta-2-ene-7,7-dioxide moiety, a pyrazole moiety, a pyridine moiety, a dihydropyridine moiety, a pyridazine moiety, or a dihydropyridazine moiety. Preferred structures for conjugation group Z are shown below as (Z1) to (Z8). [ka]

[0057]

[0063] In this specification, the functional group R in (Z3), (Z7), and (Z8) is defined as hydrogen, C1-C 24 Alkyl alkyl groups, C2-C 24 Acyl group, C3~C24 a cycloalkyl group, C2-C 24 (hetero)aryl group, C3-C 24 alkyl(hetero)aryl group, C3-C 24 (hetero)arylalkyl group and C1-C 24 can be selected from a sulfonyl group, and each of these (excluding hydrogen) may be optionally substituted and interrupted by one or more heteroatoms optionally selected from O, S and NR 32 [where R 32 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups] and may be optionally interrupted. The bond of the waveform labeled with * is connected to the biomolecule, and the bonds of the other waveforms are connected to D. Those skilled in the art will understand which R groups can be applied to each of the linking groups Z. For example, the R group connected to the nitrogen atom of (Z3) can be selected from alkyl or aryl as defined above, and the R group connected to the carbon atom of (Z3) can be selected from hydrogen, alkyl, aryl, acyl and sulfonyl as defined above.

[0058]

[0064] In a particularly preferred embodiment, Q contains a cyclic alkyne moiety, F is an azide, and Z contains a triazole moiety formed by 1,3-dipolar cycloaddition of the alkyne moiety and the azide moiety.

[0059]

[0065] As described above, the use of surfactants according to the present invention brings several unexpected advantages to bioconjugation reactions. Therefore, use according to a second embodiment may be for one or more of the following purposes: (i) increasing the conversion rate of the bioconjugation reaction; (ii) increasing the yield of the bioconjugation reaction; (iii) reducing the amount of organic cosolvent in the solvent system in which the bioconjugation reaction takes place; (iv) providing flexibility in the concentration of biomolecules during the bioconjugation reaction; (v) reducing the excess amount of alkyne or alkene functionalized payload used during the bioconjugation reaction; (vi) reducing the degree of agglomeration formation during the bioconjugation reaction; and (vii) simplifying the downstream processing of the bioconjugate. In one embodiment, use according to a second embodiment is for at least increasing the conversion rate of the bioconjugation reaction. In one embodiment, use according to a second embodiment is for at least increasing the yield of the bioconjugation reaction. In one embodiment, the use according to the second aspect is for reducing the amount of organic co-solvent in the solvent system in which the bioconjugation reaction takes place. In one embodiment, the use according to the second aspect is for providing flexibility in the concentration of biomolecules during the bioconjugation reaction. In one embodiment, the use according to the second aspect is for reducing the excess amount of cyclic alkyne or alkene functionalized payload used during the bioconjugation reaction. In one embodiment, the use according to the second aspect is for reducing the degree of aggregate formation during the bioconjugation reaction. In one embodiment, the use according to the second aspect is for simplifying the downstream processing of the bioconjugate.

[0060]

[0066] As shown in the examples, the conversion rate of the bioconjugation reaction is improved when a surfactant is present in the reaction mixture. This results in a conjugate with a higher yield and also improves the drug-antibody ratio (DAR). The DAR of the obtained conjugate is closer to the theoretical DAR, which is determined by the number of conjugation sites on the biomolecule and the number of payloads per conjugation site. Therefore, the use according to a second aspect of the present invention is, in a particularly preferred embodiment, for improving the DAR, and more particularly for obtaining a DAR closer to the theoretical DAR. A DAR closer to the theoretical DAR can mean that the average DAR of the obtained conjugate is closer to the absolute value of the theoretical DAR, and that the average DAR of the obtained conjugate has a lower standard deviation, even if the average DAR is similarly farther from the theoretical DAR. The latter is particularly relevant when, when a conjugate with a theoretical DAR of 1 is prepared, a mixture of DAR0 and DAR2 conjugates can give an average DAR close to the theoretical DAR, but with a large standard deviation. The use of surfactants provides DAR1 conjugates with a low standard deviation, as shown in Examples 15 and 16.

[0061]

[0067] Such DAR improvements are made without altering the stoichiometry of the reaction partners (the alkyne or alkene compound of structure (2) and the molecule of structure (3)) in the reaction mixture. Conjugates with a DAR closer to the theoretical DAR are desirable because they are more homogeneous, i.e., they do not have a wide probability distribution of conjugates with various DARs that deviate from the theoretical DAR, including those with DARs lower than the theoretical DAR and those with DARs higher than the theoretical DAR. Firstly, low-homogeneity conjugates contain a large number of components, which not only impairs analysis (disadvantageous from a control standpoint) but also includes components that do not contribute to the desired mechanism of action of the conjugate, contribute less to it, or even have a potentially negative impact on its efficacy. For example, an antibody conjugate with a low DAR (i.e., having an antibody as a biomolecule) competes with a preferred conjugate with a higher DAR for the target receptor, but may not reach a sufficient concentration of effective catabolite in the cell because there are fewer drugs on the conjugate than the optimal number. Furthermore, antibody conjugates with a DAR higher than the optimal level and containing a hydrophobic payload (as the most cytotoxic payload) are rapidly removed from circulation, potentially leading to enhanced hepatotoxicity. Such disadvantages of antibody conjugates with cytotoxic agents are well known with many commercially available ADCs, such as Adcetris® and Kadcyla®.

[0062]

[0068] The surfactant according to the present invention makes it possible to reduce the amount of organic co-solvent used in the solvent system in which the bioconjugation reaction takes place. The amount of organic co-solvent can be reduced by 10 to 50% compared to the same reaction carried out in the absence of the surfactant. To increase antibody stability and reduce aggregation problems during the bioconjugation reaction, it is preferable to carry out the bioconjugation reaction in as little organic solvent as possible. However, due to solubility reasons, it is usually not possible to completely avoid the use of organic solvents. In the context of the present invention, the amount of organic solvent in the solvent system can be as low as 0 to 30% by volume. Therefore, in a preferred embodiment, the reaction is carried out in a solvent system containing water and organic solvent in a ratio in the range of 50 / 50 to 100 / 0 (v / v), preferably in the range of 75 / 25 to 95 / 5 (v / v). Any organic solvent suitable for bioconjugation reactions can be used, but the organic solvent is preferably selected from dimethyl sulfoxide (DMSO), N,N-dimethylaniline (DMA), dimethylformamide (DMF), propylene glycol (PG), pyridine, and N-methyl-2-pyrrolidone (NMP). Most preferably, the organic solvent is DMF or PG.

[0063]

[0069] Therefore, the use of the surfactant according to the present invention results in a reduction of aggregate formation during the bioconjugation process, such as less than 10%, preferably less than 5%, more preferably less than 3%, and most preferably less than 1%. Typically, these values ​​represent a reduction of at least 20%, more preferably at least 30%, and most preferably at least 50% in aggregate compared to the same reaction in the absence of the surfactant. The degree of aggregate can be easily determined by size exclusion chromatography of a sample of the reaction mixture.

[0064]

[0070] The use of surfactants according to the present invention further provides flexibility in the concentration of the functionalized biomolecule of structure (3) that can be used during the bioconjugation reaction. Such flexibility can take the form of an increase or decrease, both of which may be advantageous depending on the precise properties of the biomolecule. For example, surfactants allow the use of higher concentrations of the functionalized biomolecule of structure (3). From a reaction kinetics standpoint, it is preferable that the concentration of the biomolecule be as high as possible. Therefore, in a preferred embodiment, the concentration of the azide-functionalized biomolecule is in the range of 1 to 100 mg / mL, preferably in the range of 5 to 50 mg / mL, more preferably in the range of 8 to 25 mg / mL, and most preferably in the range of 10 to 20 mg / mL.

