Saponin conjugates

Abstract

The present invention relates to saponin conjugates; an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, the effector moiety comprising a payload conjugated to at least one saponin; an antibody-drug conjugate or a ligand-drug conjugate comprising the effector moiety; a therapeutic combination or a pharmaceutical composition comprising an effector moiety and an antibody-drug conjugate or a ligand-drug conjugate and optionally a pharmaceutically acceptable excipient. An effector moiety or an antibody-drug conjugate or a therapeutic combination or a ligand-drug conjugate or a pharmaceutical composition for use as a medicament and / or for the treatment or prevention of cancer or an autoimmune disease.

Description

[0001] This application is a divisional application of an application filed on December 9, 2019, with application number 201980092610.X and invention name “Saponin Conjugate”. Technical Field

[0002] The present invention relates to an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, the effector moiety being conjugated to at least one saponin. The present invention relates to an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, the effector moiety comprising a payload conjugated to at least one saponin. The present invention also relates to an antibody-drug conjugate comprising an effector moiety according to the present invention, or a ligand-drug conjugate comprising an effector moiety according to the present invention, the effector moiety comprising a covalently coupled saponin. The present invention also relates to a therapeutic combination comprising: (a) an effector moiety according to the present invention, the effector moiety comprising at least one saponin and an optional pharmaceutically acceptable excipient; and (b) an antibody-drug conjugate or a ligand-drug conjugate, and an optional pharmaceutically acceptable excipient. In addition, the present invention relates to a pharmaceutical composition comprising an effector moiety according to the present invention or an antibody-drug conjugate according to the present invention or a ligand-drug conjugate according to the present invention (which comprises at least one saponin covalently linked to an effector molecule) and an optional pharmaceutically acceptable excipient. The invention also relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament. Finally, the invention also relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use in the treatment or prevention of cancer or an autoimmune disease. Background Art

[0003] In theory, molecules with therapeutic biological activity are suitable for application as effective therapeutic drugs for treating diseases in human patients in need, such as cancer, in many cases. A typical example is a small molecule biologically active part. However, many (if not all) potential drug-like molecules and therapeutic agents currently used in clinical practice have at least one of many shortcomings and defects. When applied to the human body, in addition to the biological activity for the potential aspects of the disease to be treated or health problems, the therapeutically active molecule can also play an off-target effect. Such off-target effects are undesirable, and there is a risk that the administered molecule induces health or even life-threatening side effects. It is the occurrence of such adverse events that causes many drug-like compounds and therapeutic parts to fail to pass Phase III clinical trials or even Phase IV clinical trials (follow-up after entering the market). Therefore, it is highly desirable to provide drug molecules, such as small molecule therapeutics, wherein the therapeutic effects of the drug molecules should, for example, (1) be highly specific to the biological factors or biological processes driving the disease, (2) be sufficiently safe, (3) be sufficiently effective, (4) be sufficiently targeted to the diseased cells and have little off-target activity to non-diseased cells, (5) have a sufficiently timely mode of action (e.g., the administered drug molecules should reach the target site in the human patient within a certain time frame and should remain at the target site for a certain time frame), and / or (6) have sufficiently long-lasting therapeutic activity in the patient's body, etc. Unfortunately, to date, despite long and intensive research and despite impressive progress in several areas that have encountered difficulties and deficiencies individually, the 'ideal' therapeutic agents having many or even all of the above-mentioned beneficial characteristics are still not available to patients.

[0004] Chemotherapy is one of the most important treatment options in cancer treatment. However, it is often associated with a low therapeutic window because it has no specificity for cancer cells compared to dividing cells in healthy tissues. The invention of monoclonal antibodies provides the possibility of using their specific binding properties as a mechanism for targeting cytotoxic agents to cancer cells while retaining normal cells. This can be achieved by chemically conjugating cytotoxic effectors (also called payloads or warheads) to antibodies to produce antibody-drug conjugates (ADCs). Typically, very effective payloads such as emtansine (DM1) are used, and their unconjugated forms have a limited therapeutic index (comparing the ratio of toxic dose to effective dose). The conjugation of DM1 to trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla, increases the DM1 tolerance dose in monkeys by at least two times. Over the past few decades, great efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, it remains challenging to introduce ADCs into the clinic. In 2000, the first ADC approved for clinical use was gemtuzumab ozogamicin (Mylotarg, CD33-targeted, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML). However, Mylotarg was withdrawn from the market at the request of the Federal Drug Administration (FDA) due to a number of issues, including its safety profile. Patients treated with Mylotarg were found to be dead more often than those treated with conventional chemotherapy. Mylotarg entered the market again in 2017 with a lower recommended dose, a different schedule in combination with chemotherapy or on its own, and in new patient populations. To date, only five ADCs have been approved for clinical use, while clinical development of approximately 55 ADCs has been discontinued. However, interest remains high, and approximately 80 ADCs are currently still in clinical development in nearly 600 clinical trials.

[0005] Although it is possible to use toxic payloads that are not generally tolerated by patients, a low therapeutic index (ratio comparing toxic dose to effective dose) is a major problem that has led to the discontinuation of many ADCs in clinical development, which may be caused by several mechanisms, such as off-target toxicity to normal cells, resistance to cytotoxic agents, and premature release of the drug in the circulation. The FDA's systematic review of ADCs found that the toxicity profile of most ADCs could be classified according to the payload used, but not according to the antibody used, indicating that toxicity was mainly determined by premature release of the payload. Of the approximately 55 ADCs that were discontinued, it is estimated that at least 23 were due to a poor therapeutic index. For example, the development of trastuzumab tesirine conjugate (ADCT-502, HER-2 targeted, ADC therapeutic) was recently discontinued due to a narrow therapeutic index, which may be due to on-target, off-tissue effects in lung tissue that expresses considerable levels of HER2. In addition, several ADCs in Phase 3 trials have been discontinued due to the lack of a primary endpoint. For example, phase 3 trials of depatuxizumab mafodotin conjugates (ABT-414, EGFR-targeted, AbbVie) tested in patients with newly diagnosed glioblastoma and mirvetuximabsoravtansine conjugates (IMGN853, folate receptor α (FRα)-targeted, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently stopped, showing no survival benefit. It is important to note that the clinically used doses of some ADCs may not be sufficient to exert their full anticancer activity. For example, the MTD of ado-trastuzumab emtansine in humans is 3.6 mg / kg. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels above 3 mg / kg, but more effective efficacy was observed at 15 mg / kg. This suggests that ado-trastuzumab emtansine may not exert its maximum potential antitumor effect at clinically administered doses.

[0006] ADCs are mainly composed of antibodies, cytotoxic parts (such as payloads) and connectors. Several novel strategies have been proposed and implemented in the design and development of new ADCs to overcome existing problems in targeting each component of ADCs. For example, by identifying and validating sufficient antigen targets for antibody components, by selecting antigens that have high expression levels in tumors and are barely expressed in normal tissues, antigens that are present on the cell surface to easily access circulating ADCs, and antigens that allow ADCs to be internalized into cells after binding; and alternative mechanisms of activity; designing and optimizing connectors that improve the solubility and drug-antibody ratio (DAR) of ADCs and overcome protein-induced resistance that can transport chemotherapeutic agents out of cells; improving the DAR ratio by including more payloads, selecting and optimizing antibodies to improve the homogeneity and developability of antibodies. In addition to the technical development of ADCs, new clinical and translational strategies are being deployed to maximize the therapeutic index, such as changing the dosing schedule by fractionated dosing; conducting biodistribution studies; including biomarkers to optimize patient selection, capture response signals early and monitor the duration and depth of response, and inform combination studies.

[0007] Examples of ADCs with clinical potential are those ADCs such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, which are being evaluated as treatment options for lymphoid malignancies and multiple myeloma. Polotuzumab vedotin, which binds to CD79b on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, are being tested in clinical trials, where each ADC is combined with co-administered rituximab (a monoclonal antibody that binds to CD20 but does not provide a payload) [B. Yu and D. Liu, Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12: 94]. Combinations of monoclonal antibodies (such as these examples) are a further approach and attempt to obtain a 'magic bullet' that combines many or even all of the above-mentioned desirable features of ADCs.

[0008] Meanwhile, over the past few decades, nucleic acid-based therapeutics are being developed. Therapeutic nucleic acids can be based on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON) and short interfering RNA (siRNA), microRNA, and DNA and RNA aptamers, and are used in methods such as gene therapy, RNA interference (RNAi). Many of them share the same basic basis of action by inhibiting DNA or RNA expression, thereby preventing the expression of abnormal proteins associated with diseases. The largest number of clinical trials are ongoing in the field of gene therapy, with nearly 2,600 ongoing or completed clinical trials worldwide, but only about 4% have entered Phase 3. Clinical trials of ASOs followed. Similar to ADCs, despite the exploration of a large number of technologies, therapeutic nucleic acids have two main problems in clinical development: delivery to cells and off-target effects. For example, ASOs such as peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), locked nucleic acids (LNAs), and bridged nucleic acids (BNAs) are being studied as attractive strategies for specifically inhibiting target genes, especially those that are difficult to target with small molecule inhibitors or neutralizing antibodies. Currently, the efficacy of different ASOs is being studied in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, as well as in several stages of cancer. The use of ASOs as potential therapeutic agents requires safe and effective methods to deliver them to the cytoplasm and / or nucleus of target cells and tissues. Although the clinical relevance of ASOs has been demonstrated, inefficient cellular uptake in vitro and in vivo limits the efficacy of ASOs and has become an obstacle to the development of therapeutic agents. Cellular uptake can be less than 2% of the dose, resulting in too low ASO concentrations at the active site to obtain effective and sustained results. Therefore, this requires an increase in the administered dose, which can induce off-target effects. The most common side effects are activation of the complement cascade, inhibition of the coagulation cascade, and stimulation of the immune system mediated by Toll-like receptors.

[0009] Chemotherapeutic agents are the most common small molecules, however, their efficacy is hampered by severe off-target side toxicities as well as poor solubility, rapid clearance, and limited tumor exposure. Scaffold-small molecule drug conjugates such as polymer-drug conjugates (PDCs) are pharmacologically active macromolecular constructs that contain one or more molecules of a small molecule drug bound to a carrier scaffold such as polyethylene glycol (PEG).

[0010] Such conjugate principles have attracted widespread attention and have been studied for decades. Most conjugates of small molecule drugs in preclinical or clinical development are used for tumor indications. However, only one drug that is not related to cancer (Movantik, a PEG oligomer conjugate of the opioid antagonist naloxone, AstraZeneca) was recently approved for opioid-induced constipation in chronic pain patients in 2014, which is a non-oncology indication. So far, there has been little clinical success in translating the application of drug-scaffold conjugates into the treatment of human subjects. For example, PK1 (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has shown great anticancer activity in both solid tumors and leukemias in mouse models and is in clinical studies for tumor indications. Although it has demonstrated significant reductions in nonspecific toxicity and improved pharmacokinetics in humans, the improvement in anticancer efficacy in patients is negligible, and further development of PK1 has therefore been stopped.

[0011] The failure of scaffold-small molecule drug conjugates is at least in part due to their poor accumulation at the tumor site. For example, although PK1 showed 45-250 times higher accumulation in tumors than in healthy tissues (liver, kidney, lung, spleen and heart) in murine models, accumulation in tumors was only observed in a small subgroup of patients in clinical trials.

[0012] A potential solution to the above problems is to apply nanoparticle systems to drug delivery, such as liposomes. Liposomes are spherical vesicles composed of one or more phospholipid bilayers, which are spontaneously formed when phospholipids are dispersed in water. The amphiphilic characteristics of phospholipids provide them with self-assembly properties, emulsification and wetting characteristics, and these properties can be used in the design of new drugs and new drug delivery systems. Compared with direct administration of drugs, drugs encapsulated in liposome delivery systems can have multiple advantages, such as improved and controlled pharmacokinetics and pharmacodynamics, tissue targeting properties, reduced toxicity and enhanced drug activity. An example of such success is the small molecule chemotherapeutic agent doxorubicin in liposome encapsulation (Doxil: a PEGylated liposome encapsulated form of doxorubicin; Myocet: a non-PEGylated liposomal doxorubicin), which has been approved for clinical use.

[0013] Therefore, there remains a need to find a solution that allows drug therapies, such as anti-tumor therapies, to be suitable for non-systemic use when needed, where the drug has, for example, an acceptable safety profile, little off-target activity, sufficient efficacy, sufficiently low clearance from the patient's body, etc. Summary of the invention

[0014] For one embodiment of the present invention, a first object is to provide improved bioactive compounds or compositions comprising such improved bioactive compounds.

[0015] One of the several goals of embodiments of the present invention is to provide a solution to the non-specificity problem encountered when administering small molecule therapeutically active compounds to human patients in need. One of the several goals of embodiments of the present invention is to provide a solution to the problem of drugs that have non-optimal specificity for biological factors or biological processes that drive disease. One of the several goals of embodiments of the present invention is to provide a solution to the problem that the safety characteristics of current drugs are insufficient when administered to human patients in need. One of the several goals of embodiments of the present invention is to provide a solution to the problem that current drugs are not as effective as desired when administered to human patients in need. One of the several goals of embodiments of the present invention is to provide a solution to the problem that current drugs are not sufficiently targeted to diseased cells and have little off-target activity to non-disease cells when administered to human patients in need. One of the several goals of embodiments of the present invention is to provide a solution to the problem that current drugs do not have a sufficiently timely mode of action when administered to human patients in need (e.g., the administered drug molecule should reach the target site of the human patient within a certain time frame and should remain at the target site for a certain time frame). One of the several goals of embodiments of the present invention is to provide a solution to the problem that current drugs do not have a sufficiently long-lasting therapeutic activity in the patient's body when administered to human patients in need.

[0016] At least one of the above objects of embodiments of the present invention is achieved by providing an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, wherein the effector moiety is conjugated to at least one saponin, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker, or is directly covalently bound to the effector moiety.

[0017] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.The embodiments of the invention described herein can be combined and operate in conjunction with each other unless otherwise indicated.

[0018] One aspect of the invention relates to an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, the effector moiety being conjugated to at least one saponin, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker, or is directly covalently bound to the effector moiety.

[0019] One aspect of the invention relates to an antibody-drug conjugate comprising an effector moiety according to the invention, or a ligand-drug conjugate comprising an effector moiety according to the invention, the effector moiety comprising a covalently coupled saponin. The effector moiety of the invention is a conjugate comprising at least one saponin and at least one effector molecule covalently coupled to each other directly or via at least one linker, optionally comprising a cleavable linker, and optionally via an oligomeric or multimeric scaffold, the at least one saponin and / or the at least one effector moiety being covalently bound to the oligomeric or multimeric scaffold.

[0020] One aspect of the invention relates to a therapeutic combination comprising: (a) an effector portion of the invention comprising at least one saponin and optionally a pharmaceutically acceptable excipient; and (b) an antibody-drug conjugate or a ligand-drug conjugate and optionally a pharmaceutically acceptable excipient.

[0021] One aspect of the invention relates to a pharmaceutical composition comprising an effector portion of the invention or an antibody-drug conjugate of the invention or a ligand-drug conjugate of the invention comprising at least one saponin covalently linked to an effector molecule and optionally a pharmaceutically acceptable excipient.

[0022] One aspect of the invention relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0023] One aspect of the invention relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use in treating or preventing cancer or an autoimmune disease.

[0024] Other aspects of the present invention may also relate to the following items:

[0025] 1. An effector moiety capable of inducing an intracellular effect when present in a mammalian cell, said effector moiety being conjugated to at least one saponin, wherein said at least one saponin is covalently bound to said effector moiety via at least one linker, or is covalently bound directly to said effector moiety.

[0026] 2. The effector portion as described in item 1, which comprises or consists of at least one oligonucleotide, nucleic acid, heterologous nucleic acid, and the oligonucleotide, nucleic acid, heterologous nucleic acid is preferably selected from any one or more of the following: a vector, a gene, a transgene that induces cell suicide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, mRNA, a minicircle DNA, a peptide nucleic acid (PNA), an aminophosphoryl morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridging nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinose nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridging nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA), or derivatives thereof, more preferably BNA, such as BNA for silencing HSP27 protein expression.

[0027] 3. An effector portion as described in item 1 or 2, which comprises at least one protein molecule, which is preferably selected from any one or more of the following: peptides, proteins, enzymes such as urease and Cre recombinase, ribosome inactivating proteins, protein toxins such as any one or more of the protein toxins selected from Table A5 and / or bacterial toxins or plant toxins, more preferably selected from any one or more of the following: viral toxins, such as apoptotic proteins; bacterial toxins, such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins, such as α-sarcin; plant toxins, including ribosome inactivating proteins and type 2 ribosomal toxins. The A chain of a ribosome inactivating protein, such as dianthin, such as dianthin-30 or dianthin-32, saporin, such as saporin-S3 or saporin-S6, bougainvillea ribosome inactivating protein or a deimmunized derivative of bougainvillea ribosome inactivating protein, de-bougainvillea ribosome inactivating protein, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, ricin, ricin A chain, abrin, abrin A chain, abrin, abrin A chain, abrin, abrin A chain, mistletoexin, mistletoexin A chain; or an animal or human toxin, such as frog RNase or granzyme B or angiogenin from humans, or any fragment or derivative thereof; preferably, the protein toxin is dianthin and / or saporin.

[0028] 4. An effector portion as described in any one of items 1-3, comprising at least one payload, the payload is preferably selected from any one or more of the following: toxins targeting ribosomes, toxins targeting elongation factors, toxins targeting tubulin, toxins targeting DNA and toxins targeting RNA, more preferably selected from any one or more of the following: maytansine, pasudotox, maytansine derivative DM1, maytansine derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mofortin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine , CC-1065 analogs, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulin, indoline benzodiazepine , AZ13599185, nostoc, rhizobactin, methotrexate, anthracycline drugs, camptothecin analogs, SN-38, DX-8951f, exotecan mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivatives, amanitin, α-amanitin, scotstatin, talanstatin, ozogamicin, teshilin, Amberstatin269 and soravtansine or their derivatives.

[0029] 5. An effector portion as described in any of items 1-4, wherein at least one saponin is a triterpene saponin and / or a disaccharide chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function at the C-23 position and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, and / or is a saponin isolated from a species of the genus Lithospermum and / or a species of the genus Saponaria and / or a species of the genus Osmanthus and / or a species of the genus Quillaja such as Quillaja spp.

[0030] 6. The effector moiety of any one of items 1-5, wherein the at least one saponin is a single specific saponin or a mixture of two or more different saponins.

[0031] 7. The effector portion of any one of items 1-6, wherein the saponin is one or more of the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Saponin, Saponin album, QS-18, Quil-A, Gyp1, gypsophila saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any one of their stereoisomers and / or any combination thereof, preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or has a sapogenin aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3β-OH group and a Glc-(1→3)-Xyl-(1→4)-Rha at the C-28-OH group. -(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent saponin, and / or 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronic acid pyranosyl saponin 28-O-β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4-OAc-β-D-quinopyranosyl-(1→4)]-β-D-fucopyranoside, more preferably, the saponin is SO1861 and / or QS-21.

[0032] 8. An effector moiety as described in any of items 1 to 7, wherein the saponin is a disaccharide chain saponin having a molecular mass of at least 1.500 Daltons and comprising an oleanane-type triterpene containing an aldehyde group at position C-23 and optionally a hydroxyl group at position C-16, and a first branched carbohydrate side chain at position C-3, the first branched carbohydrate side chain optionally containing glucuronic acid, wherein the saponin contains an ester group having a second branched carbohydrate side chain at position C-28, the second branched carbohydrate chain preferably comprising at least four carbohydrate units and optionally containing at least one acetyl residue, such as two acetyl residues and / or optionally containing one or more deoxy carbohydrates and / or quinovose and / or glucose and / or 4-methoxycinnamic acid and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprises 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester bond, or wherein at least one saponin is QS-21 or any one or more of the following: QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

[0033] 9. An effector moiety as described in any of items 1-8, wherein the at least one saponin is a disaccharide chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde functional group at the C-23 position, wherein the at least one saponin is covalently coupled to an amino acid residue of the effector moiety when present via the aldehyde functional group in the saponin, preferably the aldehyde functional group at the C-23 position, preferably via at least one linker, more preferably via at least one cleavable linker, wherein the amino acid residue is preferably selected from cysteine ​​and lysine.

[0034] 10. An effector moiety as described in any of items 1-9, wherein the at least one saponin is a disaccharide chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function at the C-23 position and comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, wherein the at least one saponin is covalently coupled to an amino acid residue of the effector moiety when present via the glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, preferably via at least one linker, wherein the amino acid residue is preferably selected from cysteine ​​and lysine.

[0035] 11. An effector portion as described in any of items 1-10, wherein the at least one linker comprises at least one non-cleavable linker and / or at least one cleavable linker, wherein optionally the cleavable linker is cleaved under acidic conditions, reducing conditions, enzymatic conditions or light-induced conditions, and preferably, the cleavable linker comprises a cleavable bond selected from the following: a hydrazone bond and a hydrazide bond that are cleaved under acidic conditions, and / or a bond that is susceptible to proteolysis, such as a bond that is susceptible to proteolysis by tissue peptone B, and / or a bond that is susceptible to cleavage under reducing conditions, such as a disulfide bond.

[0036] 12. An effector portion as described in any of items 1-11, wherein the at least one linker comprises at least one cleavable linker that is cleaved in vivo under acidic conditions such as those present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5 and more preferably at pH ≤ 5.5.

[0037] 13. An effector moiety as described in any of items 1-12, wherein the at least one saponin is covalently bound to a lysine side chain to form an amide bond, and / or covalently bound to a cysteine ​​side chain to form a thioether bond or a disulfide bond, wherein the lysine and / or cysteine ​​is comprised by the effector moiety, and wherein the saponin is directly bound to the effector moiety, or is bound to the effector moiety via at least one linker.

[0038] 14. The effector portion of any one of items 1-13, wherein the at least one saponin is covalently bound to the effector portion via at least one linker, wherein the linker is or comprises a scaffold, the scaffold comprising a polymeric or oligomeric structure and a chemical group for covalently coupling the scaffold to the effector portion.

[0039] 15. The effector portion of item 14, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the scaffold via a cleavable bond.

[0040] 16. An effector portion as described in item 15, wherein the cleavable bond is cleaved under any one of acidic conditions, reducing conditions, enzymatic conditions and light-induced conditions, more preferably, the cleavable bond is a hydrazone bond or a hydrazide bond that is cleaved under acidic conditions and / or is a bond that is susceptible to proteolysis, such as a bond that is susceptible to proteolysis by tissue peptone B, and / or is a bond that is susceptible to cleavage under reducing conditions, such as a disulfide bond.

[0041] 17. An effector portion as described in item 15 or 16, wherein the cleavable bond is cleaved in vivo under acidic conditions such as those present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5 and more preferably at pH ≤ 5.5.

[0042] 18. An effector portion as described in any of items 14-17, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the scaffold via an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond and / or an ester bond, preferably via at least one linker, preferably an amide bond, a hydrazide bond, a thioether bond and / or a hydrazone bond.

[0043] 19. An effector portion as described in any of items 14-18, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the scaffold, and the covalent bond involves the aldehyde functional group at the C-23 position of the at least one saponin (when present), and the covalent bond is preferably an imine bond, a hydrazone bond, an amide bond, a thioether bond, or a disulfide bond, and / or the covalent bond involves the glucuronic acid functional group in the carbohydrate substituent at the C-3β-OH group of the at least one saponin (when present), wherein preferably, the covalent bond is an amide bond, a disulfide bond, or a thioether bond.

[0044] 20. An effector portion as described in any of items 14-19, wherein the aldehyde functional group at the C-23 position of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, and the linker is covalently coupled to a sulfhydryl group in the polymeric or oligomeric structure of the scaffold via a thioether bond, such as a sulfhydryl group of cysteine.

[0045] 21. An effector portion as described in any of items 14-20, wherein the glucuronic acid functional group in the carbohydrate substituent at the C-3β-OH group of at least one saponin is covalently coupled to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, and the linker is covalently coupled to an amine group in the polymeric or oligomeric structure of the scaffold via an amide bond, such as an amine group of lysine or the N-terminus of the polymeric or oligomeric structure of the scaffold.

[0046] 22. An effector moiety as described in any of items 14-21, wherein the chemical group of the scaffold for covalently coupling the scaffold to the effector moiety is a click chemistry group, which is preferably selected from tetrazines, azides, olefins or alkynes or cyclic derivatives of these groups, more preferably, the click chemistry group is an azide.

[0047] 23. The effector portion of any one of items 14-22, wherein the polymeric or oligomeric structure of the scaffold comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol or an assembly of these polymeric or oligomeric structures, the assembly preferably being constructed by covalent cross-linking.

[0048] 24. The effector portion of any one of items 1-23, wherein the at least one saponin is a defined number of saponins or a defined range of saponins, preferably 1-128 saponins or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128 saponins, or any number of saponins therebetween, such as 7, 9, 12 saponins.

