Cluster of biologically active molecule

Abstract

To provide a base preferable for covalently bonding at least one biologically active molecule to a carrier molecule.SOLUTION: Provided is a base which consists of a) polymer or oligomer structure, and b) at least one saponin SO1861 covalently bonded to the polymer or an oligomer structure, the base further contains c) a first chemical group for coupling the base to a carrier molecule in a covalently bonded state, e) the base is preferable for covalently bonding the at least one saponin SO1861 to the carrier molecule, and f) the carrier molecule contains or consists of any one of protein molecule, protein, peptide, nucleic acid, oligonucleotide, lipid, fat, fatty acid, nanoparticle and carbohydrate.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a molecular backbone suitable for covalently bonding at least one bioactive molecule, such as a therapeutic drug molecule, to a carrier molecule. Specifically, the present invention relates to an antibody-drug conjugate based on a monoclonal antibody having an improved therapeutic window for a drug due to the covalent linkage of an enhancer molecule (cluster), such as a payload, such as a protein toxin or oligonucleotide, to an ADC, or alternatively, due to the co-administration of an ADC and a cell-targeting conjugate containing an enhancer molecule (cluster) to a patient in need thereof. The present invention also relates to a method for producing a backbone suitable for covalently bonding at least one bioactive molecule to a carrier molecule, and such bioactive molecule is covalently bonded to the backbone.

Background Art

[0002] Molecules with therapeutic bioactivity are often theoretically well-suited 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 bioactive moiety. However, many potential drug-like molecules and therapeutic agents, if not all, currently used clinically suffer from at least one of a plethora of drawbacks and deficiencies. When administered to the human body, a therapeutically active molecule can exert off-target effects in addition to the bioactivity directed at aspects underlying the disease or health problem to be treated. Such off-target effects are undesirable and carry the risk of inducing side effects that are detrimental to the health or even life of the administered molecule. The occurrence of such adverse events causes many drug-like compounds and therapeutic agents to fail in phase III trials or even phase IV trials (post-marketing follow-up). Thus, there is a strong desire to provide drug molecules such as small molecule therapeutics, where the therapeutic effect of the drug molecule is, for example, (1) highly specific for the biological factor or biological process driving the disease, (2) sufficiently safe, (3) sufficiently effective, (4) sufficiently targets diseased cells with little or no off-target activity against non-diseased cells, (5) has a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in a human patient within a certain time frame and remain at the target site over a certain time frame), and / or (6) should have a therapeutic activity that persists sufficiently long in the patient's body. Despite already extensive and intensive research, and despite impressive progress made in some areas addressing the individual challenges and deficiencies faced, unfortunately, to date, an "ideal" therapeutic agent having many or even all of the beneficial features outlined above is not available to patients.

[0003] Chemotherapy is one of the most important treatment options for cancer treatment. However, since it does not have specificity for cancer cells compared to the dividing cells of healthy tissues, it is often associated with a low therapeutic window. The present invention of monoclonal antibodies offers the possibility of exploiting their specific binding properties as a mechanism for the targeted delivery of cytotoxic agents to cancer cells while sparing normal cells. This can be achieved by the chemical conjugation of cytotoxic effectors (also known as payloads or warheads) to antibodies to create antibody-drug conjugates (ADCs). Typically, very potent payloads such as emtansine (DM1), which have a limited therapeutic index (the ratio of the toxic dose to the effective dose) in their unconjugated form, are used. The conjugation of DM1 to trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla, improves the tolerated dose of DM1 by at least two-fold in monkeys. Over the past few decades, enormous efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, it has remained difficult to bring ADCs into the clinic. The first ADC to be approved for clinical use was gemtuzumab ozogamicin (CD33-targeted Mylotarg, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML) in 2000. However, Mylotarg was withdrawn from the market at the request of the Federal Drug Administration (FDA) due to several concerns, including its safety profile. Patients treated with Mylotarg were found to die more often than those treated with conventional chemotherapy. Mylotarg was re-approved for market in 2017 with a lower recommended dose, different schedules in combination with chemotherapy or by itself, and in a new patient population. To date, only five ADCs have been approved for clinical use, while the clinical development of approximately 55 ADCs has been discontinued. However, the interest remains high, and currently, approximately 80 ADCs are still in clinical development by nearly 600 clinical trials.

[0004] Despite the potential to use payloads with toxicity that is generally not tolerated by patients, the low therapeutic index (the ratio of the toxic dose to the effective dose) is a major problem that explains the discontinuation of many ADCs in clinical development. This can be caused by several mechanisms, such as off-target toxicity to normal cells, the development of resistance to cytotoxic agents, and the premature release of the drug in circulation. The systematic review by the FDA of ADCs has found that the toxicity profiles of most ADCs can be categorized according to the payload used rather than the antibody used, suggesting that toxicity is largely determined by the premature release of the payload. It is estimated that at least 23 out of approximately 55 discontinued ADCs were due to poor therapeutic indices. For example, the development of trastuzumab tesirine conjugate (HER-2 targeted ADCT-502, ADC therapeutics) was recently discontinued due to a narrow therapeutic index, presumably caused by on-target normal tissue effects in lung tissue expressing a significant level of HER2. In addition, several ADCs in phase 3 trials were discontinued due to missing primary endpoints. For example, the phase 3 trials of depatuxizumab mafodotin conjugate (EGFR targeted ABT-414, AbbVie) tested in patients with newly diagnosed glioblastoma and mirvetuximab soravtansine conjugate (folate receptor alpha (FRα) targeted IMGN853, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently stopped 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 anti-cancer 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 be able to exist without exerting its maximum possible anti-tumor effect.

[0005] An ADC mainly consists of an antibody, a cytotoxic moiety such as a payload, and a linker. In the design and development of new ADCs to overcome existing problems, several new strategies have been proposed and implemented, targeting each of the ADC components. For example, by identifying and validating a satisfactory antigenic target for the antibody component, 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 and accessible to circulating ADCs, and antigens that allow internalization of the ADC into cells after binding; and alternative mechanisms of activity; designing and optimizing a linker that improves the solubility and drug-to-antibody ratio (DAR) of the ADC and overcomes resistance induced by proteins that can transport chemotherapeutic agents extracellularly; improving the DAR ratio by including more payloads and selecting and optimizing the antibody to improve the homogeneity and developability of the antibody. In addition to the technological development of ADCs, new clinical and bridging strategies are also being developed to maximize the therapeutic index, for example, changing the dosing schedule by fractionated dosing; conducting biodistribution studies; optimizing patient selection by including biomarkers, capturing response signals early, monitoring the duration and depth of response, and providing information for 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, are being tested in clinical trials. The ADCs are each combined with rituximab, a monoclonal antibody that binds to CD20 and does not provide a payload (B. Yu and D. Liu, Antibody-drug Conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12:94). Combinations of monoclonal antibodies such as these examples are yet another approach, attempting to reach the "magic bullet" that combines many or even all of the desired characteristics mentioned earlier for ADCs.

[0007] On the one hand, in the past few decades, nucleic acid-based therapeutic agents 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, as well as DNA and RNA aptamers for approaches such as gene therapy, RNA interference (RNAi). Many of them share the same underlying 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 being conducted in the field of gene therapy, with nearly 2,600 ongoing or completed trials worldwide, but only about 4% enter phase III. Next to this are the trials with ASO. Similar to ADC, despite numerous technologies being explored, therapeutic nucleic acids share two major issues during clinical development: delivery into cells and off-target effects. For example, ASOs such as peptide nucleic acid (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acid (LNA), and bridged nucleic acid (BNA) are being considered as attractive strategies for specifically inhibiting genes that are difficult to target by target genes, especially small molecule inhibitors or neutralizing antibodies. Currently, the effectiveness of different ASOs is also being studied in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in some cancer stages. The application of ASOs as potential therapeutic agents requires a safe and effective method for their delivery into the cytoplasm and / or nucleus of target cells and tissues. Although the clinical validity of ASOs has been demonstrated, inefficient cellular uptake both in vitro and in vivo limits the effectiveness of ASOs and has been a barrier to therapeutic development. Cellular uptake can be <2% of the dose, resulting in ASO concentrations that are too low at the active site for effective and sustained outcomes. This ultimately requires an increase in the administered dose, which induces 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 agents are most commonly small molecules. However, their effectiveness is hampered by severe secondary off-target toxicity, as well as their poor solubility, rapid clearance, and limited tumor exposure. Skeleton-small molecule drug conjugates, such as polymer-drug conjugates (PDCs), are macromolecular constructs with pharmacological activity that contain one or more molecules of a small molecule drug attached to a carrier skeleton (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 oncology indications. However, more recently, only one drug not related to cancer (the PEG oligomer conjugate of the opioid antagonist naloxone, Movantik, AstraZeneca) has been approved in 2014 for opioid-induced constipation in patients with chronic pain, a non-oncology indication. The translational application of drug-skeleton conjugates to the treatment of human subjects has thus far provided little clinical success. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) showed significant anti-cancer activity against both solid tumors and leukemia in a mouse model and was under clinical investigation for oncology indications. Despite demonstrating a significant reduction in non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer efficacy was found to be modest in patients, and as a result, further development of PK1 was discontinued.

[0010] The failure of skeleton-small molecule drug conjugates is at least partially attributed to their poor accumulation at the tumor site. For example, in a murine model, PK1 showed 45- to 250-fold higher accumulation in tumors than in healthy tissues (liver, kidney, lung, spleen, and heart), but accumulation in tumors was only observed in a small subset of patients in the clinical trial.

[0011] A solution as a possible way to address the aforementioned problems is the application of nanoparticle systems for drug delivery, such as liposomes. Liposomes are spherical vesicles composed of one or more phospholipid bilayers and are formed spontaneously when phospholipids are dispersed in water. The amphiphilic properties of phospholipids endow 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 liposome delivery systems can offer several advantages compared to direct drug administration, such as improved pharmacokinetics and pharmacodynamics and control, tissue targeting properties, reduced toxicity, and enhanced drug activity. An example of such success is the liposomal encapsulated form of the small molecule chemotherapeutic agent doxorubicin (Doxil: pegylated liposomal encapsulated form of doxorubicin; Myocet: non-pegylated liposomal doxorubicin), which is approved for clinical use.

[0012] Therefore, there remains a need to find a solution that allows for drug therapy, such as anti-tumor therapy, applicable for non-systemic use when desired, where the drug has, 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 one embodiment of the present invention, a first goal is to provide an improved bioactive compound or a composition comprising such an improved bioactive compound.

[0014] One of several objectives of some embodiments of the present invention is to provide a solution to the problem of non-specificity faced when administering a low molecular weight therapeutically active compound to a human patient in need thereof. One of several objectives of some embodiments of the present invention is to provide a solution to the problem of drugs having suboptimal specificity for biological factors or biological processes driving a disease. One of several objectives of some embodiments of the present invention is to provide a solution to the problem of the inadequate safety profile of current drugs when administered to a human patient in need thereof. One of several objectives of some embodiments of the present invention is to provide a solution to the problem that current drugs are not as effective as desired when administered to a human patient in need thereof. One of several objectives of some embodiments of the present invention is to provide a solution to the problem that current drugs do not sufficiently target diseased cells without or with little off-target activity against non-diseased cells when administered to a human patient in need thereof. One of several objectives of some embodiments of the present invention is to provide a solution to the problem that current drugs do not have a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in a human patient within a certain time frame and remain at the target site over a certain time frame) when administered to a human patient in need thereof. One of several objectives of some embodiments of the present invention is to provide a solution to the problem that current drugs do not have a therapeutically active duration that lasts long enough in the patient's body when administered to a human patient in need thereof.

[0015] At least one of the above objectives of embodiments of the present invention is achieved by providing a molecular backbone suitable for covalently bonding at least one bioactive molecule, such as a therapeutic drug molecule, to a carrier molecule in the present invention.

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

[0017] One aspect of the present invention relates to a backbone suitable for covalently bonding at least one bioactive molecule to a carrier molecule, the backbone comprising a polymeric or oligomeric structure, at least one of the bioactive molecules being covalently bonded to the polymeric or oligomeric structure, and the backbone further comprising a first chemical group for covalent coupling of the backbone to the carrier molecule.

[0018] One embodiment is the backbone of the present invention, wherein at least one bioactive 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] One embodiment is the backbone of the present invention, wherein at least one bioactive molecule is an amphiphilic molecule.

[0020] One embodiment is the backbone of the present invention, wherein when more than one bioactive molecule is covalently bonded to the polymeric or oligomeric structure comprised by the backbone, at least one bioactive molecule is a single specific molecule or a mixture of different molecules.

[0021] One embodiment is the backbone of the present invention, and at least one bioactive molecule is a saponin that can 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, for example, 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, quillaja saponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereomers and / or any combination thereof. Preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21, and / or a quillaja 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-glucuronopyranosyl quillaja 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 the backbone of the present invention, and at least one bioactive molecule is covalently attached to the polymeric or oligomeric structure of the backbone via a cleavable bond, and the cleavable bond is cleaved in vivo under acidic conditions such as those present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≤ 5.5.

[0023] One embodiment is the backbone of the present invention, and at least one bioactive molecule is a defined number of glycoside molecules or a defined range of glycoside molecules, preferably 1 to 128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 glycoside molecules, or any number of glycoside molecules in between, for example 7, 9, 12 glycoside molecules.

[0024] One embodiment is the backbone of the present invention, and the polymeric or oligomeric structure includes linear, branched, and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures, and this assembly is preferably assembled by covalent cross-linking.

[0025] One embodiment is the backbone of the present invention, and the carrier molecule includes 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 a molecule including or consisting of an antibody, IgG, Vhh domain or Vh domain, Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, or includes 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 the backbone of the present invention, where the carrier molecule comprises or consists of at least one effector molecule, or the carrier further comprises at least one effector molecule, and at this time the carrier also comprises, for example, an immunoglobulin. The effector molecule is any one or more of at least one of the prodrugs, such as a payload, a toxin, a drug, a polypeptide, an oligonucleotide, a nucleic acid, a xeno nucleic acid, an enzyme, such as urease and Cre recombinase, a protein toxin, and a ribosome-inactivating protein.

[0027] One aspect of the present invention relates to a method for producing a backbone suitable for covalently bonding at least one bioactive molecule to a carrier molecule. The method comprises: a) providing a polymer or oligomer structure comprising a first chemical group for covalent coupling of a polymer structure or oligomer structure to a 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 at least one bioactive molecule to the oligomer or polymer structure; and b) covalently coupling at least one bioactive molecule to the polymer or oligomer structure via the second chemical group(s). Preferably, the bioactive molecule(s) is / are any one of the bioactive molecules of the present invention, such as a saponin, a triterpenoid saponin, a bisdesmoside-type triterpene, more preferably SO1861 and / or GE1741 and / or SA1641 and / or QS-21, thereby providing a backbone.

[0028] One aspect of the present invention relates to a method for generating a backbone covalently attached to a carrier molecule, the backbone comprising at least one covalently attached bioactive molecule, the method comprising: a) providing a backbone comprising at least one bioactive molecule covalently attached to a polymeric or oligomeric structure of the backbone, preferably providing a backbone according to the present invention or a backbone obtainable by the method of the present invention or a backbone obtained by the method of the present invention; and b) covalently coupling the backbone of a) to a carrier molecule according to the present invention, thereby providing a backbone covalently attached to the carrier molecule, the backbone comprising at least one covalently attached bioactive molecule.