[0065]

[0071] The use of surfactants according to the present invention further enables the use of smaller excess amounts of functionalized payloads during bioconjugation reactions. Compared to the same bioconjugation reaction without surfactants, a reduction of at least 20%, and even up to 50% or more, of the functionalized payload of structure (2) can be achieved. Since cyclic alkynes and alkene functionalized payloads are typically expensive and labor-intensive to produce, the present invention improves the overall (cost) effectiveness of the bioconjugation process. Therefore, in a preferred embodiment, the functionalized payload of structure (2) is present in an excess of at most 5 times, preferably at most 3 times, more preferably at most 2 times, and most preferably at most 1.5 times, relative to the functionalized biomolecule. The stoichiometry of the functionalized payload of structure (2) should be at least 1, so that typically one molecule of the functionalized payload is available per click probe F. The present invention provides an optimal conjugation reaction with a functionalized payload with a stoichiometry close to 1. From a practical standpoint, the stoichiometry can be slightly greater than 1, such as at least 1.1 or at least 1.2. As is common in this field, the excess amount is determined stoichiometrically. Therefore, when x=2 and the excess amount=2x, the bioconjugation reaction is carried out at a rate of 4 moles of the functionalized payload of structure (2) per mole of the biomolecule of structure (3).

[0066]

[0072] The use of fewer co-solvents and smaller excess functionalization payloads during the bioconjugation reaction simplifies the downstream processing of the bioconjugate. Herein, downstream processing typically refers to the isolation and / or purification of the bioconjugate so that it can be used in clinical settings. For example, the filtration step to remove lower molecules from the bioconjugate can be reduced or made completely unnecessary. In other words, the production of suitable pharmaceuticals from the thus formed bioconjugate is simplified.

[0067]

[0073] Another advantage of using surfactants in click probe bioconjugation reactions is that, in contrast to bioconjugation via acylated lysine technology in the presence of surfactants, the number of conjugation sites on the biomolecule is not reduced and the relative distribution remains unchanged. In this invention, the number of conjugation sites, and therefore the theoretical drug-antibody ratio (DAR), is governed by the amount of click probe F present on the biomolecule (i.e., the x-value). The presence of surfactants during the bioconjugation reaction improves the reaction effectiveness as described above, but does not result in different products. Therefore, in the context of this invention, there is no difference between the generation of the biomolecular conjugate during initial development and the generation of the same biomolecular conjugate after process optimization. Furthermore, if the surfactant needs to be omitted for any reason (e.g., unavailability), it can be omitted without altering the structure of the final bioconjugate.

[0068] surfactant

[0074] The present invention utilizes a surfactant. Surfactants are well-known in the art. In a preferred embodiment, the surfactant comprises at least a negatively charged group, i.e., an anionic or zwitterionic surfactant. Most preferably, the surfactant is an anionic surfactant. Surprisingly, excellent results were obtained using an anionic surfactant. In the case of an anionic surfactant, the counterion is preferably an alkali metal cation, preferably Na. Zwitterionic surfactants can also have (positive and negative) counterions, but usually do not require a counterion and maintain their own charge balance. The negatively charged group is preferably selected from sulfate, carboxylate, and phosphate. If a positively charged group is present, it is preferably ammonium.

[0069]

[0075] In a preferred embodiment, the surfactant has the structure R 4 -X [in the structure, R 4 is selected from long-chain alkyl, alkylaryl, and cholane derivatives, and the alkyl and alkylaryl moieties may optionally be fluorinated, and X is COO (-) , SO3 (-) or PO3 (2-) . Preferably, X is COO (-) . In the context of R 4 , the alkyl is preferably a C8-C 100 alkyl moiety, preferably a C9-C 50 alkyl moiety, more preferably a C 10 -C 24 alkyl moiety. In a particularly preferred embodiment, R 4 is C 10 -C 12 alkyl or cholane, and X is COO (-) .

[0070]

[0076] Alternatively, the surfactant can be selected from the group consisting of decanoates, dodecanoates, dodecyl sulfates (e.g., SDS), deoxycholates, 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and 3-[(3-collamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO). Preferably, the surfactant is a decanoate, a dodecanoate, or a deoxycholate; more preferably, the surfactant is a decanoate or a deoxycholate; and most preferably, the surfactant is a deoxycholate. In one embodiment, the surfactant is sodium decanoate. In another embodiment, the surfactant is sodium deoxycholate.

[0071] Alkyne or alkene functionalized payloads with structure QLD(2)

[0077] The alkyne or alkene compound according to the present invention has structure QLD(2), and in the structure, Q includes a cyclic alkyne or cyclic alkene portion. L is a linker, D is the payload.

[0072]

[0078] Hereinafter, Q, L, and D are described further. Preferred embodiments of alkyne or alkene compounds having structure (2) are described further below. Particularly preferred embodiments in which Q is a cyclic alkyne are shown in Figures 7A-7F, and more preferably in Figures 7A-7C.

[0073] Click probe Q: cyclic alkynes or alkenes

[0079] ClickProbe Q constructs an alkyne- or alkene-payload construct from structure B-(F) x (3) is used in a bioconjugation process to connect to the biomolecule. Q can be a cyclic alkene or a cyclic alkyne moiety, both of which are reactive to the click probe F in a click reaction. Preferably, Q is a cyclic alkyne moiety.

[0074]

[0080] In a particularly preferred embodiment, click probe Q includes a cyclic alkyne moiety. The alkynyl group may also be called a (hetero)cycloalkynyl group, i.e., a heterocycloalkynyl group or a cycloalkynyl group, and the (hetero)cycloalkynyl group is optionally substituted. Preferably, the (hetero)cycloalkynyl group is a (hetero)cycloheptinyl group, a (hetero)cyclooctinyl group, a (hetero)cyclononinyl group or a (hetero)cyclodecynyl group. Most preferably, the (hetero)cycloalkynyl group is a (hetero)cyclooctinyl group, and the (hetero)cyclooctinyl group is optionally substituted. In this specification, alkynes and (hetero)cycloalkynes may be optionally substituted. Preferably, Q includes a (hetero)cyclooctinyl moiety corresponding to the following structure (Q1). In another preferred embodiment, the (hetero)cyclooctinyl group corresponds to the following structures (Q37), (Q38), or (Q39). Preferred examples of (hetero)cyclooctinyl groups include the structure also called the DIBO group (Q2), the structure also called the DIBAC group (Q3), or the structure also called the BARAC group (Q4), the structure also called the COMBO group (Q5), and the structure also called the BCN group (Q6), all of which are shown below, in which Y 1 is O or NR 11 And R 11 These are hydrogen, linear or branched C1-C 12 Alkyl alkyl group or C4-C 12 The group is independently selected from the group consisting of (hetero)aryl groups. The aromatic ring in (Q2) is optionally O-sulfonylated at one or more positions, and the rings of (Q3) and (Q4) may be halogenated at one or more positions. Particularly preferred cycloalkynyl groups are optionally substituted bicyclo[6.1.0]nona-4-in-9-yl] groups (BCN groups). Preferably, the bicyclo[6.1.0]nona-4-in-9-yl] group is given by the following formula (Q6) [wherein V is (CH2)] l(Q6) In the context of the base, l is most preferably 1. [ka]

[0075]

[0081] In another preferred embodiment, the click probe Q is selected from the group consisting of (Q7) to (Q21) shown below. [ka]

[0076]

[0082] In this specification, the connection to L shown in the waveform coupling can be a connection to any available carbon or nitrogen atom of Q.

[0077]

[0083] In another preferred embodiment, the click probe Q is selected from the group consisting of (Q22) to (Q36) shown below. [ka]

[0078]

[0084] In a particularly preferred embodiment, the click probe Q comprises a (hetero)cycloalkynyl group and matches the structure (Q37). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y 2 C(R 31 )2, O, S or NR 31 And R 31 Each is individually R 15 or -LD, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16.

[0079]

[0085] In a preferred embodiment, u+u'=4, 5, or 6, and more preferably u+u'=5. Typically, v=(u+u')×2 or [(u+u')×2]-1. In a preferred embodiment, v=8, 9, or 10, more preferably v=9 or 10, and most preferably v=10.