[0049] 25. An effector moiety as described in any of items 1-24, wherein the effector moiety comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins or any number of saponins in between, such as 7, 9, 12 saponins, which are covalently bound directly to an amino acid residue of the effector moiety, preferably cysteine ​​and / or lysine, and / or covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric scaffold or oligomeric scaffold, preferably 1-8 such scaffolds or 2-4 such scaffolds as described in any of items 14-23, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins are covalently bound to at least one scaffold.

[0050] 26. An antibody-drug conjugate comprising the effector moiety of any one of items 1-25, or a ligand-drug conjugate comprising the effector moiety of any one of items 1-25.

[0051] 27. The antibody-drug conjugate or ligand-drug conjugate of item 26, wherein the antibody is capable of binding to any one of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor body, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA 4. CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and / or wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, intuzumab, mositumomab, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, Alemtuzumab, pinatuzumab, OKT-10, an anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9 or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, Moxetumomab pasudotox and polotuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0052] 28. A therapeutic combination comprising:

[0053] (a) the effector moiety of any one of items 1-25 and optionally a pharmaceutically acceptable excipient; and

[0054] (b) antibody-drug conjugate or ligand-drug conjugate and optionally a pharmaceutically acceptable excipient.

[0055] 29. The therapeutic combination of claim 28, wherein the antibody-drug conjugate is capable of binding to any of the following: tumor cell receptors CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, body, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA 4. CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and / or wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, intuzumab, mositumomab, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, Alemtuzumab, pinatuzumab, OKT-10, an anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9 or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, Moxetumomab pasudotox and polotuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0056] 30. A pharmaceutical composition comprising the effector moiety of any one of items 1 to 25 or the antibody-drug conjugate of item 26 or 27 or the ligand-drug conjugate of item 26 or 27 and optionally a pharmaceutically acceptable excipient.

[0057] 31. The effector moiety of any one of items 1 to 25, or the antibody-drug conjugate of item 26 or 27, or the therapeutic combination of any one of items 25 to 27, or the ligand-drug conjugate of item 26 or 27, or the pharmaceutical composition of item 30, for use as a medicament.

[0058] 32. The effector portion of any one of items 1-25 or 31, or the antibody-drug conjugate of item 26 or 27, or the ligand-drug conjugate of item 26 or 27, or the therapeutic combination of any one of items 28, 29 or 31, or the pharmaceutical composition of item 30 or 31, for use in treating or preventing cancer or autoimmune diseases.

[0059] definition

[0060] The term "joint" has its conventional scientific meaning, and refers to a chemical part or linear segment of an amino acid residue compounded by a peptide bond herein, which connects a molecule or an atom to another molecule, such as being connected to a ligand or an effector molecule or a scaffold. Typically, a joint comprises a chain of atoms connected by a chemical bond. Any joint molecule or joint technology known in the art can be used in the present disclosure. In the case of indication, a joint is a joint for covalently binding a molecule by a chemical group on such a molecule that is suitable for forming a covalent bond or a covalent bond with a joint. The joint can be a non-cleavable joint, such as a joint that is stable under physiological conditions. The joint can be a cleavable joint, such as a cleavable joint in the presence of an enzyme or under a specific pH range or value or under physiological conditions (such as intracellular conditions in endosomes (such as late endosomes) and lysosomes of mammalian cells (such as human cells)). Exemplary linkers that can be used in the context of the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate or N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0061] The term "trifunctional linker" has its conventional scientific meaning, and refers to a linker connecting three molecules via a chemical group on each of the three molecules herein. Technicians can design such trifunctional linkers based on the present disclosure and common knowledge. Such trifunctional linkers can show, for example, maleimido groups, which can be used to conjugate to a targeting ligand showing a thiol group to carry out a thiol-ene reaction. In addition, the trifunctional linker can show a dibenzocyclooctyne (DBCO) group to carry out a so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with a saponin with an azido group. Finally, the trifunctional linker can obtain a third functional group, such as a trans-cyclooctene (TCO) group, to carry out a so-called inverse electron demand Diels-Alder (IEDDA) reaction with an effector molecule with a tetrazine (Tz). Technicians will understand that the chemical group of trifunctional joint can be all three identical or different, or joint can comprise two identical chemical groups for molecule being connected to trifunctional joint.The key formed between trifunctional joint can be covalent or non-covalent, and preferably covalent bond.The key formed via respective chemical group between trifunctional joint and one or two or three combined molecules can be cleavable (unstable) key, such as in cell (such as endosome and lysosome of mammalian cell (such as human cell)) cracking under acidic conditions, or can be non-cleavable.Certainly, trifunctional joint can contain one or two chemical groups for forming covalent bond, and other two or one chemical group are respectively used to form non-covalent bond.Certainly, trifunctional joint can contain one or two chemical groups for forming cleavable bond, and other two or one chemical group are respectively used to form non-cleavable key.

[0062] The term "cleavable", such as used in the term "cleavable linker" or "cleavable bond", has its conventional scientific meaning and refers herein to cleavage under acidic conditions, reducing conditions, enzymatic conditions or light-induced conditions. For example, the cleavable linker can be cleaved under acidic conditions, preferably the cleavable linker is cleaved in vivo under acidic conditions such as present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5, and more preferably at pH ≤ 5.5. As another example, the cleavable linker can be cleaved by an enzyme, such as by a cathepsin. In addition, an example of a covalent bond that is cleavable under reducing conditions is a disulfide bond.

[0063] The terms "oligomer" and "polymer" in the context of an oligomeric or polymeric scaffold have their conventional scientific meanings. A polymer herein refers to a substance whose molecular structure is mainly or completely composed of a large number of identical or similar units bonded together; an oligomer herein refers to a polymer whose molecules are composed of relatively few repeating units. For example, a structure containing 5-10 or fewer identical or similar units can be referred to as an oligomeric structure, while a structure containing 10-50 monomeric units or more can be referred to as a polymeric structure, and a structure of 10 monomeric units can be referred to as oligomeric or polymeric.

[0064] The term "binding site" has its conventional scientific meaning, and refers herein to a region or epitope on a molecule (eg, protein, DNA, or RNA) to which another molecule can bind.

[0065] The term "scaffold" has its conventional scientific meaning and refers herein to an oligomeric or polymeric template or carrier or base (base molecule or base structure) to which one or more molecules (such as ligand molecules, saponins, glycosides, effector molecules of the present invention) can be covalently bound directly or via a linker (such as a cleavable linker). The scaffold can have a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer or dendronized oligomer or have an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stable polymeric micelle or liposome, but does not include structures composed of non-covalent assemblies of monomers (such as cholesterol / phospholipid mixtures). The scaffold may comprise a polymeric or oligomeric structure, such as a poly- or oligomeric (amine), such as polyethyleneimine and poly(amidoamine); or a structure, such as polyethylene glycol, a poly- or oligomeric (ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer; or a poly(dextrin), a polysaccharide or an oligosaccharide, such as cyclodextrin or polyglucose; or a structure, such as a natural and / or artificial polyamino acid or oligoamino acid, such as polylysine or a peptide or protein, a DNA oligomer or polymer, a stable RNA polymer or a PNA (peptide nucleic acid) polymer. Preferably, the polymeric or oligomeric structure is biocompatible, wherein biocompatible means that the polymeric or oligomeric structure does not show significant acute or chronic toxicity in an organism and can be excreted as is or completely degraded into excretable compounds and / or physiological compounds by the body's metabolism.

[0066] The term "ligand" has its conventional scientific meaning and refers herein to any one or more molecules that can selectively bind to a target cell surface molecule or a target cell surface receptor expressed at a target cell (such as a target cancer cell or a target autoimmune cell). A ligand can bind to an epitope contained by a receptor or other antigen on a target cell. Preferably, the cell binding ligand is an antibody.

[0067] The term "antibody" as used herein is used in the broadest sense and may refer to an immunoglobulin (Ig) or a functional binding fragment or binding domain of an immunoglobulin defined as a protein belonging to the IgG class, IgM class, IgE class, IgA class or IgD class (or any subclass thereof). In the context of the present invention, a "binding fragment" or "binding domain" of an immunoglobulin is defined as an antigen-binding fragment or antigen-binding domain or other derivative of a parent immunoglobulin that substantially retains the antigen-binding activity of such parent immunoglobulin. Functional fragments and functional domains are antibodies within the meaning of the present invention, even if their affinity for antigen is lower than that of the parent immunoglobulin. According to the present invention, "functional fragments and functional domains" include but are not limited to F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single domain antibodies (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rIgG), miniantibodies, double-chain antibodies, three-chain antibodies, four-chain antibodies, Fc fusion proteins, nanoantibodies, variable V domains, such as VHH, Vh and other types of antigen recognition immunoglobulin fragments and domains. Fragments and domains can be engineered to minimize or completely remove the intermolecular disulfide interactions that occur between the CH1 and CL domains. Functional fragments and functional domains provide greater tumor penetration advantages due to their smaller size. In addition, compared with the entire immunoglobulin, the functional fragment or functional domain can be more evenly distributed throughout the tumor mass.

[0068] The antibodies (immunoglobulins) of the present invention can be bifunctional or multifunctional. For example, one arm of a bifunctional antibody has specificity for a receptor or antigen, while the other arm recognizes a different receptor or antigen. Alternatively, each arm of a bifunctional antibody can have specificity for a different epitope of the same receptor or antigen of a target cell.

[0069] The antibodies (immunoglobulins) of the present invention can be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, resurfaced antibodies, anti-idiotypic antibodies, mouse antibodies, rat antibodies, rat / mouse hybrid antibodies, llama antibodies, llama heavy chain antibodies only, heavy chain antibodies only, and veterinary antibodies. Preferably, the antibodies (immunoglobulins) of the present invention are monoclonal antibodies. Resurfaced, chimeric, humanized, and fully human antibodies are also more preferred because they are less likely to cause immunogenicity in humans. The antibodies of the ADC of the present invention preferably specifically bind to antigens expressed on the surface of cancer cells, autoimmune cells, diseased cells, abnormal cells, while leaving any healthy cells substantially unchanged (such as by not binding to such normal cells, or by binding to such healthy cells to a lesser extent in terms of quantity and / or affinity).

[0070] Specific antibodies that can be used in the ADC of the present invention include, but are not limited to, anti-HER2 monoclonal antibodies such as trastuzumab and pertuzumab, anti-CD20 monoclonal antibodies such as rituximab, ofatumumab, tositumomab and ibritumomab, anti-CA125 monoclonal antibodies such as oregovomab, anti-EpCAM (17-1A) monoclonal antibodies such as edrecolomab, anti-EGFR monoclonal antibodies such as cetuximab, panitumumab and nimotuzumab, anti-CD30 monoclonal antibodies such as brentuximab, anti-CD33 monoclonal antibodies such as gemtuzumab and huMy9-6, anti-vascular integrin α-vβ-3 monoclonal antibodies such as etaracizumab, and anti-CD33 monoclonal antibodies such as huMy9-6. umab), anti-CD52 monoclonal antibodies such as alemtuzumab, anti-CD22 monoclonal antibodies such as epratuzumab, anti-CEA monoclonal antibodies such as labetuzumab, anti-CD44v6 monoclonal antibodies such as bivatuzumab, anti-FAP monoclonal antibodies such as sibrotuzumab, anti-CD19 monoclonal antibodies such as huB4, anti-CanAg monoclonal antibodies such as huC242, anti-CD56 monoclonal antibodies such as huN901, anti-CD38 monoclonal antibodies such as daratumumab, anti-CA6 monoclonal antibodies such as DS6, anti-IGF-IR monoclonal antibodies such as citumumab and 3B7, anti-integrin monoclonal antibodies such as CNTO 95, and anti-syndecan-1 monoclonal antibodies such as B-B4. One embodiment is a protein-toxin conjugate of Ste mLine: ELZONRIS TM (tagraxofusp, SL-401) – ELZONRIS is a novel targeted therapy for interleukin-3 (IL-3) receptor-alpha (CD123), a target found on a wide range of malignancies.

[0071] Any other molecule besides an antibody that binds to a cell receptor or antigen of a target cell may also be used as a cell binding ligand of the ligand-drug conjugate of the present invention and a ligand provided with a covalently bound saponin according to the present invention. These ligands include, but are not limited to, proteins, polypeptides, peptides, small molecules. Examples of these non-antibody ligands are interferons (such as IFN-α, IFN-β and IFN-γ), transferrin, lectins, epidermal growth factor (EGF) and EGF-like domains, gastrin releasing peptide (GRP), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factor (IGF, such as IGF-1 and IGF-2), other suitable hormones such as thyrotropin-releasing hormone (TRH), melanocyte stimulating hormone (MSH), steroids. Hormones (such as estrogens and androgens), somatostatin, lymphokines (such as IL-2, IL-3, IL-4 and IL-6), colony stimulating factors (CSF, such as G-CSF, M-CSF and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (such as AS-1411, GBI-10, RNA aptamers against HIV glycoprotein), small molecules (such as folic acid, anisamide phenylboronic acid), vitamins (such as vitamin D), carbohydrates (such as hyaluronic acid, galactose).

[0072] "Effector molecule" or "effector moiety" or "payload" has its conventional scientific meaning and in the context of the present invention is any substance that affects cellular metabolism by interacting with an intracellular effector molecule target, wherein the effector molecule target is any molecule or structure within the cell other than the compartments of the endocytic and recycling pathways and the lumen of vesicles but including the membranes of these compartments and vesicles. Thus, said structures within the cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane and the cytoplasm.

[0073] Effector molecules or effector moieties are pharmaceutically active substances, such as toxins, such as protein toxins, drugs, polypeptides or polynucleotides. The pharmaceutically active substances in the present invention are effector molecules or effector moieties for achieving beneficial results in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / subject. Benefits include diagnosis, prognosis, treatment, cure and prevention (prevention) of diseases and / or symptoms and / or health problems. Pharmaceutically active substances may also cause undesirable and sometimes even harmful side effects (such as adverse events observed during clinical trials). In this case, the pros and cons must be weighed to determine whether the pharmaceutically active substance is suitable for a particular situation. If the effect of the pharmaceutically active substance in the cell is mainly beneficial to the whole organism, the cell is referred to as a target cell. If the effect in the cell is mainly harmful to the whole organism, the cell is referred to as an off-target cell. In artificial systems such as cell culture and bioreactors, target cells and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules and effector moieties are drugs, toxins, polypeptides (such as enzymes), polynucleotides (including polypeptides and polynucleotides comprising non-natural amino acids or nucleic acids) and any combination thereof.

[0074] Effector molecules or effector moieties as drugs may include, but are not limited to, anticancer agents, anti-inflammatory agents, and anti-infective (e.g., antifungal, antibacterial, antiparasitic, antiviral) agents. Preferably, the drug molecule of the present invention is an anticancer agent or an anti-autoimmune agent. Suitable anticancer agents include, but are not limited to, alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. The definition of "anti-cancer agent" also includes: for example, (i) anti-hormonal agents, which are used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands; (iii) anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation; (vii) ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines; topoisomerase 1 inhibitors; (ix) anti-angiogenic agents; and pharmaceutically acceptable salts, acids, solvates and derivatives of any of the above.

[0075] Effector molecules or effector moieties as toxins may include, but are not limited to, protein toxicity (such as bacterial toxins and plant toxins), toxins targeting microtubule protein filaments, toxins targeting DNA, toxins targeting RNA. Examples of protein toxins are saporin, dianthin, ricin, scutellaria toxin, abrin, scutellaria toxin, mistletoexin, Shiga toxin, Shiga-like toxin, Pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT) and cholera toxin. Examples of toxins targeting microtubule protein filaments are maytansine (such as DM1 and DM4), auristatin (such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), toxoids, tubulin, scutellaria, rhizobactin. Examples of DNA-targeted toxins are calicheamicins: N-acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycin, doxorubicin, methotrexate, benzodiazepines , camptothecin analogs and anthracyclines. Examples of DNA-targeted toxicity are amanitin, spliceostatins and thailanstatins. Toxins as used in the present invention are defined as pharmaceutically active substances that can kill or inactivate cells. Preferably, targeted toxins are toxins that are only or at least primarily toxic to target cells but non-toxic to off-target cells. The net effect of targeted toxins is preferably beneficial to the entire organism.

[0076] The effector molecule or effector part as a polypeptide can be a polypeptide that restores a lost function, such as, for example, enzyme replacement, gene regulatory function, or a toxin. Examples of polypeptides as effector molecules are, for example, Cas9; toxins (such as saporin, dianthin, gelonin, (de) bougainvillea ribosome inactivating protein ((de) bouganin), agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptosis protein, diphtheria toxin, pseudomonas exotoxin); metabolic enzymes (such as argininosuccinate lyase, argininosuccinate synthetase), coagulation cascade enzymes, repair enzymes; enzymes for cell signaling; cell cycle regulators; gene regulators (transcription factors such as NF-κB or gene repressors such as methionine repressors).

[0077] The effector molecule or effector part as a polynucleotide can be a polynucleotide such as a gene or open reading frame containing coding information such as a protein encoding. It can also contain regulatory information, such as a promoter or regulatory element binding region, or a sequence encoding a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include peptide nucleic acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes in the main chain of the molecule. Examples of nucleotides as effector molecules are, but are not limited to, DNA: single-stranded DNA (e.g., DNA of adenine phosphoribosyl transferase); linear double-stranded DNA (e.g., coagulation factor IX gene); circular double-stranded DNA (e.g., plasmid); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA; antisense oligonucleotides (ASO, AON, such as PNA, PMO, LNA and BNA).

[0078] The term "protein" as used in "protein molecules" and "protein toxins" is a molecule and toxin comprising at least one string of amino acid residues, which can be obtained as an expression product from a single mRNA. Such molecules or toxins may also include any post-translational modifications, carbohydrates such as N- or O-linked carbohydrates, disulfide bonds, phosphorylation, sulfation, etc. as a result of any post-translational modifications, and / or may also include any other modifications such as those resulting from chemical modifications (e.g., linking effector moieties, saponins, scaffolds, ligands, etc., directly linked to, e.g., amino acid side chains, or via at least one linker (covalently) bound to molecules used to chemically modify protein molecules and chemically bind (covalently) protein molecules). The term "protein" also encompasses and includes assemblies of such molecules, such as homodimers, heterotrimers, heterohexamers, or complex assemblies such as ribosomes.

[0079] The terms "specific" and "specifically" in the context of, for example, "specifically binds" and "a receptor or molecular target specifically present or expressed on the surface of a tumor cell", etc., have their normal scientific meaning as known in the art, and refer herein to a binding interaction of a first molecule with a second molecule that occurs with a higher affinity relative to any putative binding of the first molecule to another molecule different from the second molecule, or to the expression or expression to a higher degree of a cell surface receptor or molecular target on the surface of a first type of cell (e.g., a tumor cell, an autoimmune cell, a diseased cell, an abnormal cell) relative to the degree of expression of the same receptor or molecular target at a second type of cell (e.g., a healthy cell, etc.), such as when, for example, the number of receptors or molecular targets is taken into account, wherein the expression at the second type of cell may be completely absent or very low relative to any degree of expression on a tumor cell, etc. In addition, the term "specificity", for example, in "specifically binds", has its normal scientific meaning as known in the art, and has herein the meaning of indicating a molecule that can have a higher binding affinity to interact with another molecule than the background interaction between the molecules. Similarly, the term "specificity" refers to, for example, an interaction between two molecules or between a cell and a molecule that has a higher binding affinity than background interactions between molecules. A binding molecule such as an immunoglobulin binds to a binding site on a molecule, such as an epitope, a cell surface receptor, etc., via its binding site (such as an immunoglobulin variable region of an immunoglobulin) with a higher binding affinity than background interactions between molecules. In the context of the present invention, background interactions are typically those with a K of less than 10E-4 M. D Similarly, a "specific binding domain" is a domain that preferentially binds to a binding site on a molecule, such as an epitope, a cell surface receptor, etc., with a higher binding affinity than background interactions between molecules. In the context of the present invention, a "background interaction" is typically an interaction with a K of less than 10E-4M. D Preferably, the specific binding domain has a K of greater than about 10E-5M. D affinity binding.

[0080] The term "binding" is defined as an interaction between molecules that can be distinguished from background interactions.

[0081] Throughout the specification, the term "fragment" refers to an amino acid sequence that is a part of a protein domain or constitutes a complete protein domain. Binding fragments according to the invention must have binding specificity for the respective target, such as, for example, a cell surface receptor on the surface of a diseased cell such as a tumor cell.

[0082] The term "ADC" or "antibody-drug conjugate" has its conventional scientific meaning known to the skilled person, and refers herein to a class of biopharmaceutical drugs designed as targeted therapies for the treatment of, for example, cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADCs consist of antibodies linked to a biologically active cytotoxic (anti-cancer) payload or drug. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs. They are designed to distinguish between healthy cells and diseased tissues (such as tumor cells in a tumor).

[0083] The term "Saponinum album" has its normal meaning and refers herein to a saponin mixture produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, containing SA1657 and predominantly SA1641.

[0084] The term "Quillaja saponin" has its normal meaning and refers herein to the saponin fraction of Quillaja saponin and hence the source of all other QS saponins, containing primarily QS-18 and QS-21.

[0085] "QS-21" or "QS21" has its conventional scientific meaning and refers herein to a mixture of QS-21A-apio (~63%), QS-21A-xylo (~32%), QS-21B-apio (~3.3%) and QS-21B-xylo (~1.7%).

[0086] Similarly, "QS-21A" has its conventional scientific meaning and refers herein to a mixture of QS-21A-apio (-65%) and QS-21A-xylo (-35%).

[0087] Similarly, "QS-21B" has its conventional scientific meaning and refers herein to a mixture of QS-21B-apio (-65%) and QS-21B-xylo (-35%).

[0088] The term "Quil-A" refers to a commercially available semi-purified extract from the Quillaja saponaria Molina tree and contains variable amounts of more than 50 different saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3β-OH group present in QS-7, QS-17, QS18 and QS-21. The saponins found in Quil-A are listed in van Setten (1995), Table 2 [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon F. A. Kersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Vol. 9, 660-666 (1995)]. Quil-A and Quillaja saponins are fractions of saponins from the Quillaja tree, and both contain a wide variety of different saponins with mostly overlapping contents. The specific composition of the two fractions differs because the two fractions are obtained by different purification procedures.

[0089] The term "QS1861" and the term "QS1862" refer to QS-7 and QS-7api. The molecular mass of QS1861 is 1861 Daltons and the molecular mass of QS1862 is 1862 Daltons. QS1862 is described in Table 1, row 28 of Fleck et al. (2019) [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi: 10.3390 / molecules24010171]. The structure described is the api-variant QS1862 of QS-7. The molecular mass is 1862 Daltons because this mass is the formal mass including the proton at the glucuronic acid. At neutral pH, the molecule is deprotonated. When measured in mass spectrometry in negative ion mode, the measured mass is 1861 Daltons.

[0090] The terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. Where appropriate, the terms are interchangeable. Embodiments of the present invention may operate in other sequences than those described or illustrated herein.

[0091] Furthermore, various embodiments, although referred to as “preferred” or “such as” or “for example” or “particularly”, are to be interpreted as illustrative ways in which the invention may be implemented rather than limiting the scope of the invention.

[0092] The term "comprises / comprising" used in the claims should not be interpreted as being limited to the elements or steps listed thereafter; it does not exclude other elements or steps. It needs to be interpreted as specifying the presence of the features, integers, steps or components mentioned, but does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Thus, the scope of the expression "a pharmaceutical composition comprising A and B" should not be limited to a pharmaceutical composition consisting only of components A and B, but for the purposes of the present invention, the only listed components of the pharmaceutical composition are A and B, and the claims should be interpreted as including equivalents of those components. Similarly, the scope of the expression "a method comprising steps A and B" should not be limited to a method consisting only of steps A and B, but for the purposes of the present invention, the only listed steps of the method are A and B, and the claims should be interpreted as including equivalents of those steps.

[0093] Furthermore, the indefinite article "a" or "an" referring to a feature does not exclude the possibility that more than one feature (such as, for example, a component, an excipient, a saponin, etc.) is present, unless the context clearly requires one and only one feature. Thus, the indefinite article "a" or "an" generally means "at least one". BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 : HSP27BNA, HSP27BNA-SO1861 and HSP27BNA-Dendron-(SO1861) 4 HSP27 BNA gene silencing activity in A431 cancer cell line.

[0095] Figure 2 : A. MALDI-TOF-MS spectrum of BSA-SO1861. B. MALDI-TOF-MS spectrum of BSA.

[0096] Figure 3 : A. H-NMR spectrum of SO1861. B. H-NMR spectrum of SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide) conjugate.

[0097] Figure 4 : A. MALDI-TOF-MS spectrum of SO1861-EMCH conjugate. B. MALDI-TOF-MS spectrum of SO1861-EMCH-mercaptoethanol conjugate.