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

[0030] Definitions The term "linker" has its usual scientific meaning and here refers to a linear stretch of amino acid residues complexed via peptide bonds or a chemical moiety. This attaches a molecule or atom to another molecule, for example to a ligand or an effector molecule or to a backbone. Typically, a linker comprises a chain of atoms linked by chemical bonds. Any linker molecule or linker technology known in the art may be used in the present disclosure. Where indicated, the linker is a linker for covalent attachment of a molecule via a covalent linkage to the linker or via a chemical group on such a molecule that is suitable for forming a covalent bond. 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 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 can be used in the context of the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate, or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0031] The term "trifunctional linker" has its usual scientific meaning and here refers to a linker that attaches to three molecules via a chemical group of each of the three molecules. A person skilled in the art can design such a trifunctional linker based on the present disclosure and ordinary 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 a so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with a saponin having an azide. Finally, the trifunctional linker can obtain a third functional group such as a trans-cyclooctene (TCO) group and perform a so-called inverse electron demand Diels-Alder (IEDDA) reaction with an effector molecule having a tetrazine (Tz). A person skilled in the art will understand that all three chemical groups of the trifunctional linker can be the same or different, or the linker can include two of the same chemical group for linking a molecule to the trifunctional linker. The bonds formed between trifunctional linkers can be covalent or non-covalent, with covalent bonds being preferred. The bonds formed between the trifunctional linker and one or two or three binding molecules via each chemical group can be cleavable (labile) bonds that can be cleaved under acidic conditions in cells such as endosomes and lysosomes of mammalian cells such as human cells, or non-cleavable bonds. Of course, the trifunctional linker can include one or two chemical groups for forming a covalent bond, and the further two or one chemical group(s) are for forming non-covalent bonds respectively. Of course, the trifunctional linker can include one or two chemical groups for forming a cleavable bond, and the further two or one chemical group(s) are for forming non-cleavable bonds respectively.

[0032] For example, the term "cleavable" as used in the terms "cleavable linker" or "cleavable bond" has its ordinary scientific meaning and here refers to undergoing cleavage under conditions such as acidic conditions, reductive conditions, enzymatic conditions, or photoinductive conditions. For example, a cleavable linker can undergo cleavage under acidic conditions. Preferably, the cleavable linker undergoes cleavage in vivo under the acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≤ 5.5. As another example, a cleavable linker can undergo cleavage by an enzyme, such as a cathepsin, for example 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 an oligomeric or polymeric backbone have their ordinary scientific meanings. Here, a polymer refers to a substance having a molecular structure assembled mainly or completely from a number of equal or similar units joined together; here, an oligomer refers to a polymer whose molecule consists of a relatively small number of repeating units. For example, a structure containing 5 to 10 or fewer equal or similar units can be called an oligomeric structure, a structure containing 10 to 50 or more monomer units can be called a polymeric structure, and a structure of 10 monomer units can be called either oligomeric or polymeric.

[0034] The term "binding site" has its ordinary scientific meaning and here refers 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 here refers to an oligomer or polymer template or carrier or base (base molecule or base structure) to which one or more molecules, such as ligand molecules, effector molecules, can be covalently bound either directly or via a linker such as a cleavable linker. The scaffold can have a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or can have an aggregated polymer structure, such as a hydrogel, microgel, nanogel, stabilized polymer micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers such as cholesterol / phospholipid mixtures. The scaffold can include a polymer or oligomer structure, such as poly or oligo(amine), such as polyethyleneimine and poly(amidoamine); or polyethylene glycol, poly or oligo(ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, etc.; or poly(dextrin), poly or oligosaccharide, such as cyclodextrin or polydextrose; or natural and / or artificial poly or oligoamino acid, such as polylysine or peptide or protein, DNA oligo or polymer, stabilized RNA polymer or PNA (peptide nucleic acid) polymer, etc. Preferably, the polymer or oligomer structure is biocompatible, and biocompatibility means that the polymer or oligomer structure either shows no substantial acute or chronic toxicity in an organism and can be excreted as such, or can be completely degraded to excretable and / or physiological compounds by the body's metabolism.

[0036] The term "ligand" has its usual scientific meaning and here refers to any molecule(s) 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 target autoimmune cell. The ligand can bind to an epitope contained by a receptor or other antigen on the target cell. Preferably, the cell-binding ligand is an antibody.

[0037] As used herein, the term "antibody" is used in the broadest sense and can refer to an immunoglobulin (Ig) defined as a protein belonging to the class IgG, IgM, IgE, IgA, or IgD (or any of their subclasses), or 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 the parent immunoglobulin or other derivative that 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 an 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 (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rIgG), minibodies, diabodies, triabodies, tetra-bodies, Fc fusion proteins, nanobodies, variable V domains such as VHH, Vh, and other types of antigen-recognition immunoglobulin fragments and domains. Fragments and domains can be engineered to minimize or completely remove intermolecular disulfide interactions occurring between the CH1 and CL domains. Functional fragments and domains offer the advantage of greater tumor penetration due to their smaller size. In addition, a functional fragment or domain can be more uniformly distributed in tumor masses compared to the whole immunoglobulin.

[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 present invention can be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, resurfaced antibodies, anti-idiotype antibodies, mouse antibodies, rat antibodies, rat / mouse hybrid antibodies, llama antibodies, heavy-chain only llama antibodies, heavy-chain only antibodies, and veterinary antibodies. Preferably, the antibody (immunoglobulin) of the present invention is a monoclonal antibody. Resurfaced, chimeric, humanized, and fully human antibodies are also more preferred. This is because they are less likely to cause immunogenicity in humans. The antibody of the ADC of the present invention preferably binds specifically to an antigen expressed on the surface of cancer cells, autoimmune cells, diseased cells, 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 to a lesser extent in terms of number and / or affinity).

[0040] Specific antibodies that can be used in the ADC of the present invention include, but are not limited to, anti-HER2 monoclonal antibodies such as trastuzumab and pertuzumab, anti-CD20 monoclonal antibodies such as rituximab, ofatumumab, tositumomab, and ibritumomab, anti-CA125 monoclonal antibodies such as oregovomab, anti-EpCAM (17-1A) monoclonal antibodies such as edrecolomab, anti-EGFR monoclonal antibodies such as cetuximab, panitumumab, and nimotuzumab, anti-CD30 monoclonal antibodies such as brentuximab, anti-CD33 monoclonal antibodies such as gemtuzumab and huMy9-6, anti-vascular integrin 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 labelizumab, anti-CD44v6 monoclonal antibodies such as bevacizumab, 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 provides a covalently bound saponin according to the present invention. These ligands include, but are not limited to, proteins, polypeptides, peptides, and small molecules. Examples of these non-antibody ligands are interferon (e.g., IFN-α, IFN-β, and IFN-γ), transferrin, lectin, 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 (IGF, e.g., IGF-1 and IGF-2), other suitable hormones, such as thyrotropin-releasing hormone (TRH), melanocyte-stimulating hormone (MSH), steroid hormones (e.g., estrogen and androgen), somatostatin, lymphokines (e.g., IL-2, IL-3, IL-4, and IL-6), colony-stimulating factors (CSF, e.g., G-CSF, M-CSF, and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g., AS-1411, GBI-10, RNA aptamer against HIV glycoprotein), small molecules (e.g., folic acid, anisamide phenylboronic acid), vitamins (e.g., vitamin D), carbohydrates (e.g., hyaluronic acid, galactose).

[0042] "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 interaction with an effector molecule target within the cell. This effector molecule target is any molecule or structure within the cell that excludes the compartments and lumens of the endocytosis and recycling pathways but includes the membranes of these compartments and vesicles. Therefore, the said structures within the cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytoplasmic matrix.

[0043] The effector molecule or moiety is a prodrug, such as a toxin, such as a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide. The prodrug 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 the diagnosis, prognosis prediction, treatment, cure, and prevention (prophylaxis) of diseases and / or symptoms and / or health problems. The prodrug can also lead to unwanted and in some cases even harmful side effects (such as adverse events observed during clinical trials). In this case, the pros and cons must be weighed to determine whether the prodrug is suitable for a particular case. If the effect of the prodrug in a certain cell is overall overwhelmingly beneficial to the organism, the cell is called a target cell. If the effect in a certain cell is overall overwhelmingly harmful to the organism, the cell is called an off-target cell. In artificial systems such as cell cultures and bioreactors, the target cells and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules and moieties are drugs, toxins, polypeptides (such as enzymes), polynucleotides (including polypeptides and polynucleotides containing non-natural amino acids or nucleic acids), and any combination thereof.

[0044] The effector molecule or effector moiety that is a drug can include, but is not limited to, anti-cancer agents, anti-inflammatory agents, and anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral) agents. Preferably, the drug molecule of the present invention is an anti-cancer agent or an anti-autoimmune agent. Suitable anti-cancer agents include, but are not limited to, alkylating agents, metabolic inhibitors, spindle poison plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. The definition of "anti-cancer agent" includes, for example: (i) anti-hormone agents that act to control or inhibit the hormonal action on tumors, such as anti-estrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase that controls estrogen production in the adrenal gland; (iii) anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes of 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 also includes any pharmaceutically acceptable salts, acids, solvates, and derivatives of the above.

[0045] Effector molecules or moieties that are toxins can include, but are not limited to, proteinaceous toxins (such as bacterial-derived toxins 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, viscumicin, Shiga toxin, Shiga-like toxin, Pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of tubulin filament-targeting toxins are maytansinoids (such as DM1 and DM4), auristatins (such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), toxoid, tubulysin, cryptophycin, and lysocine. Examples of DNA-targeting toxins are calicheamicin: N-acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycin, doxorubicin, methotrexate, benzodiazepine, camptothecin analogs, and anthracyclines. Examples of DNA-targeting toxins are amatoxin, spliceostatin, and thailanstatin. The toxins used in the present invention are defined as prodrugs that can kill or inactivate cells. Preferably, the targeted toxin is a toxin that is toxic only or at least predominantly to the target cells, not to off-target cells. The net effect of the targeted toxin is preferably beneficial to the organism as a whole.

[0046] The effector molecule or moiety that is a polypeptide can be, for example, a polypeptide that restores a lost function such as, for example, enzyme replacement, gene regulatory function, or a toxin. Examples of polypeptides as effector molecules are, for example, Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)buganine, 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).

[0047] The effector molecule or effector moiety that is a polynucleotide can be, for example, a polynucleotide containing coding information, such as a gene or open reading frame encoding a protein. It can also contain regulatory information, such as a promoter or regulatory element binding region, or a sequence encoding a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include, for example, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally occurring DNA or RNA by a modification of the molecular backbone. Examples of nucleotides as effector molecules are, 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), but are not limited thereto.

[0048] For example, the term "proteinaceous" as used in "proteinaceous molecule" and "proteinaceous toxin" refers to molecules and toxins that contain at least one string of amino acid residues that can be obtained as an expression product from a single mRNA. Such a molecule or toxin may further include any post-translational modification, carbohydrate, such as N- or O-linked carbohydrate, disulfide bond, phosphorylation, sulphatation, etc. as a result of any post-translational modification, and / or may further include that resulting from any other modification, such as a chemical modification (e.g., directly to an amino acid side chain or via at least one linker that is (covalently) attached to the molecule and (covalently) chemically attached to the proteinaceous molecule for chemically modifying the proteinaceous molecule) of an effector moiety, saponin, backbone, ligand, etc.). The term "proteinaceous" also encompasses and includes aggregates of such molecules, such as homodimers, heterotrimers, heterohexamers, or complex aggregates, such as ribosomes.

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

[0050] The term "binding" is defined as an intermolecular interaction 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 assembles an intact protein domain. A binding fragment according to the present invention must have binding specificity for each target, such as a cell surface receptor on the surface of diseased cells such as, for example, tumor cells.

[0052] The term "ADC" or "antibody-drug conjugate" has its usual scientific meaning known to those skilled in the art and herein refers to a class of drugs of biopharmaceuticals designed, for example, as a targeted therapy for treating cancer. Different from chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. An ADC consists 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 intention of discriminating between healthy cells and diseased tissues such as tumor cells in a tumor.

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

[0054] The term "quillaja saponin" has its usual meaning and herein refers to the saponin fraction of Quillaja saponaria and thus to all other sources of QS saponins, mainly containing QS-18 and QS-21.

[0055] "QS-21" or "QS21" has its usual scientific meaning and here refers 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 usual scientific meaning and here refers to a mixture of QS-21 A-apio (~65%) and QS-21 A-xylo (~35%).

[0057] Similarly, "QS-21B" has its usual scientific meaning and here refers 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 and contains a variable quantity of more than 50 distinct (water-soluble) saponins, many of which incorporate the triterpene-trisaccharide moiety 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 F.A.Kersten,Glycosyl Compositions and Structural Characteristics of the Potential Immuno - adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY,VOL.9,660 - 666(1995)). Quil - A, and also quillaja saponin, are also fractions of saponins from Quillaja saponaria, both containing a large variety of different saponins and having largely overlapping contents. Since the two fractions are obtained by different purification procedures, the two fractions differ in their specific compositions.

[0059] The terms "QS1861" and the term "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 QS1862 of QS-7. The molecular mass is 1862 daltons. This is because this mass is the nominal mass including a proton in glucuronic acid. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in 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 rather to distinguish similar elements. The terms may be interchangeable in appropriate circumstances. Embodiments of the invention may operate in a sequence other than that described or illustrated herein.

[0061] Furthermore, although various embodiments are described as "preferred" or "e.g." or "for example" or "specifically", they should be construed as exemplary manners in which the invention may be implemented rather than as limiting the scope of the invention.

[0062] The term "comprising" as used in the claims is not to be construed as being limited to the elements or steps recited thereafter; it does not exclude other elements or steps. It is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Accordingly, the scope of the expression "a pharmaceutical composition comprising A and B" should not be limited to a pharmaceutical composition consisting only of components A and B, but rather, in the context of the present invention, the recited components of the pharmaceutical composition are only A and B, and further, the claims should be construed as encompassing equivalents of those components. Similarly, the scope of the expression "a method comprising step A and step B" should not be limited to a method consisting only of steps A and B, but rather, in the context of the present invention, the recited steps of the method are only A and B, and further, the claims should be construed as encompassing equivalents of those steps.

[0063] In addition, unless the context clearly requires that there be only one of the features, a reference to a feature by the indefinite article "a" or "an" does not exclude the possibility that there may be more than one of the feature, for example, there may be more than one component, excipient, saponin, etc. Accordingly, the indefinite article "a" or "an" usually means "at least one".

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0065] For a bioactive molecule to function, the molecule 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 enter the cytoplasmic matrix of the target cell, for example, to mediate its target regulatory effect. In many constellations, the toxin remains ineffective. This is because (1) the targeting moiety is poorly internalized and remains bound outside the cell, (2) it is recycled to the cell surface after internalization, or (3) it is transported to the endolysosome where it is degraded. These fundamental issues have been known for decades and more than 500 targeted toxins have been investigated in the past few decades, but the problem remains unsolved, and only one antibody-targeted protein toxin, moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP), has been approved by the FDA to date for relapsed or refractory hairy cell leukemia.

[0066] Numerous strategies for overcoming these problems are described, including approaches for transferring toxins to endogenous cell membrane transport complexes in the biosynthetic pathway in the endoplasmic reticulum, and techniques for blocking or weakening the membrane integrity of endosomes, i.e., compartments of the endocytosis pathway in cells, and thus facilitating endosomal escape. This includes the use of lysosome-affinity amines, carboxylic acid ionophores, calcium channel antagonists, viruses, bacteria, plants, animals, humans, and various cell-penetrating peptides of synthetic origin, other organic molecules, and photoinductive techniques. The effectiveness of targeted toxins typically increased 100-fold or 1000-fold in cell culture, and in exceptional cases more than a million-fold, but the requirement to co-administer endosomal escape enhancing factors with other substances poses new problems including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell type-dependent variability.

[0067] All strategies, including physicochemical techniques, interact more or less directly with membranes and require molecules of enhancing factors that are essentially small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not themselves target cell-specific and are distributed in a manner other than that of the targeted toxin. This is one of the major drawbacks of current approaches.

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

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

[0070] One aspect of the present invention is a backbone suitable for covalently bonding at least one bioactive molecule to a carrier molecule, the backbone comprising a polymer or oligomer structure, at least one of the bioactive molecules being covalently bonded to the polymer or oligomer structure, and the backbone further comprising a first chemical group for covalently coupling the backbone to a carrier molecule.

[0071] One embodiment is a backbone according to the present invention, wherein at least one bioactive 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, 1,700 daltons to 1,950 daltons.

