Clusters of bioactive molecules

Abstract

The present invention relates to a molecular scaffold suitable for covalently linking at least one biologically active molecule to a carrier molecule, the scaffold comprising a polymeric structure, the biologically active molecule being covalently linked to the polymeric structure, and the scaffold further comprising chemical groups for covalently coupling the scaffold to the carrier molecule. The biologically active molecule has a molecular weight of 3,000 daltons or less, e.g., 1,700 to 1,950 daltons. In some embodiments, the biologically active molecule is an amphiphilic molecule. When more than one biologically active molecule is covalently linked to a polymeric (or oligomeric) structure, the biologically active molecule is a single specific molecule or a mixture of different types of molecules. Specifically, the present invention relates to monoclonal antibody-based antibody-drug conjugates with an improved therapeutic window of a drug due to the covalent linkage of a (cluster of) enhancer molecules, e.g., a payload, e.g., a protein toxin or oligonucleotide, to the ADC, or alternatively, due to the coadministration of an ADC and a cell-targeting conjugate comprising a (cluster of) enhancer molecules to a patient in need thereof. The present invention also relates to a method for producing a scaffold suitable for covalently linking a bioactive molecule to a carrier molecule, providing a cluster of enhancer molecules. Furthermore, the present invention relates to a method for producing a scaffold covalently linked to a carrier molecule, the scaffold comprising a covalently linked bioactive molecule, the carrier molecule comprising an antibody and a payload.

Description

[Technical field]

[0001] The present invention relates to a molecular scaffold suitable for covalently linking at least one bioactive molecule, such as a therapeutic drug molecule, to a carrier molecule. In particular, the present invention relates to a monoclonal antibody-based antibody-drug conjugate with an improved therapeutic window of a drug due to the covalent linkage of a (cluster of) enhancer molecule, such as a payload, such as a protein toxin or oligonucleotide, to the ADC, or alternatively due to the co-administration of an ADC and a cell-targeting conjugate comprising a (cluster of) enhancer molecule to a patient in need thereof. The present invention also relates to a method for producing a scaffold suitable for covalently linking at least one bioactive molecule to a carrier molecule, such bioactive molecule being covalently linked to the scaffold. [Background technology]

[0002] Molecules with therapeutic biological activity are often theoretically suitable for application as effective therapeutic drugs for the treatment of diseases such as cancer in human patients in need thereof. A typical example is a small molecule biologically active moiety. However, many, if not all, potential drug-like molecules and therapeutic agents currently in clinical use suffer from at least one of a plethora of shortcomings and drawbacks. When administered to the human body, therapeutically active molecules may exert off-target effects in addition to the biological activity directed at the underlying aspects of the disease or health problem to be treated. Such off-target effects are undesirable and carry the risk of inducing health- or even life-threatening side effects of the administered molecule. It is the occurrence of such adverse events that causes many drug-like compounds and therapeutic moieties to fail phase III or even phase IV clinical trials (post-marketing follow-up). Thus, there is a strong desire to provide drug molecules, such as small molecule therapeutics, whose therapeutic effect should, for example, inter alia, (1) be highly specific for the biological factors or biological processes driving the disease, (2) be sufficiently safe, (3) be sufficiently effective, (4) be sufficiently directed to diseased cells with no or little off-target activity against non-diseased cells, (5) have a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in the human patient within a certain time frame and remain at the target site for a certain time frame), and / or (6) have a sufficiently long-lasting therapeutic activity in the patient's body. Despite already long-lasting and intensive research, and despite impressive progress made in several areas where the difficulties and shortcomings faced have been individually addressed, unfortunately, to date, no "ideal" therapeutics are available to patients that have many or even all of the beneficial characteristics outlined herein above.

[0003] Chemotherapy is one of the most important therapeutic options for cancer treatment. However, it is often associated with a low therapeutic window since it has no specificity for cancer cells compared to dividing cells of healthy tissues. The invention of monoclonal antibodies has offered the possibility to exploit their specific binding properties as a mechanism for targeted delivery of cytotoxic drugs to cancer cells while sparing normal cells. This can be achieved by chemical conjugation of cytotoxic effectors (also known as payloads or warheads) to antibodies to create antibody-drug conjugates (ADCs). Typically, highly potent payloads are used, such as emtansine (DM1), which has a limited therapeutic index (ratio comparing toxic dose to effective dose) in their unconjugated form. Conjugation of DM1 to trastuzumab (ado-trastuzumab-emtansine), also known as Kadcycla, improves the tolerable dose of DM1 at least two-fold in monkeys. In the past decades, enormous efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, bringing ADCs to the clinic remains difficult. The first ADC approved for clinical use was gemtuzumab ozogamicin (CD33-targeted Mylotarg, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML) in 2000. However, Mylotarg was removed from the market at the request of the Federal Drug Administration (FDA) due to a number of concerns encompassing its safety profile. Patients treated with Mylotarg were found to die more often than those treated with conventional chemotherapy. Mylotarg was approved for remarketing in 2017 with a lower recommended dose, a different schedule in combination with chemotherapy or on its own, and a new patient population. To date, only five ADCs have been approved for clinical use, while approximately 55 ADCs have been withdrawn from clinical development. However, interest remains high, and currently, approximately 80 ADCs are still in clinical development with nearly 600 trials.

[0004] Despite the potential to use toxic payloads that are usually not tolerated by patients, a low therapeutic index (ratio comparing toxic dose to efficacious dose) is a major issue that explains the discontinuation of many ADCs in clinical development. This can be caused by several mechanisms, such as off-target toxicity against normal cells, development of resistance to cytotoxic drugs, and premature release of the drug in circulation. A systematic review by the FDA of ADCs found that the toxicity profile of most ADCs can be categorized according to the payload used rather than the antibody used, suggesting that toxicity is mostly determined by premature release of the payload. Of the approximately 55 ADCs that have been discontinued, it is estimated that at least 23 were due to a poor therapeutic index. For example, development of a trastuzumab-tesirin conjugate (HER-2-targeting ADCT-502, ADC therapeutics) was recently discontinued due to a narrow therapeutic index possibly due to on-target normal tissue effects in lung tissues that express significant levels of HER2. In addition, several ADCs in phase 3 trials were discontinued due to missing primary endpoints. For example, phase 3 trials of depatuxizumab mafodotin conjugates (EGFR-targeted ABT-414, AbbVie) tested in patients with newly diagnosed glioblastoma and mirvetuximab soravtansine conjugates (folate receptor alpha (FRα)-targeted IMGN853, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently halted and showed no survival benefit. It is important to note that the clinically used doses of some ADCs may not be sufficient for their full anticancer activity. For example, ado-trastuzumab emtansine has an MTD of 3.6 mg / kg in humans. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels of 3 mg / kg or higher, but more potent efficacy was observed at 15 mg / kg. This suggests that at clinically administered doses, ado-trastuzumab emtansine may not exert its full potential antitumor efficacy.

[0005] ADCs are mainly composed of antibodies, cytotoxic moieties such as payloads, and linkers. In the design and development of new ADCs to overcome existing problems, several novel strategies have been proposed and implemented to target each of the components of ADCs. For example, by identifying and validating satisfactory antigenic targets of antibody components, by selecting antigens that have high expression levels in tumors and no or little expression in normal tissues, antigens that are present on the cell surface so as to be accessible to circulating ADCs, and antigens that allow internalization of ADCs into cells after binding; and alternative mechanisms of activity; designing and optimizing linkers that improve the solubility and drug-to-antibody ratio (DAR) of ADCs and overcome the resistance induced by proteins that can transport chemotherapeutic drugs out of cells; selecting and optimizing antibodies to improve the DAR ratio by inclusion of more payloads and improve the homogeneity and developability of antibodies. In addition to ADC technology developments, new clinical and translational strategies are also being evolved to maximize the therapeutic index, altering dosing schedules, for example with split dosing; conducting biodistribution studies; and incorporating biomarkers to optimize patient selection, capture response signals early, monitor duration and depth of response, and inform combination studies.

[0006] Examples of ADCs with clinical potential are ADCs being evaluated as treatment options for lymphoid malignancies and multiple myeloma, such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin. Polatuzumab vedotin, which binds to CD79b on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, have been tested in clinical trials. The ADCs were each combined with co-administration of rituximab, a monoclonal antibody that binds to CD20 and is not provided with 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 yet a further approach, attempting to arrive at a "magic bullet" that combines many or even all of the previously mentioned desirable characteristics of ADCs.

[0007] Meanwhile, in the past decades, therapeutic drugs based on nucleic acids have been under development. 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 for approaches such as gene therapy, RNA interference (RNAi). Many of them share the same fundamental basis of action by inhibiting either DNA or RNA expression, thereby preventing the expression of abnormal proteins related to diseases. The largest number of clinical trials are conducted in the field of gene therapy, with almost 2600 ongoing or completed clinical trials worldwide, but only about 4% enter phase 3. This is followed by clinical trials with ASO. Similar to ADC, therapeutic nucleic acids share two major issues during clinical development, despite the large number of technologies being explored; delivery into cells and off-target effects. For example, ASOs such as peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), and bridged nucleic acid (BNA) are being explored as an attractive strategy to specifically inhibit target genes, especially those that are difficult to target by small molecule inhibitors or neutralizing antibodies. Currently, the efficacy of different ASOs is being investigated in many neurodegenerative diseases, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in some cancer stages. The application of ASOs as potential therapeutic agents requires safe and effective methods for their delivery to the cytoplasm and / or nucleus of target cells and tissues. Although the clinical relevance of ASOs has been demonstrated, inefficient cellular uptake both in vitro and in vivo has limited their efficacy and been a barrier to therapeutic development. Cellular uptake can be <2% of the dose, resulting in ASO concentrations at the active site that are too low for effective and sustained outcomes. This consequently requires increased administration doses, which induce off-target effects. The most common side effects are activation of the complement cascade, inhibition of the coagulation cascade, and stimulation mediated by toll-like receptors of the immune system.

[0008] Chemotherapeutic drugs are most commonly small molecules. However, their effectiveness is hindered by severe secondary off-target toxicity, as well as their poor solubility, rapid clearance, and limited tumor exposure. Scaffold-small molecule drug conjugates, such as polymer-drug conjugates (PDCs), are pharmacologically active polymeric constructs that comprise one or more molecules of small molecule drugs bound to a carrier scaffold (e.g., polyethylene glycol (PEG)).

[0009] Such conjugate principles have attracted great attention and have been under investigation for decades. The majority of small molecule drug conjugates in preclinical or clinical development are for oncological indications. However, up to now, only one drug not related to cancer (a PEG oligomer conjugate of the opioid antagonist naloxone, Movantik, AstraZeneca) has been approved in 2014 for the non-oncological indication of opioid-induced constipation in chronic pain patients. The translational application of drug-scaffold conjugates to the treatment of human subjects has provided little clinical success so far. For example, PK1 (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has shown significant anticancer activity in both solid tumors and leukemia in mouse models and has been under clinical investigation for oncological indications. Although it demonstrated significantly reduced non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer efficacy proved to be marginal in patients and, as a consequence, further development of PK1 was discontinued.

[0010] The failure of scaffold-small molecule drug conjugates has been attributed, at least in part, to their poor accumulation at tumor sites. For example, in murine models, PK1 showed 45-250-fold higher accumulation in tumors than in healthy tissues (liver, kidney, lung, spleen, and heart), although accumulation in tumors was only observed in a small subset of patients in clinical trials.

[0011] A possible solution to the problems mentioned before is the application of nanoparticle systems for drug delivery, such as liposomes. Liposomes are spherical vesicles consisting of one or more phospholipid bilayers, which form spontaneously when phospholipids are dispersed in water. The amphiphilic properties of phospholipids provide them with properties of self-assembly, emulsification, and wetting characteristics, which can be used in the design of new drugs and new drug delivery systems. Drugs encapsulated in liposomal delivery systems can offer several advantages over direct administration of drugs, such as improved and controlled pharmacokinetics and pharmacodynamics, tissue targeting properties, reduced toxicity, and enhanced drug activity. An example of such success is the liposomal encapsulated form of the small molecule chemotherapy drug doxorubicin (Doxil: pegylated liposomal encapsulated form of doxorubicin; Myocet: non-pegylated liposomal doxorubicin), which has been approved for clinical use.

[0012] Thus, there remains a need to find solutions that allow drug therapies, such as antitumor therapies, to be applicable for non-systemic use when desired, where the drugs have, for example, an acceptable safety profile, little off-target activity, sufficient efficacy, a sufficiently low clearance rate from the patient's body, etc. Summary of the Invention

[0013] In certain embodiments of the present invention, it is a primary goal to provide improved bioactive compounds, or compositions containing such improved bioactive compounds.

[0014] It is one of the objectives of some embodiments of the present invention to provide a solution to the problem of non-specificity faced when administering small molecule therapeutically active compounds to human patients in need thereof. It is one of the objectives of some embodiments of the present invention to provide a solution to the problem of drugs having suboptimal specificity to biological factors or biological processes that drive disease. It is one of the objectives of some embodiments of the present invention to provide a solution to the problem of insufficient safety characteristics of current drugs when administered to human patients in need thereof. It is one of the objectives of some embodiments of the present invention to provide a solution to the problem of current drugs being less effective than desired when administered to human patients in need thereof. It is one of the objectives of some embodiments of the present invention to provide a solution to the problem of current drugs not being adequately directed to diseased cells without or with little off-target activity against non-disease cells when administered to human patients in need thereof. It is one of several objectives of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently timely mechanism of action (e.g., the administered drug molecule 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). It is one of several objectives of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently long-lasting therapeutic activity in the patient's body.

[0015] At least one of the above objects of embodiments of the present invention is achieved by providing a molecular scaffold of the present invention suitable for covalently linking at least one biologically active molecule, such as a therapeutic drug molecule, to a carrier molecule.

[0016] While the present invention will be described with respect to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

[0017] One embodiment of the present invention relates to a scaffold suitable for covalently binding at least one biologically active molecule to a carrier molecule, the scaffold comprising a polymeric or oligomeric structure, at least one of said biologically active molecules being covalently bound to said polymeric or oligomeric structure, the scaffold further comprising a first chemical group for covalent coupling of the scaffold to the carrier molecule.

[0018] One embodiment is a scaffold of the invention, wherein at least one biologically active molecule has a molecular mass of 3,000 daltons or less, preferably 2,500 daltons or less, more preferably 2,300 daltons or less, most preferably 2,000 daltons or less, for example between 1,700 daltons and 1,950 daltons.

[0019] In one embodiment is a scaffold of the invention, wherein at least one biologically active molecule is an amphipathic molecule.

[0020] One embodiment is a scaffold of the invention, where more than one biologically active molecule is covalently attached to the polymeric or oligomeric structure contained by the scaffold, wherein at least one biologically active molecule is a single specific molecule or a mixture of different molecules.

[0021] In one embodiment is a scaffold of the invention, wherein the at least one bioactive molecule is a saponin that may be isolated from Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, such as Quillaja saponaria, or a single specific saponin, or a mixture of two or more different saponins, such as one or more of the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any 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 a saponin having a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group, and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group. and / or 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilaic acid 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside. More preferably, the saponin is SO1861 and / or QS-21.

[0022] One embodiment is a scaffold of the invention, wherein at least one biologically active molecule is covalently attached to the polymeric or oligomeric structure of the scaffold via a cleavable bond, said cleavable bond undergoes cleavage in vivo under acidic conditions such as those present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦ 5.5.

[0023] One embodiment is a scaffold of the invention, wherein the at least one biologically active molecule is a defined number or range of glycoside molecules, preferably 1-128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 glycoside molecules, or any number therebetween, e.g., 7, 9, 12 glycoside molecules.

[0024] In one embodiment, the scaffold of the invention is a polymeric or oligomeric structure comprising 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, which assembly is preferably assembled by covalent crosslinks.

[0025] In one embodiment is a scaffold of the invention, wherein the carrier molecule comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, or comprises or consists of at least one non-proteinaceous ligand and / or at least one proteinaceous ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0026] One embodiment is a scaffold of the invention, wherein the carrier molecule comprises or consists of at least one effector molecule, or the carrier further comprises at least one effector molecule, where the carrier also comprises, for example, an immunoglobulin, and the effector molecule is at least one of a drug substance, for example, a payload, a toxin, a drug, a polypeptide, an oligonucleotide, a nucleic acid, a xenonucleic acid, an enzyme, for example, urease and Cre recombinase, a protein toxin, a ribosome-inactivating protein, or any one or more thereof.

[0027] An embodiment of the present invention relates to a method for producing a scaffold suitable for covalently linking at least one biologically active molecule to a carrier molecule, the method comprising: a) providing a polymer or oligomeric structure comprising a first chemical group for covalently coupling the polymer or oligomeric structure to the carrier molecule and comprising at least one second chemical group different from the first chemical group, each second chemical group for covalently coupling one of the at least one biologically active molecule to the oligomeric or polymeric structure; and b) covalently coupling at least one biologically active molecule to said polymer or oligomeric structure via the second chemical group(s), preferably the biologically active molecule(s) is any one of the biologically active molecules of the present invention, such as saponin, triterpenoid saponin, bisdesmosidic triterpene, more preferably SO1861 and / or GE1741 and / or SA1641 and / or QS-21, thereby providing a scaffold.

[0028] An aspect of the present invention relates to a method for producing a scaffold covalently linked to a carrier molecule, the scaffold comprising at least one covalently linked biologically active molecule, the method comprising: a) providing a scaffold comprising at least one biologically active molecule covalently linked to the polymeric or oligomeric structure of said scaffold, preferably providing a scaffold according to the present invention or a scaffold obtainable by a method of the present invention or a scaffold obtained by a method of the present invention; and b) covalently coupling the scaffold of a) to a carrier molecule according to the present invention, thereby providing a scaffold covalently linked to the carrier molecule, the scaffold comprising at least one covalently linked biologically active molecule.

[0029] One embodiment is a scaffold according to the invention or a method according to the invention, wherein the scaffold is capable of increasing endosomal escape and / or lysosomal escape of an effector molecule according to the invention, wherein said effector molecule is either covalently attached to the scaffold and contacted with a mammalian cell, or wherein said effector molecule is contacted with a mammalian cell in the presence of the scaffold.

[0030] definition The term "linker" has its usual scientific meaning and refers here to a linear stretch or chemical moiety of amino acid residues conjugated via peptide bonds. It attaches a molecule or atom to another molecule, for example to a ligand or to an effector molecule or to a scaffold. Typically, a linker comprises a chain of atoms linked by chemical bonds. Any linker molecule or linker technology known in the art can be used in the present disclosure. Where indicated, the linker is a linker for covalent attachment of molecules via a chemical group on such molecule suitable for forming a covalent bond or bond with the linker. The linker can be a non-cleavable linker. For example, the linker is stable under physiological conditions. The linker can be a cleavable linker, for example, a linker that is cleavable in the presence of an enzyme, or at a specific pH range or value, or under physiological conditions, such as the intracellular conditions of endosomes, such as lysosomes and late endosomes, of mammalian cells, such as human cells. Exemplary linkers that may 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 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0031] The term "trifunctional linker" has its usual scientific meaning and refers here to a linker that attaches to three molecules via their respective chemical groups. A person skilled in the art can design such a trifunctional linker based on this disclosure and common general knowledge. Such a trifunctional linker can exhibit, for example, a maleimide group that can be used for conjugation to a targeting ligand that exhibits a thiol group for performing a thiol-ene reaction. In addition, the trifunctional linker can exhibit a dibenzocyclooctyne (DBCO) group for performing the so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with an azide-bearing saponin. Finally, the trifunctional linker can obtain a third functional group, such as a trans-cyclooctyne (TCO) group, to perform the so-called inverse electron demand Diels-Alder (IEDDA) reaction with a tetrazine (Tz)-bearing effector molecule. Those skilled in the art will understand that the three chemical groups of the trifunctional linker can be the same or different, or the linker can contain two of the same chemical groups for linking a molecule to the trifunctional linker. The bond formed between the trifunctional linker can be covalent or non-covalent, with covalent bonds being preferred. The bond formed between the trifunctional linker and one or two or three binding molecules via each chemical group can be a cleavable (labile) bond, which can be cleaved under acidic conditions in cells, such as endosomes and lysosomes of mammalian cells, such as human cells, or a non-cleavable bond. It will be appreciated that the trifunctional linker can include one or two chemical groups for forming a covalent bond, and two or one additional chemical group(s) for forming a non-covalent bond, respectively. It will be appreciated that a trifunctional linker may include one or two chemical groups for forming a cleavable bond and an additional two or one chemical group(s), respectively, for forming a non-cleavable bond.

[0032] The term "cleavable", as used, for example, in the term "cleavable linker" or "cleavable bond", has its usual scientific meaning, and refers herein to cleavage under conditions such as acidic, reductive, enzymatic, or photoinduced conditions. For example, the cleavable linker may be cleaved under acidic conditions. Preferably, the cleavable linker is cleaved in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5, more preferably at pH ≦ 5.5. As another example, the cleavable linker may be cleaved by an enzyme, for example by a cathepsin, such as cathepsin B. Furthermore, an example of a covalent bond cleavable under reductive conditions is a disulfide bond.

[0033] The terms "oligomer" and "polymer" in the context of oligomer or polymer backbones have their customary scientific meanings. A polymer herein refers to a substance having a molecular structure built up primarily or entirely from a number of equal or similar units linked together; an oligomer herein refers to a polymer whose molecules consist of a relatively small number of repeating units. For example, a structure containing 5-10 or fewer equal or similar units may be referred to as an oligomeric structure, a structure containing 10-50 or more monomeric units may be referred to as a polymeric structure, and a structure of 10 monomeric units may be referred to as either oligomeric or polymeric.

[0034] The term "binding site" has its usual scientific meaning here, referring to a region or epitope on a molecule, such as a protein, DNA, or RNA, to which another molecule can bind.

[0035] The term "scaffold" has its usual 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, e.g., ligand molecules, effector molecules, can be covalently attached either directly or via a linker, such as a cleavable linker. The scaffold may have a structurally ordered formation, e.g., a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or may have an assembled polymeric structure, e.g., a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The backbone may comprise polymeric or oligomeric structures, such as poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines); or polyethylene glycols, poly- or oligo(esters), such as poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, or poly(dextrins), poly- or oligosaccharides, such as cyclodextrins or polydextrose; or natural and / or artificial poly- or oligoamino acids, such as polylysine or peptides or proteins, DNA oligo- or polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers. Preferably, the polymeric or oligomeric structures are biocompatible, where biocompatibility means that the polymeric or oligomeric structures do not show substantial acute or chronic toxicity in the organism and can either be excreted intact or can be completely degraded by the body's metabolism to excretable and / or physiological compounds.

[0036] The term "ligand" has its usual scientific meaning and refers herein to any molecule or molecules that can selectively bind to a target cell surface molecule or target cell surface receptor expressed on 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.

[0037] The term "antibody" as used herein is used in the broadest sense and may refer to an immunoglobulin (Ig), defined as a protein belonging to the classes IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), or to a functional binding fragment or binding domain of an immunoglobulin. In the context of the present invention, a "binding fragment" or "binding domain" of an immunoglobulin is defined as an antigen-binding fragment or domain of a parent immunoglobulin or other derivative, which essentially maintains the antigen-binding activity of such parent immunoglobulin. Functional fragments and functional domains are antibodies in the sense of the present invention, even if their affinity for the antigen is lower than that of the parent immunoglobulin. "Functional fragments and domains" according to the present invention include, but are not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single domain antibodies (sdAbs), monovalent IgG, scFv-Fc, reduced IgG (rIgG), minibodies, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanobodies, variable V domains such as VHH, Vh, and other types of antigen-recognizing immunoglobulin fragments and domains. The fragments and domains may be engineered to minimize or completely eliminate intermolecular disulfide interactions occurring between CH1 and CL domains. Functional fragments and domains offer the advantage of greater tumor penetration due to their smaller size. In addition, functional fragments or domains may be more uniformly distributed in the tumor mass compared to whole immunoglobulins.

[0038] The antibodies (immunoglobulins) of the present invention can be bifunctional or multifunctional. For example, a bifunctional antibody has one arm with specificity for one receptor or antigen, and 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 on a target cell.

[0039] The antibodies (immunoglobulins) of the invention can be, but are not limited to, polyclonal, monoclonal, human, humanized, chimeric, resurfaced, anti-idiotypic, mouse, rat, rat / mouse hybrid, llama, heavy chain only llama, heavy chain only, and veterinary antibodies. Preferably, the antibodies (immunoglobulins) of the invention are monoclonal. Resurfaced, chimeric, humanized, and fully human antibodies are also more preferred, as they are less likely to cause immunogenicity in humans. The antibodies of the ADCs of the invention preferably specifically bind to antigens expressed on the surface of cancer, autoimmune, diseased, abnormal cells, while leaving any healthy cells essentially unchanged (e.g., by not binding to such normal cells or by binding to such healthy cells with a lesser degree of number and / or affinity).

[0040] Specific antibodies that may be used in the ADCs of the invention include 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 alpha-v beta-3 monoclonal antibodies, such as etaracizumab, 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 cixutumumab and 3B7, anti-integrin monoclonal antibodies, such as CNTO95, and anti-syndecan-1 monoclonal antibodies, such as B-B4.

[0041] Any molecule other than an antibody that binds to a cell receptor or antigen of a target cell can also be used as a cell-binding ligand of the ligand-drug conjugate of the present invention. The ligand is provided according to the present invention with a covalently attached saponin. These ligands include, but are not limited to, proteins, polypeptides, peptides, and small molecules. Examples of these non-antibody ligands are interferons (e.g., 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 (TGF), transforming growth factor (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGFs, e.g., IGF-1 and IGF-2), other suitable hormones, e.g., thyrotropin releasing hormone (TRH), melanocyte stimulating hormone (MSH), steroids, and the like. Hormones (e.g., estrogens and androgens), somatostatin, lymphokines (e.g., IL-2, IL-3, IL-4, and IL-6), colony stimulating factors (CSFs, e.g., G-CSF, M-CSF, and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g., AS-1411, GBI-10, RNA aptamers against HIV glycoproteins), small molecules (e.g., folic acid, anisamidophenylboronic acid), vitamins (e.g., vitamin D), carbohydrates (e.g., hyaluronic acid, galactose).

[0042] An "effector molecule" or "effector moiety" or "payload" has its usual scientific meaning and, in the context of the present invention, is any substance that affects the metabolism of a cell by interacting with an effector molecule target within the cell. This effector molecule target is any molecule or structure within the cell, excluding the lumen of compartments and vesicles of the endocytic and recycling pathways, but including the membranes of these compartments and vesicles. Said structures within the cell therefore include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol.

[0043] The effector molecule or moiety is a drug substance, such as a toxin, such as a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide. A drug substance in the present invention is an effector molecule or moiety used to achieve a beneficial outcome in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / human subject. Benefits include diagnosis, prognosis, treatment, cure, and prevention (prophylaxis) of a disease and / or condition and / or health problem. A drug substance may also lead to unwanted and possibly even harmful side effects (e.g., adverse events observed during clinical trials). In this case, the pros and cons must be weighed to determine whether a drug substance is suitable in a particular case. If the effect of a drug substance in a cell is overwhelmingly beneficial to the organism as a whole, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the organism as a whole, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, the target and off-target cells are purpose-dependent and defined by the user. Examples of effector molecules and moieties are drugs, toxins, polypeptides (eg, enzymes), polynucleotides (including polypeptides and polynucleotides that contain unnatural amino acids or nucleic acids), and any combination thereof.

[0044] Effector molecules or effector moieties that are drugs may include, but are not limited to, anti-cancer drugs, anti-inflammatory drugs, and anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral) drugs. Preferably, the drug molecules of the present invention are anti-cancer drugs or anti-autoimmune drugs. Suitable anti-cancer drugs include, but are not limited to, alkylating agents, metabolic inhibitors, spindle poison plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. Also included within the definition of "anti-cancer agent" are, for example: (i) anti-hormonal agents that act to control or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands; (iii) antiandrogens; (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) angiogenesis inhibitors; and pharma- ceutically acceptable salts, acids, solvates, and derivatives of any of the above.

[0045] Effector molecules or moieties that are toxins may include, but are not limited to, proteinaceous toxins (e.g., bacterial and plant-derived toxins), toxins that target tubulin filaments, toxins that target DNA, and toxins that target RNA. Examples of proteinaceous toxins are saporin, dianthin, ricin, modeccin, abrin, volkensin, viscumin, shiga toxin, shiga-like toxins, pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of tubulin filament-targeting toxins are maytansinoids (e.g., DM1 and DM4), auristatins (e.g., monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF)), toxoids, tubulysins, cryptophycins, and rhizoxins. Examples of DNA-targeted toxins are calicheamicin: N-acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycin, doxorubicin, methotrexate, benzodiazepines, camptothecin analogs, and anthracyclines. Examples of DNA-targeted toxins are amanitin, splicostatin, and Thailanstatin. The toxins used in the present invention are defined as drug substances that can kill or inactivate cells. Preferably, targeted toxins are toxins that are only or at least predominantly toxic to target cells and not to off-target cells. The net effect of targeted toxins is preferably beneficial to the organism as a whole.

[0046] An effector molecule or moiety that is a polypeptide can be, for example, a polypeptide that restores a lost function, such as, for example, an enzyme replacement, a gene regulatory function, or a toxin. Examples of polypeptides as effector molecules are, for example, Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)bouganin, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (e.g., argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; enzymes of cell signaling; cell cycle regulators; gene regulators (transcription factors, such as NF-κB, or gene repressors, such as methionine repressor).

[0047] An effector molecule or effector moiety that is a polynucleotide can be, for example, a polynucleotide that contains coding information, such as a gene or an open reading frame that codes for a protein. It can also contain regulatory information, such as a promoter or regulatory element binding region, or a sequence that codes for a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include, for example, peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these is distinguished from naturally occurring DNA or RNA by modifications to the backbone of the molecule. Examples of nucleotides as effector molecules include, but are not limited to, for example, DNA: single-stranded DNA (e.g., DNA of adenine phosphoribosyltransferase); 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, e.g., PNA, PMO, LNA, and BNA).

[0048] The term "proteinaceous" as used, for example, in "proteinaceous molecule" and "proteinaceous toxin", are molecules and toxins that comprise at least one string of amino acid residues that can be obtained as an expression product from a single mRNA. Such molecules or toxins may further comprise any post-translational modification, carbohydrates, e.g. N- or O-linked carbohydrates, disulfide bonds, phosphorylation, sulfation, etc., as a result of any post-translational modification, and / or may further comprise any other modification, e.g. resulting from chemical modification (e.g. resulting from the attachment of an effector moiety, saponin, scaffold, ligand, etc., either directly, e.g. to an amino acid side chain, or via at least one linker that is (covalently) bound to the molecule and is (covalently) chemically bound to the proteinaceous molecule to chemically modify the proteinaceous molecule). The term "proteinaceous" also encompasses and includes assemblies of such molecules, e.g. homodimers, heterotrimers, heterohexamers, or complex assemblies, e.g. ribosomes.