[0098] Figure 5: A. Synthesis scheme of SO1861-EMCH. B. MALDI-TOF-MS spectrum of SO1861 in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: hours, and MW: molecular weight. C: MALDI-TOF-MS spectrum of SO1861-EMCH in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: hours, and MW: molecular weight.

[0099] Figure 6 : Dendron (-L-SO1861) 4 Reaction scheme of the synthesis.

[0100] Figure 7 : Dendron (-L-SO1861) 8 Reaction scheme of the synthesis.

[0101] Figure 8 : A. MALDI-TOF-MS spectrum of Cy3-PAMAM. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861, where each PAMAM is connected to 5 SO1861. C. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861, where each PAMAM is connected to 13 SO1861. D. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861, where each PAMAM is connected to 51 SO1861.

[0102] Fig. 9 : A. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 5 equivalents of feed SO1861-EMCH. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 30 equivalents of feed SO1861-EMCH.

[0103] Fig.10 : MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 (NC=stable bond (“non-cleavable”)).

[0104] Fig.11 :A. Reaction scheme for the thiolation of PAMAM using the thiolation agent 2-iminothiolane. B. MALDI-TOF-MS spectrum of native PAMAM. C. Thiolated PAMAM-(SH) 16 MALDI-TOF-MS spectrum of D. Thiolated PAMAM-(SH) 65MALDI-TOF-MS spectrum of E. Thiolated PAMAM-(SH) 108 MALDI-TOF-MS spectra.

[0105] Fig.12 :A. Use of PEGylation reagent mPEG 2k -NHS PEGylation reaction scheme of PAMAM. B. MALDI-TOF-MS spectrum of native PAMAM. C. PEGylated PAMAM-(mPEG 2k )3. D. PEGylated PAMAM-(mPEG 2k )8. E. PEGylated PAMAM-(mPEG 2k ) 18 MALDI-TOF-MS spectra.

[0106] Fig.13 : Reaction scheme of Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO). B. MALDI-TOF MS spectrum of Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO). C. MALDI-TOF-MS spectrum of Cy3-PAMAM-(SO1861)5-DBCO. D. MALDI-TOF-MS spectrum of Cy3-PAMAM-(SO1861)5-DBCO. 27 MALDI-TOF-MS spectrum of -DBCO.

[0107] Fig.14 :A. Reaction scheme of oleanolic acid-EGF-Alexa488. B. Reaction scheme of oleanolic acid-EGF-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of oleanolic acid-EGF. D. MALDI-TOF-MS spectrum of oleanolic acid-EGF-Alexa488. E. MALDI-TOF-MS spectrum of oleanolic acid-EGF-Alexa488-SS-PEG-N3.

[0108] Fig.15 :A. Reaction scheme of oleanolic acid-Alexa488. B. Reaction scheme of oleanolic acid-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of oleanolic acid. D. MALDI-TOF-MS spectrum of oleanolic acid-Alexa488. E. MALDI-TOF-MS spectrum of oleanolic acid-Alexa488-SS-PEG-N3.

[0109] Fig.16: Fluorescence image of SDS-PAGE gel performed on VersaDoc imaging system. M = marker, P = Cy3-PAMAM-(SO1861)27-DBCO, D = oleanolic acid EGF-Alexa488-SS-PEG-N3, C1 = Cy3-PAMAM-(SO1861)5-oleanolic acid-EGF-Alexa488, C2 = Cy3-PAMAM-NC-SO1861-oleanolic acid-EGF-Alexa488, and C3 = Cy3-PAMAM-(SO1861)27-oleanolic acid-EGF-Alexa488.

[0110] Fig.17 : A. Synthesis scheme of Cy3-PAMAM-NC-SO1861 via reductive amination. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 synthesized via reductive amination, with 10 SO1861 per PAMAM. C. MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 synthesized via reductive amination, with 30 SO1861 per PAMAM.

[0111] Fig.18 : A. MALDI-TOF-MS spectrum of native peptide. B. MALDI-TOF-MS spectrum of peptide-SO1861 conjugate.

[0112] Fig.19 : Structure of SO1861 with highlighted chemical groups used to conjugate saponins that enhance endosomal escape to polymeric structures. The highlighted groups are aldehydes (black circles), carboxylic acids (dashed circles), olefins (dashed pentagons), and alcohols (dashed boxes).

[0113] Fig. 20 : A. Schematic diagram of the production of a stable 'ready-to-conjugate' endosomal escape enhancer saponin. B. Schematic diagram of the production of a cleavable 'ready-to-conjugate' endosomal escape enhancer saponin.

[0114] Fig.21 : Hydrolysis of the hydrazone bond of SO1861-EMCH under acidic conditions.

[0115] Fig. 22 : A. Standard molecular structure of SO-1861-EMCH conjugate. Maleimide groups are marked with circles. B. 3D model of SO1861-EMCH conjugate. Maleimide groups are marked with circles.

[0116] Fig.23: A. MALDI-TOF-MS spectrum of SO1861-EMCH before hydrolysis in pH 3 HCl solution. B. MALDI-TOF-MS spectrum of SO1861-EMCH after hydrolysis in pH 3 HCl solution.

[0117] Fig.24 : Reaction scheme for conjugation of SO1861-EMCH to any amine-containing polymeric structure.

[0118] Fig.25 : A. Reaction scheme of SO1861-EMCH conjugated to polyamidoamine (PAMAM) G5 dendrimer labeled with cyanine 3 dye. B. Reaction scheme of SO1861-HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) conjugated to polyamidoamine (PAMAM) G5 dendrimer labeled with cyanine 3 dye.

[0119] Fig.26 : Molecular structure of a G4-dendron with protected amino groups.

[0120] Fig. 27 : Synthetic scheme for the generation of dendron-based scaffolds.

[0121] Fig.28 : A. Reaction scheme of partial dye labeling and deprotection of G4-dendrons. B. MALDI-TOF-MS spectra of deprotected and partially dye-labeled G4-dendrons.

[0122] Fig.29 : A. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 22 feed equivalents of SO1861-EMCH. B. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 10 feed equivalents of SO1861-EMCH. C. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 3 feed equivalents of SO1861-EMCH.

[0123] Fig.30:A. EGFR cell surface expression as determined by FACS analysis of HeLa cells. B. Cell viability of HeLa cells treated with SO1861 + oleanolic acid-EGF (Dia-EGF), SO1861 + oleanolic acid-EGF + 500nM chloroquine, SO1861 + oleanolic acid-EGF + 500nM PAMAM, SO1861 + oleanolic acid-EGF + 667nM dendron. C. Cell surface expression of EGFR as determined by FACS analysis of HeLa cells. B. Cell viability of HeLa cells treated with SO1861 + oleanolic acid-EGF (Dia-EGF), SO1861 + oleanolic acid-EGF + 500nM chloroquine, SO1861 + oleanolic acid-EGF + 500nM PAMAM, SO1861 + oleanolic acid-EGF + 667nM dendron. C. Cell surface expression of EGFR as determined by FACS analysis of HeLa cells. 16 、SO1861+oleanolic acid-EGF+500nM PAMAM-(SH) 65 、SO1861+oleanolic acid-EGF+500nM PAMAM-(SH) 108 D. Cell viability of HeLa cells treated with SO1861+oleanolic acid-EGF, SO1861+oleanolic acid-EGF+500nM chloroquine, SO1861+oleanolic acid-EGF+500nM PAMAM, SO1861+oleanolic acid-EGF+500nM PAMAM-(mPEG)3, SO1861+oleanolic acid-EGF+500nM PAMAM-(mPEG)8, SO1861+oleanolic acid-EGF+500nM PAMAM-(mPEG) 18 Cell viability of treated HeLa cells. DETAILED DESCRIPTION

[0124] In order for a bioactive molecule to work, the molecule must be able to bind to its target, such as in serum, outside of the cell surface, or inside a cell or organelle. For example, the active portion of almost all protein-based targeted toxins must enter the cytoplasm of the target cell to mediate its target regulation. In many constellations, the toxin remains ineffective because (1) the targeted portion is poorly internalized and remains bound to the outside of the cell, (2) after internalization, it circulates back to the cell surface or (3) is transported to endolysosomes where it is degraded. Although these basic problems have been known for decades and more than 500 targeted toxins have been studied in the past few decades, the problems remain unsolved and to date only one antibody-targeted protein toxin, moxetumomab pasudotox-tdfk ( AstraZeneca Pharmaceuticals LP) has been approved by the FDA for relapsed or refractory hairy cell leukemia. Further approved ADCs are Elzonris, Ontak.

[0125] To overcome these problems, many strategies have been described, including methods to redirect toxins to endogenous cell membrane transport complexes of the biosynthetic pathway in the endoplasmic reticulum, and techniques to disrupt or weaken the membrane integrity of endosomes (i.e., compartments of the endocytic pathway in the cell), thereby promoting endosomal escape. This includes the use of lysosomal amines, carboxylate ion carriers, calcium channel antagonists, various cell penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced technologies. Although the efficacy of targeted toxins is generally enhanced by hundreds or thousands of times in cell culture, in special cases exceeding a million times, the need to co-administer endosomal escape enhancers with other substances brings new problems, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window and cell type-dependent changes.

[0126] All strategies (including physicochemical techniques) require enhancer molecules that interact more or less directly with the membrane and essentially include small chemical molecules, secondary metabolites, peptides and proteins. A common feature of all these substances is that they are not target cell specific per se and are distributed with kinetics other than that of the targeted toxins. This is a major drawback of current approaches.

[0127] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.The embodiments of the invention described herein can be combined and operate in conjunction with each other unless otherwise indicated.

[0128] Although the present invention has been described according to several embodiments, it is expected that after reading the specification and studying the drawings and diagrams, those skilled in the art will understand its substitution, modification, replacement and equivalent. The present invention is not limited to the illustrated embodiments in any way. Changes can be made without departing from the scope defined by the appended claims.

[0129] One aspect of the invention relates to an effector moiety capable of inducing an intracellular effect when present in a mammalian cell, the effector moiety being conjugated to at least one saponin, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker, or is directly covalently bound to the effector moiety.

[0130] The inventors determined that when a conjugate of a saponin and an effector moiety is administered to a tumor-bearing mammal (mouse) (to which the conjugate is administered), the therapeutic window of the conjugate is increased, wherein the conjugate comprises at least one covalently bound saponin. The conjugate of the present invention has at least one glycoside, such as a saponin, bound thereto, preferably covalently bound, more preferably bound via a cleavable linker. Saponin may increase the therapeutic efficacy of the effector moiety bound to the saponin by enhancing the endosomal escape of the effector moiety into the sol where the activity of the effector moiety is required. In this way, the therapeutic effect is determined when a portion of the conjugate, such as an ADC or AOC, is under the influence of the presence of a saponin contained in the conjugate of the present invention, already at a dose lower than the conventional dose of the effector moiety, so that the saponin and the effector moiety are together near the target cell, at the target cell and / or in the target cell. The target cell is, for example, a diseased cell, such as a tumor cell or an autoimmune cell or a B cell associated with a B cell disease, etc. According to the present invention, the effector moiety is, for example, a toxin, such as a toxin that is part of an ADC; or an oligonucleotide, such as an antisense BNA that is part of an AOC.

[0131] An embodiment is an effector portion of the present invention, which comprises or consists of at least one oligonucleotide, nucleic acid, heterologous nucleic acid, which is preferably selected from any one or more of the following: a vector, a gene, a transgene that induces cell suicide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), an aminophosphoryl morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridging nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinose nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridging nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA), or derivatives thereof, more preferably BNA, such as BNA for silencing HSP27 protein expression.

[0132] To the surprise of the inventors, a conjugate of a covalently linked antisense BNA (e.g., for silencing HSP27 gene expression) and a saponin (e.g., SO1861) effectively silenced HSP27 expression in rapidly growing / dividing tumor cells (e.g., mouse A431 cancer cell line cells) compared to a control. The BNA-saponin conjugate resulted in a reduction of HSP27 expression in cells by about three times compared to HSP27 expression in cells exposed to the same concentration of free, unconjugated BNA in the absence of saponin. See also the examples Figure 1Thus, contacting tumor cells with antisense oligonucleotides, such as antisense BNAs, results in gene silencing, which is improved or enhanced when saponins, such as SO1861, are covalently linked to the oligonucleotides. Thus, the therapeutic window for oligonucleotides is expanded, since the same gene silencing effect in tumor cells can now be obtained with a three-fold lower dose of antisense BNAs when applying conjugates of BNAs and saponins according to the invention.

[0133] One embodiment is an effector portion of the invention, which comprises at least one protein molecule, which is preferably selected from any one or more of the following: peptides, proteins, enzymes such as urease and Cre recombinase, ribosome inactivating proteins, protein toxins such as any one or more of the protein toxins selected from Table A5 and / or bacterial toxins, plant toxins, more preferably selected from any one or more of the following: viral toxins, such as apoptotic proteins; bacterial toxins, such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins, such as α-sarcin; plant toxins, including ribosome inactivating proteins and the A chain of type 2 ribosome inactivating proteins, such as stone Bamboo toxins such as dianthin-30 or dianthin-32, saporins such as saporin-S3 or saporin-S6, bougainvillea ribosome inactivating protein or a deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, ricin, ricin A chain, abrin, abrin A chain, abrin, abrin A chain, abrin, abrin A chain, viscum, viscum A chain; or animal or human toxins, such as frog RNase or granzyme B or angiogenin from humans, or any fragments or derivatives thereof; preferably, the protein toxin is dianthin and / or saporin.

[0134] One embodiment is an effector portion of the invention comprising at least one payload, the payload preferably being selected from any one or more of the following: a toxin targeting a ribosome, a toxin targeting an elongation factor, a toxin targeting a tubulin, a toxin targeting a DNA, and a toxin targeting an RNA, more preferably being selected from any one or more of the following: maytansine, pasudotox, maytansine derivative DM1, maytansine derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mofortin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine , CC-1065 analogs, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulin, indoline benzodiazepine , AZ13599185, nostoc, rhizobactin, methotrexate, anthracycline drugs, camptothecin analogs, SN-38, DX-8951f, exotecan mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivatives, amanitin, α-amanitin, scotstatin, talanstatin, ozogamicin, teshilin, Amberstatin269 and soravtansine or their derivatives.

[0135] One embodiment is an effector portion of the invention, wherein the at least one saponin is a triterpene saponin and / or a disaccharide chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function at position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, and / or is a saponin isolated from a species of the genus Lithospermum and / or a species of the genus Saponaria and / or a species of the genus Osmanthus and / or a species of the genus Quillaja, such as Quillaja spp.

[0136] One embodiment is an effector moiety of the invention, wherein the at least one saponin is a single specific saponin or is a mixture of two or more different saponins.

[0137] One embodiment is an effector portion of the invention, wherein the saponin is one or more of the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsophila saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any one of their stereoisomers and / or any combination thereof, preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or has a sapogenin aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3β-OH group and a Glc-(1→3)-Xyl-(1→4)-Rha at the C-28-OH group. -(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent saponin, and / or 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronic acid pyranosyl saponin 28-O-β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4-OAc-β-D-quinopyranosyl-(1→4)]-β-D-fucopyranoside, more preferably, the saponin is SO1861 and / or QS-21.

[0138] Surprisingly, the inventors have now demonstrated that water-soluble saponin fractions from Quillaja saponaria (including QS-21 and its family members QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18 and Quil-A) when prepared as a mixture comprising monoclonal antibodies and at least one glycoside (as obtained from such QS-21 preparations ( For example, the ability to enhance the in vitro biological effects of nucleic acids bound to monoclonal antibodies or protein toxins bound to monoclonal antibodies, such as QS-21 and its family members saponins (such as the water-soluble fraction of Quillaja sylvestris) when administered to tumor cells of mammalian species (humans) in the form of covalent conjugates, in which the effector molecule and the glycoside (such as the saponin fraction of Quillaja sylvestris, QS-21, SO1861, SA1641, GE1741) are covalently bound to, for example, a protein molecule, directly or via a linker or via a multimeric or oligomeric scaffold, directly or via at least one linker. Without wishing to be bound by any theory, the stimulation or enhancement of, for example, antisense BNA-mediated reduction of tumor cell HSP27 expression (HSP27 gene silencing) observed in vitro in the presence of saponins derived from Quillaja sylvestris may (also) be related to the activation of inflammasomes in tumor cells by saponins, for example leading to tumor cell pyroptosis. The inventors determined that antibodies conjugated to, for example, antisense BNAs or oleanolic acid or saporin, when contacted with cells in a bio-based cell assay, fully exerted any anti-tumor cell activity in vitro or improved anti-tumor cell activity when in the presence of saponin coupled to the targeting antibody and targeted to the same (tumor) cells, whereas in the absence of saponin, no such activity on tumor cells was observed.

[0139] QS-21, and also the water-soluble saponin fractions from Quillaja saponaria comprising QS-21, have long been known and have previously been widely used for their ability to enhance immunity, such as as an adjuvant in subunit vaccines. For example, QS-21 was used in two Phase III clinical trials in human patients vaccinated with a subunit vaccine mixed with an adjuvant comprising QS-21 (Glaxo-Smith-Kline, MAGRIT trial, DERMA study), wherein the subunit is the MAGE-A3 protein specifically expressed and presented by tumor cells. QS-21 enhanced anti-tumor vaccination aims to prolong disease-free survival of cancer patients (melanoma; non-small cell lung cancer). In addition, QS-21 has been tested as an adjuvant in clinical trials for the development of anti-cancer vaccine therapy, for HIV-1 infection, for the development of a vaccine against hepatitis B, and for anti-malarial vaccine development using Glaxo-Smith-Kline's QS-21-containing adjuvants AS01 and AS02. Previous studies revealed that immune responses elicited against MAGE-A3 peptides presented on the surface of cancer cells were activated under the influence of an adjuvant (AS15; GSK) containing QS-21 saponin. To the surprise of the inventors, the saponin fraction of Quillaja saponin, and therefore probably QS-21 (as part of the water-soluble saponin fraction of Quillaja saponin), enhanced the anti-tumor cell activity of a payload such as a protein toxin (dianthin) bound to a second protein molecule such as the ligand EGF.

[0140] One embodiment is an effector moiety of the invention, wherein the saponin is a disaccharide chain saponin having a molecular mass of at least 1.500 Daltons and comprising an oleanane-type triterpene containing an aldehyde group at position C-23 and optionally a hydroxyl group at position C-16, and a first branched carbohydrate side chain at position C-3, the first branched carbohydrate side chain optionally containing glucuronic acid, wherein the saponin contains an ester group having a second branched carbohydrate side chain at position C-28, the second branched carbohydrate chain preferably comprising at least four carbohydrate units and optionally containing at least one acetyl residue, such as two acetyl residues and / or optionally containing one or more deoxy carbohydrates and / or quinovose and / or glucose and / or 4-methoxycinnamic acid and / or optionally containing 5-O-[5-O-Ara / Ap i-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprising 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester bond, or wherein at least one saponin is QS-21 or any one or more of the following: QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

[0141] The inventors show that a tumor cell targeting monoclonal antibody (both BNA and saponin coupled to the corresponding antibody (e.g. cetuximab) via a cleavable bond) provided with a covalently coupled antisense BNA (e.g. BNA (HSP27)) and contacted with tumor cells together with an antibody with coupled saponin (e.g. SO1861, QS-21) is able to silence HSP27 in vivo in tumors compared to controls and compared to AOC alone (in the absence of antibody with coupled saponin). Co-administration of an ADC or antibody-oligonucleotide conjugate (AOC) (e.g. antibody-BNA conjugate) with an antibody with linked saponin thus confers anti-tumor cell efficacy to the ADC or AOC. cytotoxicity, which was not observed with ADC alone or AOC alone at the same dose. Notably, AOC and monoclonal antibodies with covalently coupled saponin increased HSP27 expression in tumor cells when administered to tumor-bearing mice in different mouse groups, respectively, compared to the control group (only vehicle was administered). Co-administration of AOC containing only the effector portion and antibodies with covalently coupled saponin showed reduced HSP27 expression when compared to the control. Antisense BNA (HSP27) is a BNA with an oligonucleotide sequence of 5'-GGCacagccagtgGCG-3' according to Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333]. It is noteworthy that, to the best of the inventors' knowledge, BNAs are designed to be applied as free nucleic acids. The inventors are now the first to demonstrate that antisense BNAs can be covalently coupled to ligands or antibodies in a manner via (non-)cleavable linkers, such that gene silencing activity is retained in vitro and, more importantly, in vivo in tumor cells of tumor-bearing animals. This method of providing BNA-based AOCs opens up a new method for administering targeted BNAs to human (cancer) patients in need.

[0142] The inventors disclose herein that covalent coupling of saponins (e.g., saponins in the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, Table A1, Scheme I) to, e.g., antisense oligonucleotides, such as BNAs, or to, e.g., protein molecules, such as via a trifunctional linker (e.g., the trifunctional linker of Scheme II) or via an oligomeric or polymeric structure of a scaffold comprising covalently bound saponins, results in improved cytotoxicity exerted by an effector moiety, such as a toxin, comprised by the effector moiety of the invention under the influence of the covalently coupled saponin in the effector moiety of the invention.

[0143] According to the present invention, typically a saponin is a saponin listed in Table A1, Scheme I. The activity of a saponin has been shown to be beneficial, such as considering that when the effector moiety of the present invention comprising the coupled saponin enters the cell and accumulates in the cytoplasm, the intracellular endosomal escape enhancing activity is when the saponin is covalently coupled to a payload comprised by the effector moiety (e.g., BNA) involving a hydrazone bond and / or a hydrazide bond and / or a disulfide bond. Such bond types are easily cleaved under acidic conditions and / or under reducing conditions in (late) endosomes and lysosomes of mammalian cells (e.g., human cells). Alternatively, the inventors have also shown that the covalent coupling of a saponin to a payload comprised by the effector moiety of the present invention via a bond that is not easily cleaved under physiological conditions in the cell (e.g., (late) endosome, lysosome, cytoplasm) is also beneficial for the enhancing activity of the saponin on the biological effects of, for example, effector moieties such as nucleic acids (e.g., BNA silencing HSP27) and protein toxins such as saporin. Throughout the application including the claims, the terms 'cleavable linker', 'cleavable bond' and the like, also referred to as 'labile linker' ('L') and 'labile bond', e.g. in the context of cleavage of such bonds or linkers in (late) endosomes and / or lysosomes, when referring to a conjugate of the invention, such as an effector moiety optionally comprising a scaffold comprising a saponin coupled to a payload via a linker and / or via the scaffold via a hydrazone bond or a disulfide bond, such as BNA-SO1861, or an antisense oligonucleotide-saponin conjugate (the saponin being any of the saponins in Table A1, Scheme I). For example, the inventors showed in vivo HSP27 gene silencing in human tumors in mice. Tumor-bearing mice were treated with an antibody consisting of a monoclonal antibody with a saponin bound thereto via a labile linker (hydrazone bond) according to the invention, whereas a different part of the same antibody comprising a bound antisense BNA for silencing the HSP27 gene in tumor cells was covalently coupled to the monoclonal antibody (of the same type as the first monoclonal antibody) via a disulfide bond. That is, without wishing to be bound by any theory, once the therapeutic combination of the invention is internalized by, for example, endocytosis, the hydrazone bonds and disulfide bonds are cleaved in the (late) endosomes and / or lysosomes of the targeted tumor cells, which express epitopes on the targeted cell surface molecules (herein EGFR) on the cell surface. When considering that BNAs enter the cytoplasm from endosomes and / or lysosomes, the cleavage of the bonds may contribute to the endosomal escape enhancing activity of the saponin, although such cleavage is not necessary to observe the gene silencing effect of the combination of the cetuximab-SO1861 conjugate and the cetuximab-BNA conjugate of the invention.

[0144] The technician will understand that the trifunctional linker is a support of the present invention suitable for covalently coupling one, two or three saponin moieties. For the trifunctional linker, the covalent coupling of one or two saponin moieties is preferred. The second and / or third binding site is, for example, suitable for covalently coupling a useful load, so that the effector portion of the present invention is provided. In addition, the second or third binding site of the trifunctional linker is suitable for immunoglobulins such as monoclonal antibodies, preferably tumor cell-specific molecules, more preferably covalent coupling of tumor cell receptors specifically (over-) expressed on the surface of tumor cells. Similarly, immunoglobulins or any fragments and / or domains thereof covering immunoglobulin binding specificity are suitable for binding to cell surface molecules such as receptors expressed on the surface of autoimmune cells. Then, the trifunctional linker comprises a covalently coupled saponin and a covalently coupled useful load, thereby providing the effector portion of the present invention, and in addition, covalently coupled, for example, antibodies, for targeting the effector portion to selected cells, such as tumor cells expressing the tumor cell-specific binding site of the coupled antibody, for example, specific tumor cell receptors. Thus, in one embodiment, the effector moiety of the invention comprises a trifunctional linker comprising or consisting of a covalently bound saponin and an effector moiety, e.g. QS-21, SO1861 and e.g. antisense BNA, a protein toxin and a covalently bound antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, non-healthy cells, B cell diseases.