[0072] One embodiment is a backbone according to the present invention, wherein at least one bioactive molecule is an amphiphilic molecule. Such amphiphilic molecules, such as (phospho)lipids, glycosides, such as saponins, can typically be or form part of layers such as bilayers and lipid layers, such as cell membranes, and / or can be part of, for example, intracellular vesicles.

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

[0074] One embodiment is a backbone according to the present invention, and at least one bioactive molecule is a glycoside, preferably a bisdesmoside-type triterpene or triterpenoid saponin, more preferably a bisdesmoside-type triterpene saponin, most preferably a bisdesmoside-type triterpene saponin belonging to the type of 12,13-dehydrooleanane 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, for example as part of an ADC, and a specific number or range of glycoside molecules per effector molecule, simultaneously, for example via the endocytosis pathway of the cell, to the cytoplasmic matrix of the cell. The solution provided by the present invention is to immobilize and further polymerize glycoside molecules, enabling re-monomerization at intracellular sites where the mechanism of action of the glycoside is desired, for example after endocytosis. In this context, "polymerize" means the reversible and / or irreversible multiple conjugation of glycoside molecules into a polymer or oligomer structure for forming a backbone. Alternatively, it means the reversible and / or irreversible multiple conjugation of (modified) glycoside molecules, thereby forming a polymer or oligomer structure for forming a backbone. In this context, "re-monomerization" means, for example, the cleavage of glycoside molecules from the backbone after endocytosis and the re-acquisition of the (native) chemical state of the unbound glycoside molecules. These unbound glycoside molecules may or may not contain additional chemical groups, for example chemical groups for linking the glycoside to the backbone and / or (chemical) linkers. Due to the complex chemistry of glycoside molecules, the "polymerization" of glycoside molecules and their "re-monomerization" at desired locations, for example intracellularly, after endocytosis, was a difficult task. In particular, the chemical reactions (polymerization of glycosides) used to provide the backbone of the present invention, which includes covalently linked glycosides, for example triterpenoid saponins, usually occur in water-free organic solvents, while glycoside molecules and biocompatible polymers are water-soluble molecules. The chemical properties of unmodified glycoside molecules further hindered their polymerization by themselves. Also, one other solution for binding multiple glycoside molecules (directly) to effector molecules was not considered very promising because effector molecules (drugs, toxins, polypeptides, or polynucleotides) typically do not provide sufficient binding sites.Moreover, because the coupling products become highly heterogeneous and / or coupling a bioactive molecule such as a glycoside together with, for example, a peptide, a toxin, a nucleic acid, has the risk of affecting and interfering with the activity of one or even both of the molecules bound together in a conjugate containing such glycoside. Further, there was a significant risk that the effector molecule would lose its function after coupling. The present invention solves at least one of these drawbacks.

[0076] One embodiment is a backbone according to the present invention, wherein at least one bioactive molecule is a saponin that can be isolated from a Gypsophila species and / or a Saponaria species and / or an Agrostemma species and / or a Quillaja species, such as Quillaja saponaria, or a single specific saponin, or a mixture of two or more different saponins, for example, one or more of the saponins of Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 Any one and / or any combination of 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, Kiryasaponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypenoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or their stereomers. Preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21, and / or the Kiryac 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-glucuronopyranosyl Kiryac 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, Scheme I, and the above embodiments summarize a series of saponins that have been identified for their improved endosomal escape activity when contacted with mammalian cells such as human cells in free form together with a second molecule (e.g., an effector moiety or an effector molecule). Indeed, in cell-based bioassays, for the saponins represented in Table A1 and the saponins of Scheme I, in the presence of these saponins, a second molecule, e.g., a nucleic acid and / or a toxin, e.g., a protein toxin (e.g., one or more of the protein toxins listed in Table A5), has been established to be released into the cell from (late) endosomes and lysosomes with increased efficiency and / or effectiveness. That is, the endosomal and / or lysosomal escape of such a second molecule (i.e., an effector moiety, an effector molecule), e.g., a nucleic acid and / or a toxin, is less efficient in the absence of saponin.

[0078] Surprisingly, here, the inventors have found that QS-21 and its family members QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 A water-soluble saponin fraction from Quillaja saponaria, which contains B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18 and is also sometimes referred to as "Quil-A", is demonstrated to also show 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, as a single covalent conjugate containing a monoclonal antibody, an effector molecule (the second molecule mentioned previously) and at least one glycoside such as QS-21 and its family member saponins encapsulated by such QS-21 preparations, it is co-administered to tumor cells of mammalian species (human). The effector and glycoside, such as QS-21, SO1861, SA1641, GE1741, are either covalently bound to the polymer or oligomer backbone either directly or via at least one linker, or the glycoside is covalently bound to the effector molecule and the effector molecule is covalently bound to the polymer or oligomer backbone. Alternatively, the glycoside and effector molecule are covalently bound to the backbone separately, and the monoclonal antibody is also separately bound to the saponin, toxin, this conjugate of the backbone, and the backbone is a trifunctional linker encompassing a polymer or oligomer structure such as the backbone outlined in Scheme II and Structure B. Without being bound by any theory, the observed stimulation or enhancement of tumor cell killing mediated by, for example, saporin or dianthin in the presence of saponins from Quillaja saponaria may (also) relate to the activation of the inflammasome of tumor cells by the saponin, resulting in, for example, pyroptosis of tumor cells.

[0079] QS-21 and the water-soluble saponin fraction of Quillaja saponaria have also long been known and have been previously intensively applied, for example, due to their immune-enhancing ability as an adjuvant 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. The anti-tumor vaccination enhanced by QS-21 aimed to extend the disease-free survival of cancer patients (melanoma; non-small cell lung cancer). In addition, QS-21 has been tested as an adjuvant in trials for the development of anti-cancer vaccines, for vaccines against HIV-1 infection, for the development of vaccines against hepatitis B, and for the development of anti-malaria vaccines using QS-21, including Glaxo-Smith-Kline's adjuvants AS01 and AS02. Previous studies have revealed the immune response induced against the MAGE-A3 peptide presented on the cancer cell surface under the influence of QS-21 saponin containing an adjuvant (AS15; GSK). Surprisingly to the inventors, the saponins in the Quillaja saponaria fraction containing QS-21 enhance the anti-tumor cell activity of payloads such as protein toxins (dianthin). The saponin is covalently coupled, for example, to cetuximab. And when the ligand-dianthin conjugate is exposed to tumor cells in the presence of cetuximab-saponin, the toxic effect of dianthin 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 present inventors show that tumor cell-targeted monoclonal antibodies such as cetuximab provided with covalently coupled antisense BNA (HSP27) and provided with covalently coupled saponin (here SO1861) can silence in vivo HSP27 of tumors as compared to controls and as compared to ADCs without coupled saponin only. Both BNA and saponin are coupled to the antibody by a cleavable bond (here a hydrazone bond), via which both BNA and SO1861 are coupled to a trifunctional linker (here the trifunctional oligomer backbone of Scheme II). Therefore, conjugating the ADC with saponin results in a conjugate containing BNA and saponin, and this combination confers on the ADC antitumor cell activity not seen with the same dose of ADC. Notably, monoclonal antibodies and ADCs covalently coupled with saponin increase HSP27 expression in tumor cells as compared to a control group (administered vehicle only) when separately administered to mice bearing tumors in separate groups of mice. Only the ADCs containing the backbone of the present invention with covalently coupled saponin and effector molecules show reduced HSP27 expression when compared to controls. Antisense BNA (HSP27) is described in 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 A BNA having an oligonucleotide sequence 5'-GGCacagccagtgGCG-3' according to Gene Therapy (2011) 18, 326-333), which uses locked nucleic acid (LNA)-based antisense oligonucleotides for cancer targets without transfection. Notably, to the best of the inventors' knowledge, BNA is designed for application as a free nucleic acid. Here, the inventors demonstrate for the first time that antisense BNA can be covalently coupled to a ligand or antibody via a (non)-cleavable linker in such a way that gene silencing activity is retained in tumor cells of animals with tumors in vitro and more importantly in vivo. This approach opens up a new way to administer the targeted BNA to human (cancer) patients in need thereof.

[0081] One embodiment is a backbone according to the invention, wherein at least one bioactive molecule is a bisdesmoside-type saponin having a molecular mass of at least 1,500 daltons and comprising an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, having a first branched carbohydrate side chain at the C-3 position, and this first branched carbohydrate side chain optionally contains glucuronic acid. The saponin contains an ester group having a second branched carbohydrate side chain at the C-28 position, and this second branched carbohydrate chain preferably contains at least 4 carbohydrate units, optionally contains at least 1 acetyl residue, for example 2 acetyl residues, and / or optionally contains deoxycarbohydrates, and / or optionally contains quinovose, and / or optionally contains glucose, and / or optionally contains 4-methoxycinnamic acid, and / or optionally contains 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally contains 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and is linked to the carbohydrate via an ester bond. Alternatively, at least one saponin is any one or more of QS-21, or 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), Quil-A. Such saponins are further exemplified in Table A1 and Scheme I. The inventors herein demonstrate that covalently coupling a saponin, such as a water-soluble saponin fraction of QS-21, SA1641, SO1861, Quillaja saponaria, to a backbone, 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 backbone further comprises an effector molecule(s) covalently attached, for example, a proteinaceous toxin such as dianthin or saporin and / or a (tumor) cell surface molecular targeting ligand or moiety such as a monoclonal antibody that binds to a (tumor) cell surface receptor.

[0082] One embodiment is the backbone of the present invention and, as the bioactive molecule, comprises a saponin comprising one or some or all of the indicated structural features of the saponin of Structure A of Scheme I, and / or a saponin selected from any one or more of the further saponins of Scheme I:

[0083]

Chemical formula

[0084]

Chemical formula

[0085]

Chemical formula

[0086]

Chemical formula

[0087]

Chemical formula

[0088] According to the invention, a bioactive molecule having an "ideal" structure for the purpose of improving the endosomal escape of effector molecules bound to the backbone of the invention as a carrier molecule is a bidesmoside-type saponin according to structure A of Scheme I, having a molecular mass of at least 1,500 daltons, containing an oleanane-type triterpene having an aldehyde group at C-23 and optionally a hydroxyl group at C-16, having a first branched carbohydrate side chain at C-3, which first branched carbohydrate side chain optionally contains glucuronic acid. The saponin contains an ester group having a second branched carbohydrate side chain at C-28, which second branched carbohydrate chain preferably contains at least 4 carbohydrate units, optionally contains at least 1 acetyl residue, for example 2 acetyl residues, and / or optionally contains deoxycarbohydrates, and / or optionally contains quinovose, and / or optionally contains glucose, and / or optionally contains 4-methoxycinnamic acid, and / or optionally contains 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally contains 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and is bound to the carbohydrate via an ester bond.

[0089] SO1861 has only one acetyl residue on the quinovose and only has additional xylose, which is different from the "ideal structure" shown by Structure A of Scheme I. The "ideal structure" of saponin for improving endosomal escape of effector molecules or effector moieties is preferably a saponin having Structure A of Scheme I, and saponins showing improved endosomal escape activity have one or more of the structural features shown by Structure A of Scheme I. Without wishing to be bound by any theory, the inventor believes that Structure A of Scheme I corresponds to the "ideal saponin" (but not the minimum requirement saponin) for improved endosomal escape activity. This does not mean that all structures must be present or must be present on each saponin having at least sufficient improved endosomal escape activity to promote the accumulation of the effector moiety in the cytoplasmic matrix, which means that some saponins may have other structural elements such as acyl chains, and / or for other saponins showing improved endosomal escape activity, the sugars may be different from the sugars shown by Scheme I. For example, when considering the ideal structure of Structure A of Scheme I, some of the saponins in the water-soluble fraction of QS-21 saponin and Quillaja saponaria (kilaya saponin; Quil-A) have different carbohydrate modifications at C-28: for example, the presence of an acyl chain in QS-21. Saponins such as QS-7 and QS1862 in the water-soluble fraction of Quillaja saponaria are similar to the ideal Structure A and are similar to SO1861.