[0049] For example, in the context of "specific binding" and "receptors or molecular targets specifically present or expressed on the surface of tumor cells" and the like, the terms "specific" and "specifically" have their usual scientific meaning known in the art. Here, for example, it refers 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 a further molecule different from the second molecule, or, for example, when a number of receptors or molecular targets are considered, the expression or higher degree of expression of a cell surface receptor or molecular target on the surface of a first type of cell, such as 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 on a second type of cell, such as a healthy cell. Expression on a second type of cell may be completely absent or very low relative to any degree of expression on tumor cells, etc. Furthermore, for example, in "specific binding", the term "specific" has its usual scientific meaning known in the art, and here, it refers to a molecule that can have an interaction with another molecule with a higher binding affinity than background interactions between molecules. Similarly, the term "specificity" refers to an interaction, e.g., between two molecules or between a cell and a molecule, that has a higher binding affinity than background interactions between molecules. Binding molecules, such as immunoglobulins, bind via their binding sites, e.g., immunoglobulin variable regions of the immunoglobulin, to binding sites on molecules, e.g., epitopes, cell surface receptors, etc., with a higher binding affinity than background interactions between molecules. In the context of the present invention, background interactions typically have a K D Similarly, a "specific binding domain" is a domain that preferentially binds to a binding site on a molecule, e.g., an epitope, cell surface receptor, etc., with a binding affinity higher than the background interactions between molecules. In the context of the present invention, a "background interaction" is typically an interaction with an affinity lower than a K of 10E-4M. D Preferably, the specific binding domain has a K of about 10E-5M. D It binds with higher affinity than

[0050] The term "binding" is defined as an interaction between molecules that can be distinguished from background interactions.

[0051] As used herein, the term "fragment" refers to an amino acid sequence that is part of a protein domain or that makes up an intact protein domain. The binding fragments according to the invention must have binding specificity for their respective targets, e.g. cell surface receptors on the surface of diseased cells, e.g. tumor cells.

[0052] The term "ADC" or "antibody-drug conjugate" has its usual scientific meaning known to those skilled in the art and refers herein to a class of biopharmaceutical drugs designed as targeted therapies for treating, for example, cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADCs consist of an antibody linked to a bioactive cytotoxic (anticancer) payload or drug. ADCs combine the targeting ability of monoclonal antibodies with the cancer killing ability of cytotoxic drugs. They are designed with the intent to discriminate between healthy cells and diseased tissues, such as tumor cells in tumors.

[0053] The term "Saponinum album" has its ordinary meaning and herein refers to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, including SA1657 and primarily SA1641.

[0054] The term "Quillajasaponin" has its ordinary meaning and is used herein to refer to the saponin fraction of Quillaja saponaria and therefore the source of all other QS saponins, containing primarily QS-18 and QS-21.

[0055] "QS-21" or "QS21" has its ordinary scientific meaning and refers herein to a mixture of QS-21 A-apio (~63%), QS-21 A-xylo (~32%), QS-21 B-apio (~3.3%), and QS-21 B-xylo (~1.7%).

[0056] Similarly, "QS-21A" has its customary scientific meaning and refers herein to a mixture of QS-21 A-apio (~65%) and QS-21 A-xylo (~35%).

[0057] Similarly, "QS-21B" has its customary scientific meaning and is used herein to refer to a mixture of QS-21 B-apio (~65%) and QS-21 B-xylo (~35%).

[0058] The term "Quill-A" refers to a commercially available semi-purified extract from Quillaja saponaria that contains variable quantities of more than 50 distinct (water-soluble) saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3 beta-OH group found 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 FAKersten, 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 saponin are also fractions of saponins from Quillaja saponaria, both of which contain a large variety of different saponins with largely overlapping contents. The two fractions differ in their specific composition because they are obtained by different purification procedures.

[0059] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7api. QS1861 has a molecular mass of 1861 Daltons and QS1862 has a molecular mass of 1862 Daltons. QS1862 is described in Fleck et al. (2019), row no. 28 of Table 1 (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 of QS-7, QS1862. The molecular mass is 1862 Daltons, since this is the nominal mass including the protons in the glucuronic acid. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in the negative ion mode, the measured mass is 1861 Daltons.

[0060] The terms first, second, third and the like in the specification and claims are not necessarily used to describe a sequential or chronological order but are used to distinguish between similar elements. The terms are interchangeable in appropriate circumstances. Embodiments of the invention may operate in sequences other than those described or illustrated herein.

[0061] Furthermore, although various embodiments may be referred to as "preferably" or "eg" or "for example" or "specifically," these are to be construed as exemplary ways in which the invention may be implemented, rather than as limiting the scope of the invention.

[0062] The term "comprising" used in the claims should not be construed as being limited to the subsequently listed elements or steps; it does not exclude other elements or steps. It needs to be construed as specifying the presence of the recited feature, integer, step, or component as referred to, 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 "pharmaceutical composition comprising A and B" should not be limited to a pharmaceutical composition consisting of components A and B only, but rather, in the context of the present invention, the recited components of the pharmaceutical composition are only A and B, and the claims should be construed to encompass equivalents of those components. Similarly, the scope of the expression "method comprising steps A and B" should not be limited to a method consisting of steps A and B only, but rather, in the context of the present invention, the recited steps of the method are only A and B, and the claims should be construed to encompass equivalents of those steps.

[0063] Additionally, unless the context clearly requires that there is only one of the feature, reference to a feature by the indefinite article "a" or "an" does not exclude the possibility that more than one of the feature may be present, e.g., a component, excipient, saponin, etc. Thus, the indefinite article "a" or "an" normally means "at least one." [Brief description of the drawings]

[0064] [Figure 1] In vivo HSP27 expression in A431 xenograph "nude" mouse tumor model treated with 30 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA), 25 mg / kg cetuximab-(Lys-L-HSP27BNA)4, or 25 mg / kg cetuximab-(Cys-L-SO1861). [Diagram 2]Improved HSP27 gene silencing in vitro in EGFR-expressing A431 cells by treatment with cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA), cetuximab-(Lys-L-HSP27BNA)4, or cetuximab-(Cys-L-SO1861). [Figure 3-1] Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity in EGFR-expressing cells (MDA-MB-468) by cetuximab, cetuximab + 10 pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10 pM cetuximab-saporin. B. Cell killing activity of trastuzumab, trastuzumab + 50pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50pM trastuzumab-saporin in HER2-expressing cells (SK-BR-3). C. Cell killing activity of cetuximab, cetuximab + 10pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM cetuximab-saporin in EGFR-expressing cells (HeLa). D. Cell killing activity of trastuzumab, trastuzumab + 50 pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50 pM trastuzumab-saporin in HER2-expressing cells (JIMT-1). [Figure 3-2]Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity in EGFR-expressing cells (MDA-MB-468) by cetuximab, cetuximab + 10 pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10 pM cetuximab-saporin. B. Cell killing activity of trastuzumab, trastuzumab + 50pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50pM trastuzumab-saporin in HER2-expressing cells (SK-BR-3). C. Cell killing activity of cetuximab, cetuximab + 10pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM cetuximab-saporin in EGFR-expressing cells (HeLa). D. Cell killing activity of trastuzumab, trastuzumab + 50 pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50 pM trastuzumab-saporin in HER2-expressing cells (JIMT-1). [Figure 3-3]Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity in EGFR-expressing cells (MDA-MB-468) by cetuximab, cetuximab + 10 pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10 pM cetuximab-saporin. B. Cell killing activity of trastuzumab, trastuzumab + 50pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50pM trastuzumab-saporin in HER2-expressing cells (SK-BR-3). C. Cell killing activity of cetuximab, cetuximab + 10pM cetuximab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, and cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM cetuximab-saporin in EGFR-expressing cells (HeLa). D. Cell killing activity of trastuzumab, trastuzumab + 50 pM trastuzumab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, and trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 50 pM trastuzumab-saporin in HER2-expressing cells (JIMT-1). [Figure 4-1]Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR++ / CD71+ cells (MDA-MB-468). B. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2++ / CD71+ (SK-BR-3) cell line. C. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, or cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR+ / CD71+ cells (CaSki). D. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2+ / - / CD71+ cells (JIMT-1). [Figure 4-2]Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR++ / CD71+ cells (MDA-MB-468). B. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2++ / CD71+ (SK-BR-3) cell line. C. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, or cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR+ / CD71+ cells (CaSki). D. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2+ / - / CD71+ cells (JIMT-1). [Figure 4-3]Legends and axes are the same for panels A, B, C, and D. A. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron(-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR++ / CD71+ cells (MDA-MB-468). B. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2++ / CD71+ (SK-BR-3) cell line. C. Cell killing activity of cetuximab, cetuximab + 10pM CD71mab-saporin, cetuximab-Cys-(dendron(-L-SO1861)4)3,9, cetuximab-Cys-(dendron-L-SO1861)4)3,9 + 10pM CD71mab-saporin, cetuximab-Lys-(dendron(-L-SO1861)4)4,4, or cetuximab-Lys-(dendron(-L-SO1861)4)4,4 + 10pM CD71mab-saporin in EGFR+ / CD71+ cells (CaSki). D. Cell killing activity of trastuzumab, trastuzumab + 10pM CD71mab-saporin, trastuzumab-Cys-(dendron(-L-SO1861)4)4, trastuzumab-Cys-(dendron(-L-SO1861)4)4 + 10pM CD71mab-saporin, trastuzumab-Lys-(dendron(-L-SO1861)4)4,7, or trastuzumab-Lys-(dendron(-L-SO1861)4)4,7 + 10pM CD71mab-saporin in HER2+ / - / CD71+ cells (JIMT-1). [Diagram 5] Cytotoxic activity of T-DM1, T-DM1 + ​​25.6 nM trastuzumab, or T-DM1 + ​​5.3 nM trastuzumab-Cys-(Dendron(-L-SO1861)4)4 in HER2-expressing cells (SK-BR-3). [Figure 6] HSP27 gene silencing activity of HSP27BNA-dendron(-L-SO1861)4 compared to HSP27BNA alone. [Figure 7-1] A. Schematic diagram of the release of SO1861 from dendron (-L-SO1861) 4 under acidic conditions. B. UPLC UV traces (PDA) of dendron (-L-SO1861) 4 by itself (top), the reaction mixture containing dendron (-L-SO1861) 4 and 50 μL of a solution containing water / acetonitrile / TFA after 30 min (middle), and the reaction mixture after 1.5 h (bottom). C. Interpretation of the observed m / z values ​​in Figure 7B by LRMS. [Figure 7-2] A. Schematic diagram of the release of SO1861 from dendron (-L-SO1861) 4 under acidic conditions. B. UPLC UV traces (PDA) of dendron (-L-SO1861) 4 by itself (top), the reaction mixture containing dendron (-L-SO1861) 4 and 50 μL of a solution containing water / acetonitrile / TFA after 30 min (middle), and the reaction mixture after 1.5 h (bottom). C. Interpretation of the observed m / z values ​​in Figure 7B by LRMS. [Figure 7-3] A. Schematic diagram of the release of SO1861 from dendron (-L-SO1861) 4 under acidic conditions. B. UPLC UV traces (PDA) of dendron (-L-SO1861) 4 by itself (top), the reaction mixture containing dendron (-L-SO1861) 4 and 50 μL of a solution containing water / acetonitrile / TFA after 30 min (middle), and the reaction mixture after 1.5 h (bottom). C. Interpretation of the observed m / z values ​​in Figure 7B by LRMS. [Figure 8]The legends and axes for panels A and B are the same. A. Cell killing activity in EGFR-expressing A431 cells by the "naked" dendron (Dendron (NEM) 4), dendron (NEM) 4 + 10 pM EGF Gianthin, dendron (-L-SO1861) 4, or dendron (L-SO1861) 4 + 10 pM EGF Gianthin. B. Cell killing activity in EGFR-expressing HeLa cells by the "naked" dendron (Dendron (NEM) 4), dendron (NEM) 4 + 10 pM EGF Gianthin, dendron (-L-SO1861) 4, or dendron (L-SO1861) 4 + 10 pM EGF Gianthin. [Figure 9-1] Legends and axes are the same for Figures A, B, and C. A. Effect of trastuzumab on cell viability of a range of cancer cells. B. Effect of cetuximab on cell viability of a range of cancer cells. C. Effect of T-DM1 on cell viability of a range of cancer cells. [Figure 9-2] Legends and axes are the same for Figures A, B, and C. A. Effect of trastuzumab on cell viability of a range of cancer cells. B. Effect of cetuximab on cell viability of a range of cancer cells. C. Effect of T-DM1 on cell viability of a range of cancer cells. [Figure 10] Schematic diagram of monoclonal antibody-(SO1861-scaffold-antisense BNA oligo) conjugate. [Figure 11] Schematic diagram of a one-target, two-component system using a monoclonal antibody conjugated to a toxin and the same monoclonal antibody conjugated to a saponin-containing scaffold. [Figure 12] Schematic diagram of a two-target, two-component system using a monoclonal antibody conjugated to a toxin and a different monoclonal antibody with a different target conjugated to a saponin-containing scaffold. [Figure 13] Reaction scheme for the synthesis of SO1861-EMCH. [Figure 14-1] Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 14-2] Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 14-3]Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 14-4] Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 14-5] Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 14-6] Reaction scheme for the synthesis of dendron (-L-SO1861)4. [Figure 15-1] Reaction scheme for the synthesis of dendron (-L-SO1861)8. [Figure 15-2] Reaction scheme for the synthesis of dendron (-L-SO1861)8. [Figure 15-3] Reaction scheme for the synthesis of dendron (-L-SO1861)8. [Figure 15-4] Reaction scheme for the synthesis of dendron (-L-SO1861)8. [Figure 15-5] Reaction scheme for the synthesis of dendron (-L-SO1861)8. [Figure 16-1] Reaction scheme for the synthesis of SO1861-L-trifunctional linker-L-HSP27BNA. [Figure 16-2] Reaction scheme for the synthesis of SO1861-L-trifunctional linker-L-HSP27BNA. [Figure 16-3] Reaction scheme for the synthesis of SO1861-L-trifunctional linker-L-HSP27BNA. [Figure 16-4] Reaction scheme for the synthesis of SO1861-L-trifunctional linker-L-HSP27BNA. [Figure 17] Reaction scheme for the synthesis of dendron(SO1861)4-HSP27BNA oligoconjugate. [Figure 18] Reaction scheme for the synthesis of dendron(NEM)4 (the “naked” dendron). [Figure 19] A model scaffold consisting of four molecular arms for saponin binding via Schiff bases (imines) and one arm for click chemistry. The polymer structure is a first generation pentavalent polyethylene glycol-based dendrimer. [Figure 20]The inset shows the theoretically predicted mass spectrum of the model scaffold with SA1641 saponin, calculated by the isotopic pattern calculator enviPat Web 2.0. The experimental data obtained by LC-MS / ESI-MS showed almost exactly the same peaks at m / z 758-760 Da, proving successful saponin coupling. [Figure 21] Cell viability of HER14 cells after treatment with pentameric dendrimer (pentrimer), pentrimer in the presence of SA1641, gianthin-EGF, gianthin-EFG in the presence of SA1641, pentrimer in the presence of gianthin-EFG, and pentrimer in the presence of gianthin-EGF and SA1641. [Figure 22-1] A. H-NMR spectrum of SO1861. B. H-NMR spectrum of SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide) conjugate. [Figure 22-2] A. H-NMR spectrum of SO1861. B. H-NMR spectrum of SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide) conjugate. [Figure 23] A. MALDI-TOF-MS spectrum of the SO1861-EMCH conjugate. B. MALDI-TOF-MS spectrum of the SO1861-EMCH-mercaptoethanol conjugate. [Figure 24] SO1861 structure with highlighted chemical groups for conjugation of saponin to enhance endosomal escape to the polymer structure. Highlighted groups are aldehyde (black circle), carboxylic acid (dashed circle), alkene (dashed pentagon), and alcohol (dashed box). [Diagram 25] A. Schematic diagram of the production of stable "ready-to-conjugate" endosomal escape enhancing factor saponin. B. Schematic diagram of the production of cleavable "ready-to-conjugate" endosomal escape enhancing factor saponin. [Figure 26] Hydrolysis of the hydrazone bond of SO1861-EMCH under acidic conditions. [Figure 27] A. Standard molecular structure of the SO-1861-EMCH conjugate. The maleimide groups are marked with circles. B. 3D model of the SO1861-EMCH conjugate. The maleimide groups are marked with circles. [Figure 28-1] A. Synthesis scheme of SO1861-EMCH. B. MALDI-TOF-MS spectrum of SO1861 in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. C. MALDI-TOF-MS spectrum of SO1861-EMCH in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 28-2] A. Synthesis scheme of SO1861-EMCH. B. MALDI-TOF-MS spectrum of SO1861 in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. C. MALDI-TOF-MS spectrum of SO1861-EMCH in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 28-3] A. Synthesis scheme of SO1861-EMCH. B. MALDI-TOF-MS spectrum of SO1861 in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. C. MALDI-TOF-MS spectrum of SO1861-EMCH in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 29-1] A. MALDI-TOF-MS spectrum of SO1861-EMCH before hydrolysis in HCl solution at pH 3. B. MALDI-TOF-MS spectrum of SO1861-EMCH after hydrolysis in HCl solution at pH 3. [Figure 29-2]A. MALDI-TOF-MS spectrum of SO1861-EMCH before hydrolysis in HCl solution at pH 3. B. MALDI-TOF-MS spectrum of SO1861-EMCH after hydrolysis in HCl solution at pH 3. [Diagram 30] Reaction scheme for the conjugation of SO1861-EMCH to polymer structures bearing either amine. [Diagram 31] A. MALDI-TOF-MS spectrum of BSA-SO1861. B. MALDI-TOF-MS spectrum of BSA. [Diagram 32] A. Reaction scheme of SO1861-EMCH conjugation to cyanine 3 dye-labeled polyamidoamine (PAMAM) G5 dendrimer. B. Reaction scheme of SO1861-HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) conjugation to (B) cyanine 3 dye-labeled polyamidoamine (PAMAM) G5 dendrimer. [Figure 33-1] A. MALDI-TOF-MS spectrum of Cy3-PAMAM. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 5 SO1861 attached per PAMAM. C. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 13 SO1861 attached per PAMAM. D. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 51 SO1861 attached per PAMAM. [Figure 33-2] A. MALDI-TOF-MS spectrum of Cy3-PAMAM. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 5 SO1861 attached per PAMAM. C. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 13 SO1861 attached per PAMAM. D. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 51 SO1861 attached per PAMAM. [Diagram 34] A. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 5 equivalents of SO1861-EMCH. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-SO1861 with 30 equivalents of SO1861-EMCH. [Diagram 35] MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 (NC = stable bond ("non-cleavable"). [Figure 36-1] A. 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)27-DBCO. [Figure 36-2] A. 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)27-DBCO. [Figure 37-1] A. Reaction scheme of Gianthin-EGF-Alexa488. B. Reaction scheme of Gianthin-EGF-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of Gianthin-EGF. D. MALDI-TOF-MS spectrum of Gianthin-EGF-Alexa488. E. MALDI-TOF-MS spectrum of Gianthin-EGF-Alexa488-SS-PEG-N3. [Figure 37-2]A. Reaction scheme of Gianthin-EGF-Alexa488. B. Reaction scheme of Gianthin-EGF-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of Gianthin-EGF. D. MALDI-TOF-MS spectrum of Gianthin-EGF-Alexa488. E. MALDI-TOF-MS spectrum of Gianthin-EGF-Alexa488-SS-PEG-N3. [Figure 38-1] A. Reaction scheme of dianthine-Alexa488. B. Reaction scheme of dianthine-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of dianthine. D. MALDI-TOF-MS spectrum of dianthine-Alexa488. E. MALDI-TOF-MS spectrum of dianthine-Alexa488-SS-PEG-N3. [Figure 38-2] A. Reaction scheme of dianthine-Alexa488. B. Reaction scheme of dianthine-Alexa488-SS-PEG-N3. C. MALDI-TOF-MS spectrum of dianthine. D. MALDI-TOF-MS spectrum of dianthine-Alexa488. E. MALDI-TOF-MS spectrum of dianthine-Alexa488-SS-PEG-N3. [Figure 39] Fluorescent images of SDS-PAGE gels performed by VersaDoc imaging system. M=marker, P=Cy3-PAMAM-(SO1861)27-DBCO, D=gianthine-EGF-Alexa488-SS-PEG-N3, C1=Cy3-PAMAM-(SO1861)5-gianthine-EGF-Alexa488, C2=Cy3-PAMAM-NC-SO1861-gianthine-EGF-Alexa488, and C3=Cy3-PAMAM-(SO1861)27-gianthine-EGF-Alexa488. [Diagram 40]A. Synthesis scheme of Cy3-PAMAM-NC-SO1861 by reductive amination. B. MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 synthesized by reductive amination with 10 SO1861 per PAMAM. C. MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 synthesized by reductive amination with 30 SO1861 per PAMAM. [Diagram 41] Reaction scheme for the preparation of poly(SO1861) using SO1861-EMCH as the monomer, the APS / TMEDA system as the polymerization initiator, and aminopropanethiol as the radical quenching agent. [Diagram 42] A. MALDI-TOF-MS spectrum of the poly(SO1861) reaction batch of SO1861-EMCH at 60° C. B. MALDI-TOF-MS spectrum of the poly(SO1861) reaction batch of SO1861-EMCH+11-3 equivalents of APS at 60° C. C. MALDI-TOF-MS spectrum of the poly(SO1861) reaction batch of SO1861-EMCH+11-3 equivalents of APS / TMEDA at 60° C. [Diagram 43] Schematic of the DNA approach. Use of the principles of DNA origami to generate DNA-based scaffolds capable of conjugating and releasing glycoside molecules. In addition, one of the DNA strands carries a click chemistry moiety that can be used for conjugation to targeted toxins to form a functionalized scaffold. bp: base pair. [Diagram 44] Schematic of the poly(peptide-SO1861) approach. The use of peptide sequences that can conjugate and release glycoside molecules and react with them to form poly(peptide-SO1861) constructs. The termini of the poly(peptide) chains can be further modified with click chemistry moieties (e.g., BCN-NHS linkers) that can be used for conjugation to toxins. [Diagram 45]A. MALDI-TOF-MS spectrum of the native peptide. B. MALDI-TOF-MS spectrum of the peptide-SO1861 conjugate. [Diagram 46] Molecular structure of a G4-dendron with a protected amino group. [Figure 47] Synthetic scheme for the generation of dendron-based scaffolds. [Figure 48-1] A. Reaction scheme for partial dye-labeling and deprotection of G4-dendrons. B. MALDI-TOF-MS spectrum of deprotected and partially dye-labeled G4-dendrons. [Figure 48-2] A. Reaction scheme for partial dye-labeling and deprotection of G4-dendrons. B. MALDI-TOF-MS spectrum of deprotected and partially dye-labeled G4-dendrons. [Figure 49] A. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 22 equivalents of SO1861-EMCH. B. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 10 equivalents of SO1861-EMCH. C. MALDI-TOF-MS spectrum of G4-dendron-SO1861 scaffold with 3 equivalents of SO1861-EMCH. [Figure 50-1]A. EGFR cell surface expression determined by FACS analysis of HeLa cells. B. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF (Dia-EGF), SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 667 nM dendron. C. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500nM chloroquine, SO1861 + Gianthin-EGF + 500nM PAMAM, SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)16, SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)65, and SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)108. D. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)3, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)8, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)18. [Figure 50-2]A. EGFR cell surface expression determined by FACS analysis of HeLa cells. B. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF (Dia-EGF), SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 667 nM dendron. C. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500nM chloroquine, SO1861 + Gianthin-EGF + 500nM PAMAM, SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)16, SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)65, and SO1861 + Gianthin-EGF + 500nM PAMAM-(SH)108. D. Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)3, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)8, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)18. [Figure 51-1] A. Reaction scheme of thiolation of PAMAM with the thiolation reagent 2-iminothiolane. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)16. D. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)65. E. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)108. [Figure 51-2]A. Reaction scheme of thiolation of PAMAM with the thiolation reagent 2-iminothiolane. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)16. D. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)65. E. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)108. [Figure 51-3] A. Reaction scheme of thiolation of PAMAM with the thiolation reagent 2-iminothiolane. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)16. D. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)65. E. MALDI-TOF-MS spectrum of thiolated PAMAM-(SH)108. [Figure 52-1] A. Reaction scheme of PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)3. D. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)8. E. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)18. [Figure 52-2] A. Reaction scheme of PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)3. D. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)8. E. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)18. [Figure 52-3]A. Reaction scheme of PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. B. MALDI-TOF-MS spectrum of native PAMAM. C. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)3. D. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)8. E. MALDI-TOF-MS spectrum of PEGylated PAMAM-(mPEG2k)18. [Figure 53] Schematic of the basic scaffold with click chemistry functional groups for linking any desired effector molecule. [Figure 54] Schematic of a functionalized scaffold with pre-bound effector molecules and click chemistry functional groups for linking any desired ligand. Optionally, a pH-sensitive linker can be provided to release the effector molecule from the scaffold after reaching the endosome. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] For a bioactive molecule to work, it must be able to engage its target, for example in serum, outside the cell surface, or within a cell or organelle. The active moiety of almost all protein-based targeted toxins must, for example, enter the cytosol of the target cell to mediate its target-modulating effect. In many constellations, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound to the outside of the cell, (2) it is recycled to the cell surface after internalization, or (3) it is transported to the endolysosome, where it is degraded. Although these fundamental issues have been known for decades and more than 500 targeted toxins have been reviewed in the past decades, the problem is still not solved, and only one antibody-targeted protein toxin, moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP), has been approved by the FDA for relapsed or refractory hairy cell leukemia to date.

[0066] Many strategies have been described to overcome these problems, including approaches to transfer toxins to integral cell membrane transport complexes of the biosynthetic pathway in the endoplasmic reticulum, and techniques to block or weaken the membrane integrity of endosomes, i.e., compartments of the endocytic pathway in cells, thus facilitating endosomal escape. This includes the use of lysosomotropic amines, carboxylic acid ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human, and synthetic origin, other organic molecules, and light-inducible techniques. The efficacy of targeted toxins has typically been increased 100- or 1000-fold in cell cultures, and in exceptional cases more than 1 million-fold, but the requirement to co-administer endosomal escape enhancers with other substances introduces new problems, including additional side effects, loss of target specificity, difficulty in determining therapeutic windows, and cell type-dependent variability.

[0067] All strategies involving physicochemical techniques require molecules of enhancers that interact more or less directly with membranes and essentially include small chemical molecules, secondary metabolites, peptides, and proteins. The common feature of all these substances is that they are not target cell specific themselves and distribute with kinetics other than targeted toxins. This is one major drawback of current approaches.

[0068] While the present invention will be described with respect to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

[0069] While the present invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading and understanding the specification and upon studying the drawings and graphs. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0070] One embodiment of the present invention is a scaffold suitable for covalently attaching at least one biologically active molecule to a carrier molecule, the scaffold comprising a polymeric or oligomeric structure, at least one of said biologically active molecules being covalently attached to said polymeric or oligomeric structure, and the scaffold further comprising a first chemical group for covalently coupling the scaffold to the carrier molecule.

[0071] One embodiment is a scaffold according to the invention, wherein at least one biologically active molecule has a molecular mass of 3,000 Daltons or less, preferably 2,500 Daltons or less, more preferably 2,300 Daltons or less, most preferably 2,000 Daltons or less, for example between 1,700 Daltons and 1,950 Daltons.

[0072] An embodiment is a scaffold according to the invention, in which at least one biologically active molecule is an amphiphilic molecule. Such amphiphilic molecules, such as (phospho)lipids, glycosides, e.g. saponins, can typically be part of or form bilayers and lipid layers, e.g. layers such as cell membranes, and / or can be part of vesicles, e.g., within cells.

[0073] An embodiment is a scaffold according to the present invention, in which when more than one bioactive molecule is covalently bound to the polymer or oligomeric structure contained by the scaffold, at least one bioactive molecule is a single specific molecule or a mixture of different molecules. Typically, the bioactive molecule is obtained or derived from a natural source, and / or typically, the bioactive molecule comprises one, two, or more nearly identical or similar molecules. An example is the water-soluble fraction containing saponins obtained from Quillaja saponaria ("Quil-A"), which includes a series of Quillaja saponins, such as QS-21, QS-18, and QS-7. Another example is a mixture of QS-21xyl and QS-21api.

[0074] One embodiment is a scaffold according to the invention, wherein at least one biologically active molecule is a glycoside, preferably a bisdesmosidic triterpene or triterpenoid saponin, more preferably a bisdesmosidic triterpene saponin, most preferably a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group on a carbohydrate substituent at the C-3 beta-OH group of the saponin.

[0075] Prior to the present invention, it was not possible to guide single or multiple glycoside molecules to (target) cells. In particular, it was not possible to specifically guide effector molecules, e.g. as part of an ADC, and a specific number or range of glycoside molecules per effector molecule simultaneously, e.g. via the endocytic pathway of the cell, to the cytosol of the cell. The solution provided by the present invention allows for immobilization and even polymerization of glycoside molecules, e.g. after endocytosis, allowing remonomerization at the intracellular site where the glycoside's mode of action is desired. In this context, "polymerize" means reversible and / or irreversible multiple conjugation of glycoside molecules to polymeric or oligomeric structures to form a scaffold. Alternatively, it means reversible and / or irreversible multiple conjugation of (modified) glycoside molecules, thereby forming a polymeric or oligomeric structure to form a scaffold. In this context, "remonomerization" refers to the cleavage of glycoside molecules from the backbone, e.g. after endocytosis, and regaining the (native) chemical state of the unbound glycoside molecule. This unbound glycoside molecule may or may not contain additional chemical groups, e.g. chemical groups for linking glycosides to the backbone, and / or (chemical) linkers. Due to the complex chemistry of glycoside molecules, "polymerization" of glycoside molecules and their "remonomerization" at the desired location, e.g. within a cell, e.g. after endocytosis, has been a difficult task. In particular, the chemical reaction (polymerization of glycosides) used to provide the backbone of the present invention, including covalently linked glycosides, e.g. triterpenoid saponins, usually occurs in anhydrous organic solvents, whereas glycoside molecules and biocompatible polymers are water-soluble molecules. The chemical properties of unmodified glycoside molecules further prevented polymerization by themselves. And one other solution for (directly) linking multiple glycoside molecules to an effector molecule was projected to be less promising, because the effector molecule (drug, toxin, polypeptide, or polynucleotide) typically does not provide sufficient binding sites.And because the coupling products become very heterogeneous and / or the coupling together of biologically active molecules such as glycosides with, for example, peptides, toxins, nucleic acids carries the risk of affecting and hindering the activity of one or even both molecules linked together in the conjugate containing such glycosides. Moreover, there is a considerable risk that the effector molecule loses its function after coupling. The present invention solves at least one of these drawbacks.