[0080]

[0086] In a particularly preferred embodiment, the click probe Q comprises an alkynyl group and matches the structure (Q38). [ka] In this specification, R 15is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) ,C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 18 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, R 19 Hydrogen, -LD; halogen, C1~C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 The alkyl group is selected from the group consisting of (hetero)arylalkyl groups, and the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted. l is an integer in the range of 0 to 10.

[0081]

[0087] In a preferred embodiment of the reactive group corresponding to structure (Q38), R 15 is hydrogen, halogen, -OR 16 , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 15 All are H. In a preferred embodiment of the reactive group corresponding to structure (Q38), R 18 is independently selected from the group consisting of hydrogen and C1-C6 alkyl groups, most preferably R 18 Both are H. In a preferred embodiment of the reactive group corresponding to structure (Q38), R 19 is H. In a preferred embodiment of the reactive group corresponding to structure (Q38), I is 0 or 1, and more preferably l is 1. A particularly preferred embodiment of the reactive group corresponding to structure (Q38) is the reactive group corresponding to structure (Q30).

[0082]

[0088] In a particularly preferred embodiment, the click probe Q contains an alkynyl group and matches the structure (Q39). [ka] In this specification, R 15 is hydrogen, halogen, -OR 16 -NO2, -CN, -S(O)2R 16 -S(O)3 (-) , C1~C 24 Alkyl alkyl groups, C5-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24Independently selected from the group consisting of (hetero)arylalkyl groups, the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R 15 These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R 16 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24 Independently selected from the group consisting of (hetero)arylalkyl groups, Y is N or CR 15 That is the case.

[0083]

[0089] In a preferred embodiment of the reactive group corresponding to structure (Q39), R 15 is hydrogen, halogen, -OR 16 -S(O)3 (-) , independently selected from the group consisting of C1-C6 alkyl groups and C5-C6 (hetero)aryl groups, R 16 is hydrogen or a C1-C6 alkyl group, more preferably R 15 It consists of hydrogen and -S(O)3 (-) It is independently selected from the group consisting of the following. In a preferred embodiment of the reactive group corresponding to structure (Q39), Y is N or CH, and more preferably Y=N.

[0084]

[0090] In an alternative preferred embodiment, the click probe Q includes a cyclic alkene moiety. The alkenyl group Q may also be called a (hetero)cycloalkenyl group, i.e., a heterocycloalkenyl group or a cycloalkenyl group, preferably a cycloalkenyl group, and the (hetero)cycloalkenyl group is optionally substituted. Preferably, the (hetero)cycloalkenyl group is a (hetero)cyclopropenyl group, a (hetero)cyclobutenyl group, a trans-(hetero)cycloheptenyl group, a trans-(hetero)cyclooctenyl group, a trans-(hetero)cyclononenyl group, or a trans-(hetero)cyclodecynyl group, all of which may be optionally substituted. Particularly preferred are a (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctenyl group, and the (hetero)cyclopropenyl group, a trans-(hetero)cycloheptenyl group, or a trans-(hetero)cyclooctinyl group is optionally substituted. Preferably, Q includes a cyclopropenyl moiety corresponding to structure (Q40), a trans-(hetero)cycloheptenyl moiety corresponding to structure (Q41), or a trans-(hetero)cyclooctenyl moiety corresponding to structure (Q42). In another preferred embodiment, the cyclopropenyl group corresponds to structure (Q43). In another preferred embodiment, the trans-(hetero)cycloheptene group corresponds to structure (Q44) or (Q45). In another preferred embodiment, the trans-(hetero)cyclooctene group corresponds to structures (Q46), (Q47), (Q48), (Q49), or (Q50). [ka]

[0085]

[0091] In this specification, the R group(s) of Si in (Q44) and (Q45) are typically alkyl or aryl, preferably C1-C6 alkyl. Linker L

[0086]

[0092] Linkers, also called linking units, are well known in the art, and any suitable linker can be used. In the final cyclic alkyne-or alkene-linker-payload construct, the payload is chemically connected to the cyclic alkene or alkyne via a cleavable or incleavable linker. The linker may include one or more branching points for multiple payloads to attach to a single cyclic alkene or cyclic alkyne. Preparation of cyclic alkyne-or alkene-linker-drugs can be achieved by the chemical methods described herein.

[0087]

[0093] Linkers are, for example, linear or branched C1-C 200 Alkylene group, C2~C 200 Alkenylene group, C2~C 200 Alkynylene group, C3~C 200 Cycloalkylene group, C5~C 200 Cycloalkenylene group, C8~C 200 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group, C9~C 200 The group can be selected from the group consisting of arylalkylene groups.Optionally, alkylene groups, alkenylene groups, alkylylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkylene groups may be substituted, and optionally, the group may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, wherein the heteroatoms are preferably O, S(O) y and NR 12 Selected from the group consisting of, where y is 0, 1, or 2, preferably y=2, R 12 These are hydrogen, halogens, C1-C 24 Alkyl alkyl groups, C6-C 24 (hetero)aryl group, C7~C 24 Alkyl (hetero)aryl groups and C7-C 24The linker is independently selected from the group consisting of (hetero)arylalkyl groups. The linker may include (poly)ethylene glycol diamine (e.g., 1,8-diamino-3,6-dioxaoctane or equivalents containing longer ethylene glycol chains), (poly)ethylene glycol or (poly)ethylene oxide chains, (poly)propylene glycol or (poly)propylene oxide chains, and 1,z-diaminoalkanes, where z is the number of carbon atoms in the alkane, which can be, for example, in the range of 2 to 25.

[0088]

[0094] In a preferred embodiment, the linker L comprises a sulfamide group, preferably a sulfamide group corresponding to structure (L1). [ka]

[0089]

[0095] The dashed line represents the connection to the remainder of the compound, typically Q and D, via an optional spacer. Preferably, (O) a The C(O) portion is connected to Q, NR 13 The part is connected to D.

[0090]

[0096] In structure (L1), a=0 or 1, preferably a=1, and R 13 is hydrogen, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 Selected from the group consisting of (hetero)arylalkyl groups, C1~C 24 Alkyl alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (hetero)aryl group, C3~C 24 Alkyl (hetero)aryl groups and C3-C 24 (Hetero)arylalkyl groups are O, S and NR 14 [Here, R 14[is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups] and is optionally substituted and optionally interrupted by one or more heteroatoms selected from or R 13 This is the spacer portion, preferably Sp as defined below. 2 This is the second appearance of D connected to N via .

[0091]

[0097] In a preferred embodiment, R 13 is hydrogen or C1~C 20 It is an alkyl group, more preferably R 13 is hydrogen or C1~C 16 It is an alkyl group, and more preferably R 13 is hydrogen or C1~C 10 It is an alkyl group, and alkyl groups are O, S and NR 14 [Here, R 14 R is optionally substituted and optionally interrupted by one or more heteroatoms, preferably O, selected independently from the group consisting of hydrogen and C1-C4 alkyl groups. In a preferred embodiment, R 13 is hydrogen. In another preferred embodiment, R 13 C1~C 20 Alkyl groups, fuaC1~C 16 Alkyl alkyl groups, more preferably C1-C 10 The alkyl group is optionally interrupted by one or more oxygen atoms, and the alkyl group is optionally substituted with an -OH group, preferably a terminal -OH group. In this embodiment, R 13 It is more preferably a (poly)ethylene glycol chain containing terminal -OH groups. In another preferred embodiment, R 13 R is selected from the group consisting of hydrogen, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl and t-butyl, more preferably from the group consisting of hydrogen, methyl, ethyl, n-propyl and i-propyl, and even more preferably from the group consisting of hydrogen, methyl and ethyl. 13 is hydrogen or methyl, most preferably R13 It is hydrogen.