[0090] To describe the present invention in more detail, the process of cellular uptake of substances (although the inventors do not intend to be bound by any theory) and the terms used in the present invention are described. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. The aforementioned vesicle budding can be characterized by (1) receptor-dependent ligand uptake mediated by the cytoplasmic matrix 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 the next cellular process of vesicular transport and material sorting, called the endocytic pathway. The endocytic pathway is complex and not fully understood. Without intending to be bound by any theory, organelles can be formed de novo and mature into the next organelle along the endocytic pathway. However, here, a hypothesis has been proposed that the endocytic pathway involves stable compartments connected by the passage of vesicles. Compartments are complex multifunctional membrane organelles specialized for a specific set of functions essential to the cell. Vesicles are considered transient organelles, with a simpler composition and defined as containers surrounded by membranes formed de novo by budding from existing compartments. In contrast to compartments, vesicles can undergo maturation, which is a series of biochemical changes that are physiologically irreversible. Early endosomes and late endosomes correspond to stable compartments in the endocytic pathway, and primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and further lysosomes correspond to vesicles. At the cell membrane, endocytic vesicles, most notably arising from clathrin-coated pits, first fuse with the early endosome, which is a major sorting compartment at approximately pH 6.5. Most of the internalized cargo and membrane are recycled to the plasma membrane via recycling vesicles (the 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 the late endosomal compartment when needed.The resulting organelles are called hybrid organelles or endolysosomes. Lysosomes bud from the hybrid organelles by a process called lysosome reformation. Late endosomes, lysosomes, and hybrid organelles are extremely dynamic organelles, and the distinction between them is often difficult. Degradation of endocytosed molecules occurs within the endolysosome or lysosome. Endosomal escape is the active or passive release of substances from the lumen of any type of compartment or vesicle, preferably from the endocytic pathway, preferably clathrin-mediated endocytosis or the recycling pathway to the cytoplasmic matrix. Therefore, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediates and hybrid organelles. Unless specifically indicated otherwise, and particularly when referring to the endosomal escape mechanism of glycoside molecules, whenever the term "endosome" or "endosomal escape" is used herein, it includes endolysosomes and lysosomes and escape from endolysosomes and lysosomes. After entering the cytoplasmic matrix, the substance can move to other cellular units such as the nucleus. In formal terms, a glycoside is any molecule in which a sugar group is attached via its anomeric carbon to another group by a glycosidic bond. Without being bound by any theory, glycoside molecules in the context of the present invention are molecules that can further enhance the effect of effector molecules, particularly by facilitating the endosomal escape of effector molecules. Without being bound by any theory, glycoside molecules interact with the membranes of compartments and vesicles in the endocytic and recycling pathways, making them leaky for the effector molecules and resulting in increased endosomal escape.The term "the backbone can increase the endosomal escape of effector molecules" means that when both molecules are within an endosome, for example a late endosome, optionally and preferably, for example by cleavage of a cleavable bond between at least one glycoside and a polymer or oligomer structure, after at least one glycoside has been released from the polymer or oligomer structure, at least one glycoside molecule coupled to the polymer or oligomer structure of the backbone can improve the endosomal escape of the effector molecule. Even if the bond between at least one glycoside and the backbone can be a "stable bond", this does not mean that such a bond cannot be cleaved in the endosome, for example by an enzyme. For example, the glycoside can be cleaved from the remaining polymer structure together with a linker or part of the polymer structure. For example, it can happen that a protease cleaves a proteinaceous polymer structure, such as albumin, thereby releasing at least one glycoside. However, it is preferred that the glycoside molecule is released in its active form, preferably in the original form it had before it was (prepared to be) coupled to the backbone; therefore, the glycoside has its natural structure after such cleavage, or the glycoside has a chemical group or linker (part of it) attached to it after such cleavage, but the bioactivity of the glycoside, for example its activity to improve endosomal / lysosomal escape with respect to effector molecules present in the same endosome or lysosome, is maintained or restored by the bond between the glycoside and the carrier molecule, for example by the aforementioned cleavage of the backbone of the present invention. For the purposes of the present invention, the term "stable" means that for a bond between a saponin, a polymer or oligomer structure (of the backbone), a ligand, a (monoclonal) immunoglobulin or its binding domain or fragment, and / or an effector (effector moiety, effector molecule), the bond is not readily broken, or at least is not designed to be readily broken, for example by a pH difference, salt concentration, or UV light.For the present invention, the term "cleavable" means, with respect to the bonds between saponin, the polymeric or oligomeric structure of the backbone of the present invention, the ligand, the antibody, and / or the effector, that the bond is designed to be readily broken, for example, by a pH difference, a salt concentration, reducing conditions, and the like. Those skilled in the art are well aware of 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 interaction with an effector molecule target within the cell, where the effector molecule target is any molecule or structure within the cell, excluding the compartments and lumen of endocytic and recycling pathways but including the membranes of these compartments and vesicles. Accordingly, said structures within the cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytoplasmic matrix. Delivery of an effector molecule to the cytoplasmic matrix in the context of the present invention preferably means that the effector molecule can escape from endosomes (and / or lysosomes) (which also includes escaping from endolysosomes and lysosomes as defined above), and preferably can reach an effector molecule target as described herein. The present invention is a new type of molecule and serves as a backbone for simultaneously bringing both an effector molecule and at least one glycoside molecule into endosomes in a predetermined ratio. In the context of the present invention, the polymeric or oligomeric structure of the backbone is either a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or it is an aggregated polymeric structure, such as a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, excluding structures consisting of non-covalent aggregates of monomers such as cholesterol / phosopholipid mixtures. The terms "polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer" have their ordinary meanings. Specifically, a polymer is a substance having a molecular structure mainly or completely assembled from a large number of equal or similar units bonded together, and an oligomer is a polymer whose molecules consist of a relatively small number of repeating units. There is no consensus on a specific cutoff for the "large number" and "small number" used in the definitions of polymer and oligomer respectively above.However, since the backbone can include polymer or oligomer structures or both, the full range of the number of similar units that are joined together, i.e., from two monomer units to 100 monomer units, 1000 monomer units, and more, applies to such structures. For example, a structure containing five or fewer can be called an oligomer structure, and a structure containing 50 monomer units can be called a polymer structure. A structure of 10 monomer units can be called either an oligomer or a polymer. The backbone defined herein further includes at least one glycoside molecule. The backbone preferably includes a polymer or oligomer structure, such as poly or oligo(amine), such as polyethyleneimine and poly(amidoamine), and a biocompatible structure, such as polyethylene glycol, poly or oligo(ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), poly or oligosaccharide, such as cyclodextrin or polydextrose, and poly or oligoamino acid, such as polylysine or peptide or protein, or DNA oligo or polymer. The aggregated polymer structure defined herein includes at least one backbone and optionally other individual polymer or oligomer structures. The other individual polymer or oligomer structures of the aggregate can be (a) a backbone (e.g., including at least one glycoside molecule), (b) a functionalized backbone (e.g., including at least one glycoside molecule and a ligand, antibody, etc., and / or an effector molecule), (c) a polymer or oligomer structure including at least one ligand, antibody, etc., and / or at least one effector, or (d) a polymer or oligomer structure without a glycoside molecule, without a ligand, antibody, etc., and without an effector molecule. A functionalized aggregated polymer structure is an aggregated polymer structure containing (a) at least one functionalized backbone or (b) at least one backbone and at least one polymer structure including at least one ligand, antibody, etc., and / or at least one effector.Polymers or oligomeric structures on the aggregated polymer structure that do not contain any of the molecules mentioned above (i.e., without glycosides, ligands, antibodies, or effectors) are added, in particular, as structural components of the aggregated structure. This helps to assemble or stabilize the aggregated structure ("glue-like"). The inventors have found that, in particular, a backbone containing a polymer structure having many primary, secondary, and / or tertiary amine groups does not particularly improve endosomal escape after coupling to a glycoside molecule. In this regard, polymer and oligomeric structures containing secondary amine groups are particularly unfavorable. Without being bound by any theory, the acidic environment appears to be a prerequisite for the synergistic action between the glycoside and the effector, and such amine groups are believed to disrupt the acidic environment of the late endosome. Thus, a backbone that can disrupt the acidic environment due to the presence of, for example, amine groups is preferably not encompassed by the backbone according to the present invention. However, of course, primary amine groups can be blocked or shielded, for example, by thiolation or PEGylation. After appropriate blocking or shielding of the primary amine groups, which is known to those skilled in the art, such backbones are encompassed by the claims.

[0092] In a particularly preferred embodiment, the present invention provides a backbone that does not substantially inhibit the endosomal (and lysosomal) escape of effector molecules, preferably containing less than 10, more preferably less than 5, even more preferably less than 2 primary, secondary, or tertiary amine groups, and most preferably none. For clarity, it is emphasized that a primary amine group blocked, for example, by thiolation or PEGylation is no longer called an amine group.

[0093] Whether the backbone can disrupt the acidic environment and inhibit the endosomal escape function of at least one glycoside can be readily determined by assays as described in Example 4 and as known in the art. Inhibition is described as an “increase in the amount of glycoside ~-fold that is required for 50% induced cell killing”. The backbone preferably does not result in an increase that is at least the increase in the glycoside molecules required to obtain 50% cell killing as observed when using chloroquine as a positive control. Alternatively and preferably, the backbone does not result in an increase of at least 4-fold, more preferably at least 2-fold, of the glycoside molecules required to induce 50% cell killing. The ~-fold increase should be essentially measured by the assay described in Example 4, and chloroquine as a positive control induces an increase of 2-fold in the amount of glycoside to observe 50% cell killing.

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

[0095] Several favorable features can be formulated for the endosomal escape enhancing factor, i.e., the glycosides according to the present invention: (1) Preferably, they are not toxic and do not induce an immune response, (2) Preferably, they do not mediate the cytoplasmic matrix 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, (5) Preferably, they do not substantially interfere with biological processes of organisms that are irrelevant to the biological activity of the effector molecules with which the endosomal escape enhancing factor is combined, for example, do not interact with hormones. Examples of glycoside molecules that meet the previously mentioned criteria to at least some extent are bisdesmoside-type triterpenes, preferably bisdesmoside-type triterpene saponins, such as SO1861, SA1641, QS-21, GE1741.

[0096] One embodiment is a backbone according to the invention, in which at least one bioactive molecule, preferably a glycoside, is covalently bound to a polymer or oligomeric structure via a non-cleavable bond or via a cleavable bond, preferably said cleavable bond is cleaved under acidic conditions, reducing conditions, enzymatic conditions, or photoinduced conditions, more preferably the cleavable bond is a hydrazone bond or a hydrazide bond that is cleaved under acidic conditions, and / or a bond sensitive to proteolysis, such as proteolysis by cathepsin B, and / or a bond sensitive to cleavage under reducing conditions, such as a disulfide bond. According to the invention, typically the bioactive molecule is a saponin of the invention (see also Table A1, Scheme I). When the entry of an effector molecule covalently coupled to the same backbone as the saponin into cells and its accumulation in the cytoplasmic matrix are considered, for the activity of the saponin, such as the activity of improving endosomal escape in cells, it has been proven beneficial when the saponin is covalently coupled to the backbone and hydrazone bonds and / or hydrazide bonds and / or disulfide bonds are involved. Such types of bonds are easily cleaved under acidic conditions in (late) endosomes and lysosomes of mammalian cells, such as human cells, and / or under reducing conditions. Alternatively, the inventor has also demonstrated that the covalent coupling of saponin to a carrier molecule via a bond that is not easily cleavable under physiological conditions in cells, such as (late) endosomes, lysosomes, and the cytoplasmic matrix, is also beneficial for the enhancing activity of saponin on the biological effects of effector moieties such as nucleic acids (e.g., BNA silencing HSP27) and proteinaceous toxins such as saporin. In the present application including the claims, when a backbone having a conjugate of the invention, such as a saponin coupled to the backbone via a hydrazone bond or a disulfide bond, is referred to, terms such as "cleavable linker", "cleavable bond", etc. are also referred to as "labile linker" ("L") and "labile bond" in the context of cleavage of such a bond or linker in (late) endosomes and / or lysosomes.For example, Figure 1 shows in vivo HSP27 gene silencing in a mouse tumor. Mice bearing tumors were treated with a backbone according to the invention comprising an oligomeric trifunctional linker (see Scheme II and Structure B). Saponin SO1861 was covalently attached thereto via a hydrazone bond, and an antisense BNA for silencing the HSP27 gene of tumor cells was covalently coupled to the backbone via a hydrazone bond, and the monoclonal anti-EGFR antibody cetuximab was covalently coupled to the backbone via a disulfide bond. Hydrazone bonds and disulfide bonds are said to be cleavable and thus labile bonds. That is, without wishing to be bound by any theory, once a conjugate containing the backbone of the invention is internalized, for example by endocytosis, the hydrazone bond and the disulfide bond are cleaved in the (late) endosomes and / or lysosomes of target tumor cells expressing EGFR on the cell surface. When entry of the BNA from the endosome and / or lysosome into the cytoplasmic matrix is considered, cleavage of the bond will presumably contribute to the endosomal escape enhancing activity of saponin, but such cleavage is not essential for observing the gene silencing effect of the cetuximab(saponin)(BNA) conjugate comprising the trifunctional linker, i.e., the backbone of the invention.

[0097] One of ordinary skill in the art will appreciate that such trifunctional linkers are the backbone of the present invention suitable for covalently coupling one, two, or three saponin moieties. For trifunctional linkers, covalent coupling of one or two saponin moieties is preferred. The second and / or third binding sites are suitable for covalently coupling effector moieties such as payloads, e.g., protein toxins, small molecule toxins, nucleic acids. The second or third binding site of the trifunctional linker is also suitable for covalent coupling of non-proteinaceous ligands and / or proteinaceous ligands for targeting cells such as tumor cells or autoimmune cells. Typical proteinaceous ligands are EGF for targeting (tumor) cells expressing EGFR on the cell surface, and cytokines for targeting tumor cells or autoimmune cells. Further, the second or third binding site of the trifunctional linker is suitable for covalent coupling of immunoglobulins such as monoclonal antibodies for binding to cell surface molecules, e.g., tumor cell surface molecules, preferably tumor cell-specific molecules, more preferably tumor cell receptors specifically (over)expressed on the surface of tumor cells. Similarly, immunoglobulins, or any fragment(s) and / or domain(s) thereof encompassing the binding specificity of immunoglobulins are suitable for binding to cell surface molecules such as receptors expressed on the surface of autoimmune cells. Thus, in certain embodiments, the trifunctional linker comprises, or consists of, a covalently attached saponin, e.g., QS-21, SO1861, a covalently attached effector moiety, e.g., a toxin or oligonucleotide, e.g., BNA, and a covalently attached cell targeting moiety, e.g., a ligand or antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, non-healthy cells, B cell diseases.

[0098] One embodiment is the backbone of the present invention and includes an oligomeric trifunctional linker as a backbone core structure according to Scheme II:

[0099]

Chemical Structure

[0100] As an example, saponin and here as an example the effector part are covalently bound to a trifunctional linker backbone via an unstable cleavable hydrazone linker (acid sensitive) and / or when the effector part is considered optionally via a bond containing a maleimide with cysteine on a carrier molecule. The (optional) bond of the backbone to a carrier molecule such as a linker or an antibody is established via an unstable cleavable hydrazone linker (acid sensitive) and / or via a bond containing a maleimide with cysteine on the carrier molecule, for example 1, 2, 3, or 4 cysteines. Thereby, Structure B is formed:

[0101]

Chemical formula

[0102] As a result, 1 to 4 backbones are covalently bound to a single carrier molecule such as a monoclonal antibody. Therefore, a single carrier molecule, for example an antibody, a ligand, an effector part, can be covalently conjugated to a single backbone, for example the trifunctional linker of Scheme II and Structure B, or two or more backbones according to the invention, the two or more backbones being the same or different and the two or more backbones optionally containing the same (number) or different (number of) covalently bound saponins.

[0103] One embodiment is a backbone according to the present invention, wherein at least one bioactive molecule is covalently bound to a polymer or oligomer structure via a cleavable bond, and the cleavable bond is in vivo in mammalian cells, preferably in an acidic condition present in the endosome and / or lysosome of human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≤ 5.5, and undergoes cleavage. Such a cleavable bond facilitates the release of bioactive molecules such as one or more saponins when the backbone is in an acidic environment within such cells, such as a late endosome. Without wishing to be bound by any theory, when endosomal escape of an effector moiety present within an endosome or lysosome is contemplated, free saponins within the endosome and / or lysosome can contribute to the endosomal escape enhancing activity of the saponin. Such endosomal escape enhancing activity is, for example, higher or more efficient compared to the activity of saponins bound to the backbone. The inventors have established that saponins bound to a backbone and a carrier molecule via bonds such as hydrazone bonds, amide bonds, disulfide bonds, etc. can all improve the cytotoxic effects of a diverse payload on effector moieties or molecules within (tumor) cells, such as proteinaceous toxins and oligonucleotides such as antisense BNA.

[0104] One embodiment is a backbone according to the present invention, wherein at least one bioactive molecule is covalently bound to the polymer or oligomer structure of the backbone via an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thio-ethyl bond, an amide bond, a peptide bond, or an ester bond, preferably via at least one linker, and the bioactive molecule is one or more saponins of the present invention. The inventors have demonstrated that when cells such as tumor cells are exposed to both a backbone such as an oligomeric trifunctional linker or dendron having saponins covalently coupled thereto and an effector moiety such as antisense BNA or saporin, covalent coupling of, for example, saponins to the backbone using such bonds results in an improvement in the effect and activity of the effector moiety.

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

[0106] One embodiment is a backbone according to the present invention, 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, and this linker is covalently coupled via a thio-ethyl bond to a sulfhydryl group on the polymer or oligomer structure of the backbone, such as the sulfhydryl group of cysteine.

[0107] One embodiment is a backbone according to the present invention, wherein the glucuronic acid functional group on the 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-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate, and this linker is covalently coupled via an amide bond to an amine group on the polymer or oligomer structure of the backbone, such as the lysine or N-terminal amine group of a proteinaceous molecule.

[0108] One embodiment is the backbone of the present invention, the glycoside molecule is a saponin, and the linkage between the saponin and the polymer or oligomer structure on the backbone preferably occurs 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 the amino group of the polymer or oligomer structure and the aldehyde group of the saponin. Other chemical bonds that fulfill the pH conditions can also be used for aldehyde coupling. For example, specific hydrazones or acetals, which require hydrazide and hydroxyl groups as functional groups of the polymer or oligomer structure, respectively. When the bond is a cleavable bond, the saponin is preferably attached to the polymer or oligomer structure of the backbone via one of the carboxyl groups in the saponin or via an aldehyde functional group, more preferably via an aldehyde functional group, preferably the aldehyde functional group at position 23. Alternatively, the saponin is preferably attached to the polymer or oligomer structure of the backbone via a linker that joins the saponin to the polymer or oligomer structure of the backbone either via the carboxylic acid functional group of the glycoside molecule or via an aldehyde functional group.

[0109] One embodiment is the backbone of the present invention, and at least one glycoside molecule is attached to the polymer or oligomer structure via a stable bond. In a more preferred embodiment, at least one glycoside molecule is a saponin, and the stable bond between the saponin and the polymer or oligomer structure of the backbone preferably occurs by amide coupling or amine formation. This is achieved, for example, via carbodiimide-mediated amide bond formation by the amino group of the polymer or oligomer structure and the activated glucuronic acid group of the saponin. Chemical bonds that fulfill the definition of a stable bond can also be used for aldehyde coupling. For example, a specific amine is derived after reductive amination and requires a primary amino group as a functional group of the polymer or oligomer structure. When the bond is a stable bond, the saponin is preferably attached to the backbone via one of the carboxyl groups of the saponin.

[0110] One embodiment is a backbone according to the present invention, and the chemical group for covalent coupling of the backbone to a carrier molecule is a click chemistry group.