[0076] One embodiment is a scaffold according to the invention, wherein the at least one bioactive molecule is a saponin that may be isolated from Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, such as Quillaja saponaria, or a single specific saponin, or a mixture of two or more different saponins, such as one or more of the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any 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 a saponin having a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group, and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group. and / or 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilaic acid 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside. More preferably, the saponin is SO1861 and / or QS-21.

[0077] Table A1 and Scheme I and the above embodiments summarize a series of saponins that have been identified for their endosomal escape enhancing activity when contacted with mammalian cells, such as human cells, in free form together with a second molecule (e.g., an effector moiety or molecule). Indeed, cell-based bioassays have established that for the saponins tabulated in Table A1 and the saponins in Scheme I, under the influence of these saponins, second molecules, such as nucleic acids and / or toxins, such as protein toxins (e.g., one or more of the protein toxins listed in Table A5), are released from (late) endosomes and lysosomes into the cell with increased efficiency and / or effectiveness. That is, endosomal and / or lysosomal escape of such second molecules (i.e., effector moieties, effector molecules), such as nucleic acids and / or toxins, is less efficient in the absence of the saponin.

[0078] Surprisingly, the inventors now demonstrate that water-soluble saponin fractions from Quillaja saponaria, including QS-21 and its family members QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18, also referred to as "Quil-A", also exhibit the ability to enhance the in vitro biological effects of, for example, nucleic acids conjugated to monoclonal antibodies or protein toxins conjugated to monoclonal antibodies. At this time, the monoclonal antibody, the effector molecule (the second molecule mentioned above) and at least one glycoside, such as QS-21 and its family member saponin, which is encapsulated by such QS-21 preparation, are co-administered to tumor cells of a mammalian species (human). The effector and glycoside, such as QS-21, SO1861, SA1641, GE1741, are either directly or via at least one linker covalently attached to the polymer or oligomeric backbone, or the glycoside is covalently attached to the effector molecule and the effector molecule is covalently attached to the polymer or oligomeric backbone. Alternatively, the glycoside and the effector molecule are separately covalently attached to the backbone, and the monoclonal antibody is also separately attached to this conjugate of saponin, toxin, backbone, which is a trifunctional linker that encapsulates a polymer or oligomeric structure, such as the backbone outlined in Scheme II and Structure B. Without wishing to be bound by any theory, the observed stimulation or enhancement of tumor cell killing mediated, for example, by saporin or dianthin, in the presence of saponins from Quillaja saponaria may (also) be related to activation of the inflammasome of tumor cells by the saponins, resulting, for example, in pyroptosis of tumor cells.

[0079] QS-21, and also the water-soluble saponin fraction of Quillaja saponaria, have already been known for a long time and have been applied intensively before, for example, due to their immune-enhancing ability as adjuvants for subunit vaccines. For example, QS-21 has been applied in two Phase III clinical trials in human patients. They were vaccinated with a subunit vaccine mixed with an adjuvant containing QS-21 (Glaxo-Smith-Kline, MAGRIT trial, DERMA study). The subunit was the MAGE-A3 protein, which is specifically expressed and presented by tumor cells. Antitumor vaccination enhanced by QS-21 aimed at prolonging disease-free survival in 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 treatments, for vaccines against HIV-1 infection, for the development of vaccines against Hepatitis B, and for the development of anti-malarial vaccines using QS-21 with Glaxo-Smith-Kline's adjuvants AS01 and AS02. Previous studies have revealed that under the influence of QS-21 saponin with adjuvants, immune responses are elicited against MAGE-A3 peptides presented on the surface of cancer cells (AS15; GSK). To the inventor's surprise, the saponin in the Quillaja saponaria fraction containing QS-21 enhances the anti-tumor cell activity of payloads such as protein toxins (gianthin). The saponin is covalently coupled to, for example, cetuximab. And when the ligand-gianthin conjugate is exposed to tumor cells in the presence of cetuximab-saponin, the toxic effect of gianthin in several human tumor cell lines is established. Further details are outlined in the Examples section.

[0080] Similarly, saponin enhances the antitumor cell activity of nucleic acids such as oligonucleotide BNA (HSP27) for silencing HSP27 expression in (tumor) cells. The inventors show that a tumor cell targeting monoclonal antibody such as cetuximab provided with a covalently coupled antisense BNA (HSP27) and provided with a covalently coupled saponin (here SO1861) can silence HSP27 in tumors in vivo compared to a control and compared to an ADC alone without coupled saponin. Both the BNA and the saponin are attached to the antibody by a cleavable bond (here hydrazone bond), through which both the BNA and the SO1861 are coupled to a trifunctional linker (here trifunctional oligomer backbone of Scheme II). Thus, conjugating an ADC with a saponin results in a conjugate containing a BNA and a saponin, and this combination confers an antitumor cell activity to the ADC that is not seen with the same dose of ADC. Notably, the monoclonal antibody and ADC covalently coupled to saponin, when administered separately to tumor-bearing mice in separate groups of mice, increase HSP27 expression in tumor cells compared to the control group (received vehicle only). Only the ADCs comprising the scaffold of the present invention with covalently coupled saponin and effector molecules show reduced HSP27 expression when compared to the control. The antisense BNA (HSP27) was a BNA with the oligonucleotide sequence 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).Of note, to the best of the inventor's knowledge, BNAs are designed for application as free nucleic acids.Here, the inventors demonstrate for the first time that antisense BNAs can be covalently coupled to ligands or antibodies via (non)cleavable linkers in such a way that gene silencing activity is retained in vitro and, more importantly, in vivo in tumor cells of tumor-bearing animals.This approach opens a new way to administer targeted BNAs to human (cancer) patients in need.

[0081] One embodiment is a scaffold according to the invention, wherein the at least one biologically active molecule is a bisdesmosidic saponin, having a molecular mass of at least 1,500 daltons, comprising an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, and a first branched carbohydrate side chain at the C-3 position, which optionally contains glucuronic acid. The saponin comprises an ester group with a second branched carbohydrate side chain at the C-28 position, the second branched carbohydrate chain preferably comprising at least four carbohydrate units, optionally comprising at least one acetyl residue, for example two acetyl residues, and / or optionally comprising a deoxy carbohydrate, and / or optionally comprising quinovose, and / or optionally comprising glucose, and / or optionally comprising 4-methoxycinnamic acid, and / or optionally comprising 5-O-[5-O-Ara / Api-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, linked to the carbohydrate via an ester bond. Alternatively, the at least one saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS-1861, protonated QS1861 (QS1862), and Quil-A. Such saponins are further exemplified in Table A1 and Scheme I. The inventors herein demonstrate that covalently coupling a saponin, such as QS-21, SA1641, SO1861, a water soluble saponin fraction of Quillaja saponaria, to a scaffold, such as a dendron or a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B, results in improved cytotoxicity exerted by the toxin under the influence of the covalently coupled saponin.The scaffold further comprises a covalently attached effector molecule(s), e.g. a proteinaceous toxin such as dianthin or saporin and / or a (tumor) cell surface molecule targeting ligand or moiety, such as a monoclonal antibody for binding to a (tumor) cell surface receptor.

[0082] One embodiment is a scaffold of the invention that includes as a bioactive molecule a saponin that includes one or some or all of the indicated structural features of the saponin of Structure A in Scheme I, and / or a saponin selected from any one or more of the additional saponins of Scheme I:

[0083] [ka]

[0084] [ka]

[0085] [ka]

[0086] [ka]

[0087] [ka]

[0088] According to the invention, a biologically active molecule having an "ideal" structure for the purpose of enhancing endosomal escape of effector molecules attached to the scaffolds of the invention as carrier molecules is a bisdesmosidic saponin according to Structure A of Scheme I, having a molecular mass of at least 1,500 daltons, comprising an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, and a first branched carbohydrate side chain at the C-3 position, which optionally contains glucuronic acid. The saponin comprises an ester group with a second branched carbohydrate side chain at the C-28 position, the second branched carbohydrate chain preferably comprising at least four carbohydrate units, optionally comprising at least one acetyl residue, for example two acetyl residues, and / or optionally comprising a deoxy carbohydrate, and / or optionally comprising quinovose, and / or optionally comprising glucose, and / or optionally comprising 4-methoxycinnamic acid, and / or optionally comprising 5-O-[5-O-Ara / Api-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, linked to the carbohydrate via an ester bond.

[0089] SO1861 differs from the "ideal structure" shown by Scheme I, Structure A only by having only one acetyl residue on the quinovose and by having an additional xylose. The "ideal structure" of a saponin for enhancing endosomal escape of an effector molecule or moiety is preferably a saponin having Structure A of Scheme I, and saponins that exhibit endosomal escape enhancing activity have one or more of the structural features exhibited by Structure A of Scheme I. Without wishing to be bound by any theory, the inventors believe that Structure A of Scheme I represents an "ideal saponin" (and not a minimum requirement saponin) for endosomal escape enhancing activity. This means that not all of the structures may or must be present on every saponin that has at least sufficient endosomal escape enhancing activity to promote accumulation of the effector moiety in the cytosol, which means that some saponins may have other structural elements, such as acyl chains, and / or the sugars may differ from those shown by Scheme I for still other saponins that exhibit endosomal escape enhancing activity. For example, when the idealized structure of Structure A in Scheme I is considered, QS-21 saponin and some of the saponins in the water-soluble fraction of Quillaja saponaria (Quillaja saponin; Quil-A) differ in carbohydrate modifications at C-28: for example, the presence of an acyl chain in QS-21. Saponins such as QS-7, QS1862 in the water-soluble fraction of Quillaja saponaria are similar to the idealized structure A and similar to SO1861.

[0090] In order to explain the 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 terminology used in the present invention will be described. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. Said vesicle budding can be characterized as (1) receptor-dependent ligand uptake mediated by the cytosolic 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 enter into the next cellular process of vesicle trafficking and substance sorting called the endocytic pathway. The endocytic pathway is complex and not fully understood. Without wishing to be bound by any theory, organelles can be formed de novo and can mature into the next organelle along the endocytic pathway. However, it is hypothesized here that the endocytic pathway involves stable compartments connected by the trafficking of vesicles. Compartments are complex multifunctional membrane organelles that are specialized for a specific set of essential functions for the cell. Vesicles are considered to be transient organelles, simpler in composition and defined as membrane-enclosed containers that form de novo by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early and late endosomes represent stable compartments in the endocytic pathway, with primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and even lysosomes representing vesicles. At the plasma membrane, endocytic vesicles, most notably arising from clathrin-coated pits, first fuse with early endosomes, the main sorting compartment with a pH of approximately 6.5. The majority of internalized cargo and membranes are recycled to the plasma membrane via recycling vesicles (recycling pathway). Components to be degraded are transported via multivesicular bodies to acidic late endosomes (pH lower than 6). Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to late endosomal compartments when needed.The resulting organelle is called a hybrid organelle or an endolysosome. Lysosomes bud from the hybrid organelle by a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are extremely dynamic organelles, and the distinction between them is often difficult. Degradation of endocytosed molecules occurs in the endolysosome or lysosome. Endosomal escape is the active or passive release of material from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably from clathrin-mediated endocytosis or recycling pathway to the cytosol. Thus, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediates and hybrid organelles. Unless otherwise specifically indicated, and whenever the term "endosome" or "endosomal escape" is used herein, it includes escape from endolysosomes and lysosomes and from endolysosomes and lysosomes, respectively. After entering the cytosol, the material may be transferred to other cellular units, such as the nucleus. In formal terms, a glycoside is any molecule in which a sugar group is linked to another group via its anomeric carbon by a glycosidic bond. Without wishing to be bound by any theory, glycoside molecules in the context of the present invention are such molecules that can further improve the effect of effector molecules, in particular by facilitating the endosomal escape of the effector molecule. Without wishing to be bound by any theory, glycoside molecules interact with the membranes of compartments and vesicles of the endocytic and recycling pathways, making them leaky for the effector molecule, resulting in increased endosomal escape.The term "the scaffold can increase the endosomal escape of the effector molecule" means that at least one glycoside molecule coupled to the polymer or oligomeric structure of the scaffold can improve the endosomal escape of the effector molecule when both molecules are in an endosome, e.g., a late endosome, optionally and preferably after the at least one glycoside is released from the polymer or oligomeric structure, e.g., by cleavage of a cleavable bond between the at least one glycoside and the polymer or oligomeric structure. Even though the bond between the at least one glycoside and the scaffold can be a "stable bond", it does not mean that such a bond cannot be cleaved in the endosome, e.g., by an enzyme. For example, the glycoside, together with a linker or part of the polymer structure, can be cleaved from the remaining polymer structure. For example, it can be the case that a protease cleaves the proteinaceous polymer structure, e.g., albumin, thereby releasing the at least one glycoside. However, it is preferred that the glycoside molecule is released in an active form, preferably in the original form it had before it was (prepared to be) coupled to the scaffold; therefore, although the glycoside has its native structure after such cleavage or the glycoside has (part of) a chemical group or linker attached to it after such cleavage, the biological activity of the glycoside, e.g. endosomal / lysosomal escape enhancing activity towards an effector molecule present in the same endosome or lysosome, is maintained or restored by said cleavage of the bond between the glycoside and the carrier molecule, e.g. the scaffold of the invention. In the context of the present invention, the term "stable" means that the bond between the saponin, the polymeric or oligomeric structure (of the scaffold), the ligand, the (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or the effector (effector moiety, effector molecule) is not easily broken, or at least is not designed to be easily broken, e.g. by pH differences, salt concentrations, or UV light.For the present invention, the term "cleavable" means that the bond between the saponin, the scaffold polymeric or oligomeric structure of the invention, the ligand, the antibody, and / or the effector is designed such that the bond is easily broken, for example, by pH difference, salt concentration, under reducing conditions, and the like. Those skilled in the art are familiar with such cleavable bonds and how to prepare them.

[0091] In the context of the present invention, an effector molecule or effector moiety is any substance that affects the metabolism of a cell by interacting with an effector molecule target in the cell, which is any molecule or structure in the cell, excluding the compartments of the endocytosis and recycling pathways and the lumen of vesicles, but including the membranes of these compartments and vesicles. Said structures in the cell therefore include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol. Cytosolic delivery of an effector molecule in the context of the present invention preferably means that the effector molecule is able to escape endosomes (and / or lysosomes) (which also includes escaping endolysosomes and lysosomes as defined above) and preferably reach the effector molecule target described herein. The present invention is a new type of molecule, referred to as a scaffold, which serves to bring both an effector molecule and at least one glycoside molecule into endosomes simultaneously in a predetermined ratio. In the context of the present invention, a backbone polymeric or oligomeric structure 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, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The terms "polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer" have their usual meanings. In particular, a polymer is a substance with a molecular structure assembled mainly or entirely from a large number of equal or similar units linked together, and an oligomer is a polymer whose molecules consist of a relatively small number of repeating units. There is no consensus on one particular cut-off for "many" and "few" used in the above definitions of polymer and oligomer, respectively.However, since the backbone may comprise a polymeric or oligomeric structure or both, the full range of numbers of similar units linked together applies to such structures, i.e., from 2 monomeric units to 100 monomeric units, 1000 monomeric units, and more. For example, a structure containing 5 or less may be referred to as an oligomeric structure, and a structure containing 50 monomeric units may be referred to as a polymeric structure. A structure of 10 monomeric units may be referred to as either an oligomer or a polymer. The backbone as defined herein further comprises at least one glycoside molecule. The scaffold preferably includes polymeric or oligomeric structures such as poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines), as well as biocompatible structures such as polyethylene glycols, poly- or oligo(esters), such as poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, and poly(dextrins), poly- or oligosaccharides, such as cyclodextrins or polydextrose, and poly- or oligoamino acids, such as polylysine or peptides or proteins, or DNA oligo- or polymers. 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 can be (a) a scaffold (e.g., comprising at least one glycoside molecule), (b) a functionalized scaffold (e.g., comprising at least one glycoside molecule and a ligand, antibody, etc., and / or effector molecule), (c) a polymeric or oligomeric structure comprising at least one ligand, antibody, etc., and / or at least one effector, or (d) a polymeric or oligomeric structure without glycoside molecules, without ligand, antibody, etc., and without effector molecules. A functionalized aggregated polymeric structure is an aggregated polymeric structure that contains (a) at least one functionalized scaffold or (b) at least one polymeric structure comprising at least one scaffold and at least one ligand, antibody, etc., and / or at least one effector.Polymer or oligomeric structures on the assembled polymeric structures that do not contain any of the above mentioned molecules (i.e. without glycosides, ligands, antibodies or effectors) are particularly added as structural components of the assembled structures. This helps to assemble or stabilize the assembled structures ("glue-like"). The inventors have found that scaffolds that contain polymeric structures with many primary, secondary and / or tertiary amine groups, especially after coupling to glycoside molecules, do not particularly improve endosomal escape. In this regard, polymer and oligomeric structures that contain secondary amine groups are particularly not preferred. Without wishing to be bound by any theory, it is believed that an acidic environment appears to be a prerequisite for a synergistic action between glycosides and effectors, and that such amine groups disrupt the acidic environment of late endosomes. Thus, scaffolds that can disrupt the acidic environment, for example due to the presence of amine groups, are preferably not included by the scaffolds according to the present invention. However, it will be appreciated that the primary amine groups may be blocked or masked, for example by thiolation or PEGylation, and such scaffolds are encompassed by the claims after appropriate blocking or masking of the primary amine groups, which methods are known to those skilled in the art.

[0092] In a particularly preferred embodiment, the present invention provides a scaffold that does not substantially inhibit endosomal (and lysosomal) escape of the effector molecule, and preferably comprises less than 10, more preferably less than 5, more preferably less than 2, and most preferably none, primary, secondary, or tertiary amine groups. For the sake of clarity, it is emphasized that primary amine groups that are blocked, e.g., by thiolation or PEGylation, are no longer referred to as amine groups.

[0093] Whether the scaffold is capable of disrupting the acidic environment and inhibiting the endosomal escape function of at least one glycoside can be easily determined by assays as described in Example 4 and as known in the art. The inhibition is described as a "~-fold increase in the amount of glycoside required to induce 50% cell kill". It is preferred that the scaffold does not result in an increase that is at least the increase in glycoside molecules required to obtain 50% cell kill observed when chloroquine is used as a positive control. Alternatively and preferably, the scaffold does not result in at least a 4-fold increase, more preferably at least a 2-fold increase, of glycoside molecules to induce 50% cell kill. The ~-fold increase would essentially be measured by the assays described in Example 4, with chloroquine as a positive control inducing a 2-fold increase in the amount of glycoside to observe 50% cell kill.

[0094] The term "improving or enhancing the effect of an effector molecule" means that the glycoside molecule increases the functional effectiveness of the effector molecule (e.g., the therapeutic index of a toxin or drug or oligonucleotide, e.g., BNA; metabolic effectiveness of a regulator in a biotechnology process; transfection effectiveness of a gene in a cell culture research experiment), preferably by enabling or improving its target engagement. Acceleration, prolongation, or enhancement of an antigen-specific immune response is preferably not included. Therapeutic efficacy includes, but is not limited to, a stronger therapeutic effect, preferably with a lower dosage and / or with fewer side effects. "Improving the effect of an effector molecule" may also mean that an effector molecule that could not be used due to lack of efficacy (and for example was not known to be an effector molecule) becomes effective when used in combination with the present invention. Any other effect provided by the present invention that is beneficial or desirable and can be attributed to the combination of the effector molecule and the scaffold is considered to be an "improved effect". In certain embodiments, the scaffold improves the effect of the effector molecule, whose effect is intended and / or desired. In the case of a proteinaceous scaffold, the proteinaceous polymer structure itself may have an effect, for example, on colloid osmotic pressure in the bloodstream. If such an effect is not the intended or desired effect of the ultimate functionalized scaffold, then the proteinaceous structure of the scaffold is not an effector molecule as defined in the present invention. Or, for example, in the case of a DNA or RNA based scaffold, the DNA or RNA portion may have an (unintended) function, for example, by interfering with expression. If such interference is not the intended or desired effect of the ultimate functionalized scaffold, then the DNA or RNA polymer structure of the scaffold is not an effector molecule as defined in the present invention.

[0095] A number of preferred characteristics can be formulated for the endosomal escape enhancing factors, i.e., glycosides according to the present invention: (1) preferably, they are not toxic and do not induce immune responses, (2) preferably, they do not mediate cytosolic uptake of effector molecules into off-target cells, (3) preferably, their presence at the site of action is synchronized with the presence of the effector molecules, (4) preferably, they are biodegradable or excretable, and (5) preferably, they do not substantially interfere with biological processes of organisms unrelated to the biological activity of the effector molecules with which the endosomal escape enhancing factors are combined, e.g., do not interact with hormones. Examples of glycoside molecules that meet the previously mentioned criteria at least to some extent are bisdesmosidic triterpenes, preferably bisdesmosidic triterpene saponins, e.g., SO1861, SA1641, QS-21, GE1741.

[0096] An embodiment is a scaffold according to the invention, wherein at least one biologically active molecule, preferably a glycoside, is covalently attached to the polymeric or oligomeric structure via a non-cleavable bond or via a cleavable bond, preferably said cleavable bond is susceptible to cleavage under acidic, reductive, enzymatic or photoinduced conditions, more preferably said cleavable bond is a hydrazone or hydrazide bond susceptible to cleavage under acidic conditions and / or a bond susceptible to proteolysis, e.g. by cathepsin B, and / or a bond susceptible to cleavage under reductive conditions, e.g. a disulfide bond. Typically according to the invention the biologically active molecule is a saponin of the invention (see also Table A1, Scheme I). When considering the entry into cells and accumulation in the cytosol of effector molecules covalently coupled to the same scaffold as the saponin, the activity of the saponin, e.g. the endosomal escape enhancing activity in cells, has proven beneficial when the saponin is covalently coupled to the scaffold and involves hydrazone and / or hydrazide and / or disulfide bonds. 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 also demonstrate that the covalent coupling of the saponin to a carrier molecule via a bond that is not easily cleavable under physiological conditions in cells, e.g. (late) endosomes, lysosomes, cytosol, is also beneficial for the enhancing activity of the saponin on the biological effect of effector moieties, e.g. nucleic acids (e.g. BNA silencing HSP27) and proteinaceous toxins such as saporin. In this application, including the claims, when referring to the conjugates of the invention, e.g., scaffolds having a saponin coupled to the scaffold via a hydrazone bond or a disulfide bond, the terms "cleavable linker", "cleavable bond", and the like, are also referred to as "labile linkers" ("L") and "labile bonds", e.g., in the context of cleavage of such bonds or linkers in (late) endosomes and / or lysosomes.For example, Figure 1 shows in vivo HSP27 gene silencing in mouse tumors. Tumor-bearing mice were treated with a scaffold according to the present invention comprising an oligomeric trifunctional linker (see Scheme II and Structure B). Saponin SO1861 was covalently linked thereto via a hydrazone bond, antisense BNA for silencing the HSP27 gene in tumor cells was covalently coupled to the scaffold via a hydrazone bond, and monoclonal anti-EGFR antibody cetuximab was covalently coupled to the scaffold via a disulfide bond. Hydrazone and disulfide bonds are said to be cleavable and therefore labile bonds. That is, without wishing to be bound by any theory, once the conjugate comprising the scaffold of the present invention is internalized, for example by endocytosis, the hydrazone and disulfide bonds are cleaved in the (late) endosomes and / or lysosomes of target tumor cells expressing EGFR on the cell surface. Although bond cleavage likely contributes to the endosomal escape enhancing activity of saponins when considering entry of BNAs from endosomes and / or lysosomes into the cytosol, such cleavage is not essential to observe the gene silencing effect of cetuximab (saponin) (BNA) conjugates containing the trifunctional linker, i.e. scaffold, of the present invention.

[0097] The skilled artisan will understand that such trifunctional linkers are suitable scaffolds of the invention for covalently coupling one, two or three saponin moieties. In trifunctional linkers, covalent coupling of one or two saponin moieties is preferred. The second and / or third binding site is suitable for covalently coupling an effector moiety, such as a payload, e.g., a protein toxin, a small molecule toxin, a nucleic acid. The second or third binding site of the trifunctional linker is also suitable for covalently coupling a non-proteinaceous ligand and / or coupling a proteinaceous ligand, e.g., for targeting cells, such as tumor cells or autoimmune cells. Exemplary proteinaceous ligands are EGF for targeting (tumor) cells expressing EGFR on the cell surface, and cytokines for targeting tumor cells or autoimmune cells. Furthermore, the second or third binding site of the trifunctional linker is suitable for the covalent coupling of an immunoglobulin, such as a monoclonal antibody, for binding to a cell surface molecule, such as a tumor cell surface molecule, preferably a tumor cell specific molecule, more preferably a tumor cell receptor that is specifically (over)expressed on the surface of a tumor cell. Similarly, an immunoglobulin, or any fragment(s) and / or domain(s) thereof that encompasses the binding specificity of an immunoglobulin, is suitable for binding to a cell surface molecule, such as a receptor expressed on the surface of an autoimmune cell. Thus, in an embodiment, the trifunctional linker comprises or consists of a covalently linked saponin, such as QS-21, SO1861, a covalently linked effector moiety, such as a toxin or an oligonucleotide, such as BNA, and a covalently linked cell targeting moiety, such as a ligand or antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, non-healthy cells, B cell disorders.

[0098] One embodiment is a scaffold of the invention, which comprises an oligomeric trifunctional linker as the scaffold core structure according to Scheme II:

[0099] [ka]

[0100] By way of example, saponin, and here by way of example, the effector moiety, is covalently linked to the trifunctional linker backbone via a labile cleavable hydrazone linker (acid sensitive) and / or, optionally when an effector moiety is considered, via a maleimide-containing bond with a cysteine ​​on the carrier molecule. The (optional) bond of the backbone to a carrier molecule, such as a linker or antibody, is established via a labile cleavable hydrazone linker (acid sensitive) and / or via a maleimide-containing bond with a cysteine, for example 1, 2, 3 or 4 cysteines, on the carrier molecule, thereby forming structure B:

[0101] [ka]

[0102] As a result, one to four scaffolds are covalently attached to a single carrier molecule, such as a monoclonal antibody. Thus, a single carrier molecule, e.g., an antibody, ligand, effector moiety, may be covalently conjugated to a single scaffold, e.g., the trifunctional linker of Scheme II and Structure B, or to two or more scaffolds according to the invention, the two or more scaffolds being the same or different, and the two or more scaffolds optionally containing the same (number) or different (number) covalently attached saponins.

[0103] An embodiment is a scaffold according to the invention, wherein at least one bioactive molecule is covalently attached to the polymeric or oligomeric structure via a cleavable bond, said cleavable bond undergoes cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5, more preferably at pH ≦ 5.5. Such cleavable bond facilitates the release of one or more bioactive molecules, such as saponins, when the scaffold is in the acidic environment within such cells, e.g., in late endosomes. Without wishing to be bound by any theory, free saponins within endosomes and / or lysosomes may contribute to the endosomal escape enhancing activity of the saponin when endosomal escape of effector moieties present within endosomes or lysosomes is considered. Such endosomal escape enhancing activity is higher or more efficient, for example, compared to the activity of saponins bound to the scaffold. The inventors have established that saponins linked to scaffolds and carrier molecules via bonds such as hydrazone bonds, amide bonds, disulfide bonds etc. are all capable of improving the cytotoxic effect of payloads as diverse as effector moieties or molecules, e.g. proteinaceous toxins and oligonucleotides such as antisense BNAs, within (tumour) cells.

[0104] One embodiment is a scaffold according to the invention, wherein at least one biologically active molecule is covalently attached to the polymeric or oligomeric structure of the scaffold via an imine, hydrazone, hydrazide, oxime, 1,3-dioxolane, disulfide, thio-ethyl, amide, peptide or ester bond, preferably via at least one linker, the biologically active molecule being one or more saponins of the invention. The inventors have demonstrated that covalent coupling of e.g. saponin to the scaffold using such bonds results in enhanced efficacy and activity of the effector moiety when cells, such as tumor cells, are exposed to both a scaffold such as an oligomeric trifunctional linker or dendron with a covalently coupled saponin and an effector moiety such as an antisense BNA or saporin.

[0105] One embodiment is a scaffold according to the invention, wherein an aldehyde functional group at position C-23 of at least one saponin is involved in a covalent bond to the polymeric or oligomeric structure of the scaffold, and / or, if present, a glucuronic acid functional group on a carbohydrate substituent at the C-3 beta-OH group of at least one saponin is involved in a covalent bond to the polymeric or oligomeric structure of the scaffold, either via a direct bond or via at least one linker.

[0106] One embodiment is a scaffold according to the invention, in which the aldehyde functional group at position C-23 of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled to a sulfhydryl group, e.g., a sulfhydryl group of a cysteine, on the polymer or oligomer structure of the scaffold via a thio-ethyl bond.

[0107] One embodiment is a scaffold according to the invention, in which a glucuronic acid functionality on a carbohydrate substituent at the C-3 beta-OH group of 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 via an amide bond to an amine group on the polymeric or oligomeric structure of the scaffold, such as a lysine or N-terminal amine group of a proteinaceous molecule.

[0108] An embodiment is the scaffold of the invention, the glycoside molecule is a saponin, and the link between the saponin and the polymeric or oligomeric structure on the scaffold is preferably via an acid-labile bond that is stable at pH 7.4, and releases the saponin preferably below pH 6.5, more preferably between pH 6.5 and 5.0. This is achieved, for example, via an imine formed by an amino group of the polymeric or oligomeric structure and an aldehyde group of the saponin. Other chemical bonds that meet the pH conditions can also be used for aldehyde coupling, such as specific hydrazones or acetals, which require hydrazide and hydroxyl groups, respectively, as functional groups of the polymeric or oligomeric structure. When the bond is a cleavable bond, the saponin is preferably attached to the polymeric or oligomeric structure of the scaffold via one of the carboxyl groups in the saponin or via an aldehyde functional group, more preferably via an aldehyde functional group, preferably at position 23. Alternatively, the saponin is preferably attached to the backbone polymeric or oligomeric structure via a linker which joins the backbone polymeric or oligomeric structure either via a carboxylic acid functional group or via an aldehyde functional group of the glycoside molecule.

[0109] An embodiment is the scaffold of the invention, in which at least one glycoside molecule is attached to the polymer or oligomeric structure via a stable bond. In a more preferred embodiment, the at least one glycoside molecule is a saponin, and the stable bond between the saponin and the polymer or oligomeric structure of the scaffold preferably occurs by amide coupling or amine formation. This is achieved, for example, via carbodiimide-mediated amide bond formation with the amino group of the polymer or oligomeric structure and the activated glucuronic acid group of the saponin. A chemical bond that meets the definition of a stable bond can also be used for aldehyde coupling. For example, a specific amine is derivatized after reductive amination, requiring a primary amino group as a functional group of the polymer or oligomeric structure. When the bond is a stable bond, the saponin is preferably attached to the scaffold via one of the carboxyl groups of the saponin.

[0110] An embodiment is a scaffold according to the invention, wherein the chemical group for covalent coupling of the scaffold to a carrier molecule is a click chemistry group.

[0111] An embodiment is a scaffold according to the invention, where the click chemistry group is a tetrazine, azide, alkene, or alkyne, or a cyclic derivative of any of these groups, preferably an azide.