[0092]

[0098] In a preferred embodiment, the linker matches the structure (L2). [ka]

[0093]

[0099] In this specification, a, R 13 And the wavy line is defined as above, Sp 1 and Sp 2 The spacer portion is independent of the spacer portion, and b and c are independently 0 or 1. Preferably, b=0 or 1 and c=1, and more preferably b=0 and c=1. In one embodiment, spacer Sp 1 and Sp 2 These are linear or branched C1-C 200 Alkylene group, C2~C 200 Alkenylene group, C2~C 200 Alkynylene group, C3~C 200 Cycloalkylene group, C5~C 200 Cycloalkenylene group, C8~C 200 Cycloalkylene group, C7~C 200 Alkyl arylene group, C7~C 200 Arylalkylene group, C8~C 200 Arylalkenylene group and C9~C 200 Independently selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, and arylalkylene group are O, S and NR 20 [Here, R 20 is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24The group is optionally substituted and optionally interrupted by one or more heteroatoms selected from the group consisting of cycloalkyl groups, where the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted. When an alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylylene group, arylalkylene group, and arylalkylynylene group are interrupted by one or more heteroatoms as defined above, it is preferable that the group is interrupted by one or more oxygen atoms and / or one or more SS groups.

[0094]

[0100] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are linear or branched C1-C 100 Alkylene group, C2~C 100 Alkenylene group, C2~C 100 Alkynylene group, C3~C 100 Cycloalkylene group, C5~C 100 Cycloalkenylene group, C8~C 100 Cycloalkylene group, C7~C 100 Alkyl arylene group, C7~C 100 Arylalkylene group, C8~C 100 Arylalkenylene group and C9~C 100 Independently selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, and arylalkylene group are O, S and NR 20 [Here, R 20 is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group s and C3~C 24[Independently selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted] and optionally interrupted by one or more heteroatoms selected from the group consisting of cycloalkyl groups.

[0095]

[0101] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are linear or branched C1-C 50 Alkylene group, C2~C 50 Alkenylene group, C2~C 50 Alkynylene group, C3~C 50 Cycloalkylene group, C5~C 50 Cycloalkenylene group, C8~C 50 Cycloalkylene group, C7~C 50 Alkyl arylene group, C7~C 50 Arylalkylene group, C8~C 50 Arylalkenylene group and C9~C 50 Independently selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, and arylalkylene group are O, S and NR 20 [Here, R 20 is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 [Independently selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted] and optionally interrupted by one or more heteroatoms selected from the group consisting of cycloalkyl groups.

[0096]

[0102] More preferably, the spacer portion Sp 1 and Sp 2 If present, they are linear or branched C1-C 20Alkylene group, C2~C 20 Alkenylene group, C2~C 20 Alkynylene group, C3~C 20 Cycloalkylene group, C5~C 20 Cycloalkenylene group, C8~C 20 Cycloalkylene group, C7~C 20 Alkyl arylene group, C7~C 20 Arylalkylene group, C8~C 20 Arylalkenylene group and C9~C 20 Independently selected from the group consisting of arylalkylene groups, the alkylene group, alkenylene group, alkylylene group, cycloalkylene group, cycloalkenylene group, cycloalkylylene group, alkylarylene group, arylalkylene group, and arylalkylene group are O, S and NR 20 [Here, R 20 is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 [Independently selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group, and cycloalkyl group are optionally substituted] and optionally interrupted by one or more heteroatoms selected from the group consisting of cycloalkyl groups.

[0097]

[0103] In these preferred embodiments, the alkylene group, alkenylene group, alkylynylene group, cycloalkylene group, cycloalkenylene group, cycloalkylynylene group, alkylarylene group, arylalkenylene group and arylalkylynylene group are unsubstituted, and O, S and NR 20 [Here, R 20 It is even more preferable that the group is optionally interrupted by one or more heteroatoms, preferably O, which are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, and preferably hydrogen or methyl.

[0098]

[0104] Most preferably, the spacer portion Sp1 and Sp 2 If present, they are linear or branched C1-C 20 Independently selected from the group consisting of alkylene groups, the alkylene groups are O, S and NR 20 [Here, R 20 is hydrogen, C1~C 24 Alkyl alkyl groups, C2-C 24 Alkenyl group, C2~C 24 Alkynyl group and C3-C 24 [Independently selected from the group consisting of cycloalkyl groups, the alkyl group, alkenyl group, alkynyl group and cycloalkyl group are optionally substituted] and optionally interrupted by one or more heteroatoms selected from the group consisting of cycloalkyl groups. In this embodiment, the alkylene group is unsubstituted, and O, S and NR 20 [Here, R 20 It is even more preferable that the group is optionally interrupted by one or more heteroatoms, preferably O and / or SS, which are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, and preferably hydrogen or methyl.

[0099]

[0105] Another class of suitable linkers includes cleavable linkers. Cleavable linkers are well known in the art. For example, Shabat et al., Soft Matter, 2012, 6, 1073, incorporated herein by reference, disclose a cleavable linker comprising a self-destructive moiety released after a biological trigger, such as enzymatic cleavage or an oxidative event. Some examples of suitable cleavable linkers are peptide-linkers that are cleaved after specific recognition by proteases, such as cathepsin, plasmin, or metalloproteinases, or glycoside-based linkers that are cleaved after specific recognition by glycosidases, such as glucolonidase, or aromatic nitro compounds reduced in oxygen-deficient, hypoxic regions.

[0100]

[0106] Linker L may further include peptide spacers known in the art, preferably dipeptide or tripeptide spacers known in the art, preferably dipeptide spacers. Any dipeptide or tripeptide spacer can be used, but the peptide spacer is preferably selected from Val-Cit, Val-Ala, Val-Lys, Val-Arg, AcLys-Val-Cit, AcLys-Val-Ala, Phe-Cit, Phe-Ala, Phe-Lys, Phe-Arg, Ala-Lys, Leu-Cit, Ile-Cit, Trp-Cit, Ala-Ala-Asn, Ala-Asn, more preferably Val-Cit, Val-Ala, Val-Lys, Phe-Cit, Phe-Ala, Phe-Lys, Ala-Ala-Asn, and more preferably Val-Cit, Val-Ala, Ala-Ala-Asn. In one embodiment, the peptide spacer is Val-Cit. In another embodiment, the peptide spacer is Val-Ala. The peptide spacer can also be attached to the payload, and the amino terminus of the peptide spacer is conveniently used as an amine group in the method according to the first aspect of the present invention.

[0101]

[0107] In a preferred embodiment, the peptide spacer is represented by the general structure (L3). [ka]

[0102]

[0108] In this specification, R 17 =CH3(Val) or CH2CH2CH2NHC(O)NH2(Cit). The dashed lines indicate connections to the rest of the molecule, and preferably the peptide spacer matching structure (L3) is connected to Q via NH and to D via C(O).

[0103]

[0109] Linker L may further include a self-cleaving spacer, also known as a self-destructive spacer. The self-cleaving spacer can also be attached to the payload. Preferably, the self-cleaving spacer is a para-aminobenzyloxycarbonyl (PABC) derivative, more preferably a PABC derivative matching structure (L4). [ka]

[0104]

[0110] In this specification, wavy lines indicate connections to the remainder of the molecule. Typically, PABC derivatives are connected to Q via NH, typically via spacers, and to D via OC(O), typically via spacers.

[0105]

[0111] R 21 H, R 22 or C(O)R 22 And R 22 C1~C 24 (hetero)alkyl groups, C3-C 10 (hetero)cycloalkyl groups, C2-C 10 (hetero)aryl group, C3~C 10 Alkyl (hetero)aryl groups and C3-C 10 These are (hetero)arylalkyl groups, and they are O, S, and NR 23 It is optionally substituted and optionally interrupted by one or more heteroatoms selected from R 23 is independently selected from the group consisting of hydrogen and C1-C4 alkyl groups. Preferably, R 22 C3~C 10 It is a (hetero)cycloalkyl or polyalkylene glycol. The polyalkylene glycol is preferably polyethylene glycol or polypropylene glycol, more preferably -(CH2CH2O) s H or -(CH2CH2CH2O) s H is the most preferred polyalkylene glycol, preferably polyethylene glycol, preferably -(CH2CH2O) sH is an integer in the range of 1 to 10, preferably 1 to 5, most preferably s = 1, 2, 3, or 4. More preferably R 21 is H or C(O)R 22 And R 22 =4-methylpiperazine or morpholine. Most preferably, R 21 H is H.