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

[0112] One embodiment is a backbone according to the present invention, and the backbone further comprises click chemistry groups for coupling to an effector molecule and / or a ligand, antibody, binding domain, or fragment thereof. The click chemistry groups are chemical functional groups suitable for click chemistry. This is defined as a reaction that is modular, broad in scope, gives very high yields, produces only innocuous by-products, provides high selectivity and high tolerance for different functional groups, and is stereospecific. The required process characteristics include simple reaction conditions, readily available starting materials and reagents, use of a solvent-free or mild (e.g., water) or easily removable solvent, and simple product isolation. The click chemistry groups are preferably tetrazine, azide, alkene, or alkyne, or their reactive derivatives, such as methyl-tetrazine or maleimide (alkene), more preferably alkyne, or cyclic derivatives of these groups, such as cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]nona-4-yne, dibenzocyclooctyne).

[0113] Therefore, the backbone according to the present invention contains at least one glycoside molecule. By "at least one" in this context, it is meant that the backbone contains one glycoside molecule, but it can also contain several (e.g., 2, 3, or 4) glycoside molecules, or a large number (e.g., 10, 20, or 100) of glycoside molecules. Depending on the application, the backbone can be designed to contain a defined number of glycoside molecules. Preferably, the backbone according to the present invention contains 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. The defined number in this regard means that the backbone preferably contains a previously defined number of glycoside molecules. This can be achieved, for example, by designing a polymer structure having a certain number of possible moieties for attaching the glycoside(s). In an ideal situation, all of these moieties are coupled to glycoside molecules, and then the backbone contains a previously defined number of glycoside molecules. It is envisioned to provide a standard set of backbones containing, for example, 2, 4, 8, 16, 32, 64 glycoside molecules, etc., so that the optimal number can be easily tested by the user according to his needs. In one embodiment, which is the backbone of the present invention, in a non-ideal situation, for example, not all moieties present on the polymer structure bind glycoside molecules, and the glycosides are present within a defined range. Such ranges can be, for example, 2 to 4 glycoside molecules per backbone, 3 to 6 glycoside molecules per backbone, 4 to 8 glycoside molecules per backbone, 6 to 8 glycoside molecules per backbone, 6 to 12 glycoside molecules per backbone, etc. Therefore, in such a case, the backbone according to the present invention contains 2, 3, or 4 glycoside molecules when the range is defined as 2 to 4.

[0114] One embodiment is a backbone according to the present invention, and the number of monomers of the polymer or oligomer structure is a precisely defined number or range. Preferably, the polymer or oligomer structure is a structure such as poly(amine), for example polyethyleneimine and poly(amidoamine), or a structure such as polyethylene glycol, poly(ester), for example poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, poly(dextrin), or a peptide or protein, or a structure such as natural and / or artificial polyamino acids, for example polylysine, DNA polymer, stabilized RNA polymer, or PNA (peptide nucleic acid) polymer. It appears as a pure or mixed, linear, branched, or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or an aggregate of these structures. Preferably, the polymer or oligomer structure is biocompatible, and biocompatibility means that the polymer or oligomer structure shows no substantial acute or chronic toxicity in organisms and can either be excreted as it is or be completely decomposed into excretable and / or physiological compounds by the body's metabolism. The aggregates can be assembled by covalent cross-linking or non-covalent bonds and / or attractive forces. Thus, they can also form nanogels, microgels, or hydrogels. Alternatively, they can be attached to carriers, for example inorganic nanoparticles, colloids, liposomes, micelles, or particle-like structures containing cholesterol and / or phospholipids. The polymer or oligomer structure preferably has a precisely defined number or range of coupling moieties for the coupling of glycoside molecules (and / or effector molecules, and / or carrier molecules, for example 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 the precisely defined number or range of coupling moieties on the polymer or oligomer structure are occupied by glycoside molecules in the backbone according to the present invention.

[0115] Preferably, the dendron is a branched, well-defined dendritic polymer having a single chemically addressable group at the origin of the tree, called the focal point. A dendrimer is the connection of two or more dendrons at their focal points. A dendronized polymer is the connection of the focal points of one or more dendrons to a polymer. In preferred embodiments, a backbone according to the invention is provided, and the polymer or oligomer structure includes, either pure or mixed, linear, branched, or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates of these structures. The aggregates can be assembled by covalent cross-linking or non-covalent attractive forces and can form nanogels, microgels, or hydrogels. Preferably, the polymer is a derivative of poly(amine), such as polyethyleneimine and poly(amidoamine), and structures such as polyethylene glycol, 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 peptides or proteins (such as ligands or antibodies, such as any of the monoclonal antibodies of Tables A2 - A4), or DNA polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. Preferably, the polymer or oligomer structure is biocompatible.

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

[0117] The prodrug in the present invention is an effector molecule, which 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. The benefits include the diagnosis, prognosis prediction, treatment, cure, and / or prevention of the disease and / or symptoms. The prodrug can also lead to unwanted harmful side effects. In this case, the pros and cons must be weighed to determine whether the prodrug is suitable for a particular case. If the effect of the prodrug in a certain cell is overwhelmingly beneficial for the whole organism, the cell is called a target cell. If the effect in a certain cell is overwhelmingly harmful for the whole organism, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, the target cells and off-target cells depend on the purpose and are defined by the user.

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

[0119] One embodiment is a backbone according to the present invention, the backbone being a trifunctional linker, comprising a second chemical group to which at least one bioactive molecule is covalently bound, a third chemical group for covalent binding to a molecule, and a first chemical group for covalent binding to a carrier, preferably, the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0120] One embodiment is a backbone according to the present invention, wherein at least one bioactive molecule is a defined number of glycoside molecules or a defined range of glycoside molecules, preferably 1 to 128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 glycoside molecules, or any number of glycoside molecules in between, such as 7, 9, 12 glycoside molecules.

[0121] One embodiment is a backbone according to the present invention, wherein the polymer or oligomer structure comprises linear, branched, and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, polylysine, polyethylene glycol, or an assembly of these polymer or oligomer structures, which assembly is preferably assembled by covalent crosslinking.

[0122] The backbone is, in essence, independent of the type of effector molecule covalently bound to the backbone. Therefore, the backbone is a fundamental product for a new platform technology. Since at least one covalently bound glycoside mediates intracellular delivery, the backbone technology according to the present invention is the first known system to mediate controlled intracellular effector molecule delivery by glycosides.

[0123] Synchronization is the missing link between a very successful delivery strategy in mice and its application in humans. Indeed, the inventors have established that in a series of in vivo mouse tumor models, administering the dose of free saponin and the dose of the ADC separately to mice does not result in any desired anti-tumor activity, such as delayed tumor growth, tumor regression, decreased and slower tumor growth, compared to control animals not treated with the ADC and free saponin. The free saponin was administered using various routes of administration and at various time points for administering the free saponin, compared to the moment of administering the ADC (administering the free saponin before, during, and after administering the ADC). The ADCs tested in the in vivo tumor models were cetuximab-diantin (with free SO1861) or trastuzumab-saporin (with free SO1861). Varying the dose of free saponin did not enable effective anti-tumor activity. The ADCs referred to were administered at doses that by themselves did not result in any beneficial anti-tumor effect on animals bearing tumors. Surprisingly, here the inventors have established that beneficial anti-tumor 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 that includes a backbone according to the invention. The backbone is, for example, a dendron and has up to four covalently linked saponin molecules, such as SO1861, via a cleavable linker. The backbone is, for example, a trifunctional linker and has a saponin (e.g., SO1861, QS-21) covalently linked via a cleavable or non-cleavable linker, an effector moiety (e.g., diantin, silencing BNA (HSP27)) covalently linked via a non-cleavable or cleavable linker, and a covalently linked monoclonal antibody, such as cetuximab, trastuzumab, OKT-9. Alternatively, the backbone is a dendron, for example, a dendron to which four moieties, such as four saponin molecules, can be attached, or a dendron for attaching, for example, two saponins and two effector molecules, and the dendron includes chemical groups for (covalent) coupling to a ligand or antibody or a fragment or domain thereof.Reference is made to the section of examples that illustrate various ones of these skeletons according to the invention, which exhibit anti-tumor cell activity in vivo and / or in vitro, for example when cytotoxicity exerted by a proteinaceous toxin is contemplated or when gene silencing in tumor cells is contemplated.

[0124] The skeletons of the invention provide an optimized and functionally active unit that can be linked, preferably via a covalent bond and, if desired for the intended purpose, via a cleavable covalent bond, to an effector molecule and / or to a ligand, antibody, etc. at a single defined position.

[0125] Judging from the failures observed when considering the treatment of animals with tumors by an ADC together with a free saponin without being constrained by any theory, it is preferable to synchronize the presence of at least one glycoside, preferably a saponin, and an effector molecule, preferably an oligonucleotide such as a toxin or BNA, in compartments or vesicles of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. In the case of ADCs and free saponins, synchronizing the presence of molecules in late endosomes in order to obtain a synergistic effect in vivo was not beneficially achievable according to numerous attempts by the inventors. In one aspect, the present invention preferably solves at least one of the following problems with respect to combining an effector molecule and a glycoside molecule as one compound comprising the backbone of the present invention: (1) the number of glycoside molecules required 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), and as a result, a simple chemical linker is not suitable; (2) for example, the only reasonable chemical group on the saponin that can be used for (covalent) a single and cleavable retainable coupling is required for endosomal escape activity; (3) the effector molecule may be present without having a suitable counter group for coupling; (4) when the glycoside is not freely diffusible, it can lose its necessary potential to interact with cholesterol. Most probably, all of these limitations are, for example, the reason why, although the remarkable endosomal escape enhancing effect of saponins listed in Table A1 and Scheme I has been known for more than 10 years, glycosides have not been used in combination with a prodrug in clinical studies other than the application of saponins in a vaccination regimen involving the use of an immune enhancing adjuvant substance. The backbone according to the present invention solves these difficulties at least in part.Surprisingly, the saponins previously applied with respect to their immune-enhancing activities in the context of vaccination involving saponin as an adjuvant component are, here, for the (covalent) coupling to the backbone of the present invention, for the involvement in conjugates comprising a backbone having a saponin and further an effector molecule (covalently) bound thereto and optionally a cell targeting moiety, such as a ligand or an antibody, also suitable for anti-tumor activities in vitro and in vivo.

[0126] In its basic form, the backbone comprises at least one glycoside molecule, such as a polymer and / or oligomer structure having a specific saponin, such as SO1861 (Table A1, Figure 13). In a preferred embodiment, a backbone is provided, the glycoside molecule is bound to the polymer or oligomer structure via a cleavable bond, preferably the cleavable bond undergoes cleavage under acidic, reductive, enzymatic, or photoinductive conditions, more preferably under acidic conditions. Preferably, the cleavable bond is an imine, hydrazone, oxime, 1,3-dioxolane, disulfide, hydrazide, or ester.

[0127] One embodiment is a backbone according to the present invention, the glycoside molecule being any of SA1641, SO1861, GE1741, QS-21, or stereoisomers thereof, such as any of their diastereomers.

[0128] One embodiment is a backbone according to the present invention, the carrier molecule comprising or consisting of a covalent conjugate or covalent complex of any of a proteinaceous molecule, protein, peptide, nucleic acid, oligonucleotide, lipid, fat, fatty acid, nanoparticle, carbohydrate, or combinations thereof.

[0129] One embodiment is a backbone according to the present invention, and 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 a molecule comprising or consisting of an antibody, IgG, Vhh domain or Vh domain, 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 cytokine.

[0130] One embodiment is a backbone according to the present invention, and the carrier molecule comprises or consists 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 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-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected from CD71, EGFR, HER2.

[0131] One embodiment is a framework according to the present invention, wherein the carrier molecule is cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, an IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, an 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, for example any one of at least one tumor cell-specific receptor binding fragment and / or at least one tumor cell-specific receptor binding domain thereof, or consists of the same.

[0132] One embodiment is a framework according to the present invention, which is suitable for forming a covalent bond with a carrier molecule, and the covalent bond preferably involves a cysteine side chain of the carrier molecule and / or a lysine side chain of the carrier molecule, and at this time the carrier molecule contains at least cysteine and / or lysine.

[0133] One embodiment is a framework according to the present invention, wherein the carrier molecule contains or consists of at least one effector molecule, or the carrier further contains at least one effector molecule, and at this time immunoglobulins according to the present invention, binding fragments thereof, binding domains thereof, etc. are also included. The effector molecule is at least one of prodrugs, for example, payloads, toxins, drugs, polypeptides, oligonucleotides, nucleic acids, xeno nucleic acids, enzymes, such as urease and Cre recombinase, protein toxins, ribosome-inactivating proteins, etc., and is one or more of them.

[0134] One embodiment is a framework according to the present invention, and the protein toxin includes one or more of 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; a mycotoxin, such as alpha-sarcin; a ribosome-inactivating protein, and a plant toxin including the A chain of a type 2 ribosome-inactivating protein, such as dianthin, such as dianthin-30 or dianthin-32, saporin, such as saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumicin, viscumicin A chain; or an animal or human toxin, such as frog RNase, or granzyme B, or angiogenin from human, or any fragment or derivative thereof; preferably, the protein toxin is dianthin and / or saporin.

[0135] One embodiment is a backbone according to the present invention, and the oligonucleotide, xeno nucleic acid, or nucleic acid is a vector, gene, transgene that induces cell death, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), plasmid, glycol nucleic acid (GNA), and threose nucleic acid (TNA), or a derivative thereof, preferably BNA, for example, any one or more of BNA for silencing HSP27 protein expression, or consists of.

[0136] Here, the backbone of the present invention enables the design and manufacture of a non-viral clinically applicable gene delivery technology for one component. For example, the backbone of the present invention allows the development of a virus-based gene delivery technology, which improves the therapeutic efficacy with a lower therapeutic dose, thereby improving the health of the patient. In particular, the backbone of the present invention, when covalently bound to a carrier molecule such as a monoclonal antibody for binding to (tumor, autoimmune) cell surface-specific molecules, and when bound to a carrier molecule such as an oligonucleotide, for example BNA, overcomes a long-standing and major bottleneck in the field of gene delivery, that is, allows efficient, safe, and cost-effective transfer of gene therapy products from the endosomal membrane into the cytoplasmic matrix / nucleosol. In fact, 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 target cells and uptake of genes into the cytoplasmic matrix and nucleosol. One of the major problems in the field of non-viral gene therapy is the inefficient and insufficiently safe delivery of genetic material for use in therapeutic agents to patients.