[0145] One embodiment is an effector moiety according to the invention, wherein said at least one saponin is a disaccharide-chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function at position C-23, wherein said at least one saponin is covalently coupled to an amino acid residue of said effector moiety when present via the aldehyde function in said saponin, preferably at position C-23, preferably via at least one linker, more preferably via at least one cleavable linker, wherein said amino acid residue is preferably selected from cysteine ​​and lysine.

[0146] One embodiment is an effector moiety of the invention, wherein the at least one saponin is a disaccharide-chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function at position C-23 and comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, wherein the at least one saponin is covalently coupled to an amino acid residue of the effector moiety when present via the glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, preferably via at least one linker, wherein the amino acid residue is preferably selected from cysteine ​​and lysine.

[0147] As an example, according to the present invention, a BNA oligonucleotide, namely an antisense BNA oligonucleotide targeting the mRNA transcript of a cancer target (upregulated in cancer cells) heat shock protein 27 (HSP27BNA) is conjugated to SO1861-EMCH (HSP27BNA-L-SO1861) or a dendron (-L-SO1861) 4 (HSP27BNA-Dendron (-L-SO1861) 4 ), and co-administered to the A431 cancer cell line. As described, the inventors were surprised that conjugates such as antisense BNAs and saponins (such as SO1861) covalently linked together for silencing HSP27 gene expression effectively silenced HSP27 expression in rapidly growing / dividing tumor cells (such as mouse A431 cancer cell line cells) compared to controls. Covalent conjugation of any saponin of the present invention with an oligonucleotide, such as an antisense oligonucleotide, such as a BNA, such as an antisense BNA results in an increased gene silencing efficacy at the same dose of BNA when compared to free BNA in the absence of conjugated saponins and in the absence of free saponins. Compared to HSP27 expression in cells exposed to the same concentration of free unconjugated BNA in the absence of saponins, the BNA-saponin conjugate results in a reduction of about three times in HSP27 expression in cells. See also the examples Figure 1 . Therefore, contacting tumor cells with antisense oligonucleotides such as antisense BNAs leads to gene silencing, and when saponins such as SO1861 are covalently linked to oligonucleotides, gene silencing is improved or enhanced. Therefore, the therapeutic window for oligonucleotides is expanded, because when a conjugate of a BNA and a saponin according to the present invention is applied, the same gene silencing effect can now be obtained in tumor cells with a three-fold lower dose of antisense BNA. The inventors have determined that when considering gene silencing, a conjugate of BNA with one saponin is more effective than the gene silencing activity obtainable by free BNA applied to cells. In addition, the inventors have determined that when considering gene silencing, and compared with the gene silencing activity obtainable by free BNA applied to cells, a conjugate of BNA with four saponins is increasingly effective. That is, according to the present invention, the gene silencing improved with BNA (-saponin) 4 is even higher than the improved gene silencing obtainable with BNA-saponin. The linker is a cleavable linker.

[0148] One embodiment is an effector portion of the invention, wherein the at least one linker comprises at least one non-cleavable linker and / or at least one cleavable linker, wherein optionally the cleavable linker is cleaved under acidic conditions, reducing conditions, enzymatic conditions or light-induced conditions, and preferably, the cleavable linker comprises a cleavable bond selected from the group consisting of a hydrazone bond and a hydrazide bond that are cleaved under acidic conditions, and / or a bond that is susceptible to proteolysis, such as a bond that is susceptible to proteolysis by cathepsin B, and / or a bond that is susceptible to cleavage under reducing conditions, such as a disulfide bond.

[0149] One embodiment is an effector portion of the invention, wherein the at least one linker comprises at least one cleavable linker that is cleaved in vivo under acidic conditions as present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5 and more preferably at pH ≤ 5.5.

[0150] One embodiment is an effector moiety of the invention, wherein the at least one saponin is covalently bound to a lysine side chain, forming an amide bond, and / or covalently bound to a cysteine ​​side chain, forming a thioether bond or a disulfide bond, wherein the lysine and / or cysteine ​​is comprised by the effector moiety, and wherein the saponin is directly bound to the effector moiety, or is bound to the effector moiety via at least one linker.

[0151] One embodiment is an effector moiety of the invention, wherein said at least one saponin is covalently bound to said effector moiety via at least one linker, wherein said linker is or comprises a scaffold comprising a multimeric or oligomeric structure and a chemical group for covalent coupling of said scaffold to said effector moiety.

[0152] One embodiment is an effector moiety of the invention, wherein said at least one saponin is covalently bound to the multimeric or oligomeric structure of said scaffold via a cleavable bond.

[0153] One embodiment is an effector portion of the invention, wherein the cleavable bond is cleaved under any one of acidic conditions, reducing conditions, enzymatic conditions and light-induced conditions, more preferably, the cleavable bond is a hydrazone bond or a hydrazide bond that is cleaved under acidic conditions and / or is a bond that is susceptible to proteolysis, such as a bond that is proteolyzed by cathepsin B and / or is a bond that is susceptible to cleavage under reducing conditions, such as a disulfide bond.

[0154] One embodiment is an effector moiety of the invention, wherein the cleavable bond is cleaved in vivo under acidic conditions as present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5 and more preferably at pH ≤ 5.5.

[0155] One embodiment is an effector portion of the invention, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the scaffold via an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond and / or an ester bond, preferably via at least one linker, preferably an amide bond, a hydrazide bond, a thioether bond and / or a hydrazone bond.

[0156] One embodiment is an effector portion of the invention, wherein said at least one saponin is covalently bound to the multimeric or oligomeric structure of said scaffold, wherein said covalent bond involves an aldehyde function at position C-23 of said at least one saponin, when present, said covalent bond is preferably an imine bond or a hydrazone bond or an amide bond or a thioether bond or a disulfide bond, and / or wherein said covalent bond involves a glucuronic acid function in a carbohydrate substituent at the C-3β-OH group of said at least one saponin (when present), wherein preferably, said covalent bond is an amide bond or a disulfide bond or a thioether bond.

[0157] One embodiment is an effector portion of the invention, wherein the aldehyde function at position C-23 of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled to a sulfhydryl group, such as a sulfhydryl group of cysteine, in the polymeric or oligomeric structure of the scaffold via a thioether bond.

[0158] One embodiment is an effector portion of the invention, wherein the glucuronic acid functionality in the carbohydrate substituent at the C-3β-OH group of the at least one saponin is covalently coupled to a linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled to an amine group in the polymeric or oligomeric structure of the scaffold via an amide bond, such as an amine group of lysine or the N-terminus of the polymeric or oligomeric structure of the scaffold.

[0159] One embodiment is an effector moiety of the invention, wherein said chemical group of said scaffold for covalent coupling of said scaffold to said effector moiety is a click chemistry group, which is preferably selected from tetrazines, azides, olefins or alkynes or cyclic derivatives of these groups, more preferably, said click chemistry group is an azide.

[0160] One embodiment is an effector portion of the invention, wherein the polymeric or oligomeric structure of the scaffold comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol or an assembly of these polymeric or oligomeric structures, said assembly preferably being constructed by covalent cross-linking.

[0161] One embodiment is an effector portion of the invention, wherein the at least one saponin is a defined number of saponins or a defined range of saponins, preferably 1-128 saponins or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128 saponins, or any number of saponins in between, such as 7, 9, 12 saponins.

[0162] One embodiment is an effector portion of the invention, wherein the effector portion comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins or any number in between, such as 7, 9, 12 saponins, which are covalently bound directly to an amino acid residue, preferably cysteine ​​and / or lysine, of the effector portion and / or are covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one multimeric or oligomeric scaffold as described herein, preferably 1-8 such scaffolds or 2-4 such scaffolds, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins are covalently bound to at least one scaffold.

[0163] The inventors have determined that administration of free oligonucleotides such as antisense BNAs to mice is not very effective when considering gene silencing in (fast growing) tumor cells. Surprisingly, the inventors have now determined that beneficial antitumor activity in various in vitro mammalian cell-based bioassays can be achieved by treating animals with conjugates according to the invention, optionally comprising a scaffold according to the invention, such as a G4-dendron. The scaffold is for example a trifunctional linker with at least one saponin covalently bound via a cleavable or non-cleavable bond (e.g. SO1861, QS-21), and / or with a covalently bound effector moiety (e.g. antisense oligonucleotide) silencing BNA (HSP27) via a non-cleavable or cleavable bond, or the scaffold is a dendron, for example a dendron to which four moieties, such as four saponin molecules, can be bound, or a dendron for binding, for example, two saponins and two effector molecules. The reference examples section illustrates that linkers and scaffolds according to the invention show in vitro anti-tumor cell activity when considering gene silencing exerted by, for example, antisense BNAs (HSP27), and thus when considering gene silencing in tumor cells.

[0164] The term "improving or enhancing the effect of an effector moiety" means that a glycoside molecule, preferably a saponin of the present invention, preferably increases the functional efficacy of this effector moiety by enabling or improving its target engagement (e.g., therapeutic index of a toxin or drug or oligonucleotide such as a BNA; metabolic efficacy of a modifier in a biotechnological process; transfection efficacy of a gene in a cell culture research experiment). Preferably, acceleration, prolongation or enhancement of an antigen-specific immune response is not included. Therapeutic efficacy includes, but is not limited to, a stronger therapeutic effect, preferably with a lower dose and / or fewer side effects. "Improving the effect of an effector moiety" may also mean that an effector moiety that cannot be used (and such as is not referred to as an effector moiety) due to lack of effect becomes effective when used in combination with the present invention. Any other effect that is beneficial or desired and can be attributed to the combination of an effector moiety and a second or third protein molecule as provided by the present invention is considered an "improved effect". In one embodiment, a scaffold comprising a bound saponin and comprised by an effector moiety enhances the effect of a payload / effector moiety that is expected and / or desired.

[0165] A number of preferred features can be formulated for endosomal escape enhancers comprised by effector moieties, i.e., glycosides or saponins, preferably saponins according to the present invention: (1) they are preferably non-toxic and do not elicit an immune response, (2) they preferably do not mediate cytoplasmic uptake of the effector moiety into off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector moiety, (4) they are preferably biodegradable or excretable, and (5) they preferably do not substantially interfere with biological processes of the organism unrelated to the biological activity of the effector molecule to which the endosomal escape enhancer is combined, such as interactions with hormones. Examples of glycoside molecules such as saponins of the present invention that meet the above criteria at least to some extent are disaccharide-chain triterpenes, preferably disaccharide-chain triterpenoid saponins, such as SO1861, SA1641, QS-21, GE1741 and the saponins in Table A1, Scheme I.

[0166] To explain the present invention in more detail, the process of cellular uptake of substances (although the inventors do not wish to be bound by any theory) and the terms used in the present invention are described. The uptake of extracellular substances, such as the effector moiety of the present invention, into cells by vesicle budding is called endocytosis. The vesicle budding can be characterized by: (1) receptor-dependent ligand uptake mediated by the cytoplasmic protein clathrin, (2) lipid raft uptake mediated by the cholesterol binding protein caveolin, (3) non-specific fluid uptake (pinocytosis), or (4) non-specific particle uptake (phagocytosis). All types of endocytosis will enter the cellular process of vesicle transport and material sorting, which is hereinafter referred to as the endocytic pathway. The endocytic pathway is complex and is not yet fully understood. Without wishing to be bound by any theory, organelles can be formed from scratch and mature into the next organelle along the endocytic pathway. However, it is now assumed that the endocytic pathway involves stable compartments connected by vesicle transport. Compartments are complex, multifunctional membrane organelles that are specialized for a specific set of basic functions of the cell. Vesicle is considered as transient organelle, composition is simpler, and is defined as a membrane-enclosed container, which is formed de novo by budding from a pre-existing compartment. Compared with compartment, vesicle can undergo maturation, which is a series of physiologically irreversible biochemical changes. Early endosome and late endosome represent stable compartments in endocytic pathway, while primary endocytic vesicle, phagosome, multivesicular body (also referred to as endosomal carrier vesicle), secretory granules and even lysosome represent vesicle. Endocytic vesicle appears at plasma membrane, most prominently from the pits coated with clathrin, first merged with early endosome, early endosome is the main sorting compartment of about pH 6.5. Most of the internalized cargo and membrane are circulated back to plasma membrane by recycling vesicle (recycling pathway). The components that should be degraded are transported to acidic late endosome (pH is lower than 6) via multivesicular body. Lysosome is a vesicle that can store mature lysosomal enzymes and deliver them to late endosomal compartment when needed. The resulting organelle is called hybrid organelle (hybrid organelle) or endolysosome. Lysosomes bud from hybrid organelles in a process called lysosome regeneration (lysosome reformation). Late endosomes, lysosomes and hybrid organelles are extremely dynamic organelles, and it is usually difficult to distinguish them. The degradation of endocytic molecules occurs in endolysosomes or lysosomes. Endosomal escape is that substances are actively or passively released from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably from clathrin-mediated endocytosis, or recycling pathways into solutes. Therefore, endosomal escape includes but is not limited to release from endosomes, endolysosomes or lysosomes (including their intermediate and hybrid organelles).

[0167] Unless otherwise specifically stated and particularly in relation to the endosomal escape mechanism of glycoside molecules (such as the saponins of the present invention), whenever the term "endosome" or "endosome escape" is used herein, it also includes endolysosomes and lysosomes, as well as escape from endolysosomes and lysosomes, respectively. After entering the cytoplasm, the substance may move to other cell units such as the nucleus.

[0168] In formal terms, a glycoside is any molecule in which a sugar group is bound to another group through its anomeric carbon via a glycosidic bond. In the context of the present invention, glycoside molecules such as saponins are molecules that are able to further enhance the effect of the effector moiety, without wishing to be bound by any theory, in particular by promoting endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (saponins, such as those listed in Table A1 and Scheme I and those exemplified in the various embodiments) interact with the membranes of compartments and vesicles of the endocytic and recycling pathways and render them leaky for the effector moiety, leading to increased endosomal escape. The term "scaffold is capable of increasing the endosomal escape of the effector moiety" means that at least one saponin (glycoside molecule) coupled to the multimeric or oligomeric structure of the scaffold is capable of enhancing the endosomal escape of the payload comprised by the effector moiety of the invention, optionally and preferably after at least one glycoside, such as a saponin, is released from the effector moiety (such as from a linker or a multimeric or oligomeric structure comprised by said effector molecule), such as by cleavage of a cleavable bond between at least one glycoside (saponin) and the payload (e.g. via the multimeric or oligomeric structure of the scaffold and / or via a linker), when both molecules are in an endosome (such as a late endosome). Even if the bond between at least one glycoside, such as a saponin according to the invention and the payload comprised by the effector moiety of the invention (optionally via a linker or a scaffold) may be a "stable bond", this does not mean that such a bond cannot be cleaved, for example, enzymatically, in the endosome. For example, the glycoside or saponin may be cleaved from the remaining linker fragment or the oligomeric or oligomeric structure, optionally together with a part of the oligomeric or multimeric structure of the linker or the scaffold. For example, it may be that a protease cleaves a (protein) linker or a protein polymeric structure, such as albumin, thereby releasing at least one glycoside, saponin. However, it is preferred that the glycoside molecule (preferably a saponin) is released in an active form, preferably in its original form before being (prepared to) coupled to a payload comprised by the effector moiety of the invention, optionally via a linker and / or an oligomeric scaffold or a polymeric scaffold; thus, the glycoside (saponin) has its native structure after such cleavage or the glycoside (saponin) has (partial) chemical groups or linkers bound to it after such cleavage, while the glycoside biological activity (saponin biological activity), such as the endosomal / lysosomal escape enhancing activity against a payload present in the same endosome or lysosome, is maintained or restored after said cleavage of the bond between the glycoside (saponin) and, for example, a payload or carrier molecule (comprising the effector molecule of the invention, optionally comprising a linker and / or scaffold of the invention). In relation to the present invention, the term "stable" with respect to the bond between, for example, a saponin and an amino acid residue of a payload, a linker, a polymeric or oligomeric structure (of a scaffold), a ligand, a (monoclonal) immunoglobulin or a binding domain or binding fragment thereof and / or an additional effector (effector moiety, effector molecule) means that the bond is not easily cleaved or at least is not designed to be easily cleaved by, for example, pH differences, salt concentration or ultraviolet light, reducing conditions.In relation to the present invention, the term "cleavable" with respect to a bond between, e.g., a saponin and a (protein) payload, a linker, an amino acid residue, a multimeric or oligomeric structure of a scaffold, a ligand, an antibody and / or a further effector comprised by the effector moiety of the present invention means that the bond is designed to be easily cleaved by, e.g., pH differences, salt concentration, under reducing conditions, etc. The skilled person is well aware of such cleavable bonds and how to prepare them.

[0169] Prior to the present invention, one of the major obstacles to the introduction of ADCs and AOCs on the market was the small therapeutic window: therapeutically effective doses of ADCs or AOCs were accompanied by (unacceptable) side effects, hindering the development and suggestion of treating patients with ADCs. By applying the effector moiety of the present invention as a semi-finished product for the manufacture of an ADC-saponin conjugate or an AOC-saponin conjugate, it is now possible to direct one or more glycoside molecules (saponins) together with an ADC carrying a payload or together with a (monoclonal) antibody conjugated to an oligonucleotide according to the present invention, such as a BNA, to a (target) cell, whereby the antibody is provided with a covalently bound effector moiety according to the present invention, which comprises a covalently coupled saponin according to the present invention. In particular, it was previously impossible to simultaneously direct the effector moiety of the payload comprised by the effector moiety of the present invention and a (predetermined, controllable) specific number or range of glycoside molecules (saponins) per payload, such as via the endocytic pathway of the cell, specifically to the cell cytoplasm. Now, the effector moiety of the present invention can be covalently coupled to a monoclonal antibody for binding to an abnormal cell-specific epitope, such as a tumor cell receptor specifically exposed on the surface of the tumor cell, so that the effector moiety of the present invention is used as a semi-finished product to provide an ADC-saponin conjugate or AOC-saponin conjugate according to the present invention.

[0170] The solution provided by the present invention comprises at least one saponin covalently bound to a payload, thereby providing an effector portion of the present invention. Another solution provided by the present invention comprises the use of an oligomeric or polymeric scaffold (first) to polymerize glycoside molecules (saponins), and to provide a cluster of covalently bound saponins for the payload comprised by the effector portion of the present invention, so that, for example, after endocytosis, one or more saponins can be re-monomerized at the intracellular site (where the mode of action of the saponin is required). "Polymerization" in this context means the reversible and / or irreversible multiple conjugation of saponin molecules with payload molecules via a linker or directly or via a polymeric or oligomeric structure to form a scaffold, or the reversible and / or irreversible multiple conjugation of (modified) saponins to form a polymeric or oligomeric structure to form a scaffold. "Re-monomerization" in this context means, for example, cleavage of the saponin from the payload molecule (e.g., protein toxin, antisense BNA), from the linker connecting the saponin to the payload molecule in the effector moiety of the invention, or from the scaffold after endocytosis, and restoration of the (native) chemical state of the unbound saponin, which may or may not contain additional chemical groups, such as chemical groups used to connect the saponin to the linker, the amino acid residues of the payload or the scaffold, and / or (chemical) linkers that bind chemical groups of the saponin (e.g., aldehyde groups or carboxylic acid groups). Due to the complex chemical nature of saponins, for example, the 'polymerization' of saponins at the scaffold or other connecting linkers and their 're-monomerization' at the desired location (e.g., intracellularly, after endocytosis) is a challenging task. In particular, the chemical reactions used to provide linkers and scaffolds comprising covalently linked glycosides such as triterpenoid saponins (polymerization of glycosides) for covalently binding payloads typically occur in anhydrous organic solvents, although saponins and, for example, biocompatible polymers used as scaffolds for carrying bound saponins are water-soluble molecules.

[0171] Without wishing to be bound by any theory, it is preferred to have at least one saponin and an effector moiety, preferably a toxin or an oligonucleotide, present simultaneously in a compartment or vesicle of the endocytic pathway of a target cell, such as a tumor cell or an autoimmune cell. Using, for example, BNAs and free saponins, the presence of the molecules in the late endosome is synchronized so as to obtain a synergistic effect that cannot be beneficially obtained in vivo. In one aspect, the present invention preferably solves at least the following problems with combining an effector moiety comprised by the conjugate of the present invention with a saponin comprised by the conjugate of the present invention: without wishing to be bound by any theory, for example, the only reasonable chemical groups in the saponin that can be used for (covalent), in particular single and cleavable, retainable coupling are those required for endosomal escape activity. The known limitations are most likely the reason why saponins have not been used in combination with pharmaceutically active substances in clinical studies other than the use of saponins in vaccination schemes, where the use of immunopotentiating adjuvant substances is suggested, although the significant endosomal escape enhancer effect of the saponins, for example, listed in Table A1 and Scheme I, has been known for more than 10 years. These difficulties are at least partially solved by providing, for example, a conjugate of the present invention with a covalently conjugated scaffold. Surprisingly, saponins, which were previously applied for their immunopotentiating activity in the context of vaccination involving saponins as adjuvant components, are now also suitable for (covalent) coupling to the ADC or AOC of the present invention for in vitro and in vivo anti-tumor activity.

[0172] One aspect of the invention relates to an antibody-drug conjugate comprising an effector moiety according to the invention, or a ligand-drug conjugate comprising an effector moiety according to the invention, said effector moiety comprising a covalently coupled saponin. The effector moiety of the invention is a conjugate comprising at least one saponin and at least one effector molecule covalently coupled to each other directly or via at least one linker, optionally comprising a cleavable linker, and optionally via an oligomeric or multimeric scaffold, said at least one saponin and / or said at least one effector moiety being covalently bound to said oligomeric or multimeric scaffold.

[0173] One embodiment is an antibody-drug conjugate or ligand-drug conjugate of the present invention, wherein the antibody is capable of binding to any of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor body, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA 4. CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and / or wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, intuzumab, mositumomab, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, Alemtuzumab, pinatuzumab, OKT-10, anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9 or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, Moxetumomab pasudotox and polotuzumab vedotin and an antibody-drug conjugate of Table A2 and Table A3, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0174] One aspect of the invention relates to a therapeutic combination comprising: (a) an effector moiety of the invention comprising at least one saponin and optionally a pharmaceutically acceptable excipient; and (b) an antibody-drug conjugate or a ligand-drug conjugate and optionally a pharmaceutically acceptable excipient.

[0175] One embodiment is a therapeutic combination of the invention, wherein the antibody-drug conjugate is capable of binding to any of the following: tumor cell receptors CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor body, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA 4. CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and / or wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, intuzumab, mositumomab, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, Alemtuzumab, pinatuzumab, OKT-10, anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9 or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, Moxetumomab pasudotox and polotuzumab vedotin and an antibody-drug conjugate of Table A2 and Table A3, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0176] The effector moieties that can be used in the present invention preferably rely on late endosomal escape to exert their effects. Some effectors, such as, for example, Pseudomonas exotoxins, are rerouted to other organelles before the "late endosomal stage" and therefore generally do not benefit from coupling with the second protein molecule according to the present invention. However, such toxins may be suitable for use with the present invention, such as by deleting the signal peptide responsible for the reroute. In particular, toxins that are highly toxic and require only one molecule to escape from the endosome to kill cells may be modified to be less effective. If at least 2, more preferably at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and most preferably at least 100 toxin molecules escape from the endosome, then cell-killing toxins are preferably used. It is further preferred that the conjugate of the present invention comprising a linked effector moiety-saponin comprises a covalently conjugated functionalized scaffold, i.e., a scaffold comprising a covalently bound effector moiety for delivering a scaffold comprising a bound effector moiety at a target cell (such as a tumor cell or an autoimmune cell) and a covalently bound saponin. Furthermore, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0177] The term "ligand" as used in the present invention has its ordinary meaning, and preferably means a molecule or structure that can bind to another molecule or structure on the cell surface of a target cell, wherein the molecule or structure on the cell surface can be endocytosed and preferably does not exist or is less prominent on off-target cells. Preferably, the molecule or structure on the cell surface is constitutively endocytosed. More preferably, the ligand in the present invention induces endocytosis of the molecule or structure on the cell surface of the target cell after binding to the molecule or structure. This is, for example, the case of the epidermal growth factor receptor (EGFR) present on the surface of a variety of cancer cells. Examples of molecules or structures on the cell surface of constitutively endocytosed target cells are, for example, claudin-1 or major histocompatibility complex class II glycoproteins. The ligand can be, for example, an antibody, a growth factor or a cytokine. Combining the effector portion of the present invention, for example, a toxin coupled to a saponin, with a ligand into a carrier molecule is a possibility for producing a targeted toxin. A toxin that is toxic only in target cells (because it only interferes with processes occurring in target cells) can also be regarded as a targeted toxin (because it cannot exert its toxic effect in off-target cells, such as apoptotic proteins). Preferably, the targeted toxin is a toxin combined with a ligand or, for example, a monoclonal antibody, so that it is active in target cells but not in off-target cells (because it only binds to and is endocytosed by target cells). In a functionalized scaffold, for example, comprising a carrier molecule containing a ligand and an effector portion of the present invention, the ligand or monoclonal antibody directs the effector portion and the scaffold having a saponin bound thereto to the target cell. After internalization, at least one glycoside, preferably a saponin contained in the effector portion of the present invention, mediates the endosomal escape of the effector portion. Saponin is typically a saponin listed in Table A1 and Scheme I, and preferably, the saponin is SO1861 and / or QS-21, and / or SA1641 and / or GE1741.