[0112] An embodiment is a scaffold according to the invention, which further comprises a click chemistry group for coupling to an effector molecule and / or to a ligand, an antibody, a binding domain, or a fragment thereof. Click chemistry groups are chemical functional groups suitable for click chemistry, which is defined as a reaction that is modular, broad in scope, gives very high yields, produces only benign by-products, offers high selectivity and high tolerance to different functional groups, and is stereospecific. Required process features include simple reaction conditions, readily available starting materials and reagents, no solvent or the use of mild (e.g. water) or easily removed solvents, and simple product isolation. The click chemistry group is preferably a tetrazine, azide, alkene, or alkyne, or a reactive derivative thereof, such as methyl-tetrazine or maleimide (alkene), more preferably an alkyne, or a cyclic derivative of these groups, such as cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]nona-4-yne, dibenzocyclooctyne).

[0113] Thus, the scaffold according to the invention comprises at least one glycoside molecule. By "at least one" in this context it is meant that the scaffold comprises one glycoside molecule, but may also comprise several (e.g. 2, 3 or 4) glycoside molecules, or a large number (e.g. 10, 20 or 100) glycoside molecules. Depending on the application, the scaffold can be designed such that it comprises a defined number of glycoside molecules. Preferably, the scaffold according to the invention comprises a defined number or range of glycoside molecules, rather than a random number. This is particularly advantageous for drug development with respect to marketing approval. A defined number in this respect means that the scaffold preferably comprises a predefined number of glycoside molecules. This is achieved, for example, by designing the polymer structure with a certain number of possible moieties for the glycoside(s) to attach to. In an ideal situation, all of these moieties are coupled to glycoside molecules, and then the scaffold comprises a predefined number of glycoside molecules. It is envisaged to provide a standard set of scaffolds containing, for example, 2, 4, 8, 16, 32, 64 etc. glycoside molecules, so that the optimal number can be easily tested by the user according to his needs. An embodiment is a scaffold of the invention, where, for example, in non-ideal situations, not all moieties present on the polymer structure bind glycoside molecules, and the glycosides are present in a defined range. Such a range can be, for example, 2-4 glycoside molecules per scaffold, 3-6 glycoside molecules per scaffold, 4-8 glycoside molecules per scaffold, 6-8 glycoside molecules per scaffold, 6-12 glycoside molecules per scaffold, etc. Hence, in such cases, a scaffold according to the invention contains 2, 3 or 4 glycoside molecules when the range is defined as 2-4.

[0114] An embodiment is a scaffold according to the invention, in which the number of monomers of the polymeric or oligomeric structure is a precisely defined number or range. Preferably, the polymeric or oligomeric structure comprises structures such as poly(amines), e.g., polyethyleneimines and poly(amidoamines), or polyethylene glycols, structures such as poly(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins), or peptides or proteins, or structures such as natural and / or artificial polyamino acids, e.g., polylysine, DNA polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. They appear as either pure or mixed linear, branched, or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates of these structures. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not show substantial acute or chronic toxicity in the organism and can either be excreted intact or completely degraded to excretable and / or physiological compounds by the body's metabolism. The aggregates can be assembled by covalent crosslinking or non-covalent bonds and / or attractive forces. Thus, they can also form nanogels, microgels or hydrogels. Alternatively, they can be attached to carriers, such as inorganic nanoparticles, colloids, liposomes, micelles or particle-like structures containing cholesterol and / or phospholipids. The polymeric or oligomeric structures preferably have a precisely defined number or range of coupling moieties for the coupling of glycoside molecules (and / or effector molecules and / or carrier molecules, such as ligands, monoclonal antibodies or fragments thereof). Preferably, at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% of a precisely defined number or range of coupling moieties on a polymeric or oligomeric structure are accounted for by glycoside molecules in the backbone according to the invention.

[0115] Preferably, dendrons are branched, well-defined dendritic polymers with a single chemically addressable group at the origin of the tree, called the focal point. Dendrimers are the connection of two or more dendrons at their focal points. Dendronized polymers are the connection of the focal points of one or more dendrons to a polymer. In a preferred embodiment, a scaffold according to the invention is provided, the polymeric or oligomeric structure comprising linear, branched or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates of these structures, either pure or mixed. The aggregates may be assembled by covalent crosslinking or non-covalent attractive forces to form nanogels, microgels or hydrogels. Preferably, the polymer is a derivative of poly(amine), such as polyethyleneimine and poly(amidoamine), and polyethylene glycol, structures such as poly(ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), and structures such as natural and / or artificial polyamino acids, such as polylysine, or a peptide or protein (e.g., a ligand or antibody, such as any of the monoclonal antibodies of Tables A2-A4), or a DNA polymer, a stabilized RNA polymer, or a PNA (peptide nucleic acid) polymer. Preferably, the polymer or oligomeric structure is biocompatible.

[0116] An embodiment is a scaffold according to the invention, wherein the effector molecule is a drug substance, such as a toxin, a drug, a polypeptide, and / or a polynucleotide. An embodiment is a scaffold according to the invention, wherein the effector molecule is a toxin, a microRNA, or a polynucleotide encoding a protein. Typically, the carrier molecule encapsulates a proteinaceous molecule for targeting the scaffold, for example to tumor cells or autoimmune cells or cells involved in a B-cell disorder. Preferably, the carrier molecule comprises an immunoglobulin, or at least one or more binding domains thereof and / or binding fragments thereof, such immunoglobulin preferably being selected from any of the monoclonal antibodies of any of Tables A2-A4, such as cetuximab, OKT-9, trastuzumab.

[0117] A drug substance in the present invention is an effector molecule that is used to achieve a beneficial outcome 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 condition. A drug substance may also lead to unwanted adverse side effects. In this case, the pros and cons must be weighed to determine if the drug substance is suitable in a particular case. If the effect of the drug substance in a cell is overwhelmingly beneficial to the whole organism, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the whole organism, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, the target and off-target cells are purpose-dependent and defined by the user.

[0118] A polypeptide effector molecule can be, for example, a polypeptide that restores a lost function, such as an enzyme replacement, a gene regulatory function, or a toxin.

[0119] One embodiment is a scaffold according to the invention, wherein the scaffold is a trifunctional linker, comprises a second chemical group to which at least one biologically active molecule is covalently attached, comprises a third chemical group for covalent attachment to a molecule, and comprises a first chemical group for covalent attachment to a carrier, preferably the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0120] One embodiment is a scaffold according to the invention, wherein the at least one biologically active molecule is a defined number or range of glycoside molecules, preferably 1-128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 glycoside molecules, or any number therebetween, e.g., 7, 9, 12 glycoside molecules.

[0121] One embodiment is a scaffold according to the invention, wherein the polymeric or oligomeric structures include linear, branched and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, polylysine, polyethylene glycol, or assemblies of these polymeric or oligomeric structures, which assemblies are preferably assembled by covalent crosslinks.

[0122] The scaffold is fundamentally independent of the type of effector molecule covalently attached to the scaffold. Therefore, the scaffold is the foundation product for a new platform technology. Since at least one covalently attached glycoside mediates intracellular delivery, the scaffold technology according to the present invention is the first known system that mediates controlled intracellular effector molecule delivery by glycosides.

[0123] Synchronization is the missing link between the highly successful delivery strategy in mice and its application in humans. Indeed, the inventors have established in a series of in vivo mouse tumor models that administering a dose of free saponin and a dose of ADC separately to mice does not result in any desired antitumor activity, such as delayed tumor growth, tumor regression, diminished and slower tumor growth, compared to control animals not treated with ADC and free saponin. Free saponin was administered using different routes of administration and different time points of administration of free saponin compared to the moment of administration of ADC (free saponin was administered before, during and after administration of ADC). The ADCs tested in the in vivo tumor models were cetuximab-giantin (with free SO1861) or trastuzumab-saporin (with free SO1861). Varying the dose of free saponin did not allow effective antitumor activity. The referenced ADC was administered at a dose that did not by itself produce any beneficial antitumor effect on tumor-bearing animals. Surprisingly, the inventors have now established that beneficial antitumor activity in various in vitro mammalian cell-based bioassays and / or various in vivo animal tumor models can be achieved by treating animals with a conjugate according to the invention comprising a scaffold according to the invention. The scaffold is, for example, a dendron, with up to four covalently attached saponin molecules, for example SO1861, via a cleavable linker. The scaffold is, for example, a trifunctional linker, with a saponin (for example, SO1861, QS-21) covalently attached via a cleavable or non-cleavable linkage, with an effector moiety (for example, dianthin, silencing BNA (HSP27)) covalently attached via a non-cleavable or cleavable linkage, and with a covalently attached monoclonal antibody, for example, cetuximab, trastuzumab, OKT-9. Alternatively, the scaffold is a dendron, for example a dendron to which four moieties can be attached, such as four saponin molecules, or for example a dendron for attaching two saponin and two effector molecules, the dendron comprising chemical groups for (covalent) coupling to a ligand or an antibody or a fragment or domain thereof.Reference is made to the Examples section which illustrates various of these scaffolds according to the invention, showing in vivo and / or in vitro antitumor cell activity, for example when considering the cytotoxicity exerted by proteinaceous toxins or when considering gene silencing in tumor cells.

[0124] The scaffolds of the present invention provide optimized and functionally active units that can be linked to effector molecules and / or ligands, antibodies, etc. at a single, defined location, preferably via a covalent bond and, when desired for the intended purpose, via a cleavable covalent bond.

[0125] Without wishing to be bound by any theory, in view of the failures observed when considering treatment of tumor-bearing animals with ADC together with free saponin, it is preferred to synchronize the presence of both at least one glycoside, preferably saponin, and an effector molecule, preferably a toxin or an oligonucleotide such as a BNA, in a compartment or vesicle of the endocytic pathway of a target cell, e.g. a tumor cell or an autoimmune cell. With ADC and free saponin, synchronizing the presence of the molecules in late endosomes to obtain a synergistic effect in vivo could not be beneficially obtained according to the inventor's numerous attempts. In one embodiment, the present invention preferably solves at least one of the following problems with combining effector molecules and glycoside molecules as one compound comprising the scaffold of the present invention: (1) the required number of glycoside molecules per effector molecule is a defined number or range (e.g., preferably 1 or more, more preferably at least 2, more preferably at least 3, at least 5, more preferably at least 6, more preferably at least 10, more preferably at least 15, at least 20, more preferably at least 25, more preferably at least 27, most preferably at least 30, or more), so that a simple chemical linkage is not suitable; (2) for example, only reasonable chemical groups on the saponin that can be used for (covalent) particularly single and cleavable retainable coupling are required for endosomal escape activity; (3) the effector molecule may not possess a suitable corresponding group for coupling; (4) glycosides may lose their necessary potential to interact with cholesterol when not freely diffusible. Most likely, all these limitations are the reason why glycosides have not been used in combination with drug substances in clinical investigations other than the application of saponins in vaccination regimens involving the use of immune enhancing adjuvant substances, even though the significant endosomal escape enhancing effect of saponins, e.g., those listed in Table A1 and Scheme I, has been known for more than a decade. The scaffolds according to the present invention at least partially overcome these difficulties.Surprisingly, saponins previously applied for their immune enhancing activity in the context of vaccination involving saponins as adjuvant components are now also suitable for (covalent) coupling to the scaffolds of the invention, for involvement in conjugates comprising a scaffold carrying a saponin and further having an effector molecule and optionally a cell targeting moiety, such as a ligand or an antibody, (covalently) bound thereto, for in vitro and in vivo anti-tumour activity.

[0126] In its basic form, the scaffold comprises a polymeric and / or oligomeric structure bearing at least one glycoside molecule, such as a particular saponin, e.g., SO1861 (Table A1, Figure 13). In a preferred embodiment, a scaffold is provided, wherein the glycoside molecule is attached to the polymeric or oligomeric structure via a cleavable bond, preferably said cleavable bond undergoes cleavage under acidic, reductive, enzymatic or photoinduced conditions, more preferably under acidic conditions. Preferably, the cleavable bond is an imine, hydrazone, oxime, 1,3-dioxolane, disulfide, hydrazide, or ester.

[0127] An embodiment is a scaffold according to the invention, wherein the glycoside molecule is SA1641, SO1861, GE1741, QS-21, or any of their stereoisomers, such as any of their diastereomers.

[0128] An embodiment is a scaffold according to the invention, wherein the carrier molecule comprises or consists of a covalent conjugate or complex of any of a proteinaceous molecule, a protein, a peptide, a nucleic acid, an oligonucleotide, a lipid, a fat, a fatty acid, a nanoparticle, a carbohydrate, or a combination thereof.

[0129] One embodiment is a scaffold according to the invention, wherein the carrier molecule comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, or comprises or consists of at least one non-proteinaceous ligand and / or at least one proteinaceous ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0130] One embodiment is a scaffold according to the invention, wherein the carrier molecule is CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, D or consisting of at least one binding domain and / or at least one binding fragment for binding to a cell surface receptor, such as a tumor cell specific cell surface receptor selected from LL3, CD25, EphrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected from CD71, EGFR, HER2.

[0131] An embodiment is a scaffold according to the invention, wherein the carrier molecule comprises or alternatively consists of any one of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, 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 receptor binding fragment thereof or at least one tumor cell receptor binding domain thereof, such as at least one tumor cell specific receptor binding fragment thereof and / or at least one tumor cell specific receptor binding domain thereof.

[0132] One embodiment is a scaffold according to the invention, which is suitable for forming a covalent bond with a carrier molecule, said covalent bond preferably involving a cysteine ​​side chain of the carrier molecule and / or a lysine side chain of the carrier molecule, wherein the carrier molecule comprises at least cysteine ​​and / or lysine.

[0133] An embodiment is a scaffold according to the invention, wherein the carrier molecule comprises or consists of at least one effector molecule, or the carrier further comprises at least one effector molecule, which then also comprises an immunoglobulin according to the invention, a binding fragment thereof, a binding domain, etc., and the effector molecule is at least one of a drug substance, such as a payload, a toxin, a drug, a polypeptide, an oligonucleotide, a nucleic acid, a xenonucleic acid, an enzyme, such as urease and Cre recombinase, a protein toxin, a ribosome-inactivating protein, or any one or more thereof.

[0134] An embodiment is a scaffold according to the invention, wherein the protein toxin is a protein toxin selected from Table A5, and / or a viral toxin, such as apoptin; a bacterial toxin, 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 alpha-sarcin; ribosome inactivating proteins, and plant toxins including the A chain of ribosome inactivating proteins type 2, such as giantin, e.g. giantin-30 or giantin-32, saporin, e.g. saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin debouganin, shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog RNase, or granzyme B, or angiogenin from humans, or any fragment or derivative thereof; preferably the protein toxin is giantin and / or saporin.

[0135] In one embodiment is a scaffold according to the invention, wherein the oligonucleotide, xenonucleic acid, or nucleic acid comprises or consists of any one or more of a vector, a gene, a transgene inducing 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), a 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA), and a threose nucleic acid (TNA), or a derivative thereof, preferably a BNA, e.g. a BNA for silencing HSP27 protein expression.

[0136] Now, the scaffold of the present invention makes it possible to design and manufacture one-component non-viral clinically applicable gene delivery technologies. For example, the scaffold of the present invention allows the development of non-viral based gene delivery technologies, which improves therapeutic efficacy with lower therapeutic doses, thereby improving patient health. In particular, the scaffold of the present invention, when covalently attached to carrier molecules such as monoclonal antibodies for binding to (tumor, autoimmune) cell surface specific molecules, and when attached to carrier molecules such as oligonucleotides, e.g. BNAs, allows overcoming a long-standing and major bottleneck in the field of gene delivery, namely, the efficient, safe and cost-effective transfer of gene therapy products across the endosomal membrane into the cytosol / nucleosol. Indeed, gene therapy is one of the most promising treatment options for future advanced therapies in a wide range of diseases. Successful gene delivery requires recognition of the target cell and cytosolic and nucleosolic uptake of the gene. One of the major problems in the field of non-viral gene therapy is the inefficient and insufficiently safe delivery of genetic material for therapeutic use to patients.

[0137] Therefore, when applying the scaffolds of the present invention, which comprise cell-targeting carrier molecules, such as ligands or preferably antibodies (fragments, domains thereof) and oligonucleotides such as antisense BNAs, the inventors have now made it possible to overcome a long-standing and major bottleneck in the field of gene delivery: the safe transfer of gene therapy products through the endosomal membrane into the cytosol / nucleosol. The scaffolds of the present invention are designed to allow the targeting of any addressable cell type by all known genetic drugs, thereby ensuring better patient treatment, not limited to genetic diseases, but also for cancer treatment, and thus representing an important technology for a large patient population. The technology based on the scaffolds of the present invention is a polymeric or oligomeric scaffold, which serves as a carrier for endosomal escape enhancers (EEEs), such as the saponins of any of the embodiments according to Table A1 and Scheme I, for targeting ligands or (monoclonal) (tumor cell specific) antibodies, and for effector moieties, here effector genes, such as LNAs or BNAs. For example, the use of the scaffolds of the invention, including cell targeting antibodies (fragments) and oligonucleotides, such as BNAs, has the potential to bring any kind of biological macromolecule into the cytosol and nucleus. The development of new targeting ligands and monoclonal (human, humanized) antibodies is under continuous investigation by numerous research groups and companies worldwide. The same is true for oligonucleotides aimed at delivery in the cytosol of diseased cells, such as cancer cells. Thus, the scaffolds of the invention provide a molecular interface for linking current and future targeting ligands and antibodies, as well as current and future therapeutic oligonucleotides (as well as payloads, such as protein toxins), to the oligomeric or polymeric scaffold modules of the invention by click chemistry, allowing customized drug applications and future developments in the field of tissue and cell targeting technologies. The scaffolds of the invention can be combined with antibodies and ligands as covalently coupled carrier molecules.The global market for gene therapy drugs is growing rapidly and covers a wide range of disease areas, such as potential treatments for cancer, cardiovascular disease, Parkinson's, Alzheimer's, HIV, and many rare (monogenic) diseases. Current viral vector-based gene therapy technologies have significant challenges, such as safety, manufacturing logistics, and associated high costs. The scaffold of the present invention allows the use of a technology platform that represents an alternative to current viral gene delivery technologies. Thus, the scaffold of the present invention is suitable for implementation in approaches to develop non-viral gene therapy for diseases such as cancer, cardiovascular disease, Parkinson's, Alzheimer's, HIV infection, and many rare (monogenic) diseases. The scaffold of the present invention is suitable for developing novel treatments to transform the field of antibody-drug conjugates (ADCs) and oligonucleotide-based therapeutics, for example by creating non-viral vector-based gene therapy drugs based on targeted antisense BNAs. The application of the scaffold of the present invention, especially as covalent conjugates with antibodies and oligonucleotides such as BNAs, is one of many beneficial approaches made possible due to the present invention. For example, the use of the scaffolds of the invention here allows exploitation of the endocytic pathway of mammalian cells. Endocytosis is exploited for the delivery of therapeutic agents, and the scaffolds of the invention contribute to improved uptake and endosomal escape of, for example, siRNAs conjugated to the scaffolds. The scaffolds of the invention are preferably used together with small molecules acting as payloads, delivery enhancers of oligonucleotides. Thereby, the scaffolds of the invention with covalently coupled oligonucleotides, e.g. BNAs, and with covalently coupled cell targeting moieties, e.g. ligands and preferably antibodies (domains or fragments), provide a solution to the current problems found with current endosomal escape enhancers and gene therapy products, which relate to their application as two components, thus complicating the approval and clinical applicability of therapeutic agents, since such scaffolds of the invention are a single conjugated therapeutic agent molecule encompassing a gene product, e.g. saponin, BNA, and a (tumor) cell targeting moiety, e.g. a (monoclonal) antibody.Therefore, the present invention provides a non-viral gene delivery technology, in which an endosomal escape enhancer (e.g., glycosides according to Table A1, Scheme I, embodiments of the present invention), a gene therapy product (oligonucleotides according to the present invention, e.g., BNAs), and a targeting ligand or antibody (e.g., Tables A2, A3, A4, embodiments of the present invention) are all coupled to one molecular scaffold of the present invention. Therefore, such scaffolds of the present invention provide current and future macromolecular drug therapeutic opportunities for a wide range of diseases and a large patient population. The application of such scaffolds of the present invention, which comprise at least one saponin, at least one oligonucleotide, and at least one specific cell targeting moiety, e.g., immunoglobulin, addresses the problems evident with current methods of applying endosomal escape enhancers and gene therapy products separately. These current methods do not guarantee that both compounds are present at the interaction site at the same time. Now, this problem is overcome by using the scaffolds of the present invention. In other words, such scaffolds of the present invention provide a non-viral gene delivery technology with increased synchronization (in time and place) of both compounds, i.e., gene products such as saponins and BNAs.

[0138] Gene therapy can help with inherited and previously incurable diseases such as cystic fibrosis, correa, Huntington's disease, or hemophilia. However, currently, several problems remain to be overcome: for example, the therapeutic gene must precisely reach a specific target cell in the body. On the other hand, the therapeutic gene should be absorbed by the target cell, but the therapeutic gene should not be destroyed. Current gene therapy approaches use viruses as gene ferries. However, these procedures involve significant risks and cannot be transferred to the introduction of other biomolecules. An embodiment is the scaffold of the present invention, which contains (plant-derived) glycosides for use in platform technology, which not only allows the delivery of genes when bound to the scaffold as carrier molecules, but also allows the delivery of different therapeutic biomolecules to be introduced into the target cells. Thus, the scaffold of the present invention can be used to develop nucleic acid-based treatments for cystic fibrosis, correa, Huntington's disease, or hemophilia. The scaffold of the present invention thereby makes available new gene therapy strategies to improve the health of patients with genetic diseases, including those with cystic fibrosis, Huntington's disease, and hemophilia. As part of the present invention, a non-viral gene delivery technology is developed that combines plant-derived endosomal escape enhancers (glycosides), gene therapy products, and targeting ligands, all bound to a single molecular scaffold. The resulting non-viral gene therapy based on the scaffold of the present invention shows about 40-fold increased delivery efficiency at lower doses compared to currently available strategies. This allows the scaffold of the present invention to be used in clinical applications, such as for repairing or supplementing defective genes, such as in cystic fibrosis patients, and for targeted delivery of specific genes, such as to destroy cancer cells. In fact, the scaffold of the present invention is suitable for application in treatment regimens for any disease caused by genetic defects and currently incurable, such as cystic fibrosis, Huntington's disease, and hemophilia.Gene therapy utilizing the scaffolds of the present invention helps overcome two current problems: first, the scaffolds of the present invention allow for the delivery of therapeutic genes to specific target cells in the body; second, the therapeutic genes get inside these cells but are not destroyed due to the presence of targeting moieties such as saponin(s), oligonucleotide products, and antibodies for binding to target cells, all covalently linked to the oligomeric or polymeric scaffolds of the present invention.

[0139] One embodiment is a scaffold according to the invention, wherein the effector molecule is preferably any one or more of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, passudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethylauristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-gamma-calicheamicin, Pyrrolobenzodiazepine (PBD) dimers, benzodiazepines, CC-1065 analogs, duocarmycins, doxorubicin, paclitaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepines, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracyclines, camptothecin analogs, SN-38, DX-8951f, exatecan mesylate, Pseudomonas The antibody comprises or consists of at least one payload selected from one or more of a truncated form of P. aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or a derivative thereof.

[0140] One embodiment is a scaffold according to the invention, wherein the carrier molecule comprises or consists of a covalently linked combination of an effector molecule and a monoclonal antibody, preferably selected from gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and the antibody-drug conjugates of Table A2 and Table A3.

[0141] An embodiment of the present invention relates to a method for producing a scaffold suitable for covalently linking at least one biologically active molecule to a carrier molecule, the method comprising: a) providing a polymer or oligomeric structure comprising a first chemical group for covalently coupling the polymer or oligomeric structure to the carrier molecule and comprising at least one second chemical group different from the first chemical group, each second chemical group being for covalently coupling one of the at least one biologically active molecule to the oligomeric or polymeric structure; and b) covalently coupling at least one biologically active molecule to said polymer or oligomeric structure via the second chemical group(s), preferably the biologically active molecule(s) is any one of the biologically active molecules of the present invention, more preferably SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or any saponin of Table A1 and / or Scheme I, thereby providing a scaffold.

[0142] The polymer or oligomeric structure is preferably any of the polymer or oligomeric structures according to the present invention. Similarly, the first and second chemical groups for covalent coupling are chemical groups according to the embodiments of the present invention. The bioactive molecule is preferably any of the bioactive molecules of the aspects and embodiments of the present invention. Preferably, the bioactive molecule is a saponin according to the present invention, which is also preferably covalently coupled to the scaffold via a linker, such as a cleavable linker according to the present invention. Exemplary scaffolds of the present invention are trifunctional linkers and dendrons.

[0143] An embodiment is a method of the invention, wherein the at least one biologically active molecule is any one of the glycosides and saponins of the previous aspects and embodiments of the invention; and / or the at least one biologically active molecule is coupled to the scaffold via a linker according to any one of the previous aspects and embodiments of the invention; and / or the saponin is linked to the scaffold via a covalent bond according to any one of the previous embodiments and aspects of the invention; and / or the saponin is coupled to the scaffold via a cleavable bond of any one of the previous embodiments of the invention. An embodiment of the present invention relates to a method for producing a scaffold covalently linked to a carrier molecule, the scaffold comprising at least one covalently linked biologically active molecule, the method comprising: a) providing a scaffold comprising at least one biologically active molecule covalently linked to the polymeric or oligomeric structure of said scaffold, preferably a scaffold according to the present invention or a scaffold obtainable by the method of the present invention or a scaffold obtained by the method of the present invention; and b) covalently coupling the scaffold of a) to a carrier molecule according to the present invention, thereby providing a scaffold covalently linked to a carrier molecule, the scaffold comprising at least one covalently linked biologically active molecule, preferably a scaffold according to the present invention.

[0144] An embodiment is a method of the invention, wherein the at least one biologically active molecule is any one of the glycosides and saponins of the previous aspects and embodiments of the invention; and / or the at least one biologically active molecule is coupled to the scaffold via a linker according to any one of the previous aspects and embodiments of the invention; and / or the saponin is linked to the scaffold via a covalent bond according to any one of the previous embodiments and aspects of the invention; and / or the saponin is coupled to the scaffold via a cleavable bond according to any one of the previous embodiments of the invention; and / or the carrier molecule is or comprises any one of the payload and effector molecules according to any one of the previous embodiments and aspects of the invention, or is or comprises any one of the ligands and immunoglobulins, monoclonal antibodies, and / or any binding domains or fragments thereof according to any one of the previous embodiments and aspects of the invention. Typically, the biologically active molecule is any one of SO1861, SA1641, GE1741, QS-21. Typically, the carrier molecule comprises or is cetuximab, trastuzumab, OKT-9. Typically, the effector molecule or payload is a BNA, a protein toxin, dianthin, saporin, an oligonucleotide. A preferred bioactive molecule of the present invention is a saponin fraction from Quillaja saponaria, such as a water-soluble saponin fraction of Quillaja saponaria.

[0145] An embodiment is a scaffold according to the invention or a method according to the invention, in particular wherein at least one biologically active molecule is a glycoside such as a saponin according to the invention, more particularly wherein the saponin is SO1861, SA1641, GE1741, and / or QS-21. The scaffold is capable of increasing (late) endosomal escape and / or lysosomal escape of an effector molecule according to the invention, wherein said effector molecule is either covalently attached to the scaffold and contacted with a mammalian cell, in particular a tumor cell, for example of a human subject, or wherein said effector molecule is contacted with a mammalian cell, in particular a tumor cell, for example of a human subject, in the presence of a scaffold according to the invention.

[0146] Examples of polypeptides (proteinaceous molecules) as effector molecules are, for example, Cas9; toxins (e.g. saporin, dianthin, gelonin, (de)bouganin, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (e.g. argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; enzymes of cell signaling; cell cycle regulators; gene regulators (transcription factors, e.g. NF-κB or gene repressors, e.g. methionine repressor).

[0147] The effector molecule, which is a polynucleotide, may be a polynucleotide that contains coding information, such as a gene or an open reading frame that codes for a protein. It may also contain regulatory information, such as a promoter or a regulatory element binding region, or a sequence that codes for a microRNA. Such polynucleotides may include natural and artificial nucleic acids. Artificial nucleic acids include peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these is distinguished from naturally occurring DNA or RNA by modifications of the backbone of the molecule. Examples of nucleotides as effector molecules are, for example, DNA: single-stranded DNA (e.g., DNA for adenine phosphoribosyltransferase); linear double-stranded DNA (e.g., clotting factor IX gene); circular double-stranded DNA (e.g., plasmids); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA.

[0148] A toxin in the present invention is a drug substance capable of killing cells. Preferably, a targeted toxin is only, or at least predominantly, toxic to target cells and not to off-target cells.

[0149] Effector molecules useful in the present invention preferably depend on late endosomal escape to exert their effect. Some effectors, such as Pseudomonas exotoxin, are rerouted to other organelles before the "late endosomal stage" and therefore do not normally benefit from the scaffold according to the present invention. However, such toxins can be adapted for use in the present invention, for example by deleting the rerouting carried out by the signal peptide. In particular, toxins that are highly toxic and would require only one molecule to escape the endosome and kill the cell can be modified to be less potent. It is preferred to use toxins that kill cells when 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 the endosome. Furthermore, it is preferred that the functionalized scaffolds of the present invention, i.e., scaffolds comprising covalently attached effector molecule(s) and / or ligands and / or monoclonal antibodies for targeting the scaffold to target cells, such as tumor cells or autoimmune cells, comprise a ratio of glycoside molecules of at least 2:1, more preferably at least 5:1, more preferably at least 10:1, more preferably at least 20:1, and most preferably at least 50:1 per effector molecule covalently attached to the scaffold. Specifically, in functionalized scaffolds comprising assembled polymer structures, where the glycoside molecules and the effector molecules are attached to different polymer structures within the assembly, it is preferred to have a ratio of glycoside molecules of at least 10:1, more preferably at least 20:1, more preferably at least 50:1, more preferably at least 100:1, and most preferably at least 200:1 to the effector molecules in such an assembly. Furthermore, in order to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0150] The invention further provides functionalized scaffolds, comprising at least one scaffold according to the invention coupled to either a) at least one effector molecule, b) at least one ligand, c) at least one effector molecule and additionally at least one ligand (Figures 10-12, 54), d) at least one effector molecule itself carrying at least one ligand (Figure 53), or e) at least one ligand itself carrying at least one effector molecule. Such coupling in a)-e) may be achieved via a cleavable (labile) or stable (non-cleavable) bond. Preferably, the coupling in a)-e) occurs independently by click chemistry linkage. Preferably, the functionalized scaffold is capable of enhancing endosomal escape of the effector. An embodiment is a scaffold according to the invention, wherein the scaffold is a functionalized scaffold according to the invention, wherein the at least one effector molecule is a drug substance, e.g. a toxin, a drug, a polypeptide, and / or a polynucleotide. An embodiment is a functionalized scaffold according to the invention, wherein the effector molecule is a toxin, or a polynucleotide encoding a protein.