[0137] Therefore, when applying the backbone of the present invention, which contains a carrier molecule that targets cells, such as a ligand or preferably an antibody (its fragment, domain), and contains an oligonucleotide such as antisense BNA, the inventor has here enabled the overcoming of a long-standing and major bottleneck in the field of gene delivery: the safe transfer of gene therapy products from the endosomal membrane into the cytoplasmic matrix / nucleosol. The backbone of the present invention is designed to allow the targeting of any treatable cell type by all known gene agents, thereby guaranteeing better patient treatment not only for genetic diseases but also for cancer treatment, and thus corresponding to an important technology for a large patient population. The technology based on the backbone of the present invention is a polymeric or oligomeric backbone, which serves as a carrier for an endosome escape enhancing factor (EEE), such as any saponin of Table A1 and Scheme I and embodiments according to the present invention, for a targeting ligand or a (monoclonal) (tumor cell-specific) antibody, and for an effector moiety, here an effector gene, such as LNA or BNA. For example, the use of the backbone of the present invention containing a cell-targeting antibody (fragment) and an oligonucleotide, such as BNA, has the potential to bring any kind of biological macromolecule into the cytoplasmic matrix and nucleus. The development of new targeting ligands and monoclonal (human, humanized) antibodies is being continuously investigated by numerous research groups and companies worldwide. The same is true for oligonucleotides aimed at delivery into the cytoplasmic matrix of diseased cells such as cancer cells. Therefore, the backbone of the present invention provides a molecular interface for linking current and future targeting ligands and antibodies and current and future therapeutic oligonucleotides (and payloads, such as protein toxins) to the oligomeric or polymeric backbone modules of the present invention by click chemistry, allowing for customized drug application and future development in the fields of tissue and cell targeting technologies. The backbone of the present 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 the potential for treatment for a wide range of disease areas, such as cancer, cardiovascular diseases, Parkinson's, Alzheimer's, HIV, and many rare (single gene) diseases. Current gene therapy technologies based on viral vectors have significant challenges such as safety, manufacturing logistics, and associated high costs. The backbone of the present invention allows for use in a technology platform that serves as an alternative to current viral gene delivery technologies. Thus, the backbone of the present invention is suitable for implementation in an approach for developing non-viral gene treatments for diseases such as cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, HIV infection, and many rare (single gene) diseases. The backbone of the present invention is suitable for developing novel approaches for transforming the fields of antibody-drug conjugates (ADCs) and oligonucleotide-based therapeutic drugs, for example, by creating gene therapy drugs based on non-viral vectors based on targeted antisense BNA. The application of the backbone of the present invention is one of many beneficial approaches enabled by the present invention, particularly as a covalent conjugate with oligonucleotides such as antibodies and BNA. For example, here, the use of the backbone of the present invention allows for the exploitation of the endocytosis pathway of mammalian cells. Endocytosis is exploited for the delivery of therapeutic drugs, and the backbone of the present invention contributes to, for example, improved uptake and endosomal escape of siRNA conjugated to the backbone. The backbone of the present invention is preferably used together with, for example, a payload and a small molecule that acts as a delivery enhancer for oligonucleotides. Thereby, the backbone of the present invention having a covalently coupled oligonucleotide, such as BNA, and a covalently coupled cell targeting moiety, such as a ligand and preferably an antibody (domain or fragment), provides a solution to the current problems seen in current endosomal escape enhancers and gene therapy drug products that make the approval and clinical applicability of therapeutic drugs difficult. Because such a backbone of the present invention is a therapeutic drug molecule of a single conjugate that incorporates gene products such as saponin, BNA, and (tumor) cell targeting moieties such as (monoclonal) antibodies.Therefore, the present invention provides a non-viral gene delivery technology, where an endosome escape enhancing factor (e.g., the glycosides of Table A1, Scheme I, embodiments of the present invention), a gene therapy drug product (an oligonucleotide according to the present invention, e.g., BNA), and a targeting ligand or antibody (e.g., according to Tables A2, A3, A4, embodiments of the present invention) are all bound to one molecular backbone of the present invention. Therefore, such a backbone of the present invention provides therapeutic opportunities for current and future macromolecular drugs for a wide range of diseases and a large patient population. By applying such a backbone of the present invention, which includes at least one saponin, at least one oligonucleotide, and at least one specific cell targeting moiety, e.g., an immunoglobulin, problems are addressed that are apparent with current methods of separately applying an endosome escape enhancing factor and a gene therapy drug product. These current methods do not guarantee that both compounds are present at the interaction site simultaneously. Here, this problem is overcome by using the backbone of the present invention. That is, such a backbone of the present invention provides a non-viral gene delivery technology with an increased (temporal and spatial) synchronization of both compounds, i.e., a saponin and a gene product such as BNA.

[0138] Gene therapy can be useful for hereditary diseases that are currently incurable, such as cystic fibrosis, cholera, Huntington's disease, or hemophilia. However, currently, several problems have not been overcome: for example, the therapeutic gene must reach specific target cells in the body precisely. On the other hand, the therapeutic gene should be absorbed by the target cells, but the therapeutic gene should not be destroyed. Current gene therapy approaches use viruses as gene carriers. However, these procedures involve significant risks and cannot be transferred to the introduction of other biomolecules. One embodiment is the backbone of the present invention that contains (plant-derived) glycosides for use in a platform technology that not only allows the delivery of genes when bound to the backbone as a carrier molecule but also allows the delivery of different therapeutic biomolecules to be introduced into target cells. Thus, the backbone of the present invention is used to develop nucleic acid-based treatments for cystic fibrosis, cholera, Huntington's disease, or hemophilia. Thereby, a new gene therapy strategy for improving the health of patients with genetic diseases, including patients with cystic fibrosis, Huntington's disease, and hemophilia, is available by the backbone of the present invention. As part of the present invention, a non-viral gene delivery technology that combines a plant-derived endosome escape enhancer (glycoside), a gene therapy product, and a targeting ligand, all bound to a single molecular backbone, is developed. The non-viral gene therapy provided by the backbone of the present invention shows a delivery efficiency increased by approximately 40 times at a lower dose compared to currently available strategies. Thereby, the backbone of the present invention is for use in clinical applications, for example, for the repair or supplementation of defective genes as in patients with cystic fibrosis and for the targeted delivery of specific genes for, for example, destroying cancer cells. In fact, the backbone of the present invention is suitable for application to the treatment regimen of any disease caused by gene defects, such as cystic fibrosis, Huntington's disease, and hemophilia, which are currently incurable.Gene therapy using the backbone of the present invention helps to overcome two current problems: First, the backbone of the present invention enables the delivery of a therapeutic gene to specific target cells in the body; Second, although the therapeutic gene enters these cells, it is not destroyed due to the presence of saponin(s), oligonucleotide product, and targeting moieties such as antibodies for binding to target cells, all of which are covalently linked to the oligomeric or polymeric backbone of the present invention.

[0139] One embodiment is a backbone according to the present invention, and the effector molecule is preferably any one or more of toxins that target ribosomes, toxins that target elongation factors, toxins that target tubulin, toxins that target DNA, and toxins that target RNA, more preferably, emtansine, pastotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, duocarmycin, doxorubicin, paclitaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysine, indolinobenzodiazepine, AZ13599185, cryptophycin, lysocine, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, the shortened form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivative, amanitin, α-amanitin, spliceostatin, tyranstatin, ozogamicin, tesirine, ambrastatin 269, and solabuxant, or any one or more of its derivatives, or comprises at least one payload selected from or consisting of the foregoing.

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

[0141] One aspect of the present invention relates to a method for producing a backbone suitable for covalently attaching at least one bioactive molecule to a carrier molecule, the method comprising: a) providing a polymer or oligomer structure comprising a first chemical group for covalent coupling of a polymer or oligomer structure to a 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 at least one bioactive molecule to the oligomer or polymer structure; and b) covalently coupling at least one bioactive molecule to the polymer or oligomer structure via the second chemical group(s), preferably the bioactive molecule(s) is any one of the bioactive 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 backbone.

[0142] The polymer or oligomer structure is preferably any of the polymer or oligomer structures according to the present invention. Similarly, the first and second chemical groups for covalent coupling are chemical groups according to 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, and preferably the saponin is also covalently coupled to the backbone via a linker such as a cleavable linker according to the present invention. Typical backbones of the present invention are trifunctional linkers and dendrons.

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

[0144] One embodiment is a method of the invention, wherein at least one bioactive molecule is any one of the glycosides and saponins of the previous aspects and embodiments of the invention; and / or at least one bioactive molecule is coupled to the backbone via a linker according to any of the previous aspects and embodiments of the invention; and / or the saponin is linked to the backbone 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 backbone via a cleavable bond of any one of the previous embodiments of the invention; and / or the carrier molecule is any one of or comprises a payload and an effector molecule according to any of the previous embodiments and aspects of the invention, or is any one of or comprises a ligand and an immunoglobulin, monoclonal antibody, and / or any one of its binding domains or fragments according to any of the previous embodiments and aspects of the invention. Typically, the bioactive 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 BNA, protein toxin, dianthin, saporin, oligonucleotide. A preferred bioactive molecule of the invention is a saponin fraction from Quillaja saponaria, such as the water-soluble saponin fraction of Quillaja saponaria.

[0145] One embodiment is a backbone according to the invention or a method according to the invention, in particular, at this time, at least one bioactive molecule is a glycoside such as a saponin of the invention, and more specifically, at this time, the saponin is SO1861, SA1641, GE1741, and / or QS-21. The backbone can increase the (late) endosomal escape and / or lysosomal escape of an effector molecule according to the invention, at which time the effector molecule is covalently bound to the backbone and contacted with mammalian cells, particularly tumor cells of a human subject, for example, or, at this time, the effector molecule is contacted with mammalian cells, particularly tumor cells of a human subject, for example, in the presence of the backbone of the invention, either way.

[0146] Examples of polypeptides (proteinaceous molecules) as effector molecules are, for example, Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)buganine, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (e.g., argininosuccinate lyase, argininosuccinate synthetase), enzymes of the blood 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).

[0147] The effector molecule that is a polynucleotide can be, for example, a polynucleotide containing coding information such as a gene or open reading frame encoding a protein. It can also contain regulatory information such as a promoter or regulatory element binding region, or a sequence encoding a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include peptide nucleic acid (PNA), morpholino, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally occurring DNA or RNA by a modification of the molecular backbone. 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., factor IX gene); circular double-stranded DNA (e.g., plasmid); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA.

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

[0149] Effector molecules useful in the present invention preferably depend on late endosomal escape in order to exert their effects. For example, some effectors such as Pseudomonas exotoxin are rerouted to other organelles before the "late endosomal stage" and are therefore not normally utilized from the scaffolds according to the present invention. However, such toxins can be adapted for use in the present invention, for example, by deleting the rerouting carried by the signal peptide. Specifically, toxins that are highly toxic and 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 from the endosome. Furthermore, the scaffolds of the present invention, which include functionalized scaffolds, i.e., covalently attached effector molecule(s) and / or ligand and / or monoclonal antibody, etc., for targeting the scaffold to target cells such as tumor cells or autoimmune cells, preferably contain 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 a functionalized scaffold containing an aggregated polymer structure (wherein the glycoside molecules and effector molecules are attached to different polymer structures within the aggregate), 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 relative to the effector molecules in such an aggregate. Furthermore, to reduce off-target toxicity, cell membrane-impermeable low molecular weight toxins are preferred effector molecules over cell membrane-permeable toxins.

[0150] The present invention further provides a functionalized backbone, comprising at least one backbone 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 in addition at least one ligand (Figs. 10 - 12, 54), d) at least one effector molecule which itself has at least one ligand (Fig. 53), or e) at least one ligand which itself has at least one effector molecule. Such coupling in a) - e) can be achieved via a cleavable (labile) or stable (non - cleavable) bond. Preferably, the coupling in a) - e) occurs independently by click chemistry bonds. Preferably, the functionalized backbone can improve endosomal escape of the effector. Certain embodiments are the backbone of the present invention, the backbone is a functionalized backbone according to the present invention, and the at least one effector molecule is a prodrug, such as a toxin, a drug, a polypeptide, and / or a polynucleotide. Certain embodiments are the functionalized backbone of the present invention, and the effector molecule is a toxin or a polynucleotide encoding a protein.

[0151] The term "ligand" as used in the present invention has its ordinary meaning and preferably means a molecule or structure that can bind to another molecule or structure on the cell surface of a target cell. The molecule or structure on the cell surface can be endocytosed and is preferably absent or less prominent on off-target cells. Preferably, the molecule or structure on the cell surface is constitutively endocytosed. More preferably, the ligand in the present invention induces endocytosis of the molecule or structure on the cell surface of the target cell after binding to the molecule or structure. This applies, for example, to the epidermal growth factor receptor (EGFR) present on the surface of various cancer cells. Examples of molecules or structures on the cell surface of target cells 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 toxic only in target cells because it interferes with a process that occurs only in target cells (as in off-target cells, it cannot exert its toxic effect. For example, apoptin) can also be regarded as a targeted toxin. Preferably, a targeted toxin is a toxin combined with a ligand or, for example, a monoclonal antibody because it is active in target cells and not in off-target cells (because it is bound and endocytosed only by target cells). In a functionalized scaffold containing a carrier molecule comprising 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 the saponin listed in Table A1 and Scheme I, and preferably, the saponin is SO1861 and / or QS-21 and / or SA1641 and / or GE1741.

[0152] The present invention provides a backbone that is not provided with carrier molecules covalently linked to the backbone, such as effector molecules and / or cell targeting ligands or antibodies, i.e., a non-functionalized backbone. The provided backbone can be supplied, for example, to drug manufacturers. This then entails the coupling of effector molecules alone or effector molecules and ligands or antibodies to the backbone. If required, the drug manufacturer can add a cleavable unit for releasing the effector molecule from the backbone and / or ligand, antibody. For example, by inserting a disulfide bridge between the effector molecule and the ligand and / or between the effector molecule and the click position. The present invention also provides a (pre-)functionalized version of the backbone, which already has an effector molecule, such as a tumor cell killing toxin (Figure 54). With regard to endosomal effector molecule release, the activity of the backbone according to the present invention is preferably already incorporated in the backbone. The functionalized backbone can be supplied to the pharmaceutical industry, for example, for the further development of existing and future therapeutic antibodies, and to any supplier or owner of an antibody for functionalizing the targeted antibody. The functionalized backbone can also be used by biotech companies or for research purposes.

[0153] It comprises an effector molecule on a carrier molecule covalently bound to a backbone, and a cell targeting moiety on a carrier molecule covalently bound to a backbone such as an immunoglobulin listed in Tables A2 - A4, for example. Here, the inventors have, for the first time, provided conjugates having covalently bound glycosides, effector molecules, and monoclonal antibodies, as well as conjugates having covalently bound glycosides and effector molecules, and conjugates having covalently bound glycosides and cell targeting molecules such as ligands or monoclonal antibodies (fragments, domains) for the targeted delivery of effector molecules into target diseased cells such as tumor cells. It is probably administered to patients who need it in a systemic manner (site - specific local administration is preferred), but the backbone of the present invention has a binding partner for a ligand or antibody and specifically exerts its intracellular activity within the target cells that expose it. At this time, the backbone is provided with such a ligand or antibody that binds preferentially and specifically to the desired target cells. In this way, for example, when the effector molecule is also provided with the same or different target cell - specific ligands or antibodies specific for binding to the same or different cell surface molecules present on the same target cells such as target tumor cells or target autoimmune cells, the glycoside and effector molecule bound to the ligand or antibody are directed to and ideally accumulate in the same (late) endosomes, lysosomes of the same target (diseased) cells where the killing effect of the effector molecule is intended.

[0154] The new (functionalized) backbone offers several advantages: 1) The use of the functionalized backbone, i.e., the backbone of the present invention containing covalently bound effector molecules and ligands or antibodies, results in a one - component system. That is, due to the presence of a cell - targeting ligand or antibody, preferably any one of the monoclonal antibodies in Tables A2 - A4, the effector molecule and an endosome escape - enhancing factor, i.e., at least one glycoside, are simultaneously delivered to the endosome in a predetermined ratio.

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

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

[0157] 4) The present invention allows for the provision of a pre - formed backbone (functionalized backbone) loaded with effector molecules for use with any available ligand and / or (monoclonal) antibody (or at least one binding fragment and / or domain thereof). This makes the present invention optimal for platform development.

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

[0159] Another application of the present invention is, for example, gene therapy. The efficient intracellular delivery of biological macromolecules such as polynucleotides is still a major hurdle at present. In contrast to conventional non - specific DNA transfection systems, the present invention is not limited to DNA and is specific to target cells. Known viral systems are efficient and specific to 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 herein is based on its originality, flexibility, and ease of use.

[0160] One embodiment is the backbone of the present invention, the backbone being a functionalized backbone, the backbone 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 backbone being endocytosed together with the surface molecule after binding. Preferably, said target cells are diseased or disease-related cells, preferably tumor cells, tumor-related cells (e.g., tumor vascular cells), immune cells (e.g., regulatory T cells), or cells having a monogenic defect. The term "target cell-specific surface molecule" is meant to mean that the molecule is expressed, either qualitatively or quantitatively, preferably 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 also expressed (at lower levels) on, for example, skin fibroblasts, and HER2, which is overexpressed in breast cancer cells. However, many functional fragments and domains of target cell-specific surface molecules provide advantages known in the art, and one of ordinary skill in the art can very well select 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. See also Tables A2, A3, and A4 for examples of antibodies that bind to tumor cell-specific receptors. For tumor cell-specific receptors suitable for targeting by carrier molecules, such as monoclonal antibodies, included in the backbone of the present invention, see also the embodiments described above herein. As used herein, "monogenic defect" has its ordinary meaning, which is a modification of a single gene that occurs in substantially all cells of the body. The mutation can be present on one or both chromosomes (one chromosome is inherited from each parent). Although relatively rare, monogenic defects afflict millions of people worldwide. Currently, scientists estimate that over 10,000 human diseases are known to be monogenic diseases. Non-limiting examples of monogenic diseases known to date are: sickle cell disease, cystic fibrosis, polycystic kidney disease, and Tay-Sachs disease.