[0178] The payload, effector molecule or effector part of the present invention is any substance that affects cell metabolism by interacting with intracellular effector molecule target in the context of the present invention, wherein the effector molecule target is any molecule or structure in the cell except the compartment and vesicle cavity of endocytosis and recycling pathway but including the membrane of these compartments and vesicle. Therefore, the structure in the cell includes the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the inside of plasma membrane and cytoplasm. In the context of the present invention, the cytoplasmic delivery of payload or effector part preferably means that payload or effector part can escape endosome (and / or lysosome), which also includes escaping endosome and lysosome as previously defined, and preferably can reach payload or effector part target as described herein. The present invention also encompasses a novel molecule (referred to as a scaffold), when the effector part comprises payload and saponin, it is used to simultaneously bring payload and at least one glycoside molecule (such as saponin of the present invention) or effector part of the present invention into endosome at a predetermined ratio. In the context of the present invention, a polymeric or oligomeric structure of a scaffold is a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer or dendronized oligomer, or it is an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stable polymer micelle or liposome, but does not include structures consisting of non-covalent assemblies of monomers (such as cholesterol / phospholipid mixtures). The terms "polymer, oligomer, dendrimer, dendronized polymer or dendronized oligomer" have their ordinary meaning. In particular, a polymer is a substance whose molecular structure consists mainly or completely of a large number of identical or similar units bonded together, and an oligomer is a polymer whose molecules consist of a relatively small number of repeating units. There is no consensus on a specific cutoff value for "many" and "several" used in the above definitions of polymer and oligomer, respectively. In some embodiments, the scaffold comprises a plurality of monomeric units, such as a saponin of the present invention. The scaffold preferably comprises a polymeric or oligomeric structure, such as a poly(amine) or oligo(amine), such as polyethyleneimine and poly(amidoamine), and a biocompatible structure, such as polyethylene glycol, a poly(ester) or oligo(ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer and poly(dextrin), a polysaccharide or oligosaccharide, such as cyclodextrin or polydextrose, and a poly- or oligoamino acid, such as polylysine or a peptide or protein, or a DNA oligomer or polymer.An assembled polymeric structure as defined herein comprises at least one scaffold and optionally other individual polymeric or oligomeric structures. The other individual polymeric or oligomeric structures of the assembly may be (a) a scaffold (thus comprising at least one glycoside molecule, such as a saponin of the present invention), (b) a functionalized scaffold (thus comprising at least one glycoside molecule, such as a saponin and a ligand, antibody, etc., (c) a polymeric or oligomeric structure without a glycoside molecule (such as a saponin of the present invention (see, e.g., Table A1)), a polymeric or oligomeric structure without a ligand, antibody, etc. A functionalized assembled polymeric structure is an assembled polymeric structure containing (a) at least one functionalized scaffold or (b) at least one scaffold and at least one polymeric structure, which comprises at least one ligand, antibody, etc. The polymeric or oligomeric structure in the assembled polymeric structure that does not comprise any of the above-mentioned molecules (i.e., no glycoside such as saponin, no first protein molecule such as a ligand, antibody) is specifically added as a structural component of the assembled structure, which helps to build or stabilize the assembled structure ("gel-like"). Without wishing to be bound by any theory, an acidic environment appears to be a prerequisite for the synergistic effect between the glycoside (saponin) and the effector moiety.

[0179] The pharmaceutically active substance in the present invention is an effector part for achieving beneficial results in an organism, preferably a vertebrate, more preferably a human such as a cancer patient or an autoimmune patient. Benefits include diagnosis, prognosis, treatment, cure and / or prevention of a disease and / or symptom. Pharmaceutically active substances may also cause undesirable harmful side effects. In this case, the pros and cons must be weighed to determine whether the pharmaceutically active substance is suitable for a particular situation. If the effect of the pharmaceutically active substance in a cell is mainly beneficial to the entire organism, the cell is referred to as a target cell. If the effect in the cell is mainly harmful to the entire organism, the cell is referred to as an off-target cell. In artificial systems such as cell culture and bioreactors, target cells and off-target cells depend on the purpose and are defined by the user.

[0180] The effector moiety as a polypeptide may be, for example, a polypeptide that restores a lost function, such as, for example, enzyme replacement, gene regulatory function, or a toxin.

[0181] Preferably, when endocytosed, the effector moiety is separated from the saponin and / or, for example, the antibody, the effect of which is enhanced by the saponin bound thereto. This can be achieved, for example, by breaking the cleavable bond under acidic conditions, reducing conditions, enzymatic conditions, or light-induced conditions.

[0182] One aspect of the invention relates to a pharmaceutical composition comprising an effector moiety of the invention or an antibody-drug conjugate of the invention or a ligand-drug conjugate of the invention (comprising at least one saponin covalently linked to an effector molecule) and optionally a pharmaceutically acceptable excipient.

[0183] One aspect of the invention relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0184] One aspect of the invention relates to an effector moiety of the invention or an antibody-drug conjugate of the invention or a therapeutic combination of the invention or a ligand-drug conjugate of the invention or a pharmaceutical composition of the invention for use in treating or preventing cancer or an autoimmune disease.

[0185] The inventors disclose herein that covalent coupling of saponins (e.g., saponins in the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, Table A1, Scheme I) to effector molecules, such as oligonucleotides, such as antisense BNAs, such as via a trifunctional linker (e.g., the trifunctional linker of Scheme II) or via an oligomeric or multimeric structure of a scaffold comprising covalently bound saponins, such as a dendron, e.g., a G4-dendron, results in improved cytotoxicity exerted by the effector moiety, such as a toxin, preferably an antisense oligonucleotide, such as an antisense BNA, comprised by the conjugate under the influence of the covalently coupled saponin in the conjugate, i.e., the effector moiety of the present invention. In particular, the effector moiety of the present invention is an antisense oligonucleotide covalently conjugated to a dendron having a plurality of saponins covalently bound thereto, such as 2, 4, 8, 16 saponins, preferably 4 saponins.

[0186] One embodiment is a conjugate of the invention comprising an effector moiety and a saponin containing one or several or all of the specified structural features of a saponin of structure A in Scheme I, which is referred to as a saponin having an 'ideal' structure when the endosomal escape enhancing activity is directed to an effector moiety present in an endosome of a cell contacted with the conjugate of the invention, and / or any one or more of the additional saponins selected from Scheme I:

[0187] Scheme I

[0188]

[0189] Scheme I (continued)

[0190]

[0191] Scheme I (continued)

[0192]

[0193] Scheme I (continued)

[0194]

[0195] Scheme I (continued)

[0196]

[0197] According to the present invention, the glycoside (such as a saponin according to the present invention) bound to the effector moiety comprised by the conjugate of the present invention having an 'ideal' structure for the purpose of enhancing the endosomal escape of the effector molecule comprised by the conjugate of the present invention is a disaccharide chain saponin according to structure A of Scheme 1 having a molecular mass of at least 1.500 Daltons and comprising an oleanane-type triterpene containing an aldehyde group at position C-23 and optionally a hydroxyl group at position C-16 and a first branched carbohydrate side chain at position C-3, the first branched carbohydrate side chain optionally containing glucuronic acid, wherein the saponin contains an ester group having a second branched carbohydrate side chain at position C-28, the second branched carbohydrate side chain optionally containing glucuronic acid. The compound chain preferably comprises at least four carbohydrate units, optionally contains at least one acetyl residue, such as two acetyl residues and / or optionally comprises deoxy carbohydrates and / or optionally comprises quinovose and / or optionally comprises glucose and / or optionally comprises 4-methoxycinnamic acid and / or optionally comprises 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprises 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid bound to a carbohydrate via an ester bond.

[0198] SO1861 differs from the "ideal structure" shown in Scheme 1, Structure A, by having only one acetyl residue at the quinovose and having an additional xylose. The "ideal structure" of a saponin for enhancing endosomal escape of an effector molecule or effector moiety, such as an antisense oligonucleotide, such as a BNA, is preferably a saponin having Structure A in Scheme 1, and a saponin exhibiting endosomal escape enhancing activity has one or more structural features exhibited in Structure A in Scheme 1. Without wishing to be bound by any theory, the inventors believe that Structure A in Scheme 1 represents an "ideal saponin" (and not a minimally required saponin) for endosomal escape enhancing activity, meaning that not all structures (chemical groups) can or must be present in every saponin having at least sufficient endosomal escape enhancing activity to promote accumulation of the effector moiety in the cytoplasm, and that some saponins may have other structural elements such as acetyl chains, and / or for other saponins exhibiting endosomal escape enhancing activity, the sugar may be different from the sugar shown in Scheme 1. For example, when considering the ideal structure of structure A in Scheme 1: for example, the presence of an acetyl chain in QS-21, QS-21 saponins and some saponins in the water-soluble fraction of Quillaja saponin (Quil-A) differ in the carbohydrate modification at C-28. In the water-soluble fraction of Quillaja saponins, such as QS-7, QS1862 are similar to the ideal structure A, and are similar to QS1861.

[0199] One embodiment is an effector moiety of the invention comprising an oligomeric trifunctional linker according to Scheme II as scaffold core structure:

[0200]

[0201] wherein at least one saponin is covalently bound to the trifunctional linker scaffold via a labile, cleavable hydrazone linker (acid-sensitive) and / or via a maleimide-containing bond, whereas the binding of the scaffold to the effector moiety, such as an oligonucleotide, is established via a labile, cleavable hydrazone linker (acid-sensitive) and / or via a maleimide-containing bond to a cysteine ​​in the binding site, thereby forming structure B:

[0202]

[0203] Structure B,

[0204] Such as 1, 2, 3 or 4 cysteines, allowing 1-4 scaffolds to covalently bind a single effector moiety.

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] Table A2 - ADCs previously studied in the human clinical setting and subsequently withdrawn from further clinical studies

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227] Table A3 - ADCs that have reached Phase III clinical development

[0228]

[0229]

[0230]

[0231] Table A5: RIPs from plants*

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] As mentioned above, at least one saponin comprised by the effector moiety according to the invention increases the efficacy of at least the current and new effector moieties defined by the invention. Due to the reduced dose of the effector moiety comprised by the conjugate formed with the saponin, potential side effects will be reduced without reducing efficacy. Therefore, the present invention provides an effector moiety according to the invention for use in a medicament or as a medicament. Therefore, one aspect of the present invention relates to an effector moiety according to the invention for use as a medicament. Also provided is the use of an effector moiety according to the invention for the preparation of a medicament. Especially cancer medicaments and in particular classical chemotherapeutic drugs are well known for their side effects. Due to the synchronization of the time and position of the pharmaceutically active substance comprised by the effector moiety and the saponin comprised by the effector moiety, because the payload and the saponin are covalently linked, the therapeutic composition according to the invention is particularly valuable for use as a medicament, in particular for use in a method for treating cancer. Therefore, the present invention provides a therapeutic composition comprising an effector moiety according to the invention, which is used in a method for treating cancer. The present invention also provides a therapeutic composition comprising an effector moiety according to the invention, which is used in a method for treating acquired or hereditary disorders, in particular monogenic defect disorders. Therefore, the therapeutic composition comprises an effector moiety, such as a payload, such as an antisense oligonucleotide. Thus, one aspect of the invention relates to a therapeutic composition comprising an effector moiety according to the invention for use in a method of treating cancer or an autoimmune disease, wherein the payload comprises a covalently bound saponin.

[0245] The present invention is further illustrated by the following examples, which should not be construed as limiting the present invention in any manner.

[0246] Example

[0247] Example 1

[0248] Materials and methods

[0249] The following chemicals were purchased: methanol (MeOH, LiChrosolv, Merck), N-ε-maleimidocaproic acid hydrazide (EMCH, 95%, TCI Chemicals), trifluoroacetic acid (TFA, 99.8%, Carl Roth), 2-mercaptoethanol (98%, Sigma-Aldrich), poly(amidoamine) (PAMAM dendrimer, ethylenediamine core, generation 5.0 solution, Sigma-Aldrich), cyanine 3-carboxylic acid (Cy3-COOH, 95%, Lumiprobe), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (HATU, 97%, Sigma-Aldrich), bovine serum albumin fraction V (BSA, Carl Roth), dimethyl sulfoxide (DMSO, 99%, Carl Roth), 2-iminothiolane hydrochloride (98%, Sigma-Aldrich), rhodamine b (RhodB, 95%, Merck), Dulbecco's phosphate buffered saline (PBS, Gibco), hydrochloric acid (HCl, 37%, Merck), NHS-PEG 13 -DBCO(Click Chemistry Tools),Alexa Fluor TM 488 5-TFP (Thermo-Fischer), azido-PEG3-SS-NHS (Conju-Probe), sodium cyanoborohydride (NaCNBH3, 95%, Sigma-Aldrich), ammonium persulfate (APS, 98%, Sigma-Aldrich), N,N,N',N'-tetramethylethylenediamine (TMEDA, 99%, Sigma-Aldrich), custom peptide SESDDAMFCDAMDESDSK (95%, PeptideSynthetics), azido-dPEG 12-NHS (95%, QuantaBiodesign), PFd-G4-azide-NH-BOC dendron (G4-dendron, 95%, PolymerFactory), anthocyanin 5-DBCO (Cy5-DBCO, 95%, Lumiprobe), chloroform (CHCl3, 99.5%, Sigma), Amicon Ultra 0.5mL centrifugal filter (3kDa MWCO, Sigma), mPEG-SCM (mPEG 2k -NHS, 95.6%, Creative PEG Works), Amicon Ultra 15 mL centrifugal filter (10 kDa MWCO, Sigma).

[0250] MALDI-TOF-MS

[0251] MALDI-TOF spectra are recorded on MALDI-mass spectrometer (Bruker Ultrafex III). Typically, via dried-droplet-method, super-DHB (99%, Fluka) or sinapinic acid (SA, 99%, Sigma-Aldrich) is used as a matrix dissolved in acetonitrile (MADLI-TOF-MS of test, Sigma) / 0.1% TFA (7:3v / v), and the sample dissolved in the MilliQ water of nanomolar to micromolar range is spotted on the target (MTP 384 target plate polished steel TF, Bruker Dalton). PepMix (peptide calibration standard, Bruker Dalton) or ProteMass (protein calibration standard, Sigma-Aldrich) are used as calibration standards. RP mode refers to reflector positive mode. RN mode refers to reflector negative mode. LP mode refers to linear positive mode.

[0252] H-NMR

[0253] A Bruker 400 MHz NMR spectrometer was used. 1 H NMR analysis. Sample preparation was performed 24 h before measurement, where 2 mg of sample was dissolved in 0.8 mL of methanol-D4 (99%, Deutero).

[0254] Size Exclusion Chromatography

[0255] Size exclusion chromatography (SEC) was performed with Sephadex G 25 Superfin and prepacked PD10 columns from GE Healthcare (GE Healthcare, Sephadex G 25 M). Prior to chromatography, the material was activated by swelling in the corresponding eluent.

[0256] Dialysis

[0257] Regenerated cellulose membranes: MWCO = 1 and 2 kDa (Spectra / Por), and MWCO = 12-14 kDa (Carl Roth) were used to perform the dialysis. Typically, dialysis was performed for 24 h with 1 L of solvent, which was changed after the first 6 h of the process.

[0258] SO1861-EMCH Synthesis

[0259] SO1861 (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) from soapwort were placed in a round flask with a stirrer and dissolved in 13 mL of methanol. TFA (400 μL, catalyst) was added to the solution, and the reaction mixture was stirred for 3 h at 800 rpm and room temperature on a RCT B magnetic stirrer (IKA Labortechnik). After stirring for 3 h, the mixture was diluted with MilliQ water or PBS and fully dialyzed for 24 h with MilliQ water or PBS using a regenerated cellulose membrane tube (Spectra / Por 7) with a MWCO of 1 kDa. After dialysis, the solution was lyophilized to obtain a white powder. Yield: 62.4 mg (95%). The dried aliquots were further used for purification via 1 H NMR and MALDI-TOF-MS characterization.

[0260] 1 H NMR (400 MHz, methanol-D4) ( Figure 3 Figure A, SO1861): δ = 0.50–5.50 (m, saponin triterpenoid and sugar backbone protons), 9.43 (1H, s, saponin aldehyde protons, H a ).

[0261] 1 H NMR (400 MHz, methanol-D4) ( Figure 3 Figure B. SO1861-EMCH, PBS post-treatment): δ = 0.50–5.50 (m, saponin triterpenoid and sugar backbone protons s), 6.79 (2H, s, maleimide protons, H c ),7.62-7.68(1H,m,hydrazone proton,H b ).

[0262] MALDI-TOF-MS (RP mode) Figure 4 Figure A): m / z 2124Da ([M+K] + , saponin-EMCH), m / z 2109Da ([M+K] + ,SO1861-EMCH),m / z 2094Da([M+Na] + ,SO1861-EMCH).

[0263] MALDI-TOF-MS (RN mode) Figure 5 Figure C): m / z 2275Da ([MH]-, saponin-EMCH conjugate), 2244Da ([MH]-, saponin-EMCH conjugate), 2222Da ([MH]-, saponin-EMCH conjugate), 2178Da ([MH]-, saponin-EMCH conjugate), 2144Da ([MH]-, saponin-EMCH conjugate), 2122Da ([MH]-, saponin-EMCH conjugate), 2092Da ([MH]-, saponin-EMCH conjugate), 2070Da ([MH]-, SO1861-EMCH), 2038Da ([MH]-, SO1832-EMCH), 1936Da ([MH]-, SO1730-EMCH), 1861Da ([MH]-, SO1861).

[0264] BSA-SO1861 Synthesis

[0265] 2-Iminothiolane (231 μg, 1.1 μmol) dissolved in 47 μL PBS was added to the BSA-RhodB solution (10 mg, 0.15 μmol) in 200 μL PBS, and the mixture was shaken at 800 rpm and room temperature for 40 minutes on ThermoMixer C (Eppendorf). After shaking for 40 minutes, the reaction mixture was immediately passed through a Sephadex G25 ultrafine size exclusion column (16 mL column volume), and SO1861-EMCH (1 mg, 0.5 μmol) dissolved in 100 μL PBS was added to the collected BSA-SH fractions. The reaction mixture was shaken at 800 rpm and room temperature for 12 h on ThermoMixer C (Eppendorf). After shaking for 12 h, the BSA-SO1861 was concentrated using a centrifugal filtration at 4,000 rpm (15 ° C) via an Amicon Ultra 15 filter with a MWCO of 3 kDa. The conjugate was stored as a solution in a refrigerator and aliquots were taken for analysis. Yield: Not determined.

[0266] MALDI-TOF-MS( Figure 2 Figure A) (LP mode): m / z 74.2 kDa ([M+H] + , BSA-SO1861 linked to 4 SO1861), 72.2 kDa ([M+H] + , BSA-SO1861 linked to 3 SO1861), 70.2 kDa ([M+H] + , BSA-SO1861 linked to two SO1861), 37.0 kDa ([M+H] 2+ , BSA-SO1861 linked to 4 SO1861), 35.9 kDa ([M+H] 2+ , BSA-SO1861 linked to 3 SO1861), 34.7 kDa ([M+H] 2+ , BSA-SO1861 with 2 SO1861 connected).

[0267] result

[0268] The first proof of concept for conjugation of SO1861-EMCH to proteins (an example of a potential protein effector) was obtained by using the amines of bovine serum albumin (BSA). After conjugation, mass spectrometry obtained corresponding peaks at m / z ∼70, ∼72, and ∼74 kDa for BSA-SO1861 ( Figure 2 Compared with the detection mass of BSA with m / z 66 kDa ( Figure 2 The mass of BSA-SO1861 obtained corresponds to a mixture of BSA-SO1861 conjugates consisting of 2, 3 and 4 SO1861 molecules per BSA. This shows that SO1861 molecules are able to effectively conjugate with potential protein effectors.

[0269] Example 2

[0270] Materials and methods

[0271] Dendron (-L-SO1861) 4 synthesis( Figure 6 )

[0272] Intermediate 1:

[0273] Di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate

[0274] 6-Azidohexanoic acid (0.943 g, 6.00 mmol), EDCl.HCl (1.21 g, 6.30 mmol) and Oxyma Pure (0.938 g, 6.60 mmol) were dissolved in DMF (10.0 mL) and the mixture was stirred for 5 minutes. Then a solution of di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (1.82 g, 6.00 mmol) in DMF (5.00 mL) was added and the reaction mixture was stirred at room temperature. After 5 hours, the reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium hydrogen sulfate solution (50 mL), saturated sodium bicarbonate solution (2×50 mL) and brine (50 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (ethyl acetate-heptane gradient, 10:90 up to 100:0) to give the title compound (2.67 g, 100%) as a white solid. Purity based on LC-MS: 98%.

[0275] LRMS(m / z):287 / 343 / 465[M-155 / M-99 / M+23] 1+

[0276] LC-MS rt(min):2.02 2A

[0277] Intermediate 2:

[0278] N,N-Bis(2-aminoethyl)-6-azidohexanamide dihydrochloride

[0279] To di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate (2.66 g, 6.00 mmol) was added HCl in isopropanol (5-6N, 20.0 mL, 110 mmol) and the reaction mixture was stirred at room temperature. After 4 h, the reaction mixture was evaporated in vacuo and the resulting crude product was co-evaporated with DCM (3 x 20 mL) to give the crude title product (1.49 g, 79%) as a white solid.

[0280] LRMS(m / z):243[M+1] 1+

[0281] Intermediate 3:

[0282] Tetra-tert-butyl ((5S,5'S)-((((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(6-oxohexane-6,1,5-triyl))tetracarbamate

[0283] Boc-Lys(Boc)-ONp(3.69g, 7.90mmol) was added to a solution of N,N-bis(2-aminoethyl)-6-azidohexanamide dihydrochloride (1.19g, 3.76mmol) in DMF (30.0mL) and DIPEA (2.62mL, 15.1mmol), and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (100mL). The resulting solution was washed with 1N potassium hydrogen sulfate solution (100mL) and saturated sodium bicarbonate solution (5×100mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 to 0:100) to give the title product (3.07g, 91%) as a yellow solid. Purity based on LC-MS: 94%.

[0284] LRMS(m / z):800 / 900 / 922[M-99 / M+1 / M+23] 1+

[0285] LC-MS rt(min):2.17 2A

[0286] Intermediate 4:

[0287] 4-Nitrophenyl 3-(acetylthio) propionate

[0288] 4-Nitrophenyl trifluoroacetate (5.17g, 22.0mmol) and 3-(acetylthio) propionic acid (2.96g, 20.0mmol) were dissolved in DCM (50.0mL). Then, DIPEA (6.97mL, 40.0mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (50mL). The resulting solution was washed with 1N potassium hydrogen sulfate solution (50mL), saturated sodium bicarbonate solution (5×50mL) and brine (50mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 to 0:100) to give the title product (4.90g, 91%) as a slightly yellow solid. Purity based on LC-MS: 99%.

[0289] LRMS(m / z):292[M+23] 1+

[0290] LC-MS rt(min):1.94 2A

[0291] Intermediate 5:

[0292] (S)-2,6-Diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide tetrahydrochloride

[0293] Tetra-tert-butyl ((5S,5'S)-((((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(6-oxohexane-6,1,5-triyl))tetracarbamate (1.80 g, 2.00 mmol) was dissolved in isopropanol (5-6N, 50.0 mL, 275 mmol) containing HCl, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the resulting crude product was co-evaporated with DCM (3×20 mL) to give the crude title product as a white solid.

[0294] LRMS(m / z):250[M+2] 2+ ,500[M+1] 1+

[0295] Intermediate 6:

[0296] (2S)-2,6-bis[3-(acetylsulfanyl)propionamido]-N-[2-(6-azido-N-{2-[(2S)-2,6-bis[3-(acetylsulfanyl)propionamido]hexanamido]ethyl}hexanamido)ethyl]hexanamide

[0297] To a solution of (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide tetrahydrochloride (1.29 g, 2.00 mmol) in DMF (30 mL) and DIPEA (3.48 mL, 20.0 mmol) was added 4-nitrophenyl 3-(acetylthio)propanoate (2.26 g, 8.40 mmol) and the reaction mixture was stirred at room temperature over the weekend. The reaction mixture was evaporated in vacuo and the residue was dissolved in DCM / methanol (95:5, v / v, 100 mL). The resulting solution was washed with 1N potassium bisulfate solution (100 mL), 1N sodium hydroxide solution (3×100 mL) and brine (100 mL), dried over Na2SO4, filtered and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 to 0:100) to give the title product (1.33 g, 65%) as a white solid. Impurities (15%) were found on LC-MS, with m / z values ​​corresponding to the product with one deprotected thioacetate group. Impurities were formed during or after work-up. Purity based on LC-MS: 85%.