[0151] The term "ligand" as used in the present invention has its usual meaning, preferably meaning a molecule or structure that can bind to another molecule or structure on the cell surface of a target cell. Said molecule or structure on the cell surface can be endocytosed, and is preferably absent or less prominent on off-target cells. Preferably, said molecule or structure on the cell surface is constitutively endocytosed. More preferably, the ligand in the present invention induces endocytosis of said molecule or structure on the cell surface of a target cell after binding to said molecule or structure. This is the case, for example, for epidermal growth factor receptor (EGFR), which is present on the surface of various cancer cells. Examples of molecules or structures on the cell surface of a target cell that are constitutively endocytosed 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 a toxin with a ligand on a carrier molecule is one possibility for creating a targeted toxin. A toxin that is only toxic in target cells because it interferes with processes that occur only in the target cells can also be seen as a targeted toxin (it cannot exert its toxic effect as in off-target cells, e.g. apoptin). Preferably, the targeted toxin is a toxin that is combined with a ligand or, for example, a monoclonal antibody, in order to be active in target cells and not active in off-target cells (because it is only bound and endocytosed by target cells). In a functionalized scaffold that includes a carrier molecule that includes a ligand and an effector molecule, the ligand or monoclonal antibody guides the effector molecule and the scaffold to the target cell. After internalization, at least one glycoside, preferably a saponin, mediates the endosomal escape of the effector molecule. The saponin is typically a saponin listed in Table A1 and Scheme I, preferably the saponin is SO1861 and / or QS-21 and / or SA1641 and / or GE1741.

[0152] A scaffold of the invention that is not provided with carrier molecules, such as effector molecules and / or cell targeting ligands or antibodies, covalently linked to the scaffold, i.e. a non-functionalized scaffold, can be supplied, for example, to a drug manufacturer. This then takes care of coupling the effector molecule alone or the effector molecule and the ligand or antibody to the scaffold. If required, the drug manufacturer can add a cleavable unit for releasing the effector molecule from the scaffold and / or the ligand, the antibody, for example by inserting a disulfide bridge between the effector molecule and the ligand and / or the effector molecule and a click site. The invention also provides (pre-)functionalized versions of the scaffold, this functionalized scaffold already carrying an effector molecule, such as a tumor cytotoxin (Figure 54). With regard to endosomal effector molecule release, the activity of the scaffold according to the invention is preferably already included in the scaffold. Functionalized scaffolds can be supplied, for example, to the pharmaceutical industry for further development of existing and future therapeutic antibodies, and to any supplier or owner of antibodies to functionalize targeting antibodies. The functionalized scaffolds can also be used by biotechnology companies or for research purposes.

[0153] With the scaffolds of the invention comprising an effector molecule on a carrier molecule covalently attached to the scaffold and a cell targeting moiety on a carrier molecule covalently attached to the scaffold, such as the immunoglobulins listed in Tables A2-A4, the inventors now for the first time provide conjugates with covalently attached glycosides, effector molecules, and monoclonal antibodies, as well as conjugates with covalently attached glycosides and effector molecules, and conjugates with covalently attached glycosides and cell targeting molecules, such as ligands or monoclonal antibodies (fragments, domains), for targeted delivery of effector molecules in target diseased cells, such as tumor cells. Although administered to a patient in need thereof, possibly in a systemic manner (site-specific local administration is preferred), the scaffolds of the invention exert their intracellular activity specifically in target cells that bear and expose the binding partner of the ligand or antibody. The scaffolds are then provided with such ligands or antibodies that preferentially and specifically bind to the desired target cells. In this manner, for example, if the effector molecule is also provided with the same or a different target cell-specific ligand or antibody specific for binding to the same or a different cell surface molecule present on the same target cell, such as a target tumor cell or a target autoimmune cell, the glycoside and effector molecule bound to the ligand or antibody will be directed to and ideally accumulate in the same (late) endosome, lysosome of the same target (disease) cell where the cell killing effect of the effector molecule is intended.

[0154] The new (functionalized) scaffolds offer several advantages: 1) The use of a functionalized scaffold, i.e. a scaffold of the invention comprising a covalently attached effector molecule and a ligand or antibody, results in a one-component system, whereby the effector molecule and the endosomal escape enhancer, i.e. at least one glycoside, are delivered simultaneously to the endosome in a predefined ratio due to the presence of a cell-targeting ligand or antibody, preferably a monoclonal antibody such as any one of the antibodies in Tables A2-A4.

[0155] 2) Here, at least one glycoside molecule is also targeted by the joint use of a targeting ligand of an effector molecule or a monoclonal antibody; therefore, the glycoside is not distributed throughout the body and taken up randomly by cells. This helps to reduce possible side effects and widens the therapeutic window.

[0156] 3) The number of glycoside molecules per effector molecule can be precisely defined and thus reduced to the required minimum; side effects due to excess glycoside molecules can be avoided. A defined number of glycoside molecules per effector molecule also facilitates the marketing approval of a particular drug.

[0157] 4) The present invention allows to provide preformed effector molecule-loaded scaffolds (functionalized scaffolds) for use with any available ligand and / or (monoclonal) antibody (or at least one binding fragment and / or domain thereof), making the present invention ideal for platform development.

[0158] 5) When the scaffold or functionalized scaffold is attached to a carrier molecule, the carrier molecule can also carry a ligand or antibody and / or an effector molecule. In such a case, the carrier molecule is considered as a linker.

[0159] One other application of the present invention is, for example, gene therapy. Efficient intracellular delivery of biological macromolecules such as polynucleotides remains, at present, a major hurdle. In contrast to conventional non-specific DNA transfection systems, the present invention is not limited to DNA but is specific to the target cells. Known viral systems are efficient and specific to the target cells. However, they are only suitable for DNA. Moreover, they carry the risk of immune and inflammatory responses, possess potential carcinogenic activity, and require complex and expensive procedures for preparation in each individual case. The novelty of the technology presented here is based on its origin, flexibility, and ease of use.

[0160] An embodiment is a scaffold of the invention, the scaffold being a functionalized scaffold, the scaffold comprising a carrier molecule comprising at least one ligand or antibody, said at least one ligand or antibody being capable of specifically binding to a target cell-specific surface molecule or structure, preferably the functionalized scaffold being endocytosed together with the surface molecule after binding. Preferably, said target cell is a diseased or disease-associated cell, preferably a tumor cell, a tumor-associated cell (e.g. tumor vascular cell), an immune cell (e.g. regulatory T cell), or a cell with a monogenic defect. By the term "target cell-specific surface molecule" is meant that the molecule is preferably expressed, either qualitatively or quantitatively, in target cells and to a lesser extent in non-target cells. Examples of such target cell-specific surface molecules are the receptor EGFR, which is upregulated on tumor cells but is also expressed (at a lower level) on e.g. skin fibroblasts, and HER2, which is overexpressed in breast cancer cells. However, functional fragments and domains of many target cell-specific surface molecules offer advantages known in the art, and the skilled artisan can very well choose a target cell-specific surface molecule for a particular purpose, i.e., to distinguish target cells from non-target cells for a particular disease or application. For examples of antibodies that bind to tumor cell-specific receptors, see also Tables A2, A3, and A4. For tumor cell-specific receptors suitable for targeting by carrier molecules, e.g., monoclonal antibodies, included by the scaffolds of the present invention, see also the embodiments described herein above. As used herein, "monogenic defect" has its ordinary meaning, which is an alteration of a single gene that occurs in substantially all cells of the body. The mutation may be present on one or both chromosomes (one chromosome inherited from each parent). Although relatively rare, monogenic defects affect millions of people worldwide. Currently, scientists estimate that more than 10,000 human diseases are known to be monogenic. Non-limiting examples of monogenic diseases known to date are: sickle cell disease, cystic fibrosis, polycystic kidney disease, and Tay-Sachs disease.

[0161] An embodiment is a scaffold of the invention, the scaffold being a functionalized scaffold according to the invention, the scaffold comprising a carrier molecule covalently bound thereto and comprising a ligand and / or an antibody, at least one ligand being an antibody or a derivative or fragment thereof (e.g., VHH or scFv), a cytokine, a growth factor, or an antibody-like molecule, such as an aptamer or a designed ankyrin repeat protein (DARPin). DARPins are genetically engineered antibody mimetic proteins that typically display highly specific and high affinity target protein binding. They are derived from the natural ankyrin protein, which is responsible for a variety of cellular functions. They constitute a new class of potent, specific and versatile small protein (typically 14-18 kDa) therapeutics, which are used as investigational tools in various research, diagnostic and therapeutic applications. Other non-limiting examples of antibodies or derivatives thereof known to date are: (i) a monovalent fragment consisting of Fab' or Fab fragment, variable light domain, variable heavy domain, constant light domain, and constant heavy domain 1, or a monovalent antibody as described in WO2007059782; (ii) a bivalent fragment F(ab')2 fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting essentially of a variable heavy domain and a constant heavy domain 1; and (iv) an Fv fragment consisting essentially of the variable light and variable heavy domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, the variable light and variable heavy, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be made as a single protein chain using recombinant methods. This allows the variable light and variable heavy regions to pair to form a monovalent molecule (known as single chain antibody or single chain Fv (scFv)).

[0162] Preferably, the effector molecule, whose effect is enhanced by a glycoside molecule (e.g. saponin), is detached from the scaffold and / or the ligand or antibody when endocytosed. This can be achieved, for example, by a cleavable bond that breaks under acidic, reductive, enzymatic or light-induced conditions. Thus, an embodiment is a scaffold of the invention, which is a functionalized scaffold according to the invention, wherein said at least one effector molecule is attached to said scaffold and / or said at least one ligand or antibody via a cleavable bond. Preferably, said cleavable bond is subject to cleavage under acidic, reductive, enzymatic or light-induced conditions. Preferably, the cleavable bond is an imine, hydrazone, oxime, 1,3-dioxolane, disulfide or ester, more preferably a disulfide or hydrazone bond.

[0163] An embodiment is a scaffold of the invention, which is a functionalized scaffold according to the invention, and said at least one effector molecule is attached to said scaffold and / or said at least one ligand or antibody via a stable bond, for example by amide coupling or amine formation, for example this is achieved by carbodiimide mediated amide bond formation between an amino group of the polymeric or oligomeric structure of the scaffold and an activated carboxylic acid group on the effector molecule or ligand.

[0164] One embodiment is a scaffold or functionalized scaffold according to the invention, which further comprises a carrier, such as a nanoparticle, liposome, micelle, colloid, or particle-like structure comprising cholesterol and / or phospholipids.

[0165] As said before, at least one glycoside molecule contained on a scaffold according to the invention increases the efficacy of at least the current and new effector molecules defined in the present invention. Possible side effects are reduced by lowering the dosage of the effector molecule without reducing the efficacy. Thus, the present invention provides a scaffold according to the invention or a functionalized scaffold according to the invention for use in medicine or for use as a medicament. Therefore, an aspect of the present invention relates to a scaffold according to the invention for use as a medicament, the scaffold comprising at least an effector molecule of the invention and / or an antibody according to the invention, preferably both an effector molecule and an antibody. The use of a scaffold according to the invention or a functionalized scaffold according to the invention for the manufacture of a medicament is also provided. In particular, cancer medicine, especially classical chemotherapy medicines, are notorious for their side effects. Due to the synchronization and targeting in time and space of both the drug substance and the glycoside molecule, the scaffold or the functionalized scaffold according to the invention is particularly useful for use as a medicament, in particular for use in a method for treating cancer. Therefore, the present invention provides a scaffold according to the invention or a functionalized scaffold according to the invention for use in a method for treating cancer. The invention also provides a scaffold according to the invention or a functionalized scaffold according to the invention for use in a method for the treatment of an acquired or genetic disorder, in particular a monogenic deficiency.Thus, an aspect of the invention relates to a scaffold according to the invention for use in a method for the treatment of cancer or an autoimmune disease, wherein the scaffold comprises a covalently attached carrier molecule, which carrier molecule comprises at least an effector molecule of the invention and / or an antibody according to the invention, preferably both an effector molecule and an antibody.

[0166] One aspect of the invention relates to a scaffold of the invention comprising antibodies and / or effector molecules for targeting tumor cells for use in the treatment or prevention of cancer, wherein the scaffold is administered to a human subject in need thereof.

[0167] Certain aspects of the invention relate to the treatment of a human subject suffering from or at risk of developing cancer and in need of said treatment, the treatment comprising administering to the human subject an effective dose of a pharmaceutical composition comprising a scaffold of the invention or a functionalized scaffold of the invention, said scaffold comprising an effector moiety together with a saponin, or a monoclonal antibody together with a saponin, or both an effector molecule and a monoclonal antibody together with a saponin. Certain aspects of the invention relate to a scaffold of the invention for use as a medicament. Certain aspects of the invention relate to a scaffold of the invention for use in a method for the treatment or prevention of cancer or an autoimmune disease in a human subject in need thereof. Certain aspects of the invention relate to the use of a scaffold or functionalized scaffold of the invention, or a pharmaceutical composition comprising said scaffold or functionalized scaffold, for the manufacture of a medicament for anti-cancer or anti-autoimmune disease treatment. The scaffold or functionalized scaffold preferably comprises at least a carrier molecule comprising an effector molecule and / or an antibody, preferably both an antibody and an effector molecule.

[0168] A further application in medicine is the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting disease can be hereditary or acquired. In most cases, only symptomatic treatment is possible, and in a number of rare diseases, insufficient treatment options lead to a shortened life span of the patient involved. An example of such a disease is phenylketonuria, an inborn error of metabolism that results in a decreased metabolism of the amino acid phenylalanine. The disease is characterized by a genetic mutation of the liver enzyme phenylalanine hydroxylase. Phenylketonuria is not curable to date. The incidence is approximately 1:10,000, with the highest known incidence being 1:2,600 in Turkey. A functionalized scaffold with a polynucleotide encoding phenylalanine hydroxylase or phenylalanine hydroxylase can be used to target liver cells and to replace defective enzymes in liver cells by using suitable ligands; a scaffold covalently linked to a carrier molecule comprising a polynucleotide encoding phenylalanine hydroxylase or phenylalanine hydroxylase can be used to target liver cells and to replace defective enzymes in liver cells by using suitable ligands.This is an example of the use of a scaffold comprising a carrier molecule according to the present invention or a functionalized scaffold according to the present invention for replacement or gene therapy.In a preferred embodiment, a scaffold according to the present invention or a scaffold comprising a carrier molecule according to the present invention or a functionalized scaffold according to the present invention is provided for use in gene therapy or replacement therapy methods.

[0169] The invention can also be used in biotechnological processes. A possible application is the biomolecular engineering of eukaryotic intracellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, translational switches target the process of turning the mRNA signal into a protein, and post-translational switches control how proteins interact with each other to attenuate or relay the signal. When optimized, these cellular switches can turn cellular functions "on" and "off" based on cues specified by the developer. These cues include small molecules, hormones, and drugs. To apply the switch, the cue must enter the target cell. Thus, in current applications, only small diffusible molecules can be used that are neither specific for the target cell nor highly specific for the selected switch. Functionalized scaffolds, or scaffolds containing carrier molecules with more complex and therefore more specific non-diffusible effector molecules, can be used to target specific switches, and the use of suitable ligands can restrict the effect to the target cells. An embodiment is the use of scaffolds containing the carrier molecules of the invention or functionalized scaffolds according to the invention to improve the effect of the effector molecules, preferably in vitro. Preferably the use is for improving the efficiency of an in vitro transcriptional switch.

[0170] Another application is the use of the invention in basic research. For functional analysis of cellular processes, it is often required to bring proteins into cells, a method called protein transfection. For example, to investigate the molecular mechanism of the chicken virus protein apoptin leading to apoptosis of eukaryotic cells, it is required to bring purified proteins into target cells. However, existing protein transfection kits are characterized by low efficiency, lacking specificity for target cells, and high toxicity, and therefore cannot be used for several applications, especially when metabolic pathways are part of the investigation. The use of scaffolds or functionalized scaffolds comprising the carrier molecule of the invention covalently linked with apoptin, both of which have apoptin, and suitable ligands, can be used to carry out such investigations. An embodiment is a scaffold of the invention, a scaffold comprising a carrier molecule according to the invention, or a functionalized scaffold according to the invention, preferably for in vitro polypeptide transfection. The use of a scaffold, a scaffold comprising a carrier molecule according to the invention, or a functionalized scaffold according to the invention, preferably for in vitro polynucleotide transfection, is also provided.

[0171] The present invention also provides a method of treating cancer, the method comprising administering to a patient in need thereof a medicament comprising a scaffold according to the present invention, or preferably a functionalized scaffold according to the present invention, or preferably a scaffold of the present invention comprising a carrier molecule comprising an effector molecule or a monoclonal antibody according to the present invention, preferably both a monoclonal antibody and an effector molecule, preferably administering an effective dose of said medicament to a patient in need thereof, preferably a human cancer patient.

[0172] The scaffold or functionalized scaffold may be Provide an expanded therapeutic window for current and new ADCs, where the ADCs may contain payloads such as toxins, protein toxins, oligonucleotides, BNAs, etc. can provide highly efficient cytosolic delivery of macromolecules; - Facilitates cell research and biotechnology applications; May have therapeutic potential for several diseases, such as cancer and autoimmune diseases, including rheumatoid arthritis; can be used to induce cell destruction (e.g., of cancer cells); - Reducing the levels of therapeutic drugs required by diseased cells can reduce unwanted side effects; - may reduce the risk of immune responses against effector molecules (because fewer effector molecules are needed and, without wishing to be bound by any theory, perhaps also because the pathway of antigen presentation via endosomes onto MHC molecules is blocked); - Opens up the possibility of highly efficient manipulation of genes, It may revive failed drug candidates, especially ADCs, by increasing their efficacy; be made from biocompatible and degradable and / or excretable materials; Represents a platform technology that can rely on endosomal pH-triggered gentle and non-toxic release of effector molecules. Flexibility is guaranteed by the possibility to use any type of ligand (e.g. antibodies, their fragments and domains, or aptamers) and effector molecules. An elaborate implementation of click chemistry can be used to provide a user-friendly interface to apply this technology to own ligands and effector molecules. The platform technology of the present invention offers different possibilities, such as the production of clickable scaffolds as stand-alone products (Figure 53), which allows the user to simply couple any of his effector molecules and / or ligands at his discretion, or the production of scaffolds that include functionalized scaffolds or carrier molecules covalently linked to it. The basic scaffolds are already coupled to effector molecules (e.g. proteinaceous toxins in Table A5) and / or to ligands (e.g. antibodies in Tables A2, A3, A4). This allows the user to couple his ligands to guide the effector molecules to the desired target cells (Figure 54). A possible scaffold with a covalently coupled carrier molecule or a possible functionalized scaffold is a scaffold linked to a ribosome inactivating protein, e.g. dianthin, saporin. This toxic enzyme with high potential of killing target cells can be used to click on antibodies already on the market or any future antibodies designed to specifically recognize tumor cells, e.g. trastuzumab, cetuximab, rituximab, gemtuzumab, OKT-9, or obinutuzumab (next generation ADC technology), or any of the antibodies listed in the previous embodiments and in Tables A2-A4. As nucleic acid effector molecules, microRNA (miRNA, polynucleotide) or miRNA inhibitors or LNA or BNA can be used, for example, to create a functionalized scaffold for efficient and low dose cytosolic delivery. miRNA or miRNA inhibitors have high potential as novel therapeutics that can change the genetic program in cells and thereby change cell function.

[0173] The present invention further provides a method for producing a scaffold, preferably a scaffold according to the present invention, comprising at least one glycoside molecule capable of improving the effect of an effector molecule bound to a polymeric or oligomeric structure, the method comprising: providing a polymeric or oligomeric structure; and coupling at least one glycoside molecule to said polymeric or oligomeric structure. Preferably, the at least one glycoside molecule increases the endosomal escape of said effector molecule. Preferably, the glycoside is any of the saponins listed in Table A1 and Scheme I according to the present invention. In particular, the scaffold thus obtained increases the endosomal escape of said effector molecule. Preferably, the at least one glycoside molecule is a bisdesmosidic triterpene, more preferably a bisdesmosidic triterpene saponin, more preferably belonging to the type of 12,13-dehydrooleanane with an aldehyde function at position 23, more preferably a saponin that can be isolated from Gypsophila or Saponaria species, most preferably SA1641 and / or SO1861, or any of their diastereomers. Preferably, the at least one glycoside molecule is coupled to the polymeric or oligomeric structure via a cleavable bond, preferably said cleavable bond undergoes cleavage under acidic, reductive, enzymatic or photoinduced conditions. More preferably, the cleavable bond is an imine, hydrazone, oxime, 1,3-dioxolane, disulfide or ester, more preferably a disulfide or hydrazone bond. Where the bond is a cleavable bond, the saponin is preferably attached to the backbone via an aldehyde functionality at position 23 on the saponin or via one of the carboxyl groups, more preferably via an aldehyde functionality.

[0174] An embodiment is a scaffold of the invention, or a scaffold attached to a carrier molecule according to the invention, or a functionalized scaffold of the invention, in which at least one glycoside molecule is attached to the polymer or oligomeric structure via a stable bond. An embodiment is a scaffold of the invention, or a scaffold comprising a covalently attached carrier molecule according to the invention, in which at least one glycoside molecule is a saponin, and the stable bond between the saponin and the scaffold preferably occurs by amide coupling or amine formation. This is achieved, for example, via carbodiimide-mediated amide bond formation with an amino group of the polymer or oligomeric structure and an activated glucuronic acid group of the saponin. Chemical bonds that meet the stable conditions can also be used for aldehyde coupling, for example, certain amines derived after reductive amination, which require a primary amine group as a functional group of the polymer or oligomeric structure. If the bond is a stable bond, the saponin is preferably attached to the scaffold via one of the carboxyl groups of the saponin.

[0175] Preferably, the scaffold further comprises a click chemistry group for coupling to a carrier molecule, such as an effector molecule and / or a ligand and / or a monoclonal antibody (fragment, domain thereof), preferably both the effector molecule and an immunoglobulin. The immunoglobulin is preferably a monoclonal antibody of Tables A2, A3, A4. The monoclonal antibody and the effector molecule together preferably form an ADC according to the invention, such as an ADC of Table A4. Preferably, the click chemistry group is a tetrazine, azide, alkene or alkyne, or a cyclic derivative of these groups, such as a cyclooctyne (e.g. aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]nona4-yne or dibenzocyclooctyne).

[0176] An embodiment is a method according to the invention for producing a scaffold, preferably a scaffold of the invention, in which the number of glycoside molecules is a defined number or a defined range. Preferably, the polymer or oligomer structure comprises a linear, branched or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or aggregates of these structures, either pure or mixed. The aggregates may be assembled by covalent crosslinking or non-covalent attraction to form hydrogels or nanogels. Preferably, the polymer is a derivative of polyethyleneimine, polyethylene glycol, polyamino acid, or DNA polymer, or the oligomer or polymer is a derivative of dextran, lactic acid, nucleic acid, or peptide nucleic acid. In a preferred embodiment, the effector molecule is a drug substance, such as a toxin, a drug, a polypeptide, and / or a polynucleotide.

[0177] Also provided is a method for producing a scaffold comprising a carrier molecule according to the invention or a functionalized scaffold of the invention, the method comprising: providing a scaffold comprising a plurality of glycoside molecules and a polymeric or oligomeric structure, preferably a scaffold according to the invention or obtainable by a method according to the invention for producing a scaffold; and coupling to said scaffold either: a) at least one effector molecule, b) at least one ligand, e.g. a monoclonal antibody, c) at least one effector molecule and additionally at least one ligand, preferably a monoclonal antibody targeting a tumor cell receptor, d) at least one effector, itself having at least one ligand, e.g. a monoclonal antibody targeting a tumor cell receptor, itself having at least one effector. Preferably, the coupling in a) to e) occurs independently by click chemistry coupling. An embodiment is a method of the invention, wherein in c) the scaffold is coupled to at least one effector molecule and additionally at least one ligand, preferably a monoclonal antibody targeting a tumor cell receptor. Both the effector molecule and the ligand (e.g., monoclonal antibody) are linked to a linker that is itself linked to the backbone. Based on this disclosure and common general knowledge, a person skilled in the art can design such a trifunctional linker, for example the trifunctional linker of scheme II and structure B (see also FIG. 16). Such a trifunctional linker can exhibit, for example, a maleimide group that can be used for conjugation to a targeting ligand that exhibits a thiol group for performing a thiol-ene reaction. In addition, the trifunctional linker can exhibit a dibenzocyclooctyne (DBCO) group for performing the so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with an azide-bearing saponin. Finally, the trifunctional linker can obtain a third functional group, such as a trans-cyclooctyne (TCO) group, to perform the so-called inverse electron demand Diels-Alder (IEDDA) reaction with a tetrazine (Tz)-bearing effector molecule.An embodiment is a method of the invention, wherein the at least one effector molecule is a drug substance, such as a toxin, a drug, a polypeptide, or a polynucleotide. An embodiment is a method of the invention, wherein the at least one effector molecule is a toxin or a polynucleotide. Preferably, the at least one ligand, such as a monoclonal antibody for binding to a tumor cell receptor, is capable of specifically binding to a target cell-specific surface molecule or structure capable of undergoing endocytosis. Preferably, it is an antibody or a fragment thereof, a cytokine, a growth factor, an aptamer, or a designed ankyrin repeat protein. Preferably, the target cell is a diseased or disease-associated cell, preferably a tumor cell, a tumor-associated cell (e.g., tumor vascular cell), an immune cell (e.g., regulatory T cell), or a cell with a monogenic defect. An embodiment is a method according to the invention, a method for producing a scaffold comprising a functionalized scaffold or carrier molecule according to the invention, wherein the at least one effector molecule is coupled to the scaffold and / or to the at least one ligand via a cleavable bond. Preferably, said cleavable bond undergoes cleavage under acidic, reductive, enzymatic or photoinduced conditions.An embodiment is a method according to the invention for producing a scaffold or a functionalized scaffold, the method further comprising coupling said scaffold or functionalized scaffold to a carrier, said carrier being preferably a type of nanoparticle, liposome, micelle, colloid or particle-like structure comprising cholesterol and / or phospholipids.

[0178] The invention provides pharmaceutical compositions, comprising a scaffold according to the invention, or a functionalized scaffold according to the invention, or a scaffold comprising a covalently attached carrier molecule according to the invention and optionally an acceptable pharmaceutical carrier, for use in the treatment of a patient, in particular for use in the treatment of cancer or an acquired or inherited disorder, in particular a monogenic deficiency.

[0179] Another aspect of the invention features a pharmaceutical composition comprising a compound or combination of compounds according to the invention and a physiologically acceptable carrier. A "pharmaceutical composition" refers to a composition in a form suitable for administration to a mammal, preferably a human. Preferably, the pharmaceutical composition contains a sufficient amount of a compound according to the invention in a suitable pharmaceutical form to exert a therapeutic effect on a human.

[0180] Considerations regarding suitable forms for administration are known in the art and include toxicological effects, solubility, route of administration, and maintaining activity. For example, a pharmacological composition injected into the blood stream should be soluble.

[0181] Suitable dosage forms will depend in part on the use or route of entry, for example, by transdermal or injection. Such dosage forms should allow the compound to reach the target cells, whether or not they are present in a multicellular host. Other factors are known in the art and include considerations such as dosage forms that slow the compound or composition from exerting its effect and toxicity.

[0182] In one embodiment is a scaffold of the invention, the scaffold comprising a defined number or range of glycosides. In one embodiment is a scaffold of the invention, the scaffold comprising a defined number or range of glycosides, the defined range being between 1-30 glycoside(s), preferably between 1-20, more preferably between 1-10, more preferably between 1-6, more preferably between 2-6, more preferably between 2-5, more preferably between 3-5, more preferably between 3-4 glycosides.

[0183] An embodiment is a scaffold of the invention, the scaffold comprising a covalently attached carrier molecule, the carrier molecule comprising a binding site for targeting the cell, and e.g. being or comprising a (monoclonal) antibody for binding to a cell surface receptor on the target cell. An embodiment is a scaffold of the invention, the scaffold comprising a covalently attached carrier molecule, the carrier molecule comprising a binding site for targeting the cell, and e.g. being or comprising a (monoclonal) antibody for binding to a cell surface receptor on the target cell. The target cell is a diseased or disease-associated cell, preferably a tumor cell or tumor-associated cell (e.g. tumor vascular cell), or an immune cell (e.g. regulatory T cell), or an autoimmune cell.

[0184] One embodiment is a scaffold of the invention, wherein the biologically active molecule is a glycoside, said glycoside being capable of increasing the endosomal escape of an effector molecule carried by a carrier molecule covalently attached to the scaffold.

[0185] One embodiment is a scaffold of the invention, wherein the scaffold is part of a pharmaceutical composition, which further comprises at least one additional active pharmaceutical ingredient, such as an additional immunoglobulin, in addition to the scaffold.

[0186] One embodiment is a scaffold of the invention or a pharmaceutical composition according to the invention for use in a method of treating cancer or an autoimmune disease.

[0187] An embodiment is a scaffold of the invention for use in a method of treating cancer, the method comprising administering to a patient in need thereof a scaffold of the invention, the scaffold comprising a covalently attached carrier molecule comprising or consisting of an effector molecule of the invention and / or a ligand or cell-targeting antibody of the invention.

[0188] An embodiment is the scaffold of the invention for use in a method of treating cancer, the method comprising administering to a patient in need thereof a pharmaceutical composition according to the invention.

[0189] [Table 1]

[0190]

Table 2

[0191]

Table 3

[0192]

Table 4

[0193]

Table 5

[0194]

Table 6

[0195]

Table 7

[0196]

Table 8

[0197]

Table 9

[0198]

Table 10

[0199]

Table 11

[0200]

Table 12

[0201]

Table 13

[0202]

Table 14

[0203]

Table 15

[0204]

Table 16

[0205]

Table 17

[0206]

Table 18

[0207]

Table 19

[0208]

Table 20

[0209]

Table 21

[0210]

Table 22

[0211]

Table 23

[0212]

Table 24

[0213]

Table 25

[0214]

Table 26

[0215]

Table 27

[0216]

Table 28

[0217]

Table 29

[0218]

Table 30

[0219]

Table 31

[0220]

Table 32

[0221] [Table 33]

[0222] [Table 34]

[0223] [Table 35]

[0224] [Table 36]

[0225] [Table 37]

[0226] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. EXAMPLES

[0227] Example A - Treatment of mammalian tumor-bearing animals with a conjugate of the invention in combination with an ADC results in survival and tumor regression Female Balb / c nude mice were injected subcutaneously with a suspension of human A431 tumor cells. Human epidermal carcinoma was developed under the skin of the mice in a xenograft animal tumor model. After tumor cell injection, the xenograft tumors grew to approximately 170-180 mm 3 The A431 tumor cells were allowed to develop to a size of 100 μg / ml. The A431 tumor cells have the following characteristics: high EGFR expressor, intermediate CD71 expressor, and low HER2 expressor.