[0161] One embodiment is the backbone of the present invention, and the backbone is a functionalized backbone according to the present invention. The backbone includes a carrier molecule covalently attached thereto and includes a ligand and / or an antibody. At least one ligand is 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). DARPin is a genetically engineered antibody mimetic protein that typically exhibits highly specific and high-affinity target protein binding. They are derived from natural ankyrin proteins that perform diverse cellular functions. They constitute a new class of potent, specific, and versatile small proteins (typically 14 - 18 kDa) for therapy and are used as research 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 a Fab’ or Fab fragment, a variable light domain, a variable heavy domain, a constant light domain, and a constant heavy domain 1, or a monovalent antibody described in WO2007059782; (ii) an F(ab’)2 fragment of a divalent fragment containing two Fab fragments linked by disulfide bridging in the hinge region; (iii) an Fd fragment consisting essentially of a variable heavy domain and a constant heavy 1 domain; and (iv) an Fv fragment consisting essentially of the variable light and variable heavy domains of a single arm of an antibody. Furthermore, the two domains of the variable light and variable heavy Fv fragments are encoded by separate genes, but they can be linked by a synthetic linker that enables them to be made as a single protein chain using recombinant methods. Thereby, the variable light and variable heavy regions pair to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)).

[0162] Preferably, effector molecules whose effect is enhanced by glycoside molecules (such as saponins) are removed from the backbone and / or ligand or antibody when endocytosed. This can be achieved, for example, by a cleavable bond that breaks under acidic, reducing, enzymatic, or photoinductive conditions. Thus, one embodiment is the backbone of the present invention, which is a functionalized backbone according to the present invention, and said at least one effector molecule is bound to said backbone and / or said at least one ligand or antibody via a cleavable bond. Preferably, said cleavable bond is cleaved under acidic, reducing, enzymatic, or photoinductive conditions. Preferably, the cleavable bond is an imine, hydrazone, oxime, 1,3-dioxolane, disulfide, or ester, more preferably a disulfide or hydrazone bond.

[0163] One embodiment is the backbone of the present invention, which is a functionalized backbone according to the present invention, and said at least one effector molecule is bound to said backbone 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 backbone and an activated carboxylic acid group on the effector molecule or ligand.

[0164] One embodiment is a backbone or functionalized backbone according to the present invention, and further comprises a carrier, such as nanoparticles, liposomes, micelles, colloids, or a particle-like structure containing cholesterol and / or phospholipids.

[0165] As previously mentioned, at least one glycoside molecule included on the backbone according to the invention increases the effectiveness of at least the current and new effector molecules as defined in the present invention. Possible side effects can be reduced by decreasing the dosage of the effector molecule without reducing its effectiveness. Thus, the present invention provides a backbone according to the invention or a functionalized backbone according to the invention for use in medicine or for use as a medicament. Therefore, one aspect of the present invention relates to a backbone according to the invention for use as a medicament, the backbone comprising at least the effector molecule of the present invention and / or an antibody according to the invention, preferably both the effector molecule and the antibody. Also provided is the use of a backbone according to the invention or a functionalized backbone according to the invention for manufacturing a medicament. In particular, cancer medicines, especially classical chemotherapy medicines, are notorious for their side effects. Due to the temporal and local synchronization and targeting of both the prodrug and the glycoside molecule, the backbone or functionalized backbone according to the invention is particularly useful for use as a medicament, especially for use in a method of treating cancer. Thus, the present invention provides a backbone according to the invention or a functionalized backbone according to the invention for use in a method of treating cancer. The present invention also provides a backbone according to the invention or a functionalized backbone according to the invention for use in a method of treating acquired or genetic disorders, especially single-gene deficiency disorders. Therefore, one aspect of the present invention relates to a backbone according to the invention for use in a method for treating cancer or an autoimmune disease, the backbone comprising a covalently attached carrier molecule, the carrier molecule comprising at least the effector molecule of the present invention and / or an antibody according to the invention, preferably both the effector molecule and the antibody.

[0166] One aspect of the present invention relates to a backbone according to the invention comprising an antibody and / or an effector molecule for targeting tumor cells for use in treating or preventing cancer, the backbone being administered to a human subject in need thereof.

[0167] One aspect of the present invention relates to the treatment of a human subject suffering from cancer or at risk of developing cancer and in need of said treatment, the treatment comprising the step of administering to the human subject an effective dose of a pharmaceutical composition comprising a backbone of the present invention or a functionalized backbone of the present invention, said backbone 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. One aspect of the present invention relates to the backbone of the present invention for use as a medicament. One aspect of the present invention relates to the backbone of the present invention for use in a method for the treatment or prevention of cancer or an autoimmune disease in a human subject in need thereof. One aspect of the present invention relates to the use of the backbone of the present invention or a functionalized backbone, or a pharmaceutical composition comprising said backbone or functionalized backbone, for the manufacture of a medicament for anti-cancer treatment or anti-autoimmune disease treatment. The backbone or functionalized backbone preferably comprises a carrier molecule comprising at least an effector molecule and / or an antibody, preferably both an antibody and an effector molecule.

[0168] Further applications to medicine are the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting diseases can be hereditary or acquired. In most cases, only symptomatic treatment is possible, and in some rare diseases, the limited treatment options lead to a shortened lifespan for patients affected. An example of such a disease is phenylketonuria, which is an inborn metabolic disorder in which the metabolism of the amino acid phenylalanine is impaired. The disease is characterized by genetic mutations in the hepatic enzyme phenylalanine hydroxylase. Phenylketonuria is not curable to date. The incidence is approximately 1:10,000, and the highest known incidence is 1:2,600 in Turkey. A functionalized backbone having phenylalanine hydroxylase or a polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells and to replenish the defective enzyme in liver cells by use of a suitable ligand; a backbone covalently bound to a carrier molecule containing phenylalanine hydroxylase or a polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells and to replenish the defective enzyme in liver cells by use of a suitable ligand. This is one example of the use of a backbone or a functionalized backbone according to the invention containing a carrier molecule for replacement or gene therapy. In a preferred embodiment, a backbone according to the invention or a backbone containing a carrier molecule of the invention or a functionalized backbone according to the invention is provided for use in methods of gene therapy or replacement therapy.

[0169] The present invention can also be used in biotechnology processes. Possible applications are the biomolecular engineering of eukaryotic intracellular switches. Transcription switches target gene expression at the level of mRNA polymerization, translation switches target the process of turning mRNA signals into proteins, and post-translational switches control how proteins interact with each other to attenuate or relay signals. When optimized, these cellular switches can turn cell 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 that are not specific to the target cell and do not have high specificity for the selected switch can be used. A carrier molecule having a functionalized backbone, or a backbone containing a more complex and thus more specific non-diffusible effector molecule, can be used to target a specific switch, and the use of a suitable ligand can limit the effect to the target cell. One embodiment is the use of a backbone containing a carrier molecule of the present invention or a functionalized backbone according to the present invention, preferably in vitro, to improve the effect of an effector molecule. Preferably, the use is to improve the effect of an in vitro transcription switch.

[0170] Another application is the use of the present invention in basic research. For the functional analysis of cell processes, it is often required to introduce proteins into cells, a method called protein transfection. For example, to investigate the molecular mechanism of the chicken virus protein apoptin leading to apoptosis in eukaryotic cells, it is required to introduce the purified protein into the target cells. However, existing protein transfection kits are characterized by low efficacy, lacking specificity for target cells, and high toxicity, and thus cannot be used for many applications, especially when metabolic pathways are part of the investigation. Both the backbone or functionalized backbone of the present invention containing the covalently linked carrier molecule having apoptin and the use of suitable ligands can be used to carry out such investigations. Certain embodiments are preferably the backbone of the present invention, the backbone containing a carrier molecule according to the present invention, or the functionalized backbone according to the present invention for in vitro polypeptide transfection. Also provided is the use of a backbone, a backbone containing a carrier molecule according to the present invention, or a functionalized backbone according to the present invention, preferably for in vitro polynucleotide transfection.

[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 backbone according to the present invention, or preferably a functionalized backbone according to the present invention, or preferably an effector molecule or monoclonal antibody according to the present invention, preferably a backbone of the present invention comprising a carrier molecule comprising 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 backbone or functionalized backbone can · provide an extended therapeutic window for current and new ADCs where the ADC can contain payloads such as toxins, protein toxins, oligonucleotides, BNA, etc., · provide highly efficient cytoplasmic matrix delivery of macromolecules, · facilitate cell research and biotechnology applications, · may have the potential to treat a plurality of diseases, such as cancer and autoimmune diseases, rheumatoid arthritis, · can be used to induce cell destruction (e.g., of cancer cells), · can reduce unwanted side effects by reducing the level of therapeutic agent required by diseased cells, · can reduce the risk of immune response to effector molecules (because fewer effector molecules are required. And, without wishing to be bound by any theory, perhaps because the endosomal pathway of antigen presentation onto MHC molecules is blocked), · can open up the possibility of highly efficient manipulation of genes, · can revive failed drug candidates, especially ADCs, by increasing their effectiveness, · can be made from biocompatible and biodegradable and / or excretable materials, · symbolizes a platform technology that can rely on mild and non-harmful effector molecule release triggered by endosomal pH. Flexibility is ensured by the possibility of using any type of ligand (such as an antibody, its fragments and domains, or an aptamer) and an effector molecule. For providing a user-friendly interface for applying this technology to the user's own ligand and effector molecule, a sophisticated implementation of click chemistry can be used. The platform technology of the present invention provides various possibilities, such as the generation of a clickable backbone as a stand-alone product that allows the user to simply couple either his effector molecule and / or ligand at his discretion (Figure 53), or the generation of a backbone containing a functionalized backbone or a carrier molecule covalently linked thereto. The basic backbone is already coupled to an effector molecule (such as the proteinaceous toxin in Table A5) and / or a ligand (such as the antibodies in Tables A2, A3, A4). This allows the user to couple his ligand for guiding the effector molecule to the desired target cell (Figure 54). Possible backbones containing covalently coupled carrier molecules, or possible backbones containing functionalized backbones are backbones linked to ribosome-inactivating proteins, such as dianthin, saporin. This highly potent cytotoxic enzyme for killing target cells is designed to specifically recognize tumor cells and can be used to click any antibody already existing on the market or any future antibody, such as trastuzumab, cetuximab, rituximab, gemtuzumab, OKT-9, or obinutuzumab (next-generation ADC technology), or any of the antibodies listed in the previous embodiments and Tables A2 - A4. As a nucleic acid effector molecule, microRNA (miRNA, polynucleotide) or miRNA inhibitor or LNA or BNA can be used, for example, to create a functionalized backbone for efficient and low-dose cytoplasmic delivery. miRNA or miRNA inhibitor has a high potential as a novel therapeutic agent that can alter the intracellular gene program and thereby alter cell function.

[0173] The present invention further provides a method for producing a scaffold, preferably a scaffold according to the present invention, the scaffold comprising at least one glycoside molecule capable of improving the effect of an effector molecule conjugated to a polymer or oligomer structure, the method comprising: providing a polymer or oligomer structure; and coupling at least one glycoside molecule to the polymer or oligomer structure. Preferably, at least one glycoside molecule increases the endosomal escape of the 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 the effector molecule. Preferably, at least one glycoside molecule is a bisdesmoside-type triterpene, more preferably a bisdesmoside-type triterpene saponin, more preferably belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at the 23-position, 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, at least one glycoside molecule is coupled to the polymer or oligomer structure via a cleavable bond, preferably the 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. When the bond is a cleavable bond, the saponin is preferably attached to the scaffold via an aldehyde functional group at position 23 on the saponin or via one of the carboxyl groups, more preferably via the aldehyde functional group.

[0174] One embodiment is a backbone of the invention, or a backbone bound to a carrier molecule according to the invention, or a functionalized backbone of the invention, wherein at least one glycoside molecule is bound to a polymer or oligomer structure via a stable bond. One embodiment is a backbone of the invention, or a backbone comprising a covalently bound carrier molecule according to the invention, wherein at least one glycoside molecule is a saponin, and the stable bond between the saponin and the backbone preferably occurs by amide coupling or amine formation. This is achieved, for example, via carbodiimide-mediated amide bond formation by the amino group of the polymer or oligomer structure and the activated glucuronic acid group of the saponin. Chemical bonds fulfilling stable conditions can also be used for aldehyde coupling. For example, a specific amine derived after reductive amination, which requires a primary amine group as a functional group of the polymer or oligomer structure. When the bond is a stable bond, the saponin is preferably attached to the backbone via one of the carboxyl groups of the saponin.

[0175] Preferably, the backbone further comprises click chemistry groups for coupling to carrier molecules, such as effector molecules and / or ligands and / or monoclonal antibodies (fragments, domains thereof), preferably both effector molecules and immunoglobulins. The immunoglobulins are preferably monoclonal antibodies of Tables A2, A3, A4. The monoclonal antibodies and effector molecules together preferably form an ADC according to the invention, such as the ADC of Table A4. Preferably, the click chemistry groups are tetrazine, azide, alkene, or alkyne, or cyclic derivatives of these groups, such as cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]nona-4-yne, or dibenzocyclooctyne).

[0176] One embodiment is a method according to the invention for producing a backbone, preferably the backbone of the invention, wherein the number of glycoside molecules is a defined number or a defined range. Preferably, the polymer or oligomer structure comprises, either pure or mixed, a linear, branched, or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or an assembly of these structures. The assembly can be assembled by covalent cross-linking or non-covalent attractive forces and can form a hydrogel or nanogel. 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 prodrug, such as a toxin, drug, polypeptide, and / or polynucleotide.

[0177] A method for producing a backbone comprising a carrier molecule according to the present invention or a functionalized backbone of the present invention is also provided, the method comprising: providing a backbone comprising a plurality of glycoside molecules and a polymer or oligomer structure, preferably a backbone according to the present invention or a backbone obtainable by a method according to the present invention for producing a backbone; and coupling to said backbone either a) at least one effector molecule, b) at least one ligand, such as a monoclonal antibody, c) at least one effector molecule and in addition at least one ligand, preferably a monoclonal antibody targeting a tumor cell receptor, d) at least one effector having at least one ligand such as a monoclonal antibody for binding to a tumor cell receptor, or e) at least one ligand, such as a tumor cell targeting monoclonal antibody having at least one effector. Preferably, the coupling in a) - e) occurs independently by click chemistry bonds. One embodiment is the method of the present invention, wherein in c), the backbone is coupled to at least one effector molecule and in addition 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 coupled to a linker which itself is coupled to the backbone. Those skilled in the art can design such trifunctional linkers, such as the trifunctional linker of Scheme II and Structure B, based on the present disclosure and ordinary general knowledge (see also Figure 16). Such trifunctional linkers can present, for example, a maleimide group that can be used for conjugation to a targeting ligand presenting a thiol group for performing a thiol-ene reaction. In addition, the trifunctional linker can present a dibenzocyclooctyne (DBCO) group for performing a so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with a saponin bearing an azide. Finally, the trifunctional linker can acquire a third functional group such as a trans-cyclooctyne (TCO) group and perform a so-called inverse electron demand Diels-Alder (IEDDA) reaction with an effector molecule bearing a tetrazine (Tz).One embodiment is a method of the invention, wherein the at least one effector molecule is a prodrug, such as a toxin, a drug, a polypeptide, or a polynucleotide. One 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, can specifically bind to a target cell-specific surface molecule or structure that can undergo 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 cells are diseased or disease-related cells, preferably tumor cells, tumor-related cells (e.g., tumor vascular cells), immune cells (e.g., regulatory T cells), or cells having a monogenic defect. One embodiment is a method according to the invention, a method for producing a backbone comprising a backbone or a functionalized backbone according to the invention, wherein the at least one effector molecule is coupled to the backbone and / or the at least one ligand via a cleavable bond. Preferably, the cleavable bond is cleaved under acidic, reducing, enzymatic, or photoinductive conditions. One embodiment is a method according to the invention for producing a backbone or a functionalized backbone, the method further comprising binding the backbone or the functionalized backbone to a carrier, the carrier preferably being a nanoparticle, a liposome, a micelle, a colloid, or a particle-like structure comprising cholesterol and / or a phospholipid.