[0298] LRMS(m / z):510[M+2] 2+ ,1019 / 1041[M+1 / M+23] 1+

[0299] LC-MS rt(min):1.86 2B

[0300] Intermediate 7:

[0301] N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropionamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropionamide)carboxylate

[0302] Scaffold 2 (102 mg, 0.100 mmol) was dissolved in methanol (1.00 mL). Then, freshly prepared 1N sodium hydroxide solution (0.440 mL, 0.440 mmol) was added, and the reaction mixture was stirred at room temperature. After 30 minutes, THF (0.500 mL, 0.500 mmol) containing 1.0 M trimethylphosphine solution was added, and the resulting mixture was stirred at room temperature. After 30 minutes, the reaction mixture was evaporated in vacuo and co-evaporated with methanol (2×10 mL). The residue was dissolved in a mixture of methanol / water (9:1, v / v, 1.00 mL), and the resulting solution was subjected to preparative MP-LC2 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a colorless viscous oil (75.6 mg, 87%). Purity based on LC-MS: 96%.

[0303] LRMS(m / z):513[M+2] 2+ ,825[M+1] 1+

[0304] LC-MS rt(min):1.42 2A

[0305] Intermediate 8:

[0306] Dendron (-L-SO1861) 4 -amine

[0307] N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropionamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropionamide)formate (2.73 mg, 3.13 μmol) was dissolved in a mixture of 20 mM NH4HCO3 and 0.5 mM TCEP / acetonitrile (3:1 v / v, 3.00 mL). Then, SO1861-EMCH (29.2 mg, 0.014 mmol) was added and the reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative LC-MS 3B The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (12.3 mg, 43%). Purity based on LC-MS: 97%.

[0308] LRMS(m / z):1517[M-6] 6- ,1821[M-5] 5- ,2276[M-4] 4-

[0309] LC-MS rt(min):4.39 5A

[0310] Intermediate 9:

[0311] Dendron (-L-SO1861) 4 -Azide

[0312] Dendron (SO1861) 4-amine (6.81 mg, 0.748 μmol) and 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (2.90 mg, 7.48 μmol) were dissolved in DMF (1.00 mL). Then, DIPEA (1.302 μL, 7.48 μmol) was added, the mixture was shaken for 1 minute, and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (5.86 mg, 84%). Purity based on LC-MS: 90%.

[0313] LRMS(m / z):2344[M-4] 4-

[0314] LC-MS rt(min):4.78 5B

[0315] Intermediate 10:

[0316] Dendron (-L-SO1861) 4-Maleimide 1

[0317] Dendron (SO1861) 4-amine (8.12 mg, 0.891 μmol) and 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (3.94 mg, 8.91 μmol) were dissolved in DMF (1.00 mL). Then, DIPEA (1.55 μL, 8.91 μmol) was added, and the mixture was shaken for 1 minute and allowed to stand at room temperature. After 3 hours, the reaction mixture was subjected to preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (6.76 mg, 80%). Purity based on LC-MS: 66%.

[0318] LRMS(m / z):2358[M-4] 4-

[0319] LC-MS rt(min):2.13 6C

[0320] Intermediate 11:

[0321] Dendron (-L-SO1861) 4 -Maleimide 2

[0322] Scaffold 2 (5.10 mg, 5.00 μmol) was dissolved in methanol (100 μL). Then, freshly prepared 1N sodium hydroxide solution (22.0 μL, 22.0 μmol) was added, the mixture was shaken for 1 min, and allowed to stand at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (25.0 μL, 25.0 μmol) was added, the resulting mixture was shaken for 1 min, and allowed to stand at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and co-evaporated with methanol (2×5 mL). The resulting residue was dissolved in a mixture of 20 mM NH4HCO3 and 0.5 mM TCEP / acetonitrile (3:1, v / v, 3.242 mL). From this solution, 1000 μL was directly added to SO1861-EMCH (14.4 mg, 6.94 μmol, 4.5 equivalents compared to the scaffold), the mixture was shaken for 1 min, and allowed to stand at room temperature. After 10 minutes, the reaction mixture was lyophilized overnight. 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(3-(2,5-dioxo-2H-pyrrole-1(5H)-yl)propionamido)ethoxy)ethoxy)propanoate (5.84 mg, 0.014 mmol) and DMF (1.00 mL) were added to the resulting residue. Then, DIPEA (2.39 μL, 0.014 mmol) was added, the suspension was shaken for 1 minute, and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative LC-MS 3C The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (10.9 mg, 85%). Purity based on LC-MS: 80%.

[0323] LRMS(m / z):2354[M-4] 4-

[0324] LC-MS rt(min):4.16 5B

[0325] Dendron (-L-SO1861) 8 synthesis( Figure 7 )

[0326] Intermediate 1:

[0327] N-[(1S)-1-{[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis({[(tert-butyloxy

[0328] tert-butyl ester

[0329] (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide tetrahydrochloride (964 mg, 1.50 mmol) was dissolved in DMF (25.0 mL) and triethylamine (2.08 mL, 15.0 mmol). Then, Boc-Lys(Boc)-ONp (3.36 g, 7.18 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 to 0:100) to give the title product (2.71 g, 100%) as a white solid. Purity based on LC-MS: 97%.

[0330] LRMS(m / z):807[M-198] 2+

[0331] LC-MS rt(min):2.35 2B

[0332] Intermediate 2:

[0333] (2S,2'S)-N,N'-((5S,15S,22S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamido)-6,14,21-trioxo-7,10,13,20-tetraazahexacosane-1,5-diyl)bis(2,6-diaminohexanamide) octahydrochloride

[0334] Intermediate 1 (2.71 g, 1.50 mmol) was dissolved in isopropanol containing HCl (5-6N, 25.0 mL, 138 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then evaporated in vacuo and the resulting crude product was co-evaporated with DCM (3×20 mL) to give the crude title product as a white solid.

[0335] LRMS(m / z):203 / 254[M-200 / M+4] 4+ ,338[M+3] 3+ ,507[M+2] 2+ ,1012[M+1] 1+

[0336] Intermediate 3:

[0337] (2S)-2,6-bis[3-(acetylsulfanyl)propionamido]-N-[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis[3-(acetylsulfanyl)propionamido]hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis[3-(acetylsulfanyl)propionamido]hexanamido]pentyl]hexanamide

[0338] To (2S,2'S)-N,N'-((5S,15S,22S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamido)-6,14,21-trioxo-7,10,13,20-tetraazahexacosane-1,5-diyl)bis(2,6-diaminohexanamide) octahydrochloride (300 mg, 0.230 mmol) was added DMF (20.0 mL), triethylamine (320 μl, 2.30 mmol) and 4-nitrophenyl 3-(acetylthio) propionate (595 mg, 2.21 mmol). The resulting suspension was sonicated at 60° C. for 30 minutes and stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was purified by first flash chromatography (DCM-methanol / DCM (1 / 9, v / v) gradient 100:0 up to 0:100) followed by preparative MP-LC 2 Purification by HPLC ...

[0339] LRMS(m / z):685[M+3] 3+

[0340] LC-MS rt(min):1.91 2A

[0341] Intermediate 4:

[0342] (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropionamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropionamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropionamido)hexanamidocarboxylate

[0343] Scaffold 4 (10.0 mg, 4.87 μmol) was dissolved in methanol (200 μL). Then, freshly prepared 1N sodium hydroxide solution (42.9 μL, 0.043 mmol) was added, and the resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, 1.0 M trimethylphosphine solution in THF (24.4 μL, 0.024 mmol) was added, and the resulting mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was diluted with water (1 mL) and directly subjected to preparative MP-LC. 2 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (4.02 mg, 48%) as a white fluffy solid.

[0344] LRMS(m / z):564[M+3] 3+ ,846[M+2] 2+

[0345] LC-MS rt(min):1.54 2C

[0346] Intermediate 5:

[0347] Dendron (-L-SO1861) 8 -amine

[0348] Scaffold 5 (0.52 mg, 0.299 μmol) and SO1861-EMCH (29.2 mg, 0.014 mmol) were dissolved in a mixture of 20 mM NH4HCO3 and 0.5 mM TCEP / acetonitrile (3:1, v / v, 1.00 mL), and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, TCEP (0.30 mg, 1.05 μmol) was added, and the reaction mixture was shaken for 1 min. The mixture was then directly subjected to preparative LC-MS 3B The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (2.17 mg, 40%). Purity based on LC-MS: 97%.

[0349] LRMS(m / z):2282[M-8] 8- ,2607[M-7] 7-

[0350] LC-MS rt(min):4.41 5A

[0351] SO1861-BNA oligonucleotide conjugation

[0352] HSP27 BNA oligonucleotide disulfide (1.10mg, 0.187μmol) was dissolved in 20mM NH4HCO3 (500μL) with 1.0mM TCEP, and the mixture was shaken for 1min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered by using a centrifugal filter (14000×g, 30min) with a molecular weight cutoff of 3000Da. The residue solution was diluted with 20mM NH4HCO3 (500μL) with 1.0mM TCEP, and the resulting mixture was filtered again under the same conditions as above. The residue solution was diluted with 20mM NH4HCO3 / acetonitrile (3:1, v / v, 1.00mL), and the resulting mixture was added to SO1861-EMCH (3.54mg, 0.375μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 10min, the reaction mixture was subjected to preparative LC-MS 4A The fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.25 mg, 85%). Purity based on LC-MS: 100%.

[0353] LRMS (m / z): 1561[M-5] 5- ,1951[M-4] 4-

[0354] LC-MS rt(min):2.46 6B

[0355] Dendron (SO1861) 4 -BNA oligonucleotide conjugation

[0356] HSP27 BNA oligonucleotide disulfide (1.1 mg, 0.187 μmol) was dissolved in 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered by using a centrifugal filter with a molecular weight cutoff of 3000 Da (14000 × g, for 30 min). The residue solution was diluted with 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the resulting mixture was filtered again under the same conditions as above. The residue solution was diluted with 20 mM NH4HCO3 / acetonitrile (3:1, v / v, 1.0 mL), and the resulting mixture was added to the dendron (SO1861) 4-maleimide 1 (3.54 mg, 0.375 μmol). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 10 min, the reaction mixture was subjected to preparative LC-MS 4AFractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound as a white fluffy solid (1.25 mg, 85%). Purity based on LC-MS: 94%.

[0357] LRMS (m / z): 1896 [M-8] 8- , 2167[M-7] 7-

[0358] LC-MS rt(min):3.77 6B

[0359] Cell culture

[0360] The cells were seeded at 5,000 c / w in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) (100 μL / well) in a 96-well plate and incubated overnight at 37° C. and 5% CO 2 . The next day, samples were prepared in DMEM and cells were processed.

[0361] Gene silencing

[0362] RNA isolation and qPCR analysis were performed according to standard procedures and protocols.

[0363] HSP27 Primers: F: R:

[0364] HSP27BNA oligonucleotide

[0365] HSP27BNA(-thiol) oligonucleotide (sequence 5′-GGCacagccagtgGCG-3′) (Zhang et al., 2011) was ordered from Bio-synthesis Inc. (Lewisville, Texas).

[0366] result

[0367] Conjugating a BNA oligonucleotide, an antisense BNA oligonucleotide targeting the mRNA transcript of a cancer target (upregulated in cancer cells), heat shock protein 27 (HSP27BNA), to SO1861-EMCH (HSP27BNA-L-SO1861) or a dendron (-L-SO1861) 4 (HSP27BNA-Dendron (-L-SO1861) 4) and co-administered to the A431 cancer cell line according to the present invention. As a readout, gene silencing of HSP27 mRNA in A431 cells was determined. This revealed that HSP27BNA-L-SO1861 treatment resulted in an improvement in HSP27 gene silencing activity compared to HSP27BNA alone, whereas HSP27BNA-dendron (-L-SO1861) did not significantly improve HSP27 gene silencing activity compared to HSP27BNA alone. 4 (4 SO1861 molecules / BNA) are even more active (3 times) ( Figure 1 ). This shows that conjugation of one or more SO1861 molecules improves the gene silencing activity of therapeutic BNA oligonucleotides due to SO1861-mediated enhancement of endosomal escape and cytoplasmic delivery of antisense BNAs.

[0368] Example 3

[0369] Material

[0370] The following chemicals were purchased: methanol (MeOH, LiChrosolv, Merck), N-ε-maleimidocaproic acid hydrazide (EMCH, 95%, TCI Chemicals), trifluoroacetic acid (TFA, 99.8%, Carl Roth), 2-mercaptoethanol (98%, Sigma-Aldrich), poly(amidoamine) (PAMAM dendrimer, ethylenediamine core, generation 5.0 solution, Sigma-Aldrich), cyanine 3-carboxylic acid (Cy3-COOH, 95%, Lumiprobe), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethanium hexafluorophosphate N-oxide (HATU, 97%, Sigma-Aldrich), bovine serum albumin fraction V (BSA, Carl Roth), dimethyl sulfoxide (DMSO, 99%, Carl Roth), 2-iminothiolane hydrochloride (98%, Sigma-Aldrich), rhodamine b (RhodB, 95%, Merck), Dulbecco's phosphate buffered saline (PBS, Gibco), hydrochloric acid (HCl, 37%, Merck), NHS-PEG 13 -DBCO(Click Chemistry Tools),Alexa Fluor TM488 5-TFP (Thermo-Fischer), azido-PEG3-SS-NHS (Conju-Probe), sodium cyanoborohydride (NaCNBH3, 95%, Sigma-Aldrich), ammonium persulfate (APS, 98%, Sigma-Aldrich), N,N,N',N'-tetramethylethylenediamine (TMEDA, 99%, Sigma-Aldrich), custom peptide SESDDAMFCDAMDESDSK (95%, PeptideSynthetics), azido-dPEG 12 -NHS (95%, QuantaBiodesign), PFd-G4-azide-NH-BOC dendron (G4-dendron, 95%, PolymerFactory), anthocyanin 5-DBCO (Cy5-DBCO, 95%, Lumiprobe), chloroform (CHCl3, 99.5%, Sigma), Amicon Ultra 0.5mL centrifugal filter (3kDa MWCO, Sigma), mPEG-SCM (mPEG 2k -NHS, 95.6%, Creative PEG Works), Amicon Ultra 15 mL centrifugal filter (10 kDa MWCO, Sigma).

[0371] method

[0372] MALDI-TOF-MS

[0373] MALDI-TOF spectra are recorded on MALDI-mass spectrometer (Bruker Ultrafex III). Typically, via dried-droplet-method, super-DHB (99%, Fluka) or sinapinic acid (SA, 99%, Sigma-Aldrich) is used as a matrix dissolved in acetonitrile (MADLI-TOF-MS of test, Sigma) / 0.1% TFA (7:3v / v), and the sample dissolved in the MilliQ water of nanomolar to micromolar range is spotted on the target (MTP 384 target plate polished steel TF, Bruker Dalton). PepMix (peptide calibration standard, Bruker Dalton) or ProteMass (protein calibration standard, Sigma-Aldrich) are used as calibration standards. RP mode refers to reflector positive mode. RN mode refers to reflector negative mode. LP mode refers to linear positive mode.

[0374] H-NMR

[0375] A Bruker 400 MHz NMR spectrometer was used. 1 H NMR analysis. Sample preparation was performed 24 h before measurement, where 2 mg of sample was dissolved in 0.8 mL of methanol-D4 (99%, Deutero).

[0376] UV-Vis

[0377] UV-Vis measurements were performed on a NanoDrop ND-1000 spectrophotometer in the spectral range of 200–750 nm.

[0378] Size Exclusion Chromatography

[0379] Size exclusion chromatography (SEC) was performed with Sephadex G 25 Superfin and prepacked PD10 columns from GE Healthcare (GE Healthcare, Sephadex G 25 M). Prior to chromatography, the material was activated by swelling in the corresponding eluent.

[0380] Dialysis

[0381] Regenerated cellulose membranes: MWCO = 1 and 2 kDa (Spectra / Por), and MWCO = 12-14 kDa (Carl Roth) were used to perform the dialysis. Typically, dialysis was performed for 24 h with 1 L of solvent, which was changed after the first 6 h of the process.

[0382] Freeze-drying

[0383] Freeze drying was performed on an Alpha 1-2LD plus (Martin Christ Gefriertrocknungsanlagen GmbH). Typically, the samples were frozen with liquid nitrogen and placed in a freeze dryer under high vacuum.

[0384] SO1861-EMCH Synthesis

[0385] SO1861 (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) from soapwort were placed in a round flask with a stirrer and dissolved in 13 mL of methanol. TFA (400 μL, catalyst) was added to the solution, and the reaction mixture was stirred for 3 h at 800 rpm and room temperature on a RCT B magnetic stirrer (IKA Labortechnik). After stirring for 3 h, the mixture was diluted with MilliQ water or PBS and fully dialyzed for 24 h with MilliQ water or PBS using a regenerated cellulose membrane tube (Spectra / Por 7) with a MWCO of 1 kDa. After dialysis, the solution was lyophilized to obtain a white powder. Yield: 62.4 mg (95%). The dried aliquots were further used for purification via 1 H NMR and MALDI-TOF-MS characterization.

[0386] 1 H NMR (400 MHz, methanol-D4) ( Figure 3 Figure A, SO1861): δ = 0.50–5.50 (m, saponin triterpenoid and sugar backbone protons), 9.43 (1H, s, saponin aldehyde protons, H a ).

[0387] 1 H NMR (400 MHz, methanol-D4) ( Figure 3 Figure B. SO1861-EMCH, PBS post-treatment): δ = 0.50–5.50 (m, saponin triterpenoid and sugar backbone protons s), 6.79 (2H, s, maleimide protons, H c ),7.62-7.68(1H,m,hydrazone proton,H b ).

[0388] MALDI-TOF-MS (RP mode) Figure 4 Figure A): m / z 2124Da ([M+K] + , saponin-EMCH), m / z 2109Da ([M+K] + ,SO1861-EMCH),m / z 2094Da([M+Na] + ,SO1861-EMCH).

[0389] MALDI-TOF-MS (RN mode) Figure 5Figure C): m / z 2275Da ([MH]-, saponin-EMCH conjugate), 2244Da ([MH]-, saponin-EMCH conjugate), 2222Da ([MH]-, saponin-EMCH conjugate), 2178Da ([MH]-, saponin-EMCH conjugate), 2144Da ([MH]-, saponin-EMCH conjugate), 2122Da ([MH]-, saponin-EMCH conjugate), 2092Da ([MH]-, saponin-EMCH conjugate), 2070Da ([MH]-, SO1861-EMCH), 2038Da ([MH]-, SO1832-EMCH), 1936Da ([MH]-, SO1730-EMCH), 1861Da ([MH]-, SO1861).

[0390] Cy3-PAMAM

[0391] 720 μ L PAMAM dissolved in methanol (30 mg, 1.04 μ mol) is placed in a 250 mL round flask, and methanol is removed via a rotary evaporator (20 mbar, 60 ° C). Remaining PAMAM is dissolved in 9 mL DMSO. HATU (7.6 mg, 20 μ mol) dissolved in 0.5 mL DMSO is added to Cy3-COOH (0.6 mg, 1.2 μ mol) solution in DMSO, and the mixture is shaken at room temperature for 1 h with 800 rpm on ThermoMixer C (Eppendorf). After shaking for 1 h, HATU-Cy3 solution is added to the PAMAM solution stirred, and the reaction mixture is stirred at room temperature for 12 h. After stirring for 12 h, the reaction mixture is diluted with MilliQ water, and the regenerated cellulose membrane tube (Spectra / Por 6) that uses MWCO to be 2 kDa is fully dialyzed for 24 h to MilliQ water. After dialysis, the volume of the conjugate solution was reduced via a rotary evaporator (20 mbar, 60 ° C), and the concentrated conjugate solution was passed through a Sephadex G25 ultrafine size exclusion column (16 mL column volume). The first fraction was collected and lyophilized to obtain a viscous pink PAMAM-Cy3 conjugate. The formation of the PAMAM-Cy3 conjugate was confirmed by chromatography on thin layer chromatography (methanol / water, v / v 1: 1) and the appearance of faster bands on a Sephadex G 25 ultrafine column. Yield: 21.3 mg (63%). The dye / PAMAM molar ratio determined by UV-Vis spectrophotometry was 0.43.

[0392] MALDI-TOF-MS( Figure 8 Figure A) (LP mode): m / z 28.0 kDa ([M+H]+ ,Cy3-PAMAM).

[0393] Synthesis of Cy3-PAMAM-SO1861

[0394] The program for Cy3-PAMAM-(SO1861)5 is exemplarily described.The 2-iminothiolane (1mg, 6.7 μmol) dissolved in 250 μL MilliQ water is added to the PAMAM-Cy3 solution (0.5mg, 17nmol) in 125 μL MilliQ water, and the mixture is shaken 40 minutes at 800rpm and room temperature on ThermoMixer C (Eppendorf).After shaking for 40 minutes, the reaction mixture is immediately passed through the Sephadex G25 ultrafine size exclusion column (16mL column volume), and the SO1861-EMCH (176 μg, 85nmol) dissolved in 40 μL MilliQ water is added to the Cy3-PAMAM-SH fraction collected.The reaction mixture is shaken 12h at 800rpm and room temperature on ThermoMixer C (Eppendorf). After shaking for 12 h, the reaction mixture was diluted with MilliQ water and fully dialyzed for 24 h using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12-14 kDa to MilliQ water. After dialysis, the Cy3-PAMAM-SO1861 solution was concentrated using centrifugal filtration at 4000 rpm (15 ° C) via an Amicon Ultra 15 filter with a MWCO of 3 kDa. The conjugate was stored in a refrigerator as a solution and an aliquot was taken for analysis. Yield: 0.5 mg (75%).

[0395] The MALDI-TOF-MS spectrum is shown in Figure 8 Figure B-D and Fig. 9 MALDI-TOF-MS of Cy3-PAMAM-(SO1861)6 ( Figure 8 Figure B) (LP mode): m / z 38.4 kDa ([M+H] + ,Cy3-PAMAM-SO1861),17.9kDa([M+H] 2+ ,Cy3-PAMAM-SO1861).

[0396] Cy3-PAMAM-(SO1861)5, Cy3-PAMAM-(SO1861) 13 、Cy3-PAMAM-(SO1861) 51 and Cy3-PAMAM-(SO1861) 27The synthesis of SO1861-EMCH was similar to that of SO1861-EMCH, but the feed equivalents of the starting materials 2-iminothiolane and SO1861-EMCH were different. The corresponding feed equivalents of the starting materials and the corresponding masses of the conjugates are highlighted in Table 1.

[0397] Table 1: Reaction parameters for the synthesis of Cy3-PAMAM-SO1861.

[0398]

[0399]

[0400] Synthesis of Cy3-PAMAM-NC-SO1861

[0401] Cy3-PAMAM (0.5mg, 18nmol), SO1861 (2.3mg, 1.24μmol) and HATU (64.6mg, 170μmol) are dissolved in 200μL DMSO respectively. SO1861 and HATU solution are mixed and shaken at 800rpm and room temperature for 20 minutes on ThermoMixer C (Eppendorf). After shaking for 20 minutes, Cy3-PAMAM solution is added to the shaking SO1861-HATU solution, and the reaction mixture is shaken at 800rpm and room temperature for 12h on ThermoMixer C (Eppendorf). After shaking for 12h, the reaction mixture is diluted with MilliQ water, and the MilliQ water is fully dialyzed for 24h using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12-14kDa. After dialysis, the Cy3-PAMAM-NC-SO1861 solution was concentrated by centrifugal filtration at 4,000 rpm (15° C.) through an Amicon Ultra 15 filter with a MWCO of 3 kDa. 17 The conjugate was stored as a solution in a refrigerator and aliquots were taken for analysis. Yield: 0.77 mg (69%).

[0402] MALDI-TOF-MS( Fig.10 )(LP mode): m / z 62.3 kDa([M+H] + ,Cy3-PAMAM-NC-SO1861),35.7kDa([M+H] 2+ ,Cy3-PAMAM-NC-SO1861).

[0403] PAMAM Thiolation

[0404] The procedure for the highest PAMAM to 2-iminothiolane ratio is exemplarily described. 2-iminothiolane (0.53 mg, 3.84 μmol) dissolved in 128 μL MilliQ water was added to a solution of PAMAM (333 μg, 12.8 nmol) dissolved in 30 μL methanol. The reaction mixture was shaken for 12 h at 800 rpm and room temperature on a ThermoMixer C (Eppendorf). After shaking for 12 h, the reaction mixture was washed 4 times with MilliQ water via centrifugal filtration using an Amicon Ultra centrifugal filter (3 kDa MWCO) at 15 ° C and 13500 rpm. After washing, the sample was lyophilized to obtain a white solid. The yield was not determined.

[0405] The MALDI-TOF-MS spectrum is shown in Fig.11 In. PAMAM-(SH) 108 MALDI-TOF-MS( Fig.11 Figure C) (LP mode): m / z 41.5 kDa ([M+H] + ,PAMAM-[SH] 108 ).