[0228] In Table A, the results of treatment of control and tumor-bearing mice are presented. Tumor-bearing mice were treated with the indicated antibodies directed against either human Her2 / neu, human EGFR, or human CD71, cell surface receptors on xenograft tumors. Cetuximab was covalently conjugated with the saponin SO1861. First, SO1861 was provided with a linker EMCH (N-ε-maleimidocaproic acid hydrazide), which is a maleimide and hydrazide crosslinker for covalently conjugating a sulfhydryl (reduced cysteine ​​of an antibody) to a carbonyl (aldehyde or ketone; here, the carbonyl of the aldehyde at position C-23 of the saponin). The saponin-EMCH was covalently coupled to the reduced cysteine ​​of cetuximab, forming a thio-ethyl covalent bond between EMCH and the cysteine ​​side chain. The ADCs trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9, IgG)-saporin (covalent conjugate) were tested for their tumor attack efficacy in mice. Tumor volume was measured over time after the start of treatment with the ADC. The dose of the ADC was suboptimal in the tumor model; previous experiments established that at a suboptimal dose of the ADC, tumor regression or tumor growth cessation would not be observable.

[0229] [Table 38]

[0230] These results demonstrate that combination therapy of ADCs at doses that are ineffective when treatment of tumor-bearing mice with ADCs alone is considered (tumor growth, mouse death is not prevented (euthanasia)) with the conjugates of the present invention consisting of an antibody targeting a tumor cell-specific receptor covalently linked to saponin, SO1861, provides an efficient and effective treatment regimen expressed as regressing tumors and sustained survival of treated animals (over the duration of the experiment). The covalent conjugates are administered to cancer-bearing mice at doses that are ineffective when administered alone (tumor growth, mouse death is not prevented (euthanasia)). Thus, suboptimal doses of ADCs combined with conjugates containing covalently linked saponins of the present invention that have no antitumor activity when administered alone provide an effective treatment option for cancer patients, and relatively low doses of ADCs are effective. Lower doses of ADCs hold the promise of less risk of adverse events, or even no side effects at all. In addition, the stimulating effect of the conjugates with saponins of the present invention when considering the efficacy of ADCs indicates that ADCs previously found to lack efficacy when treating tumor patients are concerned may gain renewed attention and value, since their efficacy is improved in combination therapy conditions, as demonstrated by this example. Reference is made to Tables A2 and A3, which summarize ADCs that were previously examined in human clinical conditions, but then some ADCs were withdrawn from further clinical examination. In particular, ADCs whose clinical development was terminated due to an observed lack of efficacy and / or due to the occurrence of unacceptable adverse events are ADCs that may gain renewed value for cancer patients when combined with conjugates containing covalently attached saponins of the present invention, such as the tested cetuximab-saponin.

[0231] Example B - Quillaja saponaria saponin mixture containing QS-21 with endosomal / lysosomal escape enhancing activity Scheme I shows the common molecular structure of a series of QS-21 saponins (Conrado Pedebos, Laercio 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 endosomal / lysosomal escape enhancing conjugates, compositions, and combinations of the present invention based on the endosomal / lysosomal escape enhancing properties of at least one individual saponin present in the mixture, e.g., QS-21, or based on a combination of two or more saponins contained by the mixture, e.g., QS-21 and QS-7.

[0232] The inventors have demonstrated that a 2.5 microgram / ml dose of a mixture of saponins from Quillaja saponaria can enhance the endosomal escape of dianthine when tested in mammalian tumor cells by cell-based bioassay. The effector moiety exposed to the cells was dianthine covalently linked to the ligand EGF: EGF-dianthine. The cells tested were the tumor cell lines HeLa for free saponins, and A431, MDA-MB-468, CaSki, and A2058 to test saponins when covalently coupled to cetuximab.

[0233] Example 1 A trifunctional linker scaffold with specific chemical end groups (DBCO, TCO) for conjugation (labile (L) conjugation) with a SO1861 molecule on one arm and an antisense HSP27BNA oligonucleotide (targeting the cancer target hsp27 mRNA in cancer cells and inducing its degradation) on the other arm was designed and produced to produce SO1861-L-trifunctional linker-L-HSP27BNA (Figure 16). SO1861-L-trifunctional linker-L-HSP27BNA was conjugated by its third arm (maleimide) to the cysteine ​​residue (Cys) anti-EGFR antibody cetuximab (cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 4 ) was conjugated to

[0234] Conjugates containing this scaffold were tested for EGFR-mediated tumor-targeted gene silencing activity in the A431 xenograph "nude" mouse tumor model. 3 Dosing was initiated on day 12 when IL-16 was reached, and tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression compared to cellular control mRNA expression (reference gene). 3,7 One of the drugs was cetuximab (Cys-L-SO1861) 3,8 or Cetuximab-(Lys-L-HSP27BNA) 4 It was found that conjugated SO1861 leads to a 40% reduction in HSP27 gene expression compared to a single dose of monotherapy (Figure 1). Compared to vehicle control tumors, a 25% reduction in gene silencing was observed. This indicates and enables that conjugated SO1861 can efficiently induce targeted delivery of therapeutic oligonucleotides in tumors in vivo.

[0235] To further strengthen this, cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA DAR4) 4was tested for enhanced HSP27 gene silencing in EGFR expressing (A431) in vitro as illustrated in Figure 2. Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 3,7 Cetuximab-(Lys-L-HSP27BNA) 4 or Cetuximab-(Cys-L-SO1861) 3,8 Compared with riboflavin alone, it efficiently induces HSP27 gene silencing in A431 cells (Figure 2).

[0236] Example 2 The one-target, two-component system is illustrated in Figure 11. n ) n and mAb1-effector combination treatment, the two-target two-component system is mAb1-(Dendron (SO1861)) as illustrated in FIG. n ) n +mAb2-effector combination.

[0237] Dendron (-L-SO1861) 4 Cetuximab-Cys-(Dendron(-L-SO1861) 4 ) 3,9 , was conjugated to the anti-EGFR antibody cetuximab via cysteine ​​residue (Cys) conjugation by DAR3,9 and tested for enhanced cell killing activity in combination with an anti-EGFR antibody-protein toxin conjugate (cetuximab-saporin) in EGFR-expressing cells (MDA-MB-468). Cetuximab-Cys-(Dendron(-L-SO1861) 4 ) 3,9 +10 pM cetuximab-saporin efficiently induced toxin-mediated cell killing in high EGFR expressing cells, which was consistent with cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9or cetuximab (equivalent) + 10 pM cetuximab-saporin or cetuximab (Figure 3A). Similar experiments in cells expressing low levels of EGFR (HeLa) showed that cetuximab-Cys-(Dendron(-L-SO1861) 4 ) 3,9 (Fig. 3C), indicating that in the absence of sufficient EGFR receptor expression, an effective intracellular SO1861 concentration (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing is not reached.

[0238] Next, dendron (-L-SO1861) 4 Trastuzumab-Cys-(Dendron(-L-SO1861) 4 ) 4 It was conjugated to the anti-HER2 antibody trastuzumab by cysteine ​​conjugation (Cys) with DAR4 and tested for enhanced cell killing activity in HER2 expressing cells (SK-BR-3) in combination with an anti-HER2 antibody-protein toxin conjugate (trastuzumab-saporin). Trastuzumab-Cys-(Dendron(-L-SO1861) 4 ) 4 +50 pM trastuzumab-saporin efficiently induced toxin-mediated cell killing, which was consistent with trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 No induction was observed with trastuzumab (equivalent) + 50 nM trastuzumab-saporin or trastuzumab (Figure 3B). Similar experiments in cells expressing low levels of HER2 (JIMT-1) showed that trastuzumab-Cys-(Dendron(-L-SO1861) 4 ) 4 (Figure 3D), indicating that in the absence of sufficient HER2 receptor expression, an effective intracellular SO1861 concentration (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing is not reached.

[0239] Next, Cetuximab-Cys-(Dendron(-L-SO1861) 4 ) 3, 9 or Cetuximab-Lys-(Dendron (-L-SO1861) 4 ) 4,4 (Lys = dendron conjugated to lysine of antibody (-L-SO1861) 4 ) was tested in combination with 10 pM CD71mab-saporin as a two-target two-component system in EGFR++ / CD71+ cells (MDA-MB-468). This showed a strong enhancement of cell killing activity for both conjugates, which was greater than that of cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 , or Cetuximab-Lys-(Dendron(-L-SO1861) 4 ) 4,4 , or cetuximab (equivalent) + 10 pM CD71mab-saporin, or cetuximab (Figure 4A). Similar experiments in cells expressing low levels of EGFR (CaSKi, EGFR+ / CD71+) showed no activity in cells expressing cetuximab-Cys-(Dendron(-L-SO1861) compared to the activity in high expressers (Figure 4A). 4 ) 3,9 or Cetuximab-Lys-(Dendron(-L-SO1861) 4 ) 4,4 (Figure 4C) Both revealed reduced activity, indicating that in cells with lower EGFR receptor expression levels, the effective intracellular SO1861 concentration was lower, resulting in reduced toxin-mediated cell killing activity.

[0240] The same experiment was performed with trastuzumab-Cys-(Dendron(-L-SO1861) in combination with CD71mab-saporin. 4 ) 4 or Trastuzumab-Lys-(Dendron(-L-SO1861) 4 ) 4,7was performed on the HER2++ / CD71+ (SK-BR-3) cell line and revealed strong cell killing activity compared to the control (Figure 4B). 4 ) 4 or Trastuzumab-Lys-(Dendron(-L-SO1861) 4 ) 4,7 When tested in combination with 10 pM CD71mab-saporin on HER2+ / - / CD71+ (JIMT-1), no cell killing activity could be observed, indicating that in the absence of sufficient HER2 receptor expression, an effective intracellular SO1861 concentration (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing is not reached.

[0241] Next, Trastuzumab-Cys-(Dendron(-L-SO1861) 4 ) 4 +Trastuzumab-emtansine (T-DM1, an antibody-small molecule toxin conjugate) was tested in HER2-expressing cells (SK-BR-3) for enhanced cell killing activity. Because the endosomal membrane does not form a barrier for small molecules to reach the cytoplasm, no enhanced cell killing was observed with this combination compared to T-DM1 alone or T-DM1 + ​​an equivalent amount of trastuzumab (Figure 5).

[0242] Example 3 Materials and Methods Dendron (SO1861) 4-BNA oligo synthesis (Figure 17) HSP27BNA oligodisulfide (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 h, the reaction mixture was filtered by using a centrifugal filter with a molecular weight cutoff of 3000 Da (14000×g, 30 min). The residual 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 described above. The residual solution was diluted with 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 1.0 mL), and the resulting mixture was added to dendron (SO1861) 4-maleimide 1 (3.54 mg, 0.375 μmol) (Figure 17). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 10 min, the reaction mixture was analyzed by preparative LC-MS 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (1.25 mg, 85%) as a white fluffy solid. 94% purity based on LC-MS. LRMS(m / z):1896[M-8] 8- , 2167[M-7] 7- LC-MSr.t.(min):3.77 6B result HSP27BNA oligo (antisense BNA oligo targeting heat shock protein 27 of cancer target mRNA transcript (HSP27BNA)) was inserted into dendron (-L-SO1861) 4 Conjugated to (HSP27BNA-Dendron(-L-SO1861) 4 , Figure 17) and co-administered to A431 cancer cells. As a readout, gene silencing of HSP27 mRNA in A431 cells was determined. This was achieved by administering HSP27BNA-dendron (-L-SO1861) 4 We demonstrated that treatment with HSP27BNA resulted in improved HSP27 gene silencing activity compared to HSP27BNA alone (Figure 6).

[0243] Example 4 method SO1861 release assay To the dendron (SO1861)4-Cbz (0.05 mg) (Figure 7A) was added 50 μL of a solution containing water / acetonitrile / TFA (1.00 mL / 1.00 mL / 4 drops). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. SO1861 release was monitored by UPLC-MS. 4 was used to track the results over time. result Dendron (-L-SO1861) under acidic conditions 4 The release efficiency of SO1861 molecules from the dendron (-L-SO1861) was determined (Figure 7A). Figure 7B shows the release efficiency of SO1861 molecules from the dendron (-L-SO1861). 4 Shown are UPLC UV traces (PDA) of the reaction mixture as is (top), after 30 min (middle), and after 1.5 h (bottom). Figure 7C shows the interpretation of the observed m / z values ​​by LRMS. The following m / z values ​​with corresponding UVr.t. (min) were observed: rt=1.46, 2282 [M-4] 4- (A, dendron (-L-SO1861) 4 );rt=1.43, 2427[M-3] 3- (B, dendron (-L-SO1861) 3 );rt=1.38, 1812[M-3] 3- (C, dendron (-L-SO1861) 2 );rt=1.29, 1797[M+2] 2+ (D, Dendron-L-SO1861); rt=1.22, 1862[M-1] 1- (E, SO1861);rt=1.05, 1747 / 1769[M+1 / M+23] 1+ (F, dendron-).

[0244] Next, dendron (-L-SO1861) 4 was tested for enhanced delivery of the targeted toxin EGF-dianthine in EGFR-expressing cells (A431 and HeLa). 4+10 pM EGF-dianthin can induce enhanced toxin-mediated cell killing, but not "naked" dendrons (Dendron (NEM) 4 ) or dendron (-L-SO1861) 4 , or dendron (NEM) 4 +10 pM EGF-gamma did not appear to provide enhanced cell killing at these concentrations (FIGS. 8A, 8B).

[0245] Example 5 Dendron (-L-SO1861) n Synthesis (Figures 13, 14, and 15) Materials and Methods Abbreviation DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide EDCI·HCl 3-((ethylimino)methyleneamino)-N,N-dimethylpropan-1-aminium chloride EMCH.TFA N-(ε-Maleimidocaproic acid) hydrazide, trifluoroacetate min rt retention time TCEP Tris(2-carboxyethyl)phosphine hydrochloride Temp Temperature TFA Trifluoroacetic acid THF Tetrahydrofuran Analysis method LC-MS method 1, 1 Equipment: Agilent 1200 Bin. Pump: G1312A, Degasser; Autosampler, ColCom; DAD: Agilent G1316A, 210, 220, and 220-320 nm; PDA: 210-320 nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-1000; ELSD Alltech 3300 gas flow 1.5 ml / min, gas temp: 40°C; Column: Waters XSelect™ CSH C18, 30 x 2.1 mm, 3.5 μm, Temp: 35°C, Flow: 1 mL / min, Gradient: t0 = 5% A, t 1.6min = 98%A, t 3min = 98% A, post time: 1.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water

[0246] LC-MS method 2, 2 Instrument: Agilent 1260 Bin. Pump: G7112B, Multisampler, Column Comp. DAD: Agilent G7115A, 210, 220, and 220-320 nm. PDA: 210-320 nm. MSD: Agilent LC / MSD G6130B ESI. Mass range depends on product molecular weight: A pos / neg 100-1000 B pos / neg 100-1400 ;ELSD Alltech 3300 gas flow 1.5ml / min, gas temp: 40°C; Column: Waters XSelect™ C18, 30×2.1mm, 3.5μm, Temp: 40°C, Flow: 1mL / min, Gradient: t0=5%A, t1. 6min = 98%A, t 3min = 98% A, post time: 1.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water

[0247] LC-MS method 3, 3 Equipment: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, pos / neg 800-1500; ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Waters XSelect™ CSH C18, 50×2.1 mm, 2.5 μm Temp: 25°C, Flow: 0.6 mL / min, Gradient: t0=5% A, t 2.0min = 98%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0248] LC-MS method 4, 4 Equipment: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, pos / neg 1500-2500; ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Waters XSelect™ CSH C18, 50×2.1 mm, 2.5 μm Temp: 25°C, Flow: 0.6 mL / min, Gradient: t0=15% A, t 2.0min = 60%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0249] LC-MS method 5, 5 Instrument: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight: A pos / neg 1500-2500 B neg 2000-3000 ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Acquity C18, 50×2.1 mm, 1.7 μm Temp: 60°C, Flow rate: 0.6 mL / min, Gradient: t0=2%A, t 5.0min = 50%A, t 6.0min = 98% A, post time: 1.0 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0250] Preparation method Preparative MP-LC method 1, 1 Instrument type: Revelis™ prep MPLC; Column: Waters XSelect™ CSH C18 (145×25 mm, 10μ); Flow rate: 40 mL / min; Column temp: room temperature; Eluent A: 10 mM ammonium bicarbonate in water pH=9.0); Eluent B: 99% acetonitrile + 1% 10 mM ammonium bicarbonate in water; Gradient: t 0min = 5% B, t 1min = 5% B, t 2min = 10% B, t 17min =50%B,t 18min =100%B,t 23min = 100% B; detection UV: 210, 225, 285 nm.

[0251] Preparative MP-LC method 2, 2 Instrument type: Revelis™ prep MPLC; Column: Phenomenex LUNA C18(3) (150×25 mm, 10μ); Flow rate: 40 mL / min; Column temp: room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: t 0min =5% B,t 1min =5% B,t 2min =10% B,t 17min =50% B,t 18min =100% B,t 23min = 100% B; detection UV: 210, 225, 285 nm.

[0252] Preparative LC-MS method 1,3 MS instrument type: Agilent Technologies G6130B quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect™ CSH (C18, 100×30 mm, 10μ); Flow rate: 25 ml / min; Column temp: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH=9.0; lin. gradient depending on product polarity: A t0=20%A, t 2min = 20%A, t 8.5min = 60%A, t 10min = 100%A, t 13min =100%A B t0=5%A, t 2min = 5%A, t 8.5min = 40%A, t 10min = 100%A, t 13min =100%A C t0=10%A, t 2min = 10%A, t 8.5min = 50%A, t 10min = 100%A, t 13min =100%A Detection: DAD (220-320 nm); Detection: MSD (ESI pos / neg) mass range: 100-800; Fraction collection based on DAD.

[0253] Preparative LC-MS method 2, 4 MS instrument type: Agilent Technologies G6130B quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Shield (C18, 150×19 mm, 5μ); Flow rate: 25 ml / min; Column temp: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH=9.0; lin. gradient: t0=5% A, t 2.5min = 5%A, t 11min = 40%A, t 13min = 100%A, t 17min= 100%A; Detection: DAD (220-320 nm); Detection: MSD (ESI pos / neg) Mass range: 100-800; DAD based on fraction collection Flash Chromatography Grace Reveleris X2® C-815 Flash; Solvent delivery system: 3 piston pump, autopriming, 4 independent channels in one run, up to 4 solvents, auto switch lines when solvent runs out; Maximum pump flow rate 250mL / min; Maximum pressure 50bar (725psi); Detection: UV 200-400nm, combination of up to 4 UV signals, scanning full UV range, ELSD; Column size: 4-330g on instrument, Luer type, 750g to 3000g with optional holder.

[0254] SO1861-EMCH synthesis (Figure 13) To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 h, the reaction mixture was subjected to preparative MP-LC. 1 Fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. 96% purity based on LC-MS. LRMS(m / z):2069[M-1] 1- LC-MSr.t.(min):1.08 4 Dendron (-L-SO1861) 4 Synthesis (Figure 14)

[0255] Intermediate 1: Di-tert-butyl(((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate 6-Azidohexanoic acid (0.943 g, 6.00 mmol), EDCI.HCl (1.21 g, 6.30 mmol), and OxymaPure (0.938 g, 6.60 mmol) were dissolved in DMF (10.0 mL) and the mixture was stirred for 5 min. 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 h, the reaction mixture was evaporated in vacuum and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (2×50 mL), and brine (50 mL), dried over Na2SO4, filtered, and evaporated in vacuum. The residue was purified by flash chromatography (ethyl acetate-heptane gradient, 10:90 increasing to 100:0) to give the title compound (2.67 g, 100%) as a white solid. 98% purity based on LC-MS. LRMS(m / z):287 / 343 / 465 [M-155 / M-99 / M+23] 1+ LC-MSr.t.(min):2.02 2A

[0256] Intermediate 2: N,N-Bis(2-aminoethyl)-6-azidohexanamide dihydrochloride To di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate (2.66 g, 6.00 mmol) was added HCl in isopropanol (5-6 N, 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 (3×20 mL) to give the crude title product (1.49 g, 79%) as a white solid. LRMS(m / z):243[M+1] 1+

[0257] Intermediate 3: Tetra-tert-butyl ((5S,5S)-((((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(6-oxohexane-6,1,5-triyl))tetracarbamate To a solution of N,N-bis(2-aminoethyl)-6-azidohexanamide dihydrochloride (1.19 g, 3.76 mmol) in DMF (30.0 mL) and DIPEA (2.62 mL, 15.1 mmol), Boc-Lys(Boc)-ONp (3.69 g, 7.90 mmol) was added and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuum and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 1N potassium bisulfate solution (100 mL) and saturated sodium bicarbonate solution (5×100 mL), dried over Na2SO4, filtered and evaporated in vacuum. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (3.07 g, 91%) as a slightly yellowish solid. Purity 94% based on LC-MS. LRMS(m / z):800 / 900 / 922 [M-99 / M+1 / M+23] 1+ LC-MSr.t.(min):2.17 2A

[0258] Intermediate 4: 4-Nitrophenyl 3-(acetylthio)propanoate 4-Nitrophenyl trifluoroacetate (5.17 g, 22.0 mmol) and 3-(acetylthio)propionic acid (2.96 g, 20.0 mmol) were dissolved in DCM (50.0 mL). DIPEA (6.97 mL, 40.0 mmol) was then added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuum and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (5×50 mL), and brine (50 mL), dried over Na2SO4, filtered, and evaporated in vacuum. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (4.90 g, 91%) as a slightly yellowish solid. Purity 99% based on LC-MS. LRMS(m / z):292[M+23] 1+ LC-MSr.t.(min):1.94 2A

[0259] Intermediate 5: (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamide)ethyl)hexanamide)ethyl)hexanamide tetrahydrochloride 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 HCl in isopropanol (5-6N, 50.0 ml, 275 mmol) 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 x 20 mL) to give the crude title product as a white solid. LRMS(m / z):250 [M+2] 2 +, 500 [M+1] 1+

[0260] Intermediate 6: (2S)-2,6-Bis[3-(acetylsulfanyl)propanamido]-N-[2-(6-azido-N-{2-[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]ethyl}hexanamido)ethyl]hexanamido To a solution of (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamido)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 vacuum 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 vacuum. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (1.33 g, 65%) as a white solid. An impurity (15%) was found by LC-MS with an m / z value corresponding to the product with one deprotected thioacetate group. The impurity was formed during or after workup. Purity 85% based on LC-MS. LRMS(m / z):510 [M+2] 2+ , 1019 / 1041 [M+1 / M+23] 1+ LC-MSr.t.(min):1.86 2B

[0261] Intermediate 7: N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamido)formate Scaffold 2 (102 mg, 0.100 mmol) was dissolved in methanol (1.00 mL). Freshly prepared 1N sodium hydroxide solution (0.440 mL, 0.440 mmol) was then added and the reaction mixture was stirred at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (0.500 mL, 0.500 mmol) was added and the resulting mixture was stirred at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and coevaporated 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 analyzed by preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (75.6 mg, 87%) as a colorless sticky oil. 96% purity based on LC-MS. LRMS(m / z):513 [M+2] 2+ , 825 [M+1] 1+ LC-MSr.t.(min):1.42 2A

[0262] Intermediate 8: Dendron (-L-SO1861) 4 -Amine N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamido) formate (2.73 mg, 3.13 μmol) was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TECEP / 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 h, the reaction mixture was analyzed by preparative LC-MS. 3B The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (12.3 mg, 43%) as a white fluffy solid. 97% purity based on LC-MS. LRMS(m / z):1517[M-6]6- ,1821[M-5] 5- ,2276[M-4] 4- LC-MSr.t.(min):4.39 5A

[0263] Intermediate 9: Dendron (-L-SO1861) 4-Azide 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). DIPEA (1.302 μL, 7.48 μmol) was then added and the mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (5.86 mg, 84%) as a white fluffy solid. 90% purity based on LC-MS. LRMS(m / z):2344[M-4] 4- LC-MSr.t.(min):4.78 5B

[0264] Intermediate 10: Dendron (-L-SO1861) 4-Maleimide 1 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). DIPEA (1.55 μL, 8.91 μmol) was then added and the mixture was shaken for 1 min and allowed to stand at room temperature. After 3 h, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (6.76 mg, 80%) as a white fluffy solid. Purity 66% based on LC-MS. LRMS(m / z):2358[M-4] 4- LC-MSr.t.(min):2.13 6C

[0265] Intermediate 11: Dendron (-L-SO1861) 4 -Maleimide 2 Scaffold 2 (5.10 mg, 5.00 μmol) was dissolved in methanol (100 μL). Freshly prepared 1N sodium hydroxide solution (22.0 μL, 22.0 μmol) was then added, the mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, 1.0M 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 coevaporated with methanol (2×5 mL). The resulting residue was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TCEP / acetonitrile (3:1 v / v, 3.242 mL). From this solution, 1000 μL was added directly to SO1861-EMCH (14.4 mg, 6.94 μmol, 4.5 equivalents compared to the backbone), the mixture was shaken for 1 min and left to stand at room temperature. After 10 min, the reaction mixture was lyophilized overnight. To the resulting residue was added 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(3-(2,5-dioxo-2h-pyrrol-1(5h)-yl)propanamido)ethoxy)ethoxy)propanoate (5.84 mg, 0.014 mmol) and DMF (1.00 mL). DIPEA (2.39 μL, 0.014 mmol) was then added, the suspension was shaken for 1 min and left to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (10.9 mg, 85%) as a white fluffy solid. Purity 80% based on LC-MS. LRMS(m / z):2354[M-4] 4- LC-MSr.t.(min):4.16 5B Dendron (-L-SO1861) 8 Synthesis (Figure 15)

[0266] Intermediate 1: tert-Butyl N-[(1S)-1-{[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis({[(tert- 2,6-bis({[(tert-butoxy)carbonyl]amino})hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis({[(tert-butoxy)carbonyl]amino})hexanamido]pentyl]carbamoyl}-5-{[(tert-butoxy)carbonyl]amino}pentyl]carbamate (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). Boc-Lys(Boc)-ONp (3.36 g, 7.18 mmol) was then 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 increasing to 0:100) to give the title product (2.71 g, 100%) as a white solid. Purity 97% based on LC-MS. LRMS(m / z):807[M-198] 2+ LC-MSr.t.(min):2.35 2B

[0267] Intermediate 2: (2S,2'S)-N,N'-((5S,15S,22S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamide)-6,14,21-trioxo-7,10,13,20-tetraazahexacosane-1,5-diyl)bis(2,6-diaminohexanamide)octahydrochloride Intermediate 1 (2.71 g, 1.50 mmol) was dissolved in HCl in isopropanol (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. LRMS(m / z):203 / 254[M-200 / M +4 ]4+,338[M+3] 3+ , 507[M+2] 2+ , 1012[M+1] 1+

[0268] Intermediate 3: (2S)-2,6-Bis[3-(acetylsulfanyl)propanamido]-N-[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]pentyl]hexanamido To (2S,2'S)-N,N'-((5S,15S,22S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamide)-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)propanoate (595 mg, 2.21 mmol). The resulting suspension was sonicated at 60° C. for 30 min and allowed to stir at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was purified first by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) and then by preparative MP-LC. 2to give the title product (70 mg, 15%) as a white solid. 100% purity based on LC-MS. LRMS(m / z):685[M+3] 3+ LC-MSr.t.(min):1.91 2A

[0269] Intermediate 4: (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamido formate Scaffold 4 (10.0 mg, 4.87 μmol) was dissolved in methanol (200 μL). Freshly prepared 1N sodium hydroxide solution (42.9 μL, 0.043 mmol) was then added and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (24.4 μL, 0.024 mmol) was added and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was diluted with water (1 mL) and analyzed directly by preparative MP-LC. 2 The 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. LRMS(m / z):564[M+3] 3+ , 846[M+2] 2+ LC-MSr.t.(min):1.54 2C

[0270] Intermediate 5: Dendron (-L-SO1861) 8 -Amine 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 with 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 analyzed directly by preparative LC-MS. 3B The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (2.17 mg, 40%) as a white fluffy solid. 97% purity based on LC-MS. LRMS(m / z):2282[M-8] 8- ,2607[M-7] 7- LC-MSr.t.(min):4.41 5A

[0271] Dendron (NEM) 4 Synthesis (Figure 18) To benzyl bis(2-((S)-2,6-bis(3-mercaptopropanamido)hexanamido)ethyl)carbamate (1.69 mg, 2.00 μmol) and N-ethylmaleimide (1.05 mg, 8.40 μmol) was added a mixture of 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 2.00 mL) and the reaction mixture was stirred at room temperature. After 2 h, the reaction mixture was lyophilized overnight. The resulting residue was analyzed by preparative LC-MS. 3A to give the title compound (1.53 mg, 57%) as a white fluffy solid. 98% purity based on LC-MS. LRMS(m / z):1346[M+1] 1+ LC-MSr.t.(min):1.43 3A

[0272] Example 6 SO1861-trifunctional linker-BNA oligo synthesis and tumor sample gene silencing analysis (Figure 16) Materials and Methods Trifunctional Linkers Trifunctional linkers (DBCO, TCO, maleimide) were ordered from Bio-Synthesis Inc. (Louisville, Texas). HSP27BNA oligo HSP27BNA(-thiol) oligo (sequence 5′-GGCacagccagtgGCG-3′) ( Zhang et al., 2011 ) was ordered from Bio-synthesis Inc. (Louisville, Texas).