[0106] Payload D

[0112] Linker L connects a cyclic alkyne or alkene Q to payload D. The payload molecule is known in the art, particularly in the field of antibody-drug conjugates, as a portion that covalently binds to an antibody and is released therefrom after the conjugate is taken up and / or the linker is cleaved. In preferred embodiments, the payload is selected from the group consisting of an active substance, a reporter molecule, a polymer, a solid surface, a hydrogel, nanoparticles, microparticles, and biomolecules. Particularly preferred payloads are the active substance and the reporter molecule, particularly the active substance.

[0107]

[0113] In this specification, the term “active substance” refers to pharmacological and / or biological substances, i.e., substances that are biological and / or pharmaceutically active, such as drugs, prodrugs, cytotoxins, diagnostic agents, proteins, peptides, polypeptides, peptide tags, amino acids, glycans, lipids, vitamins, steroids, nucleotides, nucleosides, polynucleotides, RNA, or DNA. Examples of peptide tags include cell-permeable peptides such as human lactoferrin or polyarginine. An example of a glycan is oligomannose. An example of an amino acid is lysine.

[0108]

[0114] When the payload 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, particularly low to medium molecular weight compounds (e.g., about 200 to about 2500 Da, preferably about 300 to about 1750 Da). In another 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 alkaloids, anthracyclines, camptothecin, doxorubicin, daunorubicin, taxanes, calicheamycin, tubulicin, irinotecan, inhibitory peptides, amanitin, debuganin, duocalmycin, meitansine, auristatin, enediyne, pyrrolobenzodiazepines (PBDs) or indolinobenzodiazepine dimers (IGN) or PNU159,682 and their derivatives. Preferred payloads are selected from MMAE, MMAF, exatecan, SN-38, DXd, maytansinoid, calicheamicin, PNU159,685, and PBD dimer. Particularly preferred payloads are PBD, SN38, MMAE, exatecan, or DXd. In one embodiment, the payload is MMAE. In one embodiment, the payload is exatecan or DXd. In one embodiment, the payload is SN-38. In one embodiment, the payload is MMAE. In one embodiment, the payload is PBD dimer.

[0109]

[0115] In this specification, the term “reporter molecule” refers to a molecule whose presence is readily detectable, such as a diagnostic agent, dye, fluorophore, radioisotope label, contrast agent, magnetic resonance imaging agent, or mass label.

[0110]

[0116] A variety of fluorophores, also known as fluorescent probes, are known to those skilled in the art. Some fluorophores are described in more detail, for example, in G.T. Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition, 2013, Chapter 10: "Fluorescent probes", pages 395 - 463, which is incorporated by reference. Examples of fluorophores include all types of Alexa Fluor (e.g., Alexa Fluor 555), cyanine dyes (e.g., Cy3 or Cy5) and cyanine dye derivatives, coumarin derivatives, fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, boron dipyrromethene derivatives, pyrene derivatives, naphthalimide derivatives, phycobiliprotein derivatives (e.g., allophycocyanin), chromomycin, lanthanide chelates, and quantum dot nanocrystals.

[0111]

[0117] Examples of radioisotope labels include 99m Tc, 111 In, 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 are included. 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 ,N3 , 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 HYNIC (hydrazinonicotinamide) and the like, via a chelating moiety. Isotopic labeling techniques are known to those skilled in the art and are described in more detail, for example, in G. T. Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition, 2013, Chapter 12: "Isotopic labelling techniques", pages 507 - 534, which is incorporated herein by reference.

[0112]

[0118] Polymers suitable for use as payload D in the compounds according to the present invention are known to those skilled in the art, and some examples are described in more detail, for example, in G. T. Hermanson, "Bioconjugate Techniques", Elsevier, 3rd edition, 2013, Chapter 18: "PEGylation and synthetic polymer modification", pages 787 - 838, which is incorporated herein by reference. When payload D is a polymer, payload D is preferably selected independently from the group consisting of poly(ethylene glycol) (PEG), polyethylene oxide (PEO), polypropylene glycol (PPG), polypropylene oxide (PPO), 1,x-diaminoalkane polymers [where x is the number of carbon atoms of the alkane, preferably x is an integer in the range of 2 - 200, preferably 2 - 10], (poly)ethylene glycol diamines (e.g., 1,8-diamino-3,6-dioxaoctane and equivalents containing longer ethylene glycol chains), polysaccharides (e.g., dextran), poly(amino acids) (e.g., poly(L-lysine)) and poly(vinyl alcohol).

[0113]

[0119] Solid surfaces suitable for use as payload D are known to those skilled in the art. These solid surfaces include, for example, functional surfaces (e.g., surfaces of nanomaterials, carbon nanotubes, fullerenes, or viral capsids), metallic surfaces (e.g., titanium, gold, silver, copper, nickel, tin, rhodium, or zinc surfaces), metallic alloy surfaces (where the alloy is derived from, for example, aluminum, bismuth, chromium, cobalt, copper, gallium, gold, indium, iron, lead, magnesium, mercury, nickel, potassium, plutonium, rhodium, scandium, silver, sodium, titanium, tin, uranium, zinc, and / or zirconium), polymer surfaces (where the polymer is, for example, polystyrene, polyvinyl chloride, polyethylene, polypropylene, poly(dimethylsiloxane) or polymethyl methacrylate, polyacrylamide), glass surfaces, silicone surfaces, and chromatography holder surfaces (where the chromatography holder is, for example, silica holder, agarose holder, cellulose holder, or alumina holder). When the payload D is a solid surface, it is preferable that D is independently selected from the group consisting of functional surfaces or polymer surfaces.

[0114]

[0120] Hydrogels are known to those skilled in the art. A hydrogel is a water-swellable network formed by crosslinking between polymer components. See, for example, ASHoffman, Adv. Drug Delivery Rev., 2012, 64, 18, incorporated by reference. When the payload is a hydrogel, the hydrogel is preferably composed of poly(ethylene) glycol (PEG) as the polymer base.

[0115]

[0121] Microparticles and nanoparticles suitable for use as payload D are known to those skilled in the art. Various suitable microparticles and nanoparticles are described, for example, in G. Thermanson, "Bioconjugate Techniques," Elsevier, 3rd edition, 2013, Chapter 14: "Microparticles and nanoparticles," pp. 549-587, incorporated by reference. Microparticles or nanoparticles can take any shape, for example, spheres, rods, tubes, cubes, triangles, and cones. Preferably, microparticles or nanoparticles take a spherical shape. The chemical composition of microparticles and nanoparticles can vary. When payload D is microparticles or nanoparticles, the microparticles or nanoparticles are, for example, polymer microparticles or nanoparticles, silica microparticles or nanoparticles, or gold microparticles or nanoparticles. When the particles are polymer micro or nanoparticles, the polymer is preferably a polystyrene or costyrene polymer (e.g., a copolymer of styrene and divinylbenzene, butadiene, acrylate and / or vinyltoluene), polymethyl methacrylate (PMMA), polyvinyltoluene, poly(hydroxyethyl methacrylate (pHEMA) or poly(ethylene glycol dimethacrylate / 2-hydroxyethyl methacrylate) [poly(EDGMA / HEMA)]. Optionally, the surface of the micro or nanoparticles is modified, for example, by graft polymerization of a secondary polymer or covalent bonding of another polymer or spacer portion using a cleaning agent.

[0116]

[0122] Payload D can also be a biomolecule. Biomolecules and preferred embodiments thereof are described in more detail below. When Payload D is a biomolecule, it is preferably selected from the group consisting of proteins (including glycoproteins such as antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides.