[0406] The synthesis of other PAMAM-iminothiolane conjugates has been performed via the above method, but with different feed equivalents of the starting material 2-iminothiolane. For the lowest 2-iminothiolane feed reaction, Cy3-PAMAM has been used.

[0407] The corresponding feed equivalents of starting materials and the corresponding masses of conjugates are highlighted in Table 2.

[0408] Table 2: Reaction parameters for PAMAM-SH synthesis.

[0409]

[0410]

[0411] PAMAM PEGylation

[0412] The following is an exemplary description of the minimum PAMAM and mPEG 2k To a solution of PAMAM (333 μg, 12.8 nmol) dissolved in 10 μL DMSO was added mPEG dissolved in 13 μL DMSO. 2k-NHS (0.268mg, 128nmol). The reaction mixture was shaken on ThermoMixer C (Eppendorf) at 800rpm and room temperature for 12h. After shaking for 12h, the reaction mixture was diluted with MilliQ water, and the MilliQ water was fully dialyzed for 24 hours using a regenerated cellulose membrane tube (Spectra / Por 6) with a MWCO of 2kDa. After dialysis, the batch was concentrated via centrifugal filtration using an Amicon Ultra 15mL centrifugal filter ((10kDa MWCO). The concentrated batch was passed through a PD10 size exclusion column and then lyophilized to obtain a white fluffy powder. The yield was not determined.

[0413] The MALDI-TOF-MS spectrum is shown in Fig.12 PAMAM-(mPEG 2k )3 MALDI-TOF-MS( Fig.12 Figure C) (LP mode): m / z 33.46 kDa ([M+H] + ,PAMAM-[mPEG 2k ]3).

[0414] Other PAMAM-mPEG 2k The synthesis of conjugates, but the starting material mPEG 2k The feed equivalents of -NHS were different. The corresponding feed equivalents of starting materials and the corresponding masses of conjugates are highlighted in Table 3.

[0415] Table 3: PAMAM-mPEG 2k Reaction parameters for the synthesis.

[0416]

[0417] Synthesis of Cy3-PAMAM-SO1861-DBCO

[0418] The method for Cy3-PAMAM-(SO1861) is described as an example. 27 -(DBCO) 10 Procedure. Cy3-PAMAM-(SO1861) 27 (0.41 mg, 4.71 nmol) was freeze-dried and dissolved in 100 μL DMSO. 13-NHS ester (0.197mg, 188nmol) was added to the Cy3-PAMAM-SO1861 solution, and the mixture was shaken at 800rpm and room temperature on a ThermoMixer C (Eppendorf). After shaking for 3h, the reaction mixture was diluted with MilliQ water and fully dialyzed for 24h using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12-14kDa against MilliQ water. After dialysis, the Cy3-PAMAM-SO1861-DBCO solution was concentrated using centrifugal filtration at 4,000rpm (15°C) via an Amicon Ultra 15 filter with a MWCO of 3kDa. The conjugate was stored in a refrigerator as a solution and aliquots were taken for analysis. Yield: 0.1mg (22%).

[0419] MALDI-TOF-MS( Fig.13 D-pattern) (LP mode): m / z 92.5 kDa ([M+H] + ,Cy3-PAMAM-SO1861-DBCO),53.0kDa([M+H] 2+ ,Cy3-PAMAM-SO1861-DBCO).

[0420] Cy3-PAMAM-(SO1861)5-(DBCO) 38 and Cy3-PAMAM-(SO1861) 27 -(DBCO) 10 The corresponding feed equivalents of the starting materials and the corresponding masses of the conjugates are highlighted in Table 4.

[0421] Table 4: Reaction parameters for the synthesis of Cy3-PAMAM-SO1861-DBCO.

[0422]

[0423]

[0424] Synthesis of Cy3-PAMAM-NC-SO1861-DBCO

[0425] Cy3-PAMAM-NC-(SO1861) 17 (0.3 mg, 4.8 nmol) was freeze-dried and dissolved in 100 μL DMSO. 13-NHS ester (0.202mg, 194nmol) was added to the Cy3-PAMAM-NC-SO1861 solution, and the mixture was shaken at 800rpm and room temperature on a ThermoMixer C (Eppendorf). After shaking for 3h, the reaction mixture was diluted with MilliQ water and fully dialyzed for 24h using a regenerated cellulose membrane tube (ZelluTrans, CarlRoth) with a MWCO of 12-14kDa against MilliQ water. After dialysis, the Cy3-PAMAM-SO1861-DBCO solution was concentrated using centrifugal filtration at 4,000rpm (15°C) via an Amicon Ultra 15 filter with a MWCO of 3kDa. The conjugate was stored in a refrigerator as a solution and an aliquot was taken for analysis. Yield: 0.1mg (22%). The mass spectrum showed conjugation of 30 DBCO moieties per PAMAM molecule.

[0426] MALDI-TOF-MS( Fig.13 Figure B) (LP mode): m / z 93.2 kDa ([M+H] + ,Cy3-PAMAM-NC-SO1861-DBCO),49.6kDa([M+H] 2+ ,Cy3-PAMAM-NC-SO1861-DBCO).

[0427] EGF and dianthin expression

[0428] Plasmid DNA (His-oleanolic acid-EGF-pET11d or His-oleanolic acid-pET11d)

[20] was transformed into chemically competent Escherichia coli NiCo21 (DE3) (New England Biolabs, 1996). Inc.) and grown in 3 mL of lysogenic broth supplemented with 50 μg / mL ampicillin at 37°C at 200 rpm for 5 h. These bacteria were used to inoculate 500 mL of lysogenic broth supplemented with 50 μg / mL ampicillin for overnight culture at 37°C. Subsequently, the culture volume was expanded to 2 L and the bacteria were grown until the optical density (A600) was 0.9. Protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. The cells were grown for another 3 h at 37°C and 200 rpm. After centrifugation (5 min, 5,000 g, 4°C), the cell pellet was resuspended in 20 mL of phosphate buffered saline (containing Ca 2+ and Mg 2+Dulbecco's phosphate buffered saline (PBS), pH 7.4) and stored at -20°C. After thawing, the protein was released by an ultrasonic device (Branson Sonifier 250, G. Heinemann). The solution was centrifuged (15,800Δg, 30min, 4°C) and adjusted to a 20mM imidazole concentration. The construct contained an N-terminal His-tag and was purified by nickel nitrilotriacetic acid chromatography (Ni-NTA agarose, Qiagen, Hilden, Germany). After elution with imidazole (20-250mM), the eluate was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%). At 4°C, the fractions containing oleanolic acid-EGF or oleanolic acid were dialyzed with 2L chitin binding domain buffer (20mM tris(hydroxymethyl)-aminomethane / HCl, 500mM NaCl, 1mM EDTA, 0.1% Tween-20, pH8.0). Further purification by chitin column affinity chromatography was used to remove bacterial proteins with binding activity to Ni-NTA agarose. After elution with chitin binding domain buffer, the fractions were analyzed by SDS-PAGE (12%). At 4°C, the fractions containing oleanolic acid-EGF or oleanolic acid were dialyzed with 5L PBS. The purified protein was concentrated by Amicon centrifugal filter device (10kDa, Millipore, Eschborn, Germany). Protein concentration was determined by bicinchoninic acid assay (Pierce, Rockford, USA).

[0429] Synthesis of oleanolic acid-EGF-Alexa488

[0430] A solution of oleanolic acid-EGF (240 μg, 6.7 nmol) in PBS was placed in an Amicon Ultra15 filter with a MWCO of 3 kDa and centrifuged at 4,000 rpm and 4°C for 30 minutes three times. After each cycle, the Amicon filter was refilled with 0.1 M sodium carbonate buffer (pH 9). After the third centrifugation cycle, the volume was reduced to 0.5 mL via centrifugation. The oleanolic acid-EGF sodium carbonate solution was placed in a 2 mL reaction tube and Alexa Fluor 500 dissolved in 10 μL DMSO was added. TM488 5-TFP (50 μg, 56 nmol) was added to the protein solution. The mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 80 minutes. After shaking, the mixture was passed through a Sephadex G25 M size exclusion column (GE Healthcare, PD10 column). The oleanolic acid-EGF-Alexa488 conjugate was stored in a refrigerator in 0.1 M sodium carbonate buffer (pH 9) and aliquots were taken for analysis. Yield: 210 μg (85%).

[0431] MALDI-TOF-MS( Fig.14 D-pattern) (LP mode): m / z 36.8 kDa ([M+H] + , oleanolic acid-EGF-Alexa488), m / z 33.6 kDa ([M+H] + , oleanolic acid-EGF-Alexa488), 18.8 kDa ([M+H] 2+ , oleanolic acid-EGF-Alexa488), 16.6 kDa ([M+H] 2+ , oleanolic acid-EGF-Alexa488).

[0432] Synthesis of Alexa488

[0433] A solution of oleanolic acid (184 μg, 6.2 nmol) in PBS was placed in an Amicon Ultra 15 filter with a MWCO of 3 kDa and centrifuged at 4,000 rpm and 4°C for 30 minutes three times. After each cycle, the Amicon filter was refilled with 0.1 M sodium carbonate buffer (pH 9). After the third centrifugation cycle, the volume was reduced to 0.5 mL via centrifugation. The oleanolic acid sodium carbonate solution was placed in a 2 mL reaction tube and Alexa Fluor 500 dissolved in 3.5 μL DMSO was added. TM Alexa488 5-TFP (16.7 μg, 19 nmol) was added to the protein solution. The mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 80 minutes. After shaking, the mixture was passed through a Sephadex G25 M size exclusion column (GE Healthcare, PD10 column). The oleanolic acid-Alexa488 conjugate was stored in a refrigerator in 0.1 M sodium carbonate buffer (pH 9) and aliquots were taken for analysis. Yield: Not determined

[0434] MALDI-TOF-MS( Fig.15 D-pattern) (LP mode): m / z 30.7 kDa ([M+H] +, oleanolic acid-Alexa488).

[0435] Caryophyllein-EGF-Alexa488-SS-PEG-N3 and Caryophyllein-EGF-Alexa488-PEG 12 -N3 Synthesis

[0436] The procedure for oleanolic acid-EGF-Alexa488-SS-PEG-N3 is described exemplarily. A solution of oleanolic acid-EGF-Alexa488 (70 μg, 1.9 nmol) in sodium carbonate is placed in a 2 mL reaction tube, and azido-PEG3-SS-NHS (120 μg, 272 nmol) dissolved in 9 μL DMSO is added to the protein solution. The mixture is shaken on a ThermoMixer C (Eppendorf) at 800 rpm and 15 ° C for 12 h. After shaking, the reaction mixture is diluted with PBS and filtered by centrifugation at 4,000 rpm and 4 ° C using an Amicon Ultra 15 filter with a MWCO of 3 kDa, washed with PBS.

[0437] Yield: 54 μg (70%).

[0438] MALDI-TOF-MS( Fig.14 E-picture) (LP mode): m / z 40.8 kDa ([M+H] + , oleanolic acid-EGF-Alexa488-SS-PEG-N3), m / z 37.5 kDa ([M+H] + , oleanolic acid-EGF-Alexa488-SS-PEG-N3).

[0439] The oleanolic acid-EGF-Alexa488-SS-PEG-N3 and oleanolic acid-EGF-Alexa488-PEG 12 The synthesis of -N3 was similar to that of -N3, but with different azido-PEG linkers. The corresponding azido-PEG linkers, their feed equivalents and the corresponding masses of the conjugates are highlighted in Table 5.

[0440] Table 5: Reaction parameters for the synthesis of oleanolic acid-EGF-Alexa488-PEG-N3

[0441]

[0442]

[0443] Caryophyllein-Alexa488-SS-PEG-N3

[0444] The sodium carbonate solution of oleanolic acid-Alexa488 (24.5 μg, 0.8 nmol) was placed in a 2 mL reaction tube, and azido-PEG3-SS-NHS (34 μg, 78 nmol) dissolved in 9 μL DMSO was added to the protein solution. The mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and 15 ° C for 12 h. After shaking, the reaction mixture was diluted with PBS and filtered by centrifugation at 4,000 rpm and 4 ° C using an Amicon Ultra 15 filter with a MWCO of 3 kDa, and washed with PBS.

[0445] Yield: 10.3 μg (39%).

[0446] MALDI-TOF-MS( Fig.15 E-picture) (LP mode): m / z 32.9 kDa ([M+H] + , oleanolic acid-Alexa488-SS-PEG-N3).

[0447] Synthesis of Cy3-PAMAM-saponin-toxin conjugate

[0448] The method for Cy3-PAMAM-(SO1861) is described as an example. 27 -DBCO procedure. In a 1.5 mL reaction tube, Cy3-PAMAM-(SO1861) 27 -DBCO (17 μg, 0.184 nmol) solution was mixed with oleanolic acid--EGF-Alexa488-SS-PEG3-N3 (3.6 μg, 0.089 nmol) solution in PBS, and the reaction mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and 15°C for 2 h. After shaking, a small aliquot was taken for use in Molecular VersaDoc TM The samples were analyzed by SDS-PAGE and fluorescence imaging on the MP 4000 Imaging System (Bio-Rad). Fig.16 ).

[0449] Cy3-PAMAM-(SO1861)5-SS-oleanin-EGF-Alexa488,Cy3-PAMAM-(SO1861) 27 -SS-oleanolic acid-EGF-Alexa488,Cy3-PAMAM-NC-(SO1861) 17-SS-oleanolic acid-EGF-Alexa488,Cy3-PAMAM-NC-(SO1861) 17 -SS-oleanolic acid-Alexa488 and Cy3-PAMAM-NC-(SO1861) 17 -Synthesis of oleanolic acid-EGF-Alexa488, but with different PAMAM-saponin-DBCO batches, azido-toxin batches used and their feed equivalents. The corresponding feed equivalents of the starting materials are highlighted in Table 6.

[0450] Table 6: Reaction parameters for the synthesis of Cy3-PAMAM-saponin-toxin.

[0451]

[0452] Synthesis of Cy3-PAMAM-NC-SO1861 via reductive amination

[0453] Cy3-PAMAM (0.19 mg, 13 nmol) and SO1861 (0.73 mg, 0.39 μmol) were dissolved in 200 μL 0.1 M acetate buffer (pH 5) respectively. SO1861 and Cy3-PAMAM solution were mixed and shaken for 20 minutes at 800 rpm and room temperature on ThermoMixer C (Eppendorf). After shaking for 20 minutes, NaCNBH3 (5 mg, 81 μmol) was added to the shaking reaction solution, and the reaction mixture was shaken for 12 h at 800 rpm and room temperature on ThermoMixer C (Eppendorf). After shaking for 12 h, the reaction mixture was diluted with MilliQ water, and the MilliQ water was fully dialyzed for 24 h using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12-14 kDa. After dialysis, the Cy3-PAMAM-NC-SO1861 solution was concentrated using centrifugal filtration at 4,000 rpm (15° C.) through an Amicon Ultra 15 filter with a MWCO of 3 kDa. The conjugate was stored as a solution in a refrigerator and aliquots were taken for analysis. Yield: Not determined

[0454] MALDI-TOF-MS( Fig.17 Figure B, Figure C) (LP mode): m / z 88.7 kDa ([M+H] + ,Cy3-PAMAM-NC-SO1861),49.2kDa([M+H] 2+ ,Cy3-PAMAM-NC-SO1861).

[0455] Cy3-PAMAM-NC-(SO1861) has been carried out by the above method 30 and Cy3-PAMAM-NC-(SO1861) 10 The synthesis of , but at different times after the reducing agent NaCNBH3 was added to the reaction batch. The corresponding times of NaCNBH3 addition and the corresponding masses of the conjugates are highlighted in Table 7.

[0456] Table 7: Reaction parameters for the synthesis of Cy3-PAMAM-NC-SO1861 via reductive amination.

[0457]

[0458] SO1861-EMCH peptide conjugation

[0459] The custom peptide with the sequence SESDDAMFCDAMDESDSK (0.6 mg, 0.3 μmol) and SO1861-EMCH (0.8 mg, 0.39 μmol) were dissolved in 200 μL PBS. SO1861-EMCH and peptide solutions were mixed and mixed and shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After shaking, a small aliquot was taken for analysis. Yield: Not determined

[0460] MALDI-TOF-MS( Fig.18 Figure B) (RN mode): m / z 4.05 kDa ([M+H] - , peptide-SO1861), 3.92 kDa ([M+H] - , peptide-SO1730), 1.98 kDa ([M+H] - , peptide), 1.86 kDa ([M+H] - ,SO1861).

[0461] result

[0462] Considering the chemical groups that can be used for conjugation reaction with SO1861 molecule, four chemical groups have been identified. Alcohol and diol groups of sugar residues, aldehyde groups on triterpene backbone, carboxylic acid (glucuronic acid) on one of sugar residues and olefin groups on triterpene backbone, e.g. Fig.19 Highlighted in.

[0463] In view of the advantages and disadvantages of each identified chemical group (Table 8), aldehyde groups and alcohol groups are most suitable for reversible conjugation reactions, while olefins and carboxylic acids (glucuronic acid) are the groups most suitable for irreversible / stable conjugation reactions. However, compared with alcohol, the aldehyde group in the SO1861 molecular structure is most suitable for reversible conjugation reactions. On the one hand, because there is only one aldehyde in the structure, it allows chemical selective reactions. On the other hand, because aldehyde can be reversibly conjugated with a variety of chemical groups, such as amine, hydrazide and hydroxylamine, to form acid-cleavable parts, such as imines, hydrazones and oximes. This factor makes it possible to freely select chemical groups to carry out desired reversible conjugation reactions. On the contrary, alcohol is also a good candidate for reversible conjugation reactions via forming acetals and ketals, but lacks chemical selectivity because they are present in large quantities on the glycoside structure.

[0464] For the formation of irreversible and stable bonds, carboxylic acids are most suitable because they can form amides and esters with common tools used in peptide chemistry (e.g., reaction with amines via carbodiimide-mediated amide formation).

[0465] Table 8. Functional groups that can be used for saponin conjugation reactions

[0466]

[0467]

[0468] Therefore, in order to develop saponins that enhance endosomal escape (such as SO1861), a method has been established that allows the generation of non-cleavable and cleavable 'ready-to-conjugate' saponins using the most appropriate chemical groups present on SO1861 ( Fig. 20 ).

[0469] To produce a non-cleavable 'ready-to-conjugate' saponin, the carboxyl group of SO1861 was activated via reagents used in peptide coupling chemistry to produce an active ester (e.g., 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, HATU). The resulting active ester of SO1861 is capable of reacting with amines to form a stable amide-bonded conjugate ( Fig. 20 Figure A).

[0470] To generate a cleavable 'ready-to-conjugate' saponin, the aldehyde group of SO1861 was reacted with an EMCH (ε-maleimidocaproic acid hydrazide) linker. The hydrazide group of EMCH forms an acid-cleavable hydrazone bond with the aldehyde of SO1861. At the same time, the EMCH linker presents a maleimide group that is thiol (sulfhydryl) reactive and can therefore be conjugated with thiols ( Fig. 20 Figure B).

[0471] The maleimide group of SO1861-EMCH undergoes rapid and specific Michael addition reactions with thiols and thiol-bearing polymeric structures when carried out in the pH range of 6.5-7.5 ( Fig. 20 In addition, the acid-sensitive hydrazone bond between SO1861 and EMCH can be used to release saponin from the scaffold in an acidic environment ( Fig.21 ). Therefore, the EMCH linker meets the needs of both a pH-cleavable strategy and a conjugation strategy for polymeric structures.

[0472] Regarding the ideal EMCH spacer length for conjugation to polymeric structures, computer simulations (PerkinElmer, ChemBio3D, Ver. 13.0.0.3015) showed that the maleimide groups on SO1861-EMCH are located at the periphery of the molecule and should therefore be accessible to polymeric structures bearing thiols ( Fig. 22 ).

[0473] To synthesize SO1861-EMCH, a strategy has been developed that allows the aldehyde group on SO1861 to be converted into EMCH ( Figure 5 The SO1861-EMCH conjugate was isolated and characterized by NMR spectroscopy (see Materials and Methods, Figure 3 It was successfully characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Figure 5 Figure B and Figure 5 Figure C and Figure 4 As shown in Figure A.

[0474] To test the pH-dependent hydrolysis of the hydrazone bond, SO1861-EMCH was dissolved in HCl solution at pH 3 and MALDI-TOF-MS spectra were recorded at two different time points ( Fig.23 ).like Fig.23 Figure A and Fig.23 As shown in Figure B, the peak at m / z 2070Da corresponding to SO1861-EMCH shows a clear downward trend. Fig.23 As can be seen in Figure B of . Since SO1861 is generated during the hydrolysis process, an increase in the peak at m / z 1861 Da is recorded, accompanied by a decreasing trend at m / z 2070 Da. These results show that the hydrazone bond responds to hydrolysis and is cleaved even if it is attached to SO1861.

[0475] To conjugate SO1861-EMCH to a polymeric structure, the accessible amines of the polymeric structure are converted to thiols with the aid of the reagent 2-iminothiolane. The free thiol generated on the polymeric structure then acts as a nucleophile for a thiol-ene Michael-type addition to the maleimide group of SO1861-EMCH ( Fig.24 ). This developed method is applicable to the conjugation of SO1861-EMCH to any available polymeric structure, which has accessible amine groups and, moreover, allows controlling the number of conjugated SO1861 molecules, respectively, depending on the polymeric structure.

[0476] Proof of concept for conjugation of 'ready-made conjugated saponins' to polymeric structures was obtained by using a 5th generation (G5) dendrimer poly(amidoamine) bearing an amine (PAMAM with a covalently coupled red fluorescent dye (Cy3)). PAMAM-Cy3 was used as a polymeric structure for conjugation with SO1861-EMCH and SO1861-HATU and as a model for conjugation of SO1861 to polymeric structures ( Fig.25 ).

[0477] Using a 3-fold excess of 2-iminothiolane / Cy3-PAMAM amine, all accessible amine groups of Cy3-PAMAM were converted to thiols and then reacted with SO1861-EMCH. Three different feed equivalents (5, 20, and 57) of SO1861-EMCH were used for three reaction batches. After the reaction, the mass of the Cy3-PAMAM-SO1861 conjugate recorded on MALDI-TOF-MS showed an increase in the corresponding mass with the increase in SO1861-EMCH feed ( Figure 8 The three different feeds corresponded to the obtained masses of m / z 38.4 kDa, m / z 53.9 kDa and m / z 133.8 kDa for the Cy3-PAMAM-SO1861 conjugate corresponding to Figure 8 Panels B-D show 6, 13 and 51 SO1861 molecules attached to each PAMAM dendrimer.

[0478] In another reaction, only a certain amount of PAMAM amine was converted to thiol before reacting with SO1861-EMCH. Here, two different feed equivalents of 2-iminothiolane (8 and 32) and two different feed equivalents of SO1861-EMCH (5 and 30) were used, respectively. After the reaction, the corresponding spectrum of Cy3-PAMAM-SO1861 conjugate in MALDI-TOF-MS showed peaks at m / z 37.7 kDa (5 feed equivalents of SO1861-EMCH) and m / z 87.0 kDa (30 feed equivalents of SO1861-EMCH), as shown in Figure 2. Fig. 9The masses obtained at m / z 37.7 kDa and m / z 87.0 kDa correspond to Cy3-PAMAM-SO1861 conjugates with 5 and 30 SO1861 molecules attached, and demonstrate that nearly all of the SO1861-EMCH feed was conjugated using this method.

[0479] To generate a non-pH cleavable saponin conjugate, the carboxylic acid of SO1861 was activated with HATU and then reacted with the amine of Cy3-PAMAM to form a pH-stable amide bound between Cy3-PAMAM and SO1861. The mass of the resulting conjugate was detected by MALDI-TOF-MS, with a mass of m / z 62.3 kDa, corresponding to a Cy3-PAMAM-NC-SO1861 (NC = non-cleavable) conjugate, with 17.5 SO1861 molecules attached to each PAMAM ( Fig.25 Figure B, Fig.10 ).

[0480] The saponin-conjugated scaffolds are then conjugated to attachment points for possible conjugation with targeted therapeutics (e.g., targeted toxins) via the so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) to obtain functionalized scaffolds. For this reaction, Cy3-PAMAM-SO1861 ( Fig.13 Figures C and D) and Cy3-PAMAM-NC-SO1861 ( Fig.13 Figure B) and heterobifunctional NHS-PEG 13 -DBCO linker conjugation, the heterobifunctional NHS-PEG 13 -DBCO linker creates a strained alkyne on the surface of the conjugate ( Fig.13 Figure A). The NHS (N-hydroxysuccinimide) portion of the linker reacts with the remaining amine of the PAMAM-saponin conjugate to form an amide bond between the scaffold and the linker. The resulting DBCO (dibenzocyclooctyne) portion on the conjugate can undergo SPAAC with the corresponding azide on the targeted therapeutic agent.