[0273] Intermediate 1: SO1861-Azide To the mixture of SO1861 (60 mg, 0.032 mmol) and 1-azido-3,6,9,12-tetraoxapentadecane-15-hydrazide (39.3 mg, 0.129 mmol), methanol (extra dry, 1.00 mL) and TFA (9.86 μl, 0.129 mmol) were added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. 100% purity based on LC-MS. LRMS(m / z):2150[M-1] 1- LC-MSr.t.(min):1.10 3B

[0274] Intermediate 2: SO1861-Trifunctional Linker SO1861-azide (45 mg, 0.021 mmol) and trifunctional linker (26.5 mg, 0.022 mmol) were dissolved in DMF (2.50 mL) and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity 89% based on LC-MS. LRMS(m / z):1677[M-2]2- LC-MSr.t.(min):2.54 6A

[0275] Intermediate 3: (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide To 1-(4-formylbenzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (28.0 mg, 0.048 mmol) and EMCH.TFA (24.5 mg, 0.072 mmol) was added methanol (extra dry, 2.00 mL) and TFA (11.1 μL, 0.145 mmol) and the reaction mixture was stirred at 50° C. After 30 min, the reaction mixture was evaporated in vacuo and the resulting residue was analyzed by MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (33.4 mg, 88%) as a bright purple fluffy solid. 92% purity based on LC-MS. LRMS(m / z):394[M+2] 2+ ,789[M+1] 1+ LC-MSr.t.(min):1.28 7A

[0276] Intermediate 4: Methyltetrazine-BNA oligos HSP27 BNA oligodisulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). Then, TCEP (14.3 mg, 0.050 mmol) was added, the reaction mixture was shaken for 1 min, and allowed to stand at room temperature. The reaction mixture was filtered by using a centrifugal filter (5000×g, 30 min) with a molecular weight cut-off of 3000 Da. Then, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residual solution, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (30.0 mL) and the resulting mixture was added to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (14.8 mg, 18.8 μmol) in acetonitrile (10.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was frozen and lyophilized over the weekend to give the crude title product as a pink fluffy solid. To the crude product was added 20 mM NH4HCO3 solution (20.0 mL) and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter by the same conditions described above. Then, again, 20 mM NH4HCO3 solution (20.0 mL) was added to the residual solution and the resulting mixture was filtered again using a centrifugal filter by the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (20.0 mL) and the resulting mixture was lyophilized overnight to give the title product (90.0 mg, 115%) as a pink fluffy solid. Purity 91% based on LC-MS. LRMS(m / z):1631[M-4] 4- ,2174[M-3] 3- LC-MSr.t.(min):0.73 7B

[0277] Intermediate 5: SO1861-Trifunctional Linker-BNA Oligo Methyltetrazine-BNA oligo (90.0 mg, 0.014 mmol) and SO1861-trifunctional linker (48.6 mg, 0.014 mmol) were dissolved in a mixture of water / acetonitrile (4:1 v / v, 12.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 15 min, the mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. 92% purity based on LC-MS (two peaks, both m / z values ​​corresponding to the title compound). LRMS(m / z):1641[M-6] 6- , 1970[M-5] 5- LC-MSr.t.(min): 3.24 and 3.40 6B

[0278] Intermediate 1: SO1861-Azide To the mixture of SO1861 (60 mg, 0.032 mmol) and 1-azido-3,6,9,12-tetraoxapentadecane-15-hydrazide (39.3 mg, 0.129 mmol), methanol (extra dry, 1.00 mL) and TFA (9.86 μl, 0.129 mmol) were added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. 100% purity based on LC-MS. LRMS(m / z):2150[M-1] 1- LC-MSr.t.(min):1.10 3B

[0279] Intermediate 2: SO1861-Trifunctional Linker SO1861-azide (45 mg, 0.021 mmol) and trifunctional linker (26.5 mg, 0.022 mmol) were dissolved in DMF (2.50 mL) and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity 89% based on LC-MS. LRMS(m / z):1677[M-2] 2- LC-MSr.t.(min):2.54 6A

[0280] Intermediate 3: (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide To 1-(4-formylbenzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (28.0 mg, 0.048 mmol) and EMCH.TFA (24.5 mg, 0.072 mmol) was added methanol (extra dry, 2.00 mL) and TFA (11.1 μL, 0.145 mmol) and the reaction mixture was stirred at 50° C. After 30 min, the reaction mixture was evaporated in vacuo and the resulting residue was analyzed by MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (33.4 mg, 88%) as a bright purple fluffy solid. 92% purity based on LC-MS. LRMS(m / z):394[M+2] 2+ ,789[M+1] 1+ LC-MSr.t.(min):1.28 7A

[0281] Intermediate 4: Methyltetrazine-BNA oligos HSP27 BNA oligodisulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). Then, TCEP (14.3 mg, 0.050 mmol) was added, the reaction mixture was shaken for 1 min, and allowed to stand at room temperature. The reaction mixture was filtered by using a centrifugal filter (5000×g, 30 min) with a molecular weight cut-off of 3000 Da. Then, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residual solution, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (30.0 mL) and the resulting mixture was added to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (14.8 mg, 18.8 μmol) in acetonitrile (10.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was frozen and lyophilized over the weekend to give the crude title product as a pink fluffy solid. To the crude product was added 20 mM NH4HCO3 solution (20.0 mL) and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter by the same conditions described above. Then, again, 20 mM NH4HCO3 solution (20.0 mL) was added to the residual solution and the resulting mixture was filtered again using a centrifugal filter by the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (20.0 mL) and the resulting mixture was lyophilized overnight to give the title product (90.0 mg, 115%) as a pink fluffy solid. Purity 91% based on LC-MS. LRMS(m / z):1631[M-4] 4- ,2174[M-3] 3- LC-MSr.t.(min):0.73 7B

[0282] Intermediate 5: SO1861-Trifunctional Linker-BNA Oligo Methyltetrazine-BNA oligo (90.0 mg, 0.014 mmol) and SO1861-trifunctional linker (48.6 mg, 0.014 mmol) were dissolved in a mixture of water / acetonitrile (4:1 v / v, 12.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 15 min, the mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. 92% purity based on LC-MS (two peaks, both m / z values ​​corresponding to the title compound). LRMS(m / z):1641 [M-6] 6- , 1970 [M-5] 5- LC-MSr.t.(min): 3.24 and 3.40 6B

[0283] RNA isolation and gene expression analysis of tumor samples Total RNA was isolated from tumors using TriZol (Thermo Fisher) according to the manufacturer's instructions. The isolated RNA was resuspended in nuclease-free water (NFW). RNA samples were checked for their RNA integrity on gel. To prepare cDNA, first, 100 ng of total RNA was mixed with random hexamers (Qiagen; final concentration 2 μM) in a final volume of 12.5 μl in NFW, denatured at 70 °C for 5 min, and immediately chilled on ice. Then, 7.5 μl of cDNA synthesis mix was added, consisting of 4 μl of 5× RT buffer (Promega), 0.4 μl of 25 mM dNTPs (Promega), 1 μl of 200 U / μl MMLV RT enzyme (Promega), 0.5 μl of 40 U / μl RNAse inhibitor (Promega), and 1.6 μL NFW. The following cDNA synthesis protocol was used: 1) 10 min at 25°C, 2) 60 min at 37°C, 3) 5 min at 85°C, 4) indefinite at 4°C. For a single qPCR reaction, the following mix was prepared: 1 μL cDNA, 0.05 μL forward primer (250 μM), 0.05 μL reverse primer (250 μM), 8.9 μl LNFW, 10 μl SYBR Green (Bio-Rad). The following qPCR protocol was used: 1 cycle: 5 min 95°C, 40 cycles: 15 s 95°C + 30 s 60°C.

[0284] HSP27 gene expression was measured using 2-( Ct HSP27 - GEOMEAN ( Ct ref1 ; Ct ref2 ; Ct ref3 ; Ct ref4) was used to calculate the geNorm stability M value (M) of the tumor samples. Here, ref1, ref2, ref3, and ref4 are the reference genes IMMT, EIF2S2, GUSB, and UBC for the analysis of the tumor. To select the most ideal and stable reference genes for this tumor sample, among the nine reference genes tested, two reference genes were selected based on the performance of GeNORM analysis. To do so, the qPCR results were imported into the Qbase+ software program. This allows the calculation of two quality measures: the coefficient of variation (V) of the normalized reference gene expression level; and the geNorm stability M value (M)1. Reference genes with M<0.2 and V<0.15 are considered to be very stable. Based on this analysis, IMMT and EIF2S2 were selected as the most stable reference genes. However, UBC and GUSB were also added to the group of reference genes to further improve the accuracy of normalization. Each sample was analyzed as technical triplicates by a CFX96 real-time qPCR machine (Bio-Rad).

[0285] The primers used for qPCR are shown in Table B1 below.

[0286] [Table 39]

[0287] Example 7 Antibody-(SO1861-L-trifunctional linker-L-HSP27)(Cys) Antibody-Dendron Antibody-Saporin T-DM1 Materials and Methods Trastuzumab (Tras, Herceptin®, Roche), cetuximab (Cet, Erbitux®, Merck KGaA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, 99%, Sigma-Aldrich), Zeba™ spin desalting columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standards (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), isopropyl alcohol (IPA, 99.6%, VWR), tris(hydroxymethyl)aminomethane (Tris, 99%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane hydrochloride (Tris·HCL, Sigma-Aldrich), L-histidine (99%, Sigma-Aldrich), D-(+)-trehalose dehydrate (99%, Sigma-Aldrich), polyethylene glycol sorbitan monolaurate (TWEEN 20, Sigma-Aldrich), DPBS Dulbecco's phosphate buffered saline (Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetic acid dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filter 0.2 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), dianthine-Cys (Dia-Cys).dianthine mutant with a single C-terminal cysteine ​​functional group, Proteogenix), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP, Thermo-Fisher), and HSP27 oligonucleotides (Biosynthesis).

[0288] method UV-Vis Absorbance measurements were performed on a Perkin Elmer Lambda 25 UV-Vis or on a Thermo NanoDrop ND-2000 spectrophotometer in the spectral range of 200-750 nm.

[0289] Concentrations were determined using a Thermo Nanodrop 2000 or Lambda 25 spectrometer with the following parameters: Trastuzumab OD 280 =1.5 (mg / ml) -1 cm -1 Cetuximab OD 280 =1.4 (mg / ml) -1 cm -1 HSP27 Oligonucleotide OD 260 =153,000 M -1 cm -1 ;Rz 260:280 =1.819 Dia-Cys OD280=0.57 (mg / ml) -1 cm -1 PEG4-SPDP (PDT) OD 343 =8,080 M -1 cm -1 SAMSA-Fluorescein OD 495 =64,500M -1 cm -1 ;Rz 280:495 =0.232 Elman(TNB) OD412 =14,150 M -1 cm -1 Size Exclusion Chromatography Size exclusion chromatography (SEC) was performed on an AKTA purification system. Samples were analyzed by SEC using either a Biosep SEC-S3000 column or a Sephadex G50M column (10 x 40 cm). Elution was with TBS / isopropyl alcohol solution (85:15 v / v). Sample purity was determined by integration of the antibody sample peak against the aggregate peak of the trace.

[0290] Ellman assay Antibody-Dendron (-L-SO1861) 4 (Cys and Lys) Synthesis of trastuzumab-(Ld(SO1861)4)4 and cetuximab-(Ld(SO1861)4)4 by DAR4 (FBR706 STB22 / 9-10) Trastuzumab and cetuximab are referred to hereafter as "Ab". Ab was conjugated to dendritic SO1861-EMCH (d(SO1861)4-EMCH) via a labile (L) tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP) linker. The procedure for cetuximab-L-(d(SO1861)4)4 is exemplarily described: Cetuximab was desalted in DPBS pH 7.5 buffer and then normalized to 2.50 mg / ml. To an aliquot of Ab (9.19 mg, 61 nmol), an aliquot of freshly prepared PEG4-SPDP solution (5.0 mg / ml, 6.70 molar equivalents, 411 nmol) was added, the mixture was vortexed briefly, and then incubated for 60 min at 20°C with roller mixing. After incubation, the reaction was quenched by the addition of glycine (20 mg / ml, 7.7 μl), and then the SPDP moiety was reduced in situ by the addition of TCEP (5.0 mg / ml, 4.0 molar equivalents per SPDP, 1.64 μmol). The mixture was roller mixed for 15 min at 20°C with roller mixing. The resulting Ab-SH was purified by gel filtration into TBS pH 7.5 using a zeba spin desalting column. Ab-SH was characterized by UV-vis analysis and Ellman's assay (SH to Ab ratio = 5.4). To bulk Ab-SH (7.41 mg, 1.93 mg / ml, 49 nmol) was added an aliquot of freshly prepared d(SO1861)4-EMCH solution in DMSO (10 mg / ml, 8.0 molar equivalents per Ab, 0.4 μmol, 3.16 mg, 0.32 ml), the mixture was vortexed briefly and then incubated overnight at 20°C. Besides the conjugation reaction, two aliquots (0.25 mg, 1.67 nmol) of desalted Ab-SH were set aside prior to conjugation and reacted with NEM (8.0 molar equivalents per Ab, 13.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) overnight at 20° C. as positive and negative controls, respectively. After 18 h incubation (before the addition of NEM), the crude conjugation mixture was briefly centrifuged and a 100 μl aliquot was removed for analysis by UV-vis and characterized by Ellman's assay alongside the positive and negative controls to obtain d(SO1861)4 incorporation.To the bulk Ab-d(SO1861)4 mixture, an aliquot of freshly prepared NEM solution (2.5 mg / ml, 5.0 molar equivalents, 0.25 μmol) was added and the mixture was purified by a 1.6×30 cm Sephadex G50 column eluted with DPBS pH 7.5 to give the purified cetuximab-(Ld(SO1861)4)4 conjugate. The product was clarified by 0.2 μm filtration and then carefully concentrated (3,000 g, 5 min interval, 5° C.) to ca. 3 mg / ml using a vivaspin T15 concentrator to give the final cetuximab-(Ld(SO1861)4)4 conjugate. Yield: 4.41 mg, 48% (1.64 mg / ml). d(SO1861)4 to Ab ratio=4.4 (see Table B2).

[0291] [Table 40]

[0292] Antibody-L-HSP27BNA(Cys) Synthesis of trastuzumab-(L-HSP27)4, cetuximab-(L-HSP27)4, and PEG4-SPDP by DAR4, and cetuximab-(L-HSP27)2 via PEG4-SPDP by DAR2 Trastuzumab, cetuximab, trastuzumab-L-SO1861, and cetuximab-L-SO1861 are hereafter referred to as "Ab." Ab was conjugated to HSP27 via a labile (L) tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP) linker.

[0293] HSP27 (2.7 mg, 470 nmol, 6.10 mg / ml) was reacted with TCEP (10 molar equivalents, 4.7 μmol, 1.34 mg, 50 mg / ml) for 30 min at 20° C. by roller mixing. Oligo-SH was then purified by PD10 G25 desalting column eluted with TBS pH 7.5 and used immediately. Oligo-SH was obtained (2.48 mg, 90%, 1.24 mg / ml, SH to oligo ratio=0.8). Trastuzumab-(L-SO1861)4 (1.3 mg, 8.7 nmol, 2.50 mg / ml) was reacted with an aliquot (1 mg / ml) of freshly prepared PEG4-SPDP solution in DMSO (9.26 molar equivalents, 80.3 nmol, 45 μmol) by roller mixing at 20°C for 60 min. The reaction was then quenched with glycine (15.1 μl of a freshly prepared solution of 2 mg / ml in TBS pH 7.5) and then desalted by a zeba desalting column eluted with TBS pH 7.5. An aliquot of the resulting Tras-(L-SO1861)-(L-PEG4-SPDP) was removed and examined by UV-Vis analysis. SPDP incorporation was determined using TCEP to release pyridyl-2-thione (PDT) and by UV-vis analysis at 343 nm (SPDP to Ab ratio: 4). The remaining Tras-(L-SO1861)-(L-PEG4-SPDP) was reacted with an aliquot of freshly prepared HSP27 oligonucleotide (oligo-SH) (8 molar equivalents, 54.8 nmol, 0.32 mg, 1.24 mg / ml) and incubated overnight at 20° C. with roller mixing. After 17 hours, the conjugate was analyzed by UV-vis analysis to confirm HSP27 incorporation by the amount of pyridyl-2-thione (PDT) released at 343 nm. The crude conjugate was purified using a 1.6×33 cm Sephadex G50 column eluted with DPBS pH 7.5. The resulting trastuzumab-(L-SO1861)4-(L-HSP27)4 was obtained as a single fraction. Yield: 0.47 mg, 45% (0.49 mg / ml), HSP27 to Ab ratio = 3.5 (see Table B3).

[0294] Total yield: STB17 / 1-8 required

[0295] [Table 41]

[0296] Example 8 Materials and Methods In our current work, we considered a model scaffold consisting of four molecular arms for saponin binding via Schiff bases (imines) and one arm for click chemistry. The polymer structure (Figure 19) is a pentavalent polyethylene glycol-based dendrimer of the first generation (i.e., the number of repeated branching cycles). It was purchased from Iris Biotech GmbH (Marktredwitz, Germany). The saponin (in this example SA1641) was purified from a complex raw extract of saponins from Gypsophila species called Saponinum album, obtained from Merck (Darmstadt, Germany). The powdered raw extract (2.5 g) was hydrolyzed with sodium hydroxide (0.2 g) in water (100 mL). The solution was stirred at 40 °C for 20 h, and then glacial acetic acid was added until a pH of 5.0 was reached. To remove tannins, the solution was shaken with 30 mL of butanol in a separatory funnel. The aqueous phase was recaptured and the butanol extraction was repeated twice. The butanol phase was supplemented with anhydrous sodium sulfate, filtered and pooled. The butanol was evaporated and the remaining saponin powder was dissolved in 20% methanol at a final concentration of 30 mg / mL. After a short sonication, the different saponins were separated by high performance liquid chromatography (HPLC). The tube (column effluent) was rinsed with warm water (40 °C) at a flow rate of 1.5 mL / min and then with isopropanol (100%) encompassing a Eurospher RP-C18 column (5 μm, 250 × 8 mm). The saponins were loaded onto the column and eluted with a methanol gradient (20% methanol to 70% methanol within 30 min at 1.5 mL / min in water supplemented with 0.01% trifluoroacetic acid, then 70% methanol for another 60 min) (Sama et al., 2018). Aliquots of fractions were analyzed for their SA1641 content by electrospray ionization mass spectrometry (ESI-MS). Fractions containing pure SA1641 were pooled and the methanol was evaporated. The aqueous solution was frozen as a thin film in a rotating round-bottom flask by using dry ice. After 16 h storage at -80°C, the samples were lyophilized.To produce the scaffolds defined in the present invention, the polymer structure (0.2 mM) and SA1641 (3.2 mM) were dissolved in water (pH approx. 8), mixed in equal volumes and shaken at 26° C. for 24 h. Sodium cyanoborohydride (NaCNBH3; 0.1 M) was then added in a 4-fold molar excess with reference to SA1641, and the sample was incubated for another 24 h. The structure was then verified by ultra-performance high-resolution liquid chromatography (UPLC) / ESI-MS. The sample was applied to a RP-C4 column and eluted with a methanol gradient (25% methanol to 80% methanol in water supplemented with 0.01% trifluoroacetic acid within 15 min, then 80% methanol for another 10 min). Fractions were analyzed using a LC-Time of Flight (LC-TOF) mass spectrometer from Waters Corporation by using LockSpray™, an ion source specifically designed for accurate mass measurement by electrospray ionization.

[0297] result The inset of Figure 20 shows the theoretically expected mass spectrum obtained from calculations with the isotope pattern calculator enviPat Web 2.0. The pattern takes into account the charge of the molecule and the natural abundance of the isotopes. This is why more than one peak is expected for a single substance. The experimental data obtained by UPLC / ESI-MS (Figure 20) showed almost exactly the same peak at m / z 758-760 with the same intensity as predicted, therefore proving successful SA1641 coupling to the polymer structure.

[0298] Example 9 Materials and Methods As an example of a drug substance, we used the targeted toxin Gianthin-Epidermal Growth Factor (Gianthin-EGF). The plasmid His-Gianthin-EGF-pET11d (Weng et al, 2009) (100 ng) was added to 20 μL of Escherichia coli Rosetta™2(DE3)pLysS competent cells (Novagen, San Diego, CA, USA). The cells were transformed by heat shock (30 min on ice, 90 s at 42°C, 1 min on ice). Afterwards, 300 μL of lysogeny medium (LB) was added and the suspension was incubated at 37°C for 1 h with shaking at 200 rpm. A pre-warmed lysogeny medium agar plate with 50 μg / mL ampicillin was inoculated with 100 μl of the bacterial suspension and the plate was incubated at 37°C overnight. Lysogeny medium (3 mL) with 50 μg / mL ampicillin was inoculated with a colony from the plate and the bacteria were incubated at 37° C. and 200 rpm for 8 h. The suspension (50 μL) was added to 500 mL of lysogeny medium with 50 μg / mL ampicillin and incubated overnight at 37° C. and 200 rpm. The volume was then scaled up to 2.0 L and the bacteria were grown under the same conditions until an optical density at 600 nm of 0.9 was reached. Protein expression was then induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. Protein expression continued for 3 h at 37° C. and 200 rpm. Finally, the bacterial suspension was centrifuged at 5,000g and 4°C for 5 min, resuspended in 20 mL PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4) and stored at 20°C until use. For purification, the bacterial suspension was thawed and lysed by sonication. The lysate was centrifuged (15,800g, 4°C, 30 min) and imidazole was added to a final concentration of 20 mM. The supernatant was incubated with 2 mL Ni-nitrilotriacetic acid agarose in the presence of 20 mM imidazole for 30 min at 4°C under continuous shaking.The material was then loaded onto a 20 mL column, washed three times with 10 mL washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole), and Gianthin-EGF was eluted with 10 mL portions of increasing concentrations of imidazole (31, 65, 125, and 250 mM) in the washing buffer. Elution fractions (2 mL) were dialyzed overnight at 4° C. against 2.0 L of PBS. The desalted Gianthin-EGF was concentrated by Amicon® Ultra-15 (10 kDa) and the protein concentration was quantified.

[0299] To introduce suitable click chemistry groups into Gianthin-EGF, an 8-fold molar excess of alkyne-PEG5-N-hydroxysuccinimidyl ester with reference to Gianthin-EGF was dissolved in dimethylsulfoxide and added to 9 volumes of Gianthin-EGF (1 mg in 0.2 M NaH2PO4 / Na2HPO4, pH 8). After 4 h of incubation at room temperature, the unbound alkyne was separated by using a PD10 column (GE-Healthcare, Freiburg, Germany). Click chemistry with the polymer structure was carried out by copper(I)-catalyzed alkyne-azide cycloaddition. Alkyne-dianthin-EGF (0.02 mM), dendrimer (0.05 mM), CuSO4 (0.1 mM), tris(3-hydroxypropyltriazolylmethyl)amine (0.5 mM), and sodium ascorbate (5 mM) were incubated under gentle agitation at room temperature for 1 h in 0.1 M NaH2PO4 / Na2HPO4, pH 8. The low molecular mass species were then separated using a PD10 column.

[0300] To test the efficacy of the present invention, we performed a viability assay with HER14 cells. These cells are fibroblasts stably transfected with human epidermal growth factor receptor and thus are the target cells for the targeted toxin dianthin-EGF. HER14 cells (2,000 cells / 100 μL / well) were seeded into wells of a 96-well cell culture plate and incubated for 24 h at 37° C., 5% CO2, and 98% humidity in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Then, the different test substances (see Results and Figure 21) were added in triplicates in a volume of 25 μL and additional 25 μL of medium was added. After 72 h of incubation, 30 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg / mL in water) was added per well and incubated for 2 h. Afterwards, the medium was carefully removed and replaced with an aqueous solution containing 10% (v / v) isopropanol, 5% (w / v) sodium dodecyl sulfate, and 400 mM HCl, and incubated for 5 min. Solubilized formazan was quantified photometrically at 570 nM by a microplate reader (Spectra MAX 340PC, Molecular Devices, Sunnyvale, CA, USA). Untreated cells were normalized to 1, and all samples were referred to the untreated control. Significance was determined by unpaired two-sample t-test.

[0301] result The example polymer structure, a pentameric dendrimer (pentrimer), does not have any cytotoxic effect on target cells in the absence or presence of SA1641 (Figure 21, columns 2 and 3). In the absence of the scaffold, the targeted toxin (Gianthin-EGF) shows half-maximal toxicity at a concentration of 0.1 nM (column 4). In the presence of SA1641, the same concentration results in the death of all cells, indicating the general ability of SA1641 to act as an enhancer of endosomal escape (column 5). The presence of the polymer structure does not affect the toxicity of Gianthin-EGF in the presence or absence of SA1641 (columns 6 and 7), indicating that the scaffold does not affect the toxicity of Gianthin-EGF. To couple the model polymer structure to the example drug substance Gianthin-EGF by click chemistry, the material had to be previously coupled to an alkyne group. The drug substance manufacturer can directly introduce click positions into the substance during synthesis at positions of his choice where the activity of the substance remains unaffected. There is no additional loss of activity when the alkyne-modified drug substance is clicked with the polymer structure, indicating that the polymer structure itself is not toxic.

[0302] Example 10 material The following chemicals were used as purchased: methanol (MeOH, LiChrosolv, Merck), N-ε-maleimidocaproic acid hydrazide (EMCH, 95%, TCI Chemicals), trifluoroacetic acid (TFA, 99.8%, Carlsbad Chemicals), and 1,2-dimethylphenylsulfonyl ether (DMSO, 99.8%, Carlsbad Chemicals). 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-methylmethanaminium 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™ 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%, Quanta Biodesign), PFd-G4-azido-NH-BOC dendron (G4-dendron, 95%, Polymer Factory), cyanine 5-DBCO (Cy5-DBCO, 95%, Lumiprobe), chloroform (CHCl3, 99.5%, Sigma), Amicon Ultra 0.5 mL centrifugal filter (3 kDa MWCO, Sigma), mPEG-SCM (mPEG 2k -NHS, 95.6%, Creative PEG Works), Amicon Ultra 15mL centrifugal filter (10kDa MWCO, Sigma).

[0303] method MALDI-TOF-MS MALDI-TOF spectra were recorded on a MALDI mass spectrometer (Bruker Ultrafex III). Samples, typically dissolved in MilliQ water in the nanomolar to micromolar range, were spotted by the droplet drying method onto a target (MTP384 target plate polished steel TF, Bruker Daltons) using either super-DHB (99%, Fluka) or sinapinic acid (SA, 99%, Sigma-Aldrich) as matrix dissolved in acetonitrile (MADLI-TOF-MS tested, Sigma) / 0.1% TFA (7:3 v / v). PepMix (peptide calibration standard, Bruker Daltons) or ProteMass (protein calibration standard, Sigma-Aldrich) served as calibration standards. RP mode refers to the reflector positive mode. RN mode refers to the reflector negative mode. LP mode refers to the linear positive mode.

[0304] H-NMR 1 H NMR analysis was performed using a Bruker 400 MHz NMR spectrometer. Sample preparation was performed 24 h prior to the measurement, whereby 2 mg of sample was dissolved in 0.8 mL of methanol-D4 (99%, Deutero).

[0305] UV-Vis UV-Vis measurements were performed on a NanoDrop ND-1000 spectrophotometer in the spectral range of 200–750 nm.

[0306] Size Exclusion Chromatography Size exclusion chromatography (SEC) was performed with Sephadex G25 Superfine from GE Healthcare and on a pre-packed PD10 column (GE Healthcare, Sephadex G25M). Prior to chromatography, the material was activated by swelling in the respective eluent.

[0307] dialysis Dialysis was performed using regenerated cellulose membranes: MWCO = 1 and 2 kDa (Spectra / Por) and MWCO = 12-14 kDa (Carl Roth). Typically, dialysis was performed for 24 h with 1 L of solvent, which was changed after the first 6 h of the process.

[0308] Freeze drying Freeze-drying was performed in an Alpha 1-2 LD plus (Martin Christ Gefriertrocknungsanlagen GmbH). Typically, samples were frozen in liquid nitrogen and placed in a freeze-dryer under high vacuum.

[0309] SO1861-EMCH synthesis SO1861 from Saponaria officinalis L (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) were placed in a round flask with a stirrer and dissolved in 13 mL methanol. TFA (400 μL, cat.) was added to the solution and the reaction mixture was stirred with a RCT B magnetic stirrer (IKA Labortechnik) at 800 rpm and room temperature for 3 h. After 3 h of stirring, the mix was diluted with either MilliQ water or PBS and dialyzed extensively against either MilliQ water or PBS for 24 h. Regenerated cellulose membrane tubing (Spectra / Por7) with MWCO of 1 kDa was used. After dialysis, the solution was lyophilized to obtain a white powder. Yield 62.4 mg (95%). The dried aliquots were further 1 It was used for characterization by 1 H NMR and MALDI-TOF-MS.

[0310] 1 H NMR (400 MHz, methanol-D4) (Figure 22A, SO1861): δ = 0.50-5.50 (m, saponin triterpenoid and sugar backbone protons), 9.43 (1H,s, aldehyde protons of saponin, H a ).

[0311] 1 H NMR (400 MHz, methanol-D4) (Figure 22B. SO1861-EMCH, PBS workup): δ = 0.50-5.50 (m, saponin triterpenoid and sugar backbone protons), 6.79 (2 H, s, maleimide protons, H c ), 7.62-7.68 (1 H, m, hydrazone proton, H b ).

[0312] MALDI-TOF-MS (RP mode) (Figure 23A): m / z 2124 Da ([M+K] + , Saponin-EMCH), m / z 2109 Da ([M+K] + ,SO1861-EMCH),m / z 2094 Da([M+Na] + ,SO1861-EMCH) MALDI-TOF-MS (RN mode) (Figure 28C): m / z 2275 Da ([MH] - , saponin-EMCH conjugate), 2244 Da ([MH] - , saponin-EMCH conjugate), 2222 Da ([MH] - , saponin-EMCH conjugate), 2178 Da ([MH] - , saponin-EMCH conjugate), 2144 Da ([MH] - , saponin-EMCH conjugate), 2122 Da ([MH] - , saponin-EMCH conjugate), 2092 Da ([MH] - , saponin-EMCH conjugate), 2070 Da ([MH] - ,SO1861-EMCH),2038 Da([MH] - ,SO1832-EMCH),1936 Da([MH] - ,SO1730-EMCH),1861 Da ([MH] - ,SO1861).

[0313] SO1861-EMCH-Mercaptoethanol To SO1861-EMCH (0.1 mg, 48 nmol), 200 μL mercaptoethanol (18 mg, 230 μmol) was added, and the solution was shaken for 1 h at 800 rpm and room temperature on a ThermoMixer C (Eppendorf). After shaking for 1 h, the solution was diluted with methanol and dialyzed extensively against methanol for 4 h using a regenerated cellulose membrane tube (Spectra / Por7) with a MWCO of 1 kDa. After dialysis, an aliquot was removed and analyzed by MALDI-TOF-MS.

[0314] MALDI-TOF-MS (Figure 23B) (RP mode); m / z 2193 Da ([M+K] + ,SO1861-EMCH-mercaptoethanol), m / z 2185 Da ([M+K] + ,SO1861-EMCH-mercaptoethanol), m / z 2170 Da ([M+Na]+ ,SO1861-EMCH-Mercaptoethanol).

[0315] BSA-SO1861 synthesis 2-Iminothiolane (231 μg, 1.1 μmol) dissolved in 47 μL PBS was added to a BSA-RhodB solution (10 mg, 0.15 μmol) in 200 μL PBS, and the mix was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 40 min. After 40 min of shaking, the reaction mix was immediately passed through a Sephadex G25 Superfine 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 fraction. The reaction mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After 12 h of shaking, the BSA-SO1861 was concentrated using centrifugal filtration through an Amicon Ultra 15 filter with a MWCO of 3 kDa at 4,000 rpm (15° C.). The conjugate was stored in the refrigerator as a solution and an aliquot was taken for analysis. Yield: Not determined.