[0117]

[0123] In the context of the present invention, a cytotoxic payload is particularly preferred. Therefore, D is preferably a cytotoxin, and more preferably selected from the group consisting of colchicine, vinca alkaloids, anthracyclines, camptothecin, doxorubicin, daunorubicin, taxanes, calicheamicin, tubulicin, irinotecan, inhibitory peptides, amanitin, amatoxin, debuganin, duocalmycin, epothilon, mitomycin, combretastatin, meitansine, auristatin, enediyne, pyrrolobenzodiazepine (PBD) or indolinobenzodiazepine dimer (IGN), or PNU159,682. In a particularly preferred embodiment, D is MMAE or exatecan.

[0118]

[0124] A cyclic alkyne or alkene compound of structure QLD(2) is preferably represented by a structure selected from the group consisting of (2a) to (2t). [ka]

[0119]

[0125] In another preferred embodiment, the cyclic alkyne or alkene compound of structure QLD(2) is represented by a structure selected from the group consisting of (2aa) to (2ba). [ka]

[0120]

[0126] As described above, the definitions of L and D and preferred embodiments apply equally to compounds of structures (2a) to (2t) and (2aa) to (2ba). The R group(s) of Si in (2aq) and (2av) are typically alkyl or aryl, preferably C1 to C6 alkyl. Particularly preferred cyclic alkyne or alkene compounds of structure QLD(2) in the context of the present invention are shown in Figures 7A to 7F, and more preferably in Figures 7A to 7C. [Examples]

[0121]

[0127] The present invention will be explained by the following examples. General Procedure for Analytical RP-HPLC

[0122]

[0128] Prior to RP-HPLC analysis, IgG (10 μL, 1 mg / mL in PBS, pH 7.4) was added to 12.5 mM DTT, 100 mM TrisHCl pH 8.0 (40 μL) and incubated at 37°C for 15 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, and 2% formic acid (50 μL). RP-HPLC analysis was performed using an Agilent 1100 series (Hewlett Packard). The sample (10 μL) was injected at 0.5 mL / min into a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm (Waters) column at a column temperature of 70°C. A linear gradient of 0.1% TFA and acetonitrile in water from 30% to 54% was applied over 16.8 minutes.

[0123] General Procedures for Analytical SECs

[0129] HPLC-SEC analysis was performed using an Agilent 1100 series HPLC system (Hewlett Packard) with an Xbridge BEH200A column (3.5 μM, 7.8 × 300 mM, PN 186007640 Waters). Samples were diluted to 1 mg / mL in PBS and measured for 16 minutes using the isocratic method at 0.86 mL / min (0.1 M sodium phosphate buffer containing 10% isopropanol, pH 6.9 (NaHPO4 / Na2PO4)).

[0124] General procedure for mass spectrometry analysis of monoclonal antibodies

[0130] Prior to mass spectrometry, IgG was treated with IdeS. This enabled the analysis of Fc / 2 fragments. For Fc / 2 fragment analysis, a solution of 20 μg of (modified) IgG was incubated in 10 μL total volume with IdeS / Fabricator™ (1.25 U / μL) pH 6.6 in PBS at 37°C for 1 hour. The sample was diluted to 80 μL and then subjected to electrospray ionization time-of-flight (ESI-TOF) analysis using JEOL AccuTOF. Deconvolution spectra were obtained using Magtran software.

[0125] General Procedure for Analytical RP-UPLC

[0131] Prior to RP-UPLC analysis, IgG (10 μL, 1 mg / mL in PBS, pH 7.4) was added to 12.5 mM DTT, 100 mM Tris.HCl pH 8.0 (40 μL) and incubated at 37°C for 15 minutes. The reaction was quenched by adding 49% acetonitrile, 49% water, and 2% formic acid (50 μL). RP-UPLC analysis was performed using a Waters Acquity UPLC-SQD. The sample (5 μL) was injected at 0.4 mL / min into a Bioresolve RP mAb 2.1 × 150 mm 2.7 μm column (Waters) at a column temperature of 70°C. A linear gradient of 0.1% TFA and acetonitrile in water from 30% to 54% was applied over 9 minutes.

[0126] General procedure for analytical RP-UPLC: pre-quenching with 1-azidomethylpyrene.

[0132] Prior to RP-UPLC analysis, 5 μL of a DMF solution of 1 mM 1-azidomethylpyrene (TCI Europe) was added to the IgG solution (50 μL, 1 mg / mL in PBS, pH 7.4), and the mixture was incubated at room temperature for 4 hours. The mixture was spin-filtered into PBS, and RP-UPLC analysis of the intact sample was performed using a Waters Acquity UPLC-SQD. The sample (5 μL) was injected at 0.4 mL / min onto a BioResolve RP mAb 2.1 × 150 mm 2.7 μm (Waters) column at a column temperature of 70 °C. A linear gradient of 30→54% acetonitrile in 0.1% TFA and water was applied over 9 minutes.

[0127] Example 1. Synthetic Preparation

[0133] Compounds X1 and X2 were prepared according to Verkade et al., Antibodies, 2018, 12, doi:10.3390 / antib7010012. Compounds X5A, X9 and X10 were prepared according to International Publication No. 2019 / 110725 (compounds 150, 140 and 157, respectively). Compound X6 was prepared according to International Publication No. 2018 / 146189 (compound 4). Compound X8 was prepared according to International Publication No. 2017 / 137457 (compound 56). Compounds X11 and X12 were prepared according to International Publication No. 2021 / 144313 (compounds 137 and 304, respectively).

[0128] Example 2. Enzymatic Remodeling of Rituximab to Rituximab-(6-N3-GalNAc)2

[0134] Rituximab (15 mg / mL) was incubated with EndoSH (1% (w / w)) as described in international application PCT / EP2017 / 052792 (International Publication No. 2017 / 137459), His-TnGalNAcT (5% (w / w)) as described in international application PCT / EP2016 / 059194 (International Publication No. 2016 / 170186), and UDP 6-N3-GalNAc (25 equivalents compared to IgG) prepared according to international application PCT / EP2016 / 059194 (International Publication No. 2016 / 170186) in a TBS containing 10 mM MnCl2 for 16 hours at 30°C. The functionalized IgG was then purified using a HiTrap MabSelect Sure 5 mL column. After loading the reaction mixture, the column was washed with TBS + 0.2% Triton and TBS. IgG was eluted with 0.1M glycine-HCl pH 2.7 and neutralized with 1M Tris-HCl pH 8.8. After dialyzing three times to PBS, IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0129] Example 3. Enzymatic remodeling of trastuzumab to trastuzumab-(6-N3-GalNAc)2

[0135] Trastuzumab (15 mg / mL) was incubated with EndoSH (1% (w / w)) as described in international application PCT / EP2017 / 052792 (International Publication No. 2017 / 137459), His-TnGalNAcT (5% (w / w)) as described in international application PCT / EP2016 / 059194 (International Publication No. 2016 / 170186), and UDP 6-N3-GalNAc (25 equivalents compared to IgG) prepared according to international application PCT / EP2016 / 059194 (International Publication No. 2016 / 170186) in TBS containing 10 mM MnCl2 for 16 hours at 30°C. The functionalized IgG was then purified using a HiTrap MabSelect Sure 5 mL column. After loading the reaction mixture, the column was washed with TBS + 0.2% Triton and TBS. IgG was eluted with 0.1M glycine-HCl pH 2.7 and neutralized with 1M Tris-HCl pH 8.8. After dialyzing three times to PBS, IgG was concentrated to 15-20 mg / mL using a Vivaspin Turbo 15 ultrafiltration unit (Sartorius).

[0130] Example 4. Screening of various surfactants at an antibody concentration of 10 mg / mL.

[0136] Rituximab-(6-N3-GalNAc)2 (10 mg / mL, 0.2 mg) was incubated overnight with compound X1 or compound X2 (0.125-0.2 mM (2-3 equivalents)) and 10% DMF. Optionally, sodium deoxycholate (11 mM), sodium decanoate (37.5 mM), or CHAPS (12 mM) was added. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the drug-to-antibody ratio (DAR). The results are shown in Figure 6.