[0481] Olein-EGF was used as a model targeted toxin and olein was used as a non-targeted toxin. Tetrafluorophenyl ester (TFP) derivatives of the dye were used with Alexa Fluor TM 488 labeled both toxins. The dye-labeled proteins were then conjugated to heterobifunctionalized NHS-SS-PEG3-azide linkers to obtain the corresponding chemical moieties of SPAAC and PAMAM-saponin conjugates. MALDI-TOF-MS measurements showed that each oleanolic acid-EGF molecule was attached with one Alexa Fluor TM 488 dyes and 9 NHS-SS-PEG3-azide molecules ( Fig.14 , Fig.15 ). In addition, Alexa Fluor TM 488-labeled oleanolic acid-EGF conjugated to heterobifunctionalized NHS-PEG 12 -Azide linker, the heterobifunctionalized NHS-PEG 12 -azide linkers lack a disulfide bond and therefore will result in a non-toxin cleavable construct.

[0482] Cy3-PAMAM-NC-SO1861-DBCO and Cy3-PAMAM-SO1861-DBCO conjugates with AlexaFluor TM 488-labeled azide-toxin reaction for strain-promoted alkyne-azide cycloaddition. Cy3 attached only to PAMAM polymer and Alexa Fluor attached only to toxin were detected by gel electrophoresis and after gel electrophoresis. TM The colocalization of the fluorescence signal of 488 on the polyacrylamide gel indicates the conjugation between the reaction reagents ( Fig.16 ).

[0483] As an alternative polymeric structure to PAMAM dendrimers, G4-dendrons with 16 functionalized amino end groups and an azide group at the focal point (PFd-G4-azide-NH-BOC, Polymer Factory) were used for conjugation with SO1861 ( Fig.26 The advantage of using dendrons over dendrimers is the focal point exhibited by the dendron structure. This focal point allows direct conjugation to the targeted toxin without the need for post-modification with orthogonal click functions ( Fig. 27 ).like Fig. 27 As shown, the dendrons were subjected to the same method as described for PAMAM dendrons. Briefly, after partial dye labeling and deprotection ( Fig.28 ), the amino group of the dendron was converted to thiol using the thiolation reagent, 2-iminothiolane, and then conjugated with SO1861-EMCH. For the conjugation with SO1861-EMCH, three different feed equivalents of SO1861-EMCH were used. The dendron-SO1861 conjugates were analyzed via MALDI-TOF-MS. As expected, when low SO1861-EMCH feed equivalents of 3 and 10 were used, conjugate materials with 1 and 2 SO1861 molecules per dendron molecule were obtained ( Fig.29 When 22 feed equivalents of SO1861-EMCH molecules per dendron molecule were used, higher dendron-SO1861 conjugate materials with up to 9 SO1861 molecules per dendron were obtained ( Fig.29In further experiments, saponin-functionalized dendrons will be conjugated with target toxins at their focal points to generate functionalized scaffolds and will be evaluated biologically.

[0484] To further test other conjugation methods of SO1861 to polymeric structures, a reductive amination approach was used. To this end, the aldehyde group of SO1861 was directly conjugated to the PAMAM amine to form an imine bond. The imine bond formation was followed by a reductive amination step by adding the reducing agent sodium cyanoborohydride, thereby forming a pH-stable amine bond between SO1861 and PAMAM ( Fig.17 Similar to the EMCH and HATU methods, this method allows control of the number of saponins conjugated to each polymer, such as Fig.17 Figure B Fig.17 Here, a PAMAM-saponin conjugate was produced, which obtained 10 ( Fig.17 Figure B) and 30 ( Fig.17 Figure C) SO1861 molecule.

[0485] Example 4

[0486] Cell viability assay

[0487] HeLa cells were seeded at 5,000 c / w in 96-well plates (100 μL / well) containing DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) and incubated overnight at 37°C and 5% CO2. The next day, 20x concentrated stock solutions of PAMAM, PAMAM-conjugate, G4-dendron (prepared in Example 3) or chloroquine (Sigma Aldrich) samples were prepared in DMEM. The medium was removed from the cell culture plate and replaced with 160 μL of medium, followed by the addition of 10 μL of sample / well (from the 20x concentrated stock solution) and incubated at 37°C for 45 minutes. During this incubation, a SO1861 concentration curve was prepared. The SO1861 pre-stock solution was heated at 50°C for 10 minutes while shaking at 1,250 rpm. This was followed by 15 seconds of sonication and brief reheating at 50°C for 1 minute while shaking at 1,250 rpm. Serial dilutions of SO1861 were then prepared in PBS. The SO1861 concentration curve was prepared as a 10x concentrated stock solution, with 20 μL added to each well. After incubation at 37°C for 15 minutes, 10 μL of oleanolic acid-EGF (prepared in Example 2) diluted to 30 pM in DMEM or DMEM containing an equal amount of PBS was added to each well to obtain a final oleanolic acid-EGF concentration of 1.5 pM and the specified SO1861 and different polymeric structures or chloroquine concentrations, with a final volume of 200 μL / well.

[0488] After treatment, cells were incubated at 37°C for 72 hours and then cultured according to the manufacturer's instructions (CellTiter AQueous One Solution Cell Proliferation Assay, Promega) was used to determine cell viability by MTS-assay. In short, the MTS solution was diluted 20x in DMEM (PAN-Biotech GmbH) supplemented with 10% FBS without phenol red. The cells were washed once with 200 μL / PBS wells, and then 100 μL of diluted MTS solution was added to each well. The plate was incubated at 37°C for about 20-30 minutes. Subsequently, the OD at 492nm was measured on a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" well was subtracted from all other wells, and then the cell viability percentage of the treated / untreated cells was calculated by dividing the background correction signal of the treated well by the background correction signal of the untreated well (x100).

[0489] FACS analysis

[0490] HeLa cells were seeded at 500,000 c / plate in 10 cm culture dishes containing DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) and incubated for 48 h (5% CO2, 37°C) until 90% confluence was reached. Then, the cells were trypsinized (TrypE Express, Gibco Thermo Scientific) into single cells. 0.75 × 10 6 The cells were transferred to a 15 mL falcon tube and centrifuged (1,400 rpm, 3 min). The supernatant was discarded, leaving the cell pellet submerged. The pellet was separated by gently tapping the falcon tube on a vortex shaker and washed with 4 mL of cold PBS (without Mg). 2+ and Ca 2+ After washing, resuspend the cells in 3 mL of cold PBS (without Mg 2+ and Ca 2+ , 2% FBS) and evenly divided into 3 round-bottom FACS tubes (1 mL / tube). The cells were centrifuged again and resuspended in 200 μL cold PBS (without Mg). 2+ and Ca 2+ , 2% FBS) or 200 μL of antibody solution containing 2+ and Ca 2+ , 2% FBS). APC mouse IgG1, κ isotype Ctrl FC (#400122, Biolegend) was used as an isotype control, and APC anti-human EGFR #352906, Biolegend) was used to stain EGFR receptors. The samples were incubated at 4°C on a tube roller mixer for 30 minutes. Then, the cells were washed with cold PBS (without Mg). 2+ and Ca 2+ The cells were washed 3 times with 2% FBS (1% FBS) and fixed with 2% PFA solution in PBS for 20 minutes at room temperature. The cells were washed 2 times with cold PBS and resuspended in 250-350 μL cold PBS for FACS analysis. The samples were analyzed using BD FACSCantoII flow cytometry system (BD Biosciences) and FlowJo software.

[0491] result

[0492] It has been previously described that the amino groups in amine-containing polymeric structures such as PAMAM and PEI (polyethyleneimine) can be +Buffering capacity blocks endosomal acidification. Since the endosomal escape enhancing property of SO1861 is only exposed at low endosomal pH (<pH5), the scaffold or functionalized scaffold should not contain chemical groups that can interfere with endosomal acidification and thus block the activity of SO1861.

[0493] Amine-containing polymeric structures of G5 PAMAM (128 primary amines and approximately 126 tertiary amines) and G4-dendron (16 primary amines) were tested to determine whether these molecules inhibit the endosomal escape enhancing ability of SO1861. Co-application experiments of PAMAM (native or thiolated) or dendron (native) with caryophyllene-EGF and various concentrations of SO1861 were performed on HeLa cells. Chloroquine was used as a control to inhibit endosomal acidification.

[0494] HeLa cells showed sufficient EGFR cell surface levels ( Fig.30 Figure A). It was observed that both 'native' PAMAM and chloroquine inhibited the endosomal escape of SO1861-mediated targeted toxin and subsequent cell killing (in HeLa cells) ( Fig.30 Figure B). PAMAM at 500 nM inhibited even to the same extent as the endosomal acidification inhibitor chloroquine, while 667 nM dendron had no effect at all. To further address whether the inhibitory activity of 'native' PAMAM was due to the availability of amino groups in PAMAM, the primary amino groups of PAMAM were partially thiolated by reaction with 2-iminothiolane ( Fig.11 ), resulting in blocked primary amines of 16 / 128 ( Fig.11 Figure C), 65 / 128 ( Fig.11 Figure D), and 108 / 128 ( Fig.11 Figure E). It was observed that thiolation of 65 and 108 primary amines overcame the inhibition of SO1861-mediated endosomal escape, while thiolation of up to 16 amine groups still showed inhibition of SO1861-mediated endosomal escape of the targeted toxin ( Fig.30 Figure C). The primary amino groups of PAMAM were also partially polyethylene glycolylated by reaction with mPEG2k-NHS ( Fig.12 ), giving blocked primary amines of 3 / 128 ( Fig.12 Figure C), 8 / 128 ( Fig.12 Figure D), and 18 / 128 ( Fig.12 Figure E). Blocking only 3 primary amines by polyethylene glycolylation was sufficient to reverse the inhibition of SO1861-mediated endosomal escape ( Fig.11 Figure D). The shielding effect of polyethylene glycolylation is likely to extend beyond the small number of transformed amines, as it is known that polyethylene glycolylation introduces a hydrated layer that can shield the entire molecule (if a sufficient level is reached).

[0495] These results suggest that the presence of a certain number of free amino groups on polymeric structures such as PAMAM can block endosomal acidification and thus inhibit the endosomal escape activity of SO1861 or other glycosides. The inhibitory effect is reversed when the number of amino groups is lower, as shown for the G4-dendron, or if the amino groups have been shielded, such as by thiolation or PEGylation.

[0496] Example A - Saponin mixture of Quillaja saponin containing QS-21 having endosomal / lysosomal escape enhancing activity

[0497] Scheme 1 shows a series of common molecular structures of QS-21 saponins (partially adapted from: Conrado Pedebos, Laércio Pol-Fachin, Ramon Pons, Cilaine V. Teixeira Hugo Verli, Atomic Model and Micelle Dynamics of QS-21 Saponin, Molecules 2014, 19, 3744-3760). A mixture of water-soluble saponins obtained from Quillaja saponaria (Sigma-Aldrich, Product No.: S4521; Roth, Item No.: 6857; InvivoGen, Product 'Quil-A') can be applied to the effector portion comprising at least one saponin according to the present invention or to the endosomal / lysosomal escape enhancing conjugates, compositions, combinations according to the present invention based on the endosomal / lysosomal escape enhancing properties of at least one individual saponin present in the mixture (such as QS-21) or based on the combination of two or more saponins contained in the mixture (such as QS-21 and QS-7).

[0498] The inventors have demonstrated that a mixture of saponins from Quillaja salsa at a dose of 2.5 micrograms / ml is able to enhance the endosomal escape of oleanolic acid when tested in a cell-based bioassay with mammalian tumor cells. The effector moiety exposed to the cells is oleanolic acid covalently coupled to the ligand EGF: EGF-oleanolic acid. The cells tested were the tumor cell line HeLa for free saponins, and A431, MDA-MB-468, CaSki and A2058 for testing saponins from Quillaja salsa when covalently coupled to cetuximab.

[0499] References

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[0503] triterpenes from Gypsophila elegans M.Bieb.enable non-toxic deliveryof gene

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[0505] Kolb,H.C.;Finn,M.G.;Sharpless,K.B.Click Chemistry:Diverse ChemicalFunction from a Few Good Reactions.Angewandte Chemie 2001,40,2004-2021.

[0506] Bird,R.E.;Hardmann,K.D.;Jacobson,J.W.;Johnson,S.;Kaufman,B.M.;Lee,S.M.;Lee,T.;Pope,S.H.;Riordan,G.S.;Whitlow,M.Single-chain antigen-bindingproteins.Science 1988,242,423-426.

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Claims

1. An effector moiety conjugated to at least one saponin, wherein the effector moiety is capable of inducing an intracellular effect when present in a mammalian cell, wherein the at least one saponin is a triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and / or a disaccharide chain triterpene saponin, selected from a saponin having a sapogenin aglycone core and a saponin having a gypsophilic acid aglycone core, wherein the at least one saponin comprises a branched carbohydrate side chain at position C-3, wherein the carbohydrate side chain is Gal-(1→2)-[Xyl-(1→3)]-GlcA-, and wherein the saponin comprises a branched carbohydrate side chain containing a deoxy carbohydrate at position C-28, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker comprising a hydrazone bond, or is directly covalently bound to the effector moiety, and wherein the effector moiety comprises or consists of at least one oligonucleotide, a nucleic acid, a heterologous nucleic acid.

2. The effector moiety of claim 1 conjugated to at least one saponin, wherein the saponin is SO1861 and / or QS-21.

3. The effector moiety conjugated to at least one saponin as claimed in claim 1, wherein the effector moiety is selected from any one or more of the following: a vector, a gene, a transgene that induces cell suicide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinose nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA), or a derivative thereof.

4. An effector moiety conjugated to at least one saponin as described in claim 1 or 2, wherein the effector moiety is a BNA.

5. The effector moiety conjugated to at least one saponin according to claim 1 or 2, wherein the effector moiety is a BNA for silencing the expression of HSP27 protein.

6. The effector moiety of any one of claims 1 to 5 conjugated to at least one saponin, wherein the at least one saponin comprises a glucuronic acid functionality in the carbohydrate substituent at the C-3β-OH group of the saponin.

7. The effector moiety conjugated to at least one saponin according to any one of claims 1 to 6, wherein the at least one saponin is a single specific saponin or a mixture of two or more different saponins.

8. The effector moiety conjugated to at least one saponin as described in any one of claims 1-7, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker, wherein the at least one linker comprises at least one cleavable linker, wherein the cleavable linker comprises a hydrazone bond.

9. The effector moiety conjugated to at least one saponin according to claim 8, wherein the linker is N-ε-maleimidocaproic acid hydrazide (EMCH).

10. An effector moiety conjugated to at least one saponin according to any one of claims 1 to 9, wherein the at least one saponin is covalently bound to the effector moiety via at least one linker, wherein the linker is or comprises a scaffold, the scaffold comprising a polymeric or oligomeric structure and a chemical group for covalently coupling the scaffold to the effector moiety, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the scaffold via the aldehyde functional group at the C-23 position of the at least one saponin, and the covalent bond is a hydrazone bond.

11. An effector moiety conjugated to at least one saponin as claimed in claim 10, wherein the aldehyde functional group at the C-23 position of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, and the linker is covalently coupled to a sulfhydryl group in the polymeric or oligomeric structure of the scaffold via a thioether bond.

12. The effector moiety conjugated to at least one saponin as claimed in claim 10, wherein the aldehyde functional group at the C-23 position of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, and the linker is covalently coupled to the sulfhydryl group of cysteine ​​in the polymeric or oligomeric structure of the scaffold via a thioether bond.

13. The effector moiety conjugated to at least one saponin according to any one of claims 10 to 12, wherein the chemical group of the scaffold for covalent coupling of the scaffold to the effector moiety is a click chemistry group.

14. The effector moiety conjugated to at least one saponin according to claim 13, wherein the click chemistry group is selected from tetrazines, azides, olefins or alkynes or cyclic derivatives of these groups.

15. The effector moiety conjugated to at least one saponin as claimed in claim 13 or 14, wherein the click chemistry group is an azide.

16. The effector moiety conjugated to at least one saponin according to any one of claims 10 to 15, wherein the polymeric or oligomeric structure of the scaffold comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol or an assembly of these polymeric or oligomeric structures.

17. The effector moiety conjugated to at least one saponin according to claim 16, wherein the assembly is preferably constructed by covalent cross-linking.

18. The Effector Moiety conjugated to at least one saponin of any one of claims 1-17, wherein the at least one saponin is a defined number of saponins or a defined range of saponins.

19. The effector moiety conjugated to at least one saponin of any one of claims 1-18, wherein the at least one saponin is 1-128 saponins or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128 saponins, or any number of saponins therebetween.

20. The Effector Moiety conjugated to at least one saponin as claimed in any one of claims 1 to 19, wherein the at least one saponin is 7, 9, 12 saponins.

21. An antibody-drug conjugate comprising the effector moiety of any one of claims 1-20 conjugated to at least one saponin, or a ligand-drug conjugate comprising the effector moiety of any one of claims 1-20 conjugated to said at least one saponin.

22. The antibody-drug conjugate or ligand-drug conjugate of claim 21, wherein the antibody is capable of binding to any of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, and / or wherein the antibody of the antibody-drug conjugate is or comprises any of the following: intuzumab, mosimo Monoclonal antibody, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, Herceptin, pinatuzumab, OKT-10, or anti-HER2 monoclonal antibody, anti-CD20 monoclonal antibody, anti-CA125 monoclonal antibody, anti-EpCAM (17-1A) monoclonal antibody, anti-EGFR monoclonal antibody, anti-CD30 monoclonal antibody, anti-CD33 monoclonal antibody, anti-vascular integrin α-vβ-3 monoclonal antibody, anti-CD52 monoclonal antibody, anti-CD22 monoclonal antibody, anti-CEA monoclonal antibody, anti-CD44v6 monoclonal antibody, anti-FAP monoclonal antibody Antibodies, anti-CD19 monoclonal antibodies, anti-CanAg monoclonal antibodies, anti-CD56 monoclonal antibodies, anti-CD38 monoclonal antibodies, anti-CA6 monoclonal antibodies, anti-IGF-IR monoclonal antibodies, anti-integrin monoclonal antibodies and anti-syndecan-1 monoclonal antibodies, or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of the following: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, moxetumomab pasudotox and polotuzumab vedotin, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule.

23. The antibody-drug conjugate or ligand-drug conjugate of claim 22, wherein the antibody is capable of binding to any one of CD71, HER2, and EGFR.

24. The antibody-drug conjugate or ligand-drug conjugate of claim 22, wherein the anti-HER2 monoclonal antibody is trastuzumab or pertuzumab, the anti-CD20 monoclonal antibody is rituximab, ofatumumab, tositumomab or isobemomab, the anti-CA125 monoclonal antibody is oregol monoclonal antibody, the anti-EpCAM (17-1A) monoclonal antibody is edrecolomab, the anti-EGFR monoclonal antibody is cetuximab, panitumumab or nimotuzumab, the anti-CD30 monoclonal antibody is brentuximab, the anti-CD33 monoclonal antibody is gemtuzumab or huMy9-6, the anti-vascular integrin α-vβ-3 monoclonal antibody is The monoclonal antibody is edarizumab, the anti-CD52 monoclonal antibody is alemtuzumab, the anti-CD22 monoclonal antibody is epratuzumab, the anti-CEA monoclonal antibody is labetuzumab, the anti-CD44v6 monoclonal antibody is bivacizumab, the anti-FAP monoclonal antibody is sirolimus, the anti-CD19 monoclonal antibody is huB4, the anti-CanAg monoclonal antibody is huC242, the anti-CD56 monoclonal antibody is huN901, the anti-CD38 monoclonal antibody is daratumumab, the anti-CA6 monoclonal antibody is DS6, the anti-IGF-IR monoclonal antibodies are citumumab and 3B7, the anti-integrin monoclonal antibody is CNTO 95 and the anti-syndecan-1 monoclonal antibody is B-B4.

25. The antibody-drug conjugate or ligand-drug conjugate of claim 22, wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab or trastuzumab and OKT-9.

26. The antibody-drug conjugate or ligand-drug conjugate of claim 22, wherein the ligand-drug conjugate comprises or consists of EGF and / or cytokines for binding to cell surface molecules.

27. A therapeutic combination comprising: (a) an effector moiety conjugated to at least one saponin as claimed in any one of claims 1 to 20 and optionally a pharmaceutically acceptable excipient; and (b) antibody-drug conjugate or ligand-drug conjugate and optionally a pharmaceutically acceptable excipient.

28. The therapeutic combination of claim 27, wherein the antibody-drug conjugate is capable of binding to any of the following: tumor cell receptors CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, and / or wherein the antibody of the antibody-drug conjugate is or comprises any of the following: intuzumab, mosimo Monoclonal antibody, polotuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, Herceptin, pinatuzumab, OKT-10, or anti-HER2 monoclonal antibody, anti-CD20 monoclonal antibody, anti-CA125 monoclonal antibody, anti-EpCAM (17-1A) monoclonal antibody, anti-EGFR monoclonal antibody, anti-CD30 monoclonal antibody, anti-CD33 monoclonal antibody, anti-vascular integrin α-vβ-3 monoclonal antibody, anti-CD52 monoclonal antibody, anti-CD22 monoclonal antibody, anti-CEA monoclonal antibody, anti-CD44v6 monoclonal antibody, anti-FAP monoclonal antibody Antibodies, anti-CD19 monoclonal antibodies, anti-CanAg monoclonal antibodies, anti-CD56 monoclonal antibodies, anti-CD38 monoclonal antibodies, anti-CA6 monoclonal antibodies, anti-IGF-IR monoclonal antibodies, anti-integrin monoclonal antibodies and anti-syndecan-1 monoclonal antibodies, or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of the following: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, intuzumab ozogamicin, moxetumomab pasudotox and polotuzumab vedotin, and / or wherein the ligand-drug conjugate comprises or consists of at least one non-protein ligand and / or at least one protein ligand for binding to a cell surface molecule.

29. The therapeutic combination of claim 28, wherein the antibody-drug conjugate is capable of binding to any one of CD71, HER2, and EGFR.

30. The therapeutic combination of claim 28, wherein the anti-HER2 monoclonal antibody is trastuzumab or pertuzumab, the anti-CD20 monoclonal antibody is rituximab, ofatumumab, tositumomab or isobemomab, the anti-CA125 monoclonal antibody is oregol, the anti-EpCAM (17-1A) monoclonal antibody is edrecolomab, the anti-EGFR monoclonal antibody is cetuximab, panitumumab or nimotuzumab, the anti-CD30 monoclonal antibody is brentuximab, the anti-CD33 monoclonal antibody is gemtuzumab or huMy9-6, the anti-vascular integrin α-vβ-3 monoclonal antibody is edrecolomab The anti-CD52 monoclonal antibody is alemtuzumab, the anti-CD22 monoclonal antibody is epratuzumab, the anti-CEA monoclonal antibody is labetuzumab, the anti-CD44v6 monoclonal antibody is bivacizumab, the anti-FAP monoclonal antibody is sirolimus, the anti-CD19 monoclonal antibody is huB4, the anti-CanAg monoclonal antibody is huC242, the anti-CD56 monoclonal antibody is huN901, the anti-CD38 monoclonal antibody is daratumumab, the anti-CA6 monoclonal antibody is DS6, the anti-IGF-IR monoclonal antibody is citumumab and 3B7, the anti-integrin monoclonal antibody is CNTO 95 and the anti-syndecan-1 monoclonal antibody is B-B4.

31. The therapeutic combination of claim 28, wherein the antibody of the antibody-drug conjugate is or comprises any one of the following: cetuximab or trastuzumab and OKT-9.

32. The therapeutic combination of claim 28, wherein the ligand-drug conjugate comprises or consists of EGF and / or a cytokine for binding to a cell surface molecule.

33. A pharmaceutical composition comprising an effector moiety conjugated to at least one saponin as claimed in any one of claims 1 to 20 or an antibody-drug conjugate as claimed in any one of claims 21 to 26 or a ligand-drug conjugate as claimed in any one of claims 21 to 26 or a therapeutic combination as claimed in any one of claims 27 to 32 and optionally a pharmaceutically acceptable excipient.

34. Use of the effector moiety conjugated to at least one saponin as claimed in any one of claims 1 to 20, or the antibody-drug conjugate as claimed in any one of claims 21 to 26, or the therapeutic combination as claimed in any one of claims 27 to 32, or the ligand-drug conjugate as claimed in any one of claims 21 to 26, or the pharmaceutical composition as claimed in claim 33 in the preparation of a medicament.

35. Sambucus alpha from Sambucus sambucus.

36. A method for synthesizing di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate, comprising: Dissolve 6-azidohexanoic acid, EDCl.HCl and Oxyma Pure in DMF and stir for 5 minutes; adding a solution of di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate in DMF to obtain a reaction mixture, and stirring the reaction mixture for 5 hours; Evaporate the reaction mixture in vacuo to obtain a residue; The residue was dissolved in ethyl acetate, washed with potassium bisulfate solution, saturated sodium bicarbonate solution and brine, dried over Na2SO4, filtered and evaporated in vacuo to obtain di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate.

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