[0316] MALDI-TOF-MS (Figure 15A) (LP mode): m / z 74.2 kDa ([M+H] + , BSA-SO1861 with four SO1861 attached, 72.2 kDa ([M+H] 2+ , BSA-SO1861 with three SO1861 attached), 70.2 kDa ([M+H] + , BSA-SO1861 with two SO1861 attached), 37.0 kDa ([M+H] 2+ , BSA-SO1861 with four SO1861 units attached), 35.9 kDa ([M+H] 2+ , BSA-SO1861 with three SO1861 attached, 34.7 kDa ([M+H] 2+ ,BSA-SO1861 with two SO1861s installed).

[0317] Cy3-PAMAM 720 μL of PAMAM (30 mg, 1.04 μmol) dissolved in methanol was placed in a 250 mL round flask and the methanol was removed by rotary evaporation (20 mbar, 60 °C). The remaining PAMAM was dissolved in 9 mL of DMSO. HATU (7.6 mg, 20 μmol) dissolved in 0.5 mL of DMSO was added to the Cy3-COOH (0.6 mg, 1.2 μmol) solution in DMSO and the mix was shaken at 800 rpm and room temperature for 1 h on a ThermoMixer C (Eppendorf). After 1 h of shaking, the HATU-Cy3 solution was added to the stirred PAMAM solution and the reaction mix was stirred at room temperature for 12 h. After 12 h of stirring, the reaction mix was diluted with MilliQ water and dialyzed extensively against MilliQ water for 24 h using a regenerated cellulose membrane tube (Spectra / Por6) with a MWCO of 2 kDa. After dialysis, the volume of the conjugate solution was reduced by rotary evaporation (20 mbar, 60° C.) and the concentrated conjugate solution was passed through a Sephadex G25 Superfine size-exclusion column (16 mL column volume). The first fraction was collected and lyophilized to give a viscous pink PAMAM-Cy3 conjugate. PAMAM-Cy3 conjugate formation was confirmed by chromatography with thin layer chromatography (methanol / water, v / v 1:1) and the appearance of a faster band on a Sephadex G25 Superfine column. Yield 21.3 mg (63%). The dye molar ratio per PAMAM, determined by UV-Vis spectrophotometry, was 0.43.

[0318] MALDI-TOF-MS (Figure 33A) (LP mode): m / z 28.0 kDa ([M+H] + ,Cy3-PAMAM).

[0319] Cy3-PAMAM-SO1861 synthesis The procedure for Cy3-PAMAM-(SO1861)5 is described by way of example. 2-Iminothiolane (1 mg, 6.7 μmol) dissolved in 250 μL of MilliQ water was added to PAMAM-Cy3 solution (0.5 mg, 17 nmol) in 125 μL of MilliQ water, and the mix was shaken at 800 rpm and room temperature for 40 min on a ThermoMixer C (Eppendorf). After 40 min of shaking, the reaction mix was immediately passed through a Sephadex G25 Superfine size-exclusion column (16 mL column volume), and SO1861-EMCH (176 μg, 85 nmol) dissolved in 40 μL of MilliQ water was added to the collected Cy3-PAMAM-SH fraction. The reaction mixture was shaken at 800 rpm and room temperature for 12 h on a ThermoMixer C (Eppendorf). After stirring for 12 h, the reaction mix was diluted with MilliQ water and dialyzed extensively for 24 h against MilliQ water using regenerated cellulose membrane tubing (ZelluTrans, Carl Roth) with a MWCO of 12-14 kDa. After dialysis, the Cy3-PAMAM-SO1861 solution was concentrated using centrifugal filtration at 4000 rpm (15 °C) through an Amicon Ultra 15 filter with a MWCO of 3 kDa. The conjugate was stored in the refrigerator as a solution and an aliquot was taken for analysis. Yield: 0.5 mg (75%).

[0320] The MALDI-TOF-MS spectra are illustrated in Figures 33B-D and 34. MALDI-TOF-MS of Cy3-PAMAM-(SO1861)6 (Figure 33B) (LP mode): m / z 38.4 kDa ([M+H] + ,Cy3-PAMAM-SO1861),17.9 kDa([M+H] 2+ ,Cy3-PAMAM-SO1861).

[0321] Cy3-PAMAM-(SO1861)5, Cy3-PAMAM-(SO1861) 13 , Cy3-PAMAM-(SO1861) 51 , and Cy3-PAMAM-(SO1861) 27The synthesis of was carried out according to the methodology described above, but with different equivalents of starting materials 2-iminothiolane and SO1861-EMCH. The respective equivalents of starting materials and respective masses of the conjugates are highlighted in Table 1.

[0322] [Table 42]

[0323] Synthesis of Cy3-PAMAM-NC-SO1861 Cy3-PAMAM (0.5 mg, 18 nmol), SO1861 (2.3 mg, 1.24 μmol), and HATU (64.6 mg, 170 μmol) were dissolved in 200 μL DMSO separately. The SO1861 and HATU solutions were mixed and shaken for 20 min at 800 rpm and room temperature on a ThermoMixer C (Eppendorf). After 20 min of shaking, the Cy3-PAMAM solution was added to the shaking SO1861-HATU solution and the reaction mixture was allowed to shake for 12 h at 800 rpm and room temperature on a ThermoMixer C (Eppendorf). After 12 h of shaking, the reaction mix was diluted with MilliQ water and dialyzed extensively for 24 h against MilliQ water using a regenerated cellulose membrane tube with a MWCO of 12-14 kDa (ZelluTrans, Carl Roth). 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. Cy3-PAMAM-NC-(SO1861) 17 The conjugate was stored in solution in the refrigerator and an aliquot was taken for analysis. Yield: 0.77 mg (69%).

[0324] MALDI-TOF-MS (Figure 35) (LP mode): m / z 62.3 kDa ([M+H] + ,Cy3-PAMAM-NC-SO1861),35.7 kDa ([M+H] 2+ ,Cy3-PAMAM-NC-SO1861).

[0325] G4-dendron dye labeling and deprotection PFd-G4-Azide-NH-BOC (G4-Dendron) (9.75 mg, 2.11 μmol) was placed in a 2 mL reaction tube (Eppendorf) and dissolved in 200 μL DMSO. 100 μL of Cy5-DBCO solution (1.72 μmol) in DMSO was added. * mL -1 , 170 nmol) was added to the G4-dendron solution and the mix was shaken for 12 h at room temperature and 800 rpm on a ThermoMixer C (Eppendorf). After 12 h of stirring, the reaction mix was diluted with MilliQ water and dialyzed extensively against MilliQ water for 24 h using a regenerated cellulose membrane tubing with 1 kDa MWCO (Spectra / Por7). After dialysis, the solution was lyophilized to obtain a blue powder. The crude product was used for the deprotection step as obtained from lyophilization.

[0326] The partially Cy5-labeled lyophilized G4-dendron was dissolved in 12 mL CHCl3 in a 50 mL round flask with a stir bar. 12 mL TFA was added and the reaction mix was stirred for 3 h at 800 rpm and room temperature on a RCT B magnetic stirrer (IKA Labortechnik). After 3 h of stirring, the solvent was removed under reduced pressure (50 °C, 30 mbar) on a rotary evaporator (Heidolph WB 2000). After evaporation, the batch was dissolved in MilliQ water and passed through a PD10 size exclusion column. The G4-dendron conjugate formation was confirmed by chromatography by thin layer chromatography (methanol / water, v / v 1:1) and the appearance of a faster band by the PD10 column. The resulting fractions of the size exclusion chromatography were lyophilized to give a blue powder.

[0327] Yield 5.7 mg (93%). The dye molar ratio per G4-dendron determined by UV-Vis spectrophotometry was 0.012.

[0328] MALDI-TOF-MS (Figure 32B) (RP mode): m / z 3956 Da ([M + Na] + , Cy5-G4-dendron + PF6-counterion), 3820 Da ([M+Na] + ,Cy5-G4-dendron - PF6- counterion), 3617 Da ([M+H] + ,G4-dendron impurity), 3017([M+H] + ,G4-dendron).

[0329] G4-dendron-SO1861 synthesis The procedure for the lowest G4-dendron to SO1861-EMCH ratio is exemplarily described. 2-Iminothiolane (2.65 mg, 19.2 μmol) dissolved in 300 μL of MilliQ water was added to the partially Cy5-labeled G4-dendron solution (0.577 mg, 192 nmol) in 252 μL of MilliQ water, and the mix was shaken at 800 rpm and room temperature for 40 min on a ThermoMixer C (Eppendorf). After 40 min of shaking, the reaction mix was immediately passed through a PD10 size-exclusion column, and SO1861-EMCH (1.19 mg, 575 nmol) dissolved in 100 μL of MilliQ water was added to the collected G4-dendron-SH fraction. The reaction mixture was shaken at 800 rpm and room temperature for 12 h on a ThermoMixer C (Eppendorf). After shaking for 12 h, the reaction mix was concentrated by centrifugal filtration using an Amicon Ultra centrifugal filter (3 kDa MWCO). The conjugate was stored in the refrigerator as a solution and an aliquot was taken for analysis. Yield: 90 nmol (47%).

[0330] The MALDI-TOF-MS spectrum is illustrated in FIG. MALDI-TOF-MS of G4-dendron-SO1861 (Figure 33C) (LP mode): m / z 10.19 kDa ([M+H]+, Cy5-G4-dendron-[SO1861]3), 9.27 kDa ([M+H]+, G4-dendron-[SO1861]3), 7.92 kDa ([M+H]+, Cy5-G4-dendron-[SO1861]2), 7.14 kDa ([M+H]+, G4-dendron-[SO1861]2), 5.86 kDa ([M+H]+, Cy5-G4-dendron-[SO1861]1), 5.07 kDa ([M+H]+, G4-dendron-[SO1861]1).

[0331] The synthesis of other G4-dendron-(SO1861)n conjugates was carried out by the methodology described above, but with different equivalents of the starting material SO1861-EMCH. The respective equivalents of the starting materials and the respective masses of the conjugates are highlighted in Table 2.

[0332] [Table 43]

[0333] PAMAM thiolation The procedure is exemplarily described for the highest PAMAM to 2-iminothiolane ratio. To a solution of PAMAM (333 μg, 12.8 nmol) dissolved in 30 μL of methanol, 2-iminothiolane (0.53 mg, 3.84 μmol) dissolved in 128 μL of MilliQ water was added. The reaction mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After shaking for 12 h, the reaction mix was washed four times with MilliQ water by 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.

[0334] The MALDI-TOF-MS spectrum is illustrated in Figure 51. 108 MALDI-TOF-MS (Figure 51C) (LP mode): m / z 41.5 kDa ([M+H] + ,PAMAM-[SH] 108 ).

[0335] The synthesis of other PAMAM-iminothiolane conjugates was carried out by the methodology described above, but with different equivalents of 2-iminothiolane starting material charged. For the reaction with the lowest 2-iminothiolane charge, Cy3-PAMAM was used.

[0336] The respective charge equivalents of the starting materials and respective masses of the conjugates are highlighted in Table 3.

[0337] [Table 44]

[0338] PAMAM PEGylation Lowest PAMAM vs mPEG 2k The procedure for the ratio is described as an example. PAMAM (333 μg, 12.8 nmol) solution dissolved in 10 μL of DMSO was mixed with mPEG (13 μL) dissolved in DMSO. 2k -NHS (0.268 mg, 128 nmol) was added. The reaction mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After 12 h of shaking, the reaction mix was diluted with MilliQ water and dialyzed extensively against MilliQ water for 24 h using regenerated cellulose membrane tubing with 2 kDa MWCO (Spectra / Por6). After dialysis, the batch was concentrated by centrifugal filtration using an Amicon Ultra 15 mL centrifugal filter (10 kDa 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.

[0339] The MALDI-TOF-MS spectrum is illustrated in Figure 52. 2k MALDI-TOF-MS of 3 (Figure 52C) (LP mode): m / z 33.46 kDa ([M+H] + ,PAMAM-[mPEG 2k ]3).

[0340] Other PAMAM-mPEG 2k The synthesis of the conjugate was carried out by the methodology described above, except that the starting material mPEG 2k The respective equivalents of starting materials and respective masses of the conjugates are highlighted in Table 4.

[0341] [Table 45]

[0342] Cy3-PAMAM-SO1861-DBCO synthesis Cy3-PAMAM-(SO1861) 27 -(DBCO) 10 The procedure for Cy3-PAMAM-(SO1861) is described below. 27 (0.41 mg, 4.71 nmol) was freeze-dried and dissolved in 100 μL of DMSO. 13-NHS ester (0.197 mg, 188 nmol) was added to ...

Claims

1. The base is a) a polymeric or oligomeric structure, and b) at least one biologically active molecule covalently attached to said polymeric or oligomeric structure, said base further comprising: c) a first chemical group for covalently coupling said base to a carrier molecule; a) the polymeric or oligomeric structure is: poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines); - polyethylene glycol, - poly- or oligo(esters), e.g. poly(lactide); - poly(lactam), - polylactide-co-glycolide copolymers, poly- or oligosaccharides, such as cyclodextrins and polydextrose; - poly- or oligo(amino acids), such as proteins, peptides, and polylysine; and - selected from the group consisting of DNA oligomers or polymers, RNA polymers, stabilized RNA polymers, and PNA (peptide nucleic acid) polymers; b) said at least one biologically active molecule is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23, selected from the group consisting of saponins having a quillaic acid aglycone core and saponins having a gypsogenin aglycone core, said saponins comprising a branched carbohydrate side chain at the C-3 position, said carbohydrate side chain comprising a glucuronic acid functional group, said saponins comprising a branched carbohydrate side chain at the C-28 position comprising a deoxy carbohydrate, said aldehyde functional group at position C-23 is covalently coupled to said polymeric or oligomeric structure via a hydrazone bond; said base being suitable for covalently binding said at least one biologically active molecule to said carrier molecule; The base, wherein the carrier molecule comprises or consists of any of proteinaceous molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, fats, fatty acids, nanoparticles, and carbohydrates.

2. 2. The base according to claim 1, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 has a molecular mass of less than or equal to 3,000 Daltons.

3. 3. The base according to claim 1 or 2, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 is an amphiphilic molecule.

4. A base according to any one of claims 1 to 3, in which the bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 is a single specific molecule or a mixture of different molecules when more than one biologically active molecule is covalently bound to the polymeric or oligomeric structure contained by the base.

5. 5. The base according to any one of claims 1 to 4, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 contains a glucuronic acid function on a carbohydrate substituent at the C-3 beta-OH group of said saponin.

6. 6. The base according to any one of claims 1 to 5, wherein the at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function in position C-23 is a saponin that can be isolated from Gypsophila species and / or Saponaria species and / or Agrostenma species and / or Quillaja species, for example Quillaja saponaria.

7. The at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 may be a single specific saponin or a mixture of two or more different saponins, such as saponins SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-apio, QS-21 A-xylo, QS-21B, QS-21 B-apio, QS-21 6. The base according to any one of claims 1 to 5, which is one or more of B-xylo, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereoisomers and / or any combination thereof.

8. At least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 is selected from the group consisting of SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group.

6. The base according to any one of claims 1 to 5, which is a saponin having a hydrate substituent and / or 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilayic acid, 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside.

9. The base according to any one of claims 1 to 5, wherein the at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 is SO1861 and / or QS-21.

10. 10. The base according to claim 1, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane carrying an aldehyde function at position C-23 has a molecular mass of at least 1.500 Daltons.

11. 10. The base according to claim 1, wherein the at least one bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function at position C-23 is QS-21 or one or more of QS-21A, QS-21 A-apio, QS-21 A-xylo, QS-21B, QS-21 B-apio, QS-21 B-xylo, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS-1861, Quil-A.

12. 11. The base according to any one of claims 1 to 10, wherein the aldehyde functional group at position C-23 of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled to a sulfhydryl group on the polymer or oligomer structure of the base via a thio-ether bond.

13. The base according to any one of claims 1 to 12, wherein the chemical group for the covalent coupling of the base to a carrier molecule is a click chemistry group.

14. 14. The base of claim 13, wherein the click chemistry group is a tetrazine, an azide, an alkene, or an alkyne, or a cyclic derivative of any of these groups.

15. The base of claim 14, wherein the click chemistry group is an azide.

16. 16. The base according to any one of claims 1 to 15, wherein the base is a trifunctional linker, comprises a second chemical group to which at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function in position C-23 is covalently attached, comprises a third chemical group for covalent attachment to a molecule, and comprises a first chemical group for covalent attachment to a carrier.

17. 17. The base according to any one of claims 1 to 16, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 is a defined number or range of glycoside molecules.

18. 18. The base of claim 17, wherein at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 is a defined number or range of glycoside molecules, selected from 1 to 128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 glycoside molecules, or any number therebetween.

19. The polymer or oligomer structure is: poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines); - polyethylene glycol, Poly- or oligo(esters), such as poly(lactides), and A base according to any one of claims 1 to 18, which is selected from the group consisting of poly- or oligo(amino acids), such as proteins, peptides and polylysine.

20. The polymer or oligomer structure is: A pentavalent polyethylene glycol-based dendrimer of the following structure: 【Chemistry 1】 Generation 5 (G5) poly(amidoamine) dendrimers with ethylenediamine cores derivatized with 2-iminothiolane; A polyester dendron with 16 functional amino end groups and a focal azide group, which in its NH-BOC protected form corresponds to the following structure: 【Chemistry 2】 and which is derivatized with 2-iminothiolane). Bovine serum albumin (BSA), and - a peptide having the sequence SESDDAMFCDAMDESDSK.

21. 21. The base according to any one of claims 1 to 20, wherein the carrier molecule comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, or comprises or consists of at least one non-proteinaceous ligand and / or at least one proteinaceous ligand for binding to a cell surface molecule, such as EGF or a cytokine.

22. The carrier molecule is an antibody, an IgG, a Vhh domain or a molecule comprising or consisting of a Vh domain, a Fab, a scFv, a Fv, a dAb, a F(ab) ２ 21. The base according to any one of claims 1 to 20, comprising or consisting of an Fcab fragment.

23. Carrier molecules include CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25 23. The base according to any one of claims 1 to 22, comprising or consisting of at least one binding domain and / or at least one binding fragment for binding to a cell surface receptor, such as a tumor cell specific cell surface receptor selected from the group consisting of: Ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2.

24. The carrier molecule may be cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody buntuzumab, borsetuzumab, denintuzumab, sofituzumab, lorvotuzumab, indusa 24. The base according to any one of claims 1 to 23, comprising or consisting of any one of the following: tumour cell receptor binding fragments and / or at least one tumour cell receptor binding domains thereof: tumour cell receptor binding fragments and / or at least one tumour cell receptor binding domains thereof.

25. 24. The base according to any one of claims 1 to 23, wherein the carrier molecule comprises or consists of any one of cetuximab or trastuzumab or OKT-9 or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof, such as at least one tumor cell specific receptor binding fragment thereof and / or at least one tumor cell specific receptor binding domain thereof.

26. The base according to any one of claims 1 to 25, wherein the base is suitable for forming a covalent bond with a carrier molecule.

27. The base according to any one of claims 1 to 26, wherein the carrier molecule comprises or consists of at least one effector molecule or, when dependent on any one of claims 21 to 26, further comprises at least one effector molecule, said effector molecule being at least one of a drug substance, such as a payload, a toxin, a drug, a polypeptide, an oligonucleotide, a nucleic acid, a xenonucleic acid, an enzyme, such as urease and Cre recombinase, a protein toxin, a ribosome-inactivating protein or more.

28. Protein toxins include Ebulitin α, Ebulitin β, Ebulitin γ, Ebulin f, Ebulin l, Ebulin r1, Ebulin r2, SEA, SEAII, SELfd, SELld, SELlm, α-Nigritin, β-Nigritin, γ-Nigritin, Niglitin f1, Niglitin f2, basic Nigrin b, Nigrin b (SNA-V), Nigrin f (SNA-Vf), Nigrin l1, Nigrin l2, Nigrin s, SNA-I, SNA-I', SNA-If, and SNAf lu-I, SNLRP1, SNLRP2, SNA-ld, SNA-lm, SNA-II, SNA-III, SNA-IV (SNA-IVf), SNA-IVl, SNApol-I, SNApol-II, TrSNA-I, TrSNA-If, basic racemosin b, SRA, SRLbm (SRAbm), SSA (SSA-b-1), Sieboldin-b (SSA-b-2), SSA-b-3, SSA-b-4, RIP1, amaranthin (ACA), ACL, A. leucocarpus lectin, amaramangin, AAP-27, amaranthin, beetin-27 (BE27), beetin-29 (BE29), betavulgin, CCP-25, CCP-27, CAP30, SoRIP1 (BP31), SoRIP2, aralin, panaxagin, quinqueginsin, asparin 1, asparin 2, charybdin, musarmin 1, musarmin 2, musarmin 3, musarmin 4, PMRIPm, PMRIPt, yucca leaf protein (YLP), Basella RIP 2a, Basella RIP 2b, Basella RIP 3, Agrostin 2, Agrostin 5, Agrostin 6, Agrostin, Dianthin 29, Dianthin 30, Dianthin 32, D. sinensisRIP, Gypsophilin, Lychnin, Petroglaucin 1, Petroglaucin 2, Petroglaudin, Ocymoidin, Saporin-L1 (SO-L1), Saporin-L2 (SO-L2), Saporin-L3 (SO-L3), Saporin-l (SO-l, SO -4), saporin-R1 (SO-R1), saporin-R2 (SO-R2), saporin-R3 (SO-R3), SO3a, SO3b, saporin-S5 (saporin 5, SO-S5), saporin-S6 (saporin 6, SO-6, SO-S6), saporin-S8 (SO-S8), saporin-S9 (saporin 9, SO-S9), SAP-C, SAP-S, Stellarin, RIP Q3, Pyramidatin, Hispin, α-benincasin, β-benincasin, Bryodin 1 (BD1), Bryodin 2, Bryodin-L, Bryodin-R, BDA, Colocin 1, Colocin 2, Fortidissimin, Fortidissimin II, Cucumis figarei RIP (CF-RIP), Cucurmoschin, Cucurmosin, Cucurmosin 2, C. Moschata RIP, Moschatin, PRIP 1, PRIP 2, α-moschin, β-moschin, Pepocin, Texanin, Gynostemmin, Lagenin, Luffaculin-1, Luffaculin-2, Luffangulin, Pakistani Luffaacutangula fruit lectin, Luffin, Luffin-a, Luffin-b, α-luffin, β-luffin, LRIP, Luffacylin, Luffin P1, Luffin-S, Luffin S(1), Luffin S(2) (Luffin S2), Luffin S(3), MOR-I, MOR-II, Balsamin, MbRIP-1, Momordin II, MAP 30, α-momorcharin (α-MC, α-MMC), β-momorcharin (β-MC, β-MMC), δ-momorcharin (δ-MMC), momordin, momordin (bitter melon (Momordica charantia inhibitor), momordin II, momordin-a, momordin-b, gamma-momorcharin (gamma-MMC), Charantin, RIP 1 candidate, MCL (M. charantia lectin), anti-H lectin, Momordica agglutinin, momordin, protein fraction 1, protein fraction 2, MCL (Momordica charantia seed lectin, Momordica charantia lectin), MCL1, Cochinin B, Momorcochin, Momorcochin-S, Momorgrosvin, Sechiumin, Sechium edule fruit lectin, trichoanguin, SGSL, TCA-I, TCA-II, TCSL, β-trichosanthin (β-TCS), α-kirilowin, β-kirilowin, TAP 29, TK-35, Trichobitacin, Trichokirin, Trichomislin (TCM), Trichosanthin (Trichosanthi antiviral protein, TAP, TCS), α-trichosanthin (α-TCS, GLQ223), β-trichosanthi (β-TCS), γ-trichosanthi (γ-TCS), Trichokirin S1, S-trichokirin, Trichosanthrip, TKL-1 (Trichosanthi antiviral protein, TAP, TCS),kirilowii lectin-1), TK-I, TK-II, TK-III, Trichomaglin lectin, Karasurin-A, Karasurin-B, Karasurin-C, Trichomaglin, TDSL, Trichobakin, Arborvitae RIP, Crotin I, Crotin 2, E. characias lectin, Gelonin (GAP 31), Hura crepitans RIP, Hura crepitans RIP-5, Hura crepitans latex lectin, Crepitin, Hurin, Hura crepitans seed lectin, Curcin, Curcin 2, Curcin-L, Jc-SCRIP, Mapalmin, Manutin 1, Manutin 2, Ricin (crystalline ricin, ricin D), Ricin E, RCA (Ricinus communis agglutinin, RCAI, RCA120, Castor bean hemagglutinin, RCB-PHA I, RCAII, RCA60, RCB-PHA II), ricin 1, ricin 2, ricin 3, ricin I, ricin II, ricin III, ricin 11, ricin 12, ricin 2, abrin, abrin-a (abrin C, abrin-III), abrin-b, abrin-c (abrin A, abrin-I), abrin-d, abrin-II, APA (Abrus oryzae)precatorius agglutinin, Abrus lectin, AAG), APA-I, APA-II, Pulchellin, Pulchellin PI, Pulchellin PII, Pulchellin PIII, α-pisavin, β-pisavin, Sativin, IrisRIP (IRIP), IrisRIP. A1, IrisRIP. A2, IrisRIP. A3, IRA, IRAb, IRAr, CA-SRI, CIP-29, CIP-34, Leonurin, Bodinierin, Camphorin, Cinnamomin, Cinnamomin 1, Cinnamomin 2, Cinnamomin 3, Cinphorin, Porrectin, Abelesculin, Boerhavia Inhibitor, BAPI, Bougainvillea RIP I), BBP-24, BBP-28, BBAP1, ME1, ME2, MAP, MAP-2, MAP-3, MAP-4, MAP-S, Malanin, Riproximin (Rpx, Rpx-I, Rpx-II), Modeccin (Modeccin 4B, Modeccin 6B), A. ellenbeckii lectin, A. fruticosa lectin, A. glauca lectin, A. goetzei lectin, A. keramanthus lectin, Lanceolin, A. racemosa lectin, A. spinosa lectin, stenodactylin, A. venenata lectin, Volkensin, α-PAP, PAP (Phytolaccaamericana protein, pokeweed antiviral protein), PAP-I, PAP-II, PAP-III, PAP-C, PAP-H, PAP-R, PAP-S, PAP-S1, PAP-S2, Diocin 1, Diocin 2, PD-L1, PD-L2, PD-L3, PD-L4, PD-S1, PD-S2, PD-S3, Dodecandrin, Dodecandrin C, Heterotepalin 4, Heterotepalin 5b, Insularin (PIP, Phytolacca insularis antiviral protein), PIP2 (P. insularis antiviral protein 2), Barley toxin (Barley translation inhibitor, Barley protein synthesis inhibitor, BPSI, RIP 30), Barley toxin I (barley translation inhibitor I), Barley toxin II (barley translation inhibitor II, barley protein synthesis inhibitor II, BPSI II), Barley toxin III (barley translation inhibitor III), JIP60, rice (Oryza sativa) RIP, RPSI, Tritin, Tritin 1, Tritin 2, Tritin 3, Tritin-S, Tritin-L, b-32 (maize RIP, maize proRIP1), maize proRIP2, EHL, PCL, HmRip, HmRip 1, HmRip 2, HmRip 3, HmRip 4. ML-I (Mistletoe lectin I, Viscumin, Eu-ML, EML-1, VAA-I), ML-II (Mistletoe lectin II, VAA-II), ML-III (Mistletoe lectin III, VAA-III), Articulatin-D, KML, KML-C, KML-IIL, KML-IIU, VCA, CIP31, TRIP, and P. macrocarpa RIP, and / or viral toxins, such as apoptin; bacterial toxins, such as Shiga toxin, Shiga toxin-like toxin, Pseudomonas28. The base of claim 27, which comprises or consists of any one or more of: P. aeruginosa exotoxin (PE), or exotoxin A of PE, full length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins, such as alpha-sarcin; ribosome inactivating proteins, and plant toxins including the A chain of ribosome inactivating proteins type 2, such as giantin, such as giantin-30 or giantin-32, saporin, such as saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin debouganin, shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog RNase, or granzyme B, or angiogenin from humans, or any fragment or derivative thereof.

29. 29. The base of claim 27 or 28, wherein the oligonucleotide, xenonucleic acid or nucleic acid comprises or consists of any one or more of a vector, a gene, a transgene inducing 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), a 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA), or a derivative thereof.

30. The base according to any one of claims 27 to 29, wherein the effector molecule comprises or consists of at least one payload.

31. The base according to any one of claims 1 to 26, wherein the carrier molecule comprises or consists of a covalently linked combination of an effector molecule and a monoclonal antibody.

32. 1. A method for producing a base suitable for covalently binding at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 to a carrier molecule, said method comprising: a) providing a polymeric or oligomeric structure, comprising a first chemical group for covalently coupling said polymeric or oligomeric structure to said carrier molecule and comprising at least one second chemical group different from said first chemical group, each second chemical group for covalently coupling one of said at least one bisdesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 to said oligomeric or polymeric structure; b) covalently coupling, via said second chemical group(s), at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 to said polymeric or oligomeric structure, said bisdesmosidic triterpene saponin(s) belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 being any one of the bisdesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 according to any one of claims 1 to 11; The polymer or oligomer structure is: poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines); - polyethylene glycol, - poly- or oligo(esters), e.g. poly(lactide); - poly(lactam), - polylactide-co-glycolide copolymers, poly- or oligosaccharides, such as cyclodextrins and polydextrose; - selected from the group consisting of poly- or oligo(amino acids), such as proteins, peptides, and polylysine, and - DNA oligomers or polymers, RNA polymers, stabilized RNA polymers, and PNA (peptide nucleic acid) polymers; said aldehyde functional group at position C-23 is covalently coupled to said polymeric or oligomeric structure via a hydrazone bond; Thereby providing said base.

33. A method for producing a base covalently bound to a carrier molecule, said base comprising at least one covalently bound bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23, said method comprising: a) providing a base comprising at least one bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23, covalently bonded to a polymeric or oligomeric structure of said base; b) covalently coupling the base of a) to a carrier molecule according to any one of claims 21 to 31, This provides said base covalently bound to a carrier molecule, said base comprising at least one covalently bound bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function at position C-23, The polymer or oligomer structure is: poly- or oligo(amines), such as polyethyleneimines and poly(amidoamines); - polyethylene glycol, - poly- or oligo(esters), e.g. poly(lactide); - poly(lactam), - polylactide-co-glycolide copolymers, - poly- or oligosaccharides, such as cyclodextrins and polydextrose; - selected from the group consisting of poly- or oligo(amino acids), such as proteins, peptides, and polylysine, and - DNA oligomers or polymers, RNA polymers, stabilized RNA polymers, and PNA (peptide nucleic acid) polymers; said aldehyde functional group at position C-23 is covalently coupled to said polymeric or oligomeric structure via a hydrazone bond; The at least one covalently linked bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 is selected from the group consisting of saponins having a quillaic acid aglycone core and saponins having a gypsogenin aglycone core, the saponin comprising a branched carbohydrate side chain at the C-3 position, the carbohydrate side chain comprising a glucuronic acid functional group, and the saponin comprising a branched carbohydrate side chain at the C-28 position comprising a deoxy carbohydrate.

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