[0131] Example 5. Comparison with compound X1 (structure in Figure 7A) at 15 mg / mL and 10% DMF.

[0137] Rituximab-(6-N3-GalNAc)2 (15 mg / mL, 0.2 mg) was incubated overnight with X1 (0.26 mM, 3 equivalents) and 10% DMF, and either sodium decanoate (37.5 mM) or sodium deoxycholate (11 mM) was added. After 16 hours, the reaction was analyzed by (reduced) RP-HPLC to determine the DAR. The results are shown in the table below. [Table 1]

[0132] Example 6. Comparison with compound X2 (structure in Figure 7A) at 15 mg / mL and 10% DMF.

[0138] Rituximab-(6-N3-GalNAc)2 (15 mg / mL, 0.2 mg) was incubated overnight with X2 (0.3 mM, 3 equivalents) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reaction was analyzed by (post-reduction) RP-HPLC to determine the DAR. The results are shown in the table below. [Table 2]

[0133] Example 7. Conjugation with X2 in propylene glycol (PG)

[0139] Trastuzumab-(6-N3-GalNAc)2 (10 mg / mL, 0.2 mg) was incubated overnight with 0.4 mM (6 equivalents) or 0.33 mM (5 equivalents) of X2 and 30% PG, either without additives or with 11 mM sodium deoxycholate. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. [Table 3]

[0134] Example 8. Comparison with compound X1 at 15 mg / mL, 10 mg / mL, and 5% DMF.

[0140] Trastuzumab-(6-N3-GalNAc)2 (10 mg / mL, 15 mg / mL, 0.2 mg) was incubated overnight with X1 (0.2-0.3 mM, 3 equivalents) and 5% DMF, and sodium deoxycholate (22 mM) was added. After 16 hours, the reaction was analyzed by (reduced) RP-HPLC to determine the DAR. The results are shown in the table below. [Table 4]

[0135] Example 9. Conjugation with X2 in propylene glycol (PG)

[0141] Trastuzumab-(6-N3-GalNAc)2 (10 mg / mL, 0.2 mg) was incubated overnight with 0.33 mM (5 equivalents) of X2, 20%, 25%, and 30% of PG, and 11 mM and 22 mM of sodium deoxycholate. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. [Table 5]

[0136] Example 10. Comparison with compound X5A at 15 mg / mL and 10% DMF.

[0142] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 0.3 mg) was incubated overnight with X5A (2 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. The results are shown in the table below. A clear improvement in DAR was observed when sodium deoxycholate was used during conjugation. [Table 6]

[0137] Example 11. Comparison with compound X6 at 10 mg / mL and 10% DMF.

[0143] Trastuzumab-(6-N3-GalNAc)2 (10 mg / mL, 0.3 mg) was incubated overnight with X6 (2 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. The results are shown in the table below. A clear improvement in DAR was observed when sodium deoxycholate was used during conjugation. [Table 7]

[0138] Example 12. Comparison with compound X8 at 15 mg / mL and 10% DMF.

[0144] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 0.3 mg) was incubated overnight with X8 (2 or 3 equivalents relative to the antibody) and 10% DMF. CHAPS (12 mM), sodium deoxycholate (11 mM), or sodium decanoate (37.5 mM) were added at will. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the drug-antibody ratio (DAR). The results are shown in the table below. Significant improvement in DAR was observed when sodium deoxycholate or sodium decanoate was used during conjugation. [Table 8]

[0139] Example 13. Comparison with compound X9 at 15 mg / mL and 10% DMF.

[0145] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 0.3 mg) was incubated overnight with X9 (2 or 3 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. The results are shown in the table below. A significant improvement in DAR was observed when sodium deoxycholate was used during conjugation. [Table 9]

[0140] Example 14. Comparison with compound X10 at 15 mg / mL and 10% DMF.

[0146] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 0.3 mg) was incubated overnight with X10 (2 or 3 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR. The results are shown in the table below. A clear improvement in DAR was observed when sodium deoxycholate was used during conjugation. [Table 10]

[0141] Example 15. Comparison with compound X11 at 5 mg / mL and 10% DMF.

[0147] Trastuzumab-(6-N3-GalNAc)2 (5 mg / mL, 0.3 mg) was incubated overnight with X11 (1.5 or 2.5 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reactions were analyzed by RP-HPLC analysis (after DTT reduction) to determine the DAR, and by RP-HPLC analysis (of intact samples) to determine the relative amounts of "DAR0" (non-conjugate antibody), "DAR1" (closed DAR1 conjugate where two click reactions occurred between both azide moieties of the single antibody and both BCN moieties of the single compound X11; and open DAR1 conjugate where one click reaction occurred between the azide moiety and the BCN moiety), and "DAR2" conjugate (where both azide moieties of the single antibody reacted with two different BCN moieties of compound X11). The results are shown in the table below. A clear improvement in %DAR1 was observed when sodium deoxycholate was used during conjugation. [Table 11]

[0142] Example 16. Comparison with compound X12 at 5 mg / mL and 10% DMF.

[0148] Trastuzumab-(6-N3-GalNAc)2 (5 mg / mL, 0.3 mg) was incubated overnight with X12 (1.5 or 2.5 equivalents relative to the antibody) and 10% DMF, and sodium deoxycholate (11 mM) was optionally added. After 16 hours, the reactions were analyzed by RP-UPLC analysis (of intact samples after quenching with 1-azidomethylpyrene) to determine the relative amounts of "DAR0" (non-conjugate antibody), "DAR1" (closed DAR1 conjugate where two click reactions occurred between the azide moieties of the single antibody and the BCN moieties of the single compound X12), and "other" conjugates (open DAR1 conjugate where a single click reaction occurred between the azide moiety and the BCN moiety; and DAR2 conjugate where the azide moieties of the single antibody reacted with two different BCN moieties of compound X12). The results are shown in the table below. A clear improvement in %DAR1 was observed when sodium deoxycholate was used during conjugation. [Table 12]

[0143] Example 17. Conjugation of trastuzumab-(6-N3-GalNAc)2 and compound X1 in the absence of sodium deoxycholate (Comparative example)

[0149] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 7 mg) was incubated overnight with X1 (7 equivalents) and 25% DMF. After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR (3.7). Subsequently, the reaction was diluted to 2.5 mL with TBS, then concentrated to 1 mL using an Amicon 10 kDa spin filter, and then purified using an AKTA Purifier-10 (GE Healthcare) equipped with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column to obtain the conjugate in 80% yield.

[0144] Example 18. Conjugation of trastuzumab-(6-N3-GalNAc)2 and compound X1 in the presence of sodium deoxycholate.

[0150] Trastuzumab-(6-N3-GalNAc)2 (15 mg / mL, 10 mg) was incubated overnight with X1 (3 equivalents), 10% DMF, and sodium deoxycholate (11 mM). After 16 hours, the reaction was analyzed by RP-HPLC (after DTT reduction) to determine the DAR (3.7). Subsequently, the reaction product was directly purified using an AKTA Purifier-10 (GE Healthcare) equipped with a Superdex200 Increase 10 / 300 GL (GE Healthcare) column to obtain the conjugate in 89% yield.

[0145]

[0151] The results of Examples 17 and 18 demonstrate that the presence of a surfactant increases the yield. Furthermore, since the dialysis step using the Amicon 10kDa spin filter is not required for the conjugation reaction in the presence of a surfactant, the downstream processing (work-up, purification) of the conjugate after the conjugation reaction is simplified.

Claims

1. Structure B-(Z-LD) ｘ (1) A method for preparing the bioconjugate, (i) Alkyne or alkene compounds of structure Q-L-D(2) [In the structure, Q is a click probe that includes a cyclic alkyne portion or a cyclic alkene portion. L is a linker, D is the payload. (ii) Structure B-(F) ｘ (3) Molecules [In the structure, B is a biomolecule functionalized with x click probes F, F is a click probe that can react to Q, x is an integer in the range of 1 to 10. A method comprising the step of reacting in the presence of a surfactant containing a negatively charged moiety to form a bioconjugate in which the payload is covalently bonded to a biomolecule via a connecting group Z formed by a click reaction between Q and F.

2. The method according to claim 1, wherein the surfactant is an anionic surfactant.

3. The method according to claim 1 or 2, wherein the surfactant is selected from the group consisting of sodium decanoate, sodium dodecanoate, sodium lauryl sulfate (SDS), and sodium deoxycholate, and preferably the surfactant is sodium decanoate or sodium deoxycholate.

4. The method according to any one of claims 1 to 3, wherein the reaction is carried out in a solvent system containing water and an organic solvent in a ratio of 50 / 50 to 100 / 0, preferably in the range of 75 / 25 to 95 / 5.

5. The method according to any one of claims 1 to 4, wherein the concentration of the molecule of structure (3) is in the range of 1 to 100 mg / mL, preferably in the range of 5 to 50 mg / mL, and more preferably in the range of 10 to 20 mg / mL.

6. The method according to any one of claims 1 to 5, wherein the click probe Q comprises a cyclic alkyne moiety, and the click probe F is selected from the group consisting of azides, tetrazines, triazines, nitrones, nitrile oxides, nitrile imines, diazo compounds, orthoquinones, dioxothiophenes, and cydonones, and preferably the click probe F is an azide moiety.

7. Click probe Q is selected from the group consisting of (Q22) to (Q36), 【Chemistry 1】 Or, the (hetero)cycloalkynyl moiety Q matches structure (Q37), 【Chemistry 2】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ alkyl(hetero)aryl group and C ７ ~C ２４ (hetero)arylalkyl group, independently selected from the group consisting of, said alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ may be linked to form a fused cycloalkyl optionally substituted or a fused (hetero)arene substituent optionally substituted, R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ alkyl(hetero)aryl group and C ７ ~C ２４ (hetero)arylalkyl group, independently selected from the group consisting of, Y ２ is C(R ３１ ) ２ , O, S or NR ３１ And R ３１ Each is individually R １５ Or -LD, u is 0, 1, 2, 3, 4, or 5. u' is 0, 1, 2, 3, 4, or 5, and u + u' = 4, 5, 6, 7, or 8. v is an integer in the range of 8 to 16. Preferably, the cyclooctinyl moiety Q corresponds to structure (Q38), 【Transformation 3】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ Alkyl (hetero)aryl groups and C ７ ~C ２４ A (hetero)arylalkyl group is independently selected from the group consisting of (hetero)arylalkyl groups, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ These may be linked to form optionally substituted condensed cycloalkyl or optionally substituted condensed (hetero)arene substituents, R １６ is hydrogen, halogen, 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 selected from the group consisting of (hetero)arylalkyl groups, R １８ is hydrogen, halogen, 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 selected from the group consisting of (hetero)arylalkyl groups, R １９ This is hydrogen, -LD, halogen, 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 A group consisting of (hetero)arylalkyl groups is selected, and the alkyl group is optionally interrupted by one or more heteroatoms selected from the group consisting of O, N, and S, and the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group, and (hetero)arylalkyl group are independently optionally substituted. l is an integer in the range of 0 to 10. Or, the (hetero)cyclooctinyl moiety Q matches structure (Q39) 【Chemistry 4】 [In the structure, R １５ is hydrogen, halogen, -OR １６ , -NO ２ , -CN, -S(O) ２ R １６ , -S(O) ３ （－） , C １ ~C ２４ alkyl group, C ５ ~C ２４ (hetero)aryl group, C ７ ~C ２４ alkyl(hetero)aryl group and C ７ ~C ２４ (hetero)arylalkyl group, independently selected from the group consisting of, wherein the alkyl group, (hetero)aryl group, alkyl(hetero)aryl group and (hetero)arylalkyl group are optionally substituted, and two substituents R １５ may be linked to form a optionally substituted fused cycloalkyl or an optionally substituted fused (hetero)arene substituent, and R １６ is hydrogen, halogen, C １ ~C ２４ alkyl group, C ６ ~C ２４ (hetero)aryl group, C ７ ~C ２４ alkyl(hetero)aryl group and C ７ ~C ２４ (hetero)arylalkyl group, independently selected from the group consisting of Y is N or CR １５ [is] The method according to any one of claims 1 to 6.

8. Click probe Q is selected from the group consisting of optionally substituted (hetero)cyclopropenyl groups, (hetero)cyclobutenyl groups, trans-(hetero)cycloheptenyl groups, trans-(hetero)cyclooctenyl groups, trans-(hetero)cyclononenyl groups, or trans-(hetero)cyclodecynyl groups, preferably click probe Q is selected from the group consisting of (Q40) to (Q50). 【Transformation 5】 The method according to any one of claims 1 to 6, wherein in (Q44) and (Q45), the R group(s) of Si are alkyl or aryl.

9. The method according to any one of claims 1 to 8, wherein payload D is a cytotoxin, preferably colchicine, vinca alkaloid, anthracycline, camptothecin, doxorubicin, daunorubicin, taxane, calicheamicin, tubulicin, irinotecan, inhibitory peptide, amanitin, debouganin, duocalmycin, meitansine, auristatin, engine, pyrrolobenzodiazepine (PBD) or indolinobenzodiazepine dimer (IGN), or a cytotoxin selected from PNU-159, 682 and their derivatives, more preferably calicheamicin, PBD dimer, SN-38, MMAE, or exatecan.

10. The method according to any one of claims 1 to 9, wherein the biomolecule is selected from the group consisting of proteins (including glycoproteins such as antibodies), polypeptides, peptides, glycans, lipids, nucleic acids, oligonucleotides, polysaccharides, oligosaccharides, enzymes, hormones, amino acids, and monosaccharides, more preferably from the group consisting of proteins, polypeptides, peptides, and glycans, and most preferably the biomolecule is a protein.

11. The method according to claim 10, wherein the biomolecule is selected from the group consisting of mAb, Fab, VHH, scFv, diabody, minibody, afibody, affin, affimer, atrimer, finomer, Cys-not, DARPin, adnectin / centinin, notchin, antikalin®, FN3, Knitz domain, bicyclic peptide, and tricyclic peptide.

12. The method according to any one of claims 1 to 11, wherein the click probe F is attached to a monosaccharide portion, preferably to the terminal monosaccharide portion of the glycoprotein glycan, and most preferably to the terminal monosaccharide portion of the antibody glycan.

13. Structure B-(Z-LD) ｘ (1) The use of a surfactant containing a negatively charged moiety in a bioconjugation reaction for preparing the bioconjugate, wherein x payloads D are covalently bonded to biomolecule B via connecting groups Z formed by a click reaction between click probe Q and click probe F, and the reaction proceeds (i) Alkyne or alkene compounds of structure Q-L-D(2) [In the structure, Q is a click probe that includes a cyclic alkyne portion or a cyclic alkene portion. L is a linker, D is the payload. (ii) Structure B-(F) ｘ (3) Molecules [In the structure, B is a biomolecule functionalized with x click probes F, F is a click probe that can react to Q, x is an integer in the range of 1 to 10.

14. (i) To increase the conversion rate of the bioconjugation reaction, (ii) To increase the yield of the bioconjugation reaction, (iii) Reduce the amount of organic co-solvent in the solvent system in which the bioconjugation reaction takes place. (iv) To provide flexibility in the concentration of biomolecules during bioconjugation reactions. (v) Reduce the excess amount of alkyne or alkene functionalized payload used during the bioconjugation reaction. (vi) Reduce the degree of aggregate formation during the bioconjugation reaction. (vii) Simplify downstream processing of bioconjugates. The use according to claim 13 for one or more of the following.

15. The use according to claim 13 or 14 for improving the drug-antibody ratio (DAR) of a bioconjugate.

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