[0178] The present invention provides a pharmaceutical composition comprising a backbone according to the invention, or a functionalized backbone according to the invention, or a backbone comprising a covalently bound carrier molecule according to the invention and an optionally acceptable pharmaceutical carrier. Such pharmaceutical composition is for use in the treatment of a patient, in particular for use in the treatment of cancer or an acquired or genetic disorder, in particular a monogenic deficiency disorder.

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

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

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

[0182] One embodiment is the backbone of the present invention, and the backbone contains a defined number or a defined range of glycosides. One embodiment is the backbone of the present invention, and the backbone contains a defined number or a defined range of glycosides, where the defined range is glycoside(s) between 1 and 30, preferably between 1 and 20, more preferably between 1 and 10, more preferably between 1 and 6, more preferably between 2 and 6, more preferably between 2 and 5, more preferably between 3 and 5, more preferably between 3 and 4.

[0183] One embodiment is the backbone of the present invention, and the backbone includes a covalently linked carrier molecule, which includes a binding site for targeting cells and is, for example, a (monoclonal) antibody for binding to a cell surface receptor on a target cell or includes the same. One embodiment is the backbone of the present invention, and the backbone includes a covalently linked carrier molecule. This carrier molecule includes a binding site for targeting cells and is, for example, a (monoclonal) antibody for binding to a cell surface receptor on a target cell or includes the same. The target cells are diseased cells or disease-related cells, preferably tumor cells or tumor-related cells (e.g., tumor vascular cells), or immune cells (e.g., regulatory T cells), or autoimmune cells.

[0184] One embodiment is the backbone of the present invention, and the bioactive molecule is a glycoside, and the glycoside can increase the endosomal escape of an effector molecule contained by a carrier molecule covalently linked to the backbone.

[0185] One embodiment is the backbone of the present invention, and the backbone is part of a pharmaceutical composition, and the pharmaceutical composition further includes at least one additional active pharmaceutical ingredient such as an additional immunoglobulin in addition to the backbone.

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

[0187] One embodiment is the backbone of the present invention for use in a method of treating cancer, and the method includes administering the backbone of the present invention to a patient in need thereof, and the backbone includes a covalently linked carrier molecule including the effector molecule of the present invention and / or the ligand or cell targeting antibody of the present invention or consisting of the same.

[0188] One embodiment is the backbone of the present invention for use in a method of treating cancer, and the method includes administering a pharmaceutical composition according to the present invention to a patient in need thereof.

[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 in no way be construed as limiting the present invention.

Examples

[0227] Example A - Treating animals with mammalian tumors with the conjugate of the present invention in combination with an ADC results in survival and tumor regression. Female Balb / c nude mice were subcutaneously injected with a suspension of human A431 tumor cells. A human epidermal carcinoma was generated in a xenograft animal tumor model under the skin of the mice. After injection of the tumor cells, the xenograft tumors were allowed to grow to a size of approximately 170 - 180 mm 3 . The A431 tumor cells have the following characteristics: high EGFR expressors, moderate CD71 expressors, and low HER2 expressors.

[0228] Table A presents the results of the treatment of control mice and mice with tumors. Mice with tumors were treated with an antibody directed against either human Her2 / neu, human EGFR, or human CD71, which are cell surface receptors on xenograft tumors. Cetuximab was covalently conjugated to saponin SO1861. First, linker EMCH (N-ε-maleimidocaproic acid hydrazide) was provided to SO1861. This EMCH is a maleimide and hydrazide crosslinking agent for covalently conjugating sulfhydryl (reduced cysteine of the antibody) to carbonyl (aldehyde or ketone; here, the carbonyl of the aldehyde at position C-23 of the saponin). 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. It was measured as the tumor volume over time after the start of treatment with the ADC. The dose of the ADC was sub-optimal in the tumor model. That is, it was established from previous experiments at which sub-optimal dose of the ADC tumor regression or arrest of tumor growth would not be observable.

[0229]

Table 38

[0230] These results demonstrate that the combination therapy of an ADC with the conjugate of the present invention, which consists of an antibody targeting a tumor cell-specific receptor covalently linked to saponin, i.e., SO1861, provides an efficient and effective treatment regimen manifested as regression of the treated animals' tumors and prolonged survival when treatment with ADC alone is considered ineffective (tumor growth and mouse death are not prevented (euthanasia)) at the dose of the ADC. The covalent conjugate is administered to cancer-bearing mice at a dose that is ineffective (tumor growth and mouse death are not prevented (euthanasia)) when administered alone. Therefore, the sub-optimal dose of the ADC combined with the conjugate containing the covalently linked saponin of the present invention, which has no anti-tumor activity when administered alone, provides an effective treatment option for cancer patients and the relatively low dose of the ADC is effective. The lower dose of the ADC holds the promise of a lower risk of adverse events or even no side effects at all. Additionally, the stimulatory effect of the conjugate with saponin of the present invention when the effectiveness of the ADC is considered indicates that ADCs, which have been found to lack effectiveness when it comes to treating tumor patients, may gain renewed attention and value. Because, as demonstrated in this example, the effectiveness of the ADC is improved under combination therapy conditions. Reference is made to Tables A2 and A3 which summarize the ADCs. These were previously considered in human clinical settings but some of the ADCs were then withdrawn from further clinical consideration. In particular, ADCs that ended clinical development due to the observed lack of effectiveness and / or the occurrence of unacceptable adverse events may gain renewed value for cancer patients when combined with the conjugate containing the covalently linked saponin of the present invention, such as the tested cetuximab-saponin.

[0231] Example B - The saponin mixture of Quillaja saponaria containing QS-21 with enhanced endosome / lysosome escape activity Scheme I shows the common molecular structure of a series of QS-21 saponins (extracted from 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") is based on the endosome / lysosome escape enhancing properties of at least one individual saponin present in the mixture, such as QS-21, or a combination of two or more saponins contained by the mixture, such as QS-21 and QS-7, and can be applied to the endosome / lysosome escape enhancing conjugates, compositions, combinations of the present invention.

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

[0233] Example 1 Design and production of a trifunctional linker backbone with specific chemical end groups (DBCO, TCO) for conjugation (labile (L) conjugation) of SO1861 molecules on one arm and antisense HSP27 BNA oligonucleotides (targeting the cancer target hsp27 mRNA of cancer cells and inducing its degradation) on the other arm to produce SO1861-L-trifunctional linker-L-HSP27BNA (Figure 16). SO1861-L-trifunctional linker-L-HSP27BNA was conjugated to cetuximab (cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 4 ) via its third arm (maleimide) to a cysteine residue (Cys).

[0234] Conjugates containing this backbone were tested for gene silencing activity targeting EGFR-mediated tumors in an A431 xenograft "nude" mouse tumor model. Dosing was initiated on day 12 when tumors reached a size of ~170 mm 3 . Tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression compared to control mRNA expression of cells (reference gene). This revealed that a single dose of 25 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 3,7 resulted in a 40% reduction in HSP27 gene expression compared to a single dose of either cetuximab-(Cys-L-SO1861) 3,8 or cetuximab-(Lys-L-HSP27BNA) 4 alone (Figure 1). A 25% reduction in gene silencing was observed compared to vehicle control tumors. This indicates and enables that conjugated SO1861 can efficiently induce targeted delivery of therapeutic oligonucleotides in in vivo tumors.

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

[0236] Example 2 The 1-target 2-component system is the combined treatment of mAb1-(dendron(SO1861) n ) n and mAb1-effector, and the 2-target 2-component system is the combination of mAb1-(dendron(SO1861) n ) n + mAb2-effector.

[0237] Dendron(-L-SO1861) 4 was conjugated to the anti-EGFR antibody cetuximab via cysteine residue (Cys) conjugation by DAR3,9 as Cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 and tested for improved cytotoxic 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 induces toxin-mediated killing in highly EGFR-expressing cells, which is Cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9Or cetuximab (equivalent) + 10 pM cetuximab - saporin or not induced by cetuximab (Figure 3A). Similar experiments in cells expressing low levels of EGFR (HeLa) were cetuximab - Cys - (dendron (-L - SO1861) 4 ) 3,9 did not reveal the activity of (Figure 3C), indicating that in the absence of sufficient EGFR receptor expression, an effective intracellular SO1861 concentration (threshold) for endosomal protein toxin escape and toxin - mediated cell killing induction is not reached.

[0238] Next, dendron (-L - SO1861) 4 was conjugated to the anti - HER2 antibody trastuzumab by cysteine conjugation (Cys) with DAR4 to form trastuzumab - Cys - (dendron (-L - SO1861) 4 ) 4 and tested for enhanced cell killing activity in HER2 - expressing cells (SK - BR - 3) in combination with the 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 not induced by trastuzumab - Cys - (dendron (-L - SO1861) 4 ) 4 or trastuzumab (equivalent) + 50 nM trastuzumab - saporin or trastuzumab (Figure 3B). Similar experiments in cells expressing low levels of HER2 (JIMT - 1) were trastuzumab - Cys - (dendron (-L - SO1861) 4 ) 4 did not reveal the activity of (Figure 3D), indicating that in the absence of sufficient HER2 receptor expression, an effective intracellular SO1861 concentration (threshold) for endosomal protein toxin escape and toxin - mediated cell killing induction is not reached.

[0239] Next, cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3, 9 or cetuximab-Lys-(dendron(-L-SO1861) 4 ) 4,4 (Lys = dendron(-L-SO1861) conjugated to the lysine of the antibody 4 ) was tested as a two-target two-component system in combination with 10 pM CD71mab-saporin in EGFR++ / CD71+ cells (MDA-MB-468). This showed a strong improvement in cytotoxic activity for both conjugates, which was not induced by 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 that, compared to the activity in high expressors (Figure 4A), cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 or cetuximab-Lys-(dendron(-L-SO1861) 4 ) 4,4 (Figure 4C) both showed reduced activity, indicating that in cells with lower EGFR receptor expression levels, the effective intracellular SO1861 concentration is low, resulting in cytotoxic activity mediated by the reduced toxin.

[0240] The same experiment was performed with trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 or trastuzumab-Lys-(dendron(-L-SO1861) 4 ) 4,7This was performed on the HER2++ / CD71+(SK-BR-3) cell line, revealing strong cytotoxic activity compared to the control (Figure 4B). Trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 or Trastuzumab-Lys-(dendron(-L-SO1861) 4 ) 4,7 When tested on HER2+ / - / CD71+(JIMT-1) in combination with 10 pM CD71mab-saporin, no cytotoxic activity was observed, indicating that in the absence of sufficient HER2 receptor expression, an effective intracellular SO1861 concentration (threshold) for endosomal protein toxin escape and toxin-mediated 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 improved cytotoxic activity. Since the endosomal membrane does not form a barrier for small molecules to reach the cytoplasm, no improved killing was observed with this combination compared to T-DM1 alone or T-DM1 + equivalent trastuzumab (Figure 5).

[0242] Example 3 Materials and Methods Dendron(SO1861) 4-BNA Oligo Synthesis (Figure 17) HSP27BNA oligosulfide (1.1 mg, 0.187 μmol) was dissolved in 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered by using a centrifugal filter with a molecular weight cut-off of 3000 Da (14000×g, 30 min). The residue solution was diluted with 20 mM NH4HCO3 with 1.0 mM TCEP (500 μL), and the resulting mixture was filtered again under the same conditions described above. The residue solution was diluted with 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 1.0 mL), and the resulting mixture was added to 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 subjected to preparative LC-MS 4A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to give the title compound (1.25 mg, 85%) as a white fluffy solid. Purity based on LC-MS 94% LRMS (m / z): 1896 [M-8] 8- , 2167 [M-7] 7- LC-MS r.t. (min): 3.77 6B Results HSP27BNA oligo (antisense BNA oligo (HSP27BNA) targeting heat shock protein 27 of cancer target mRNA transcript) was conjugated to dendron (-L-SO1861) 4 (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 revealed that the treatment with HSP27BNA-dendron (-L-SO1861) 4 resulted in an improvement in 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), 50 μL of a solution containing water / acetonitrile / TFA (1.00 mL / 1.00 mL / 4 drops) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. SO1861 release was monitored over time using UPLC-MS 4 . Results Release efficiency of SO1861 molecules from the dendron (-L-SO1861) 4 under acidic conditions (Figure 7A) was determined (Figure 7A). Figure 7B shows the UPLC UV traces (PDA) of the dendron (-L-SO1861) 4 itself (top), the reaction mixture after 30 min (center), and the reaction mixture after 1.5 h (bottom). Figure 7C shows the interpretation of the observed m / z values by LRMS. The following m / z values were observed at the corresponding UV r.t. (min): r.t. = 1.46, 2282 [M - 4] 4- (A, dendron (-L-SO1861) 4 ); r.t. = 1.43, 2427 [M - 3] 3- (B, dendron (-L-SO1861) 3 ); r.t. = 1.38, 1812 [M - 3] 3- (C, dendron (-L-SO1861) 2 ); r.t. = 1.29, 1797 [M + 2] 2+ (D, dendron -L-SO1861); r.t. = 1.22, 1862 [M - 1] 1- (E, SO1861); r.t. = 1.05, 1747 / 1769 [M + 1 / M + 23] 1+ (F, dendron -).

[0244] Next, the dendron (-L-SO1861) 4 was tested for improved delivery of the targeted toxin EGF dianthrin in EGFR-expressing cells (A431 and HeLa). This is because the dendron (L-SO1861) 4 +10 pM EGF dianthrin can induce cell killing mediated by the improved toxin, while the "naked" dendron (dendron (NEM)4 ) or dendron (-L-SO1861) 4 , or dendron (NEM) 4 +10 pM EGF diantin did not attempt to show enhanced cell killing at these concentrations (Figs. 8A, 8B).

[0245] Example 5 dendron (-L-SO1861) n Synthesis (Figs. 13, 14, 15) Materials and Methods Abbreviations DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide EDCI·HCl 3-((Ethylimino)methylenamino)-N,N-dimethylpropan-1-aminium chloride EMCH.TFA N-(ε-Maleimidocaproic acid) hydrazide, trifluoroacetate min minute r.t. 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, degassing device; 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, 30×2.1 mm, 3.5 μm, Temp: 35°C, flow rate: 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 Equipment: Agilent 1260 Bin. Pump: G7112B, Multi-sampler, 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 the molecular weight of the product: A pos / neg 100 - 1000 B pos / neg 100 - 1400 ; ELSD Alltech 3300 Gas flow 1.5 ml / min, Gas temp: 40 °C; Column: Waters XSelect™ C18, 30×2.1 mm, 3.5 μm, Temp: 40 °C, Flow rate: 1 mL / 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 rate: 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; Binary 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 rate: 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 Equipment: Waters IClass; Binary pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210 - 320 nm, SQD: ACQ - SQD2 ESI, mass range depends on the molecular weight of the product: 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 Machine type: Reveleris (trademark) prep MPLC; Column: Waters XSelect (trademark) CSH C18 (145×25mm, 10μ); Flow rate: 40mL / min; Column temp: Room temperature; Eluent A: 10mM ammonium bicarbonate in water pH = 9.0); Eluent B: 99% acetonitrile + 1% 10mM 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, 285nm.

[0251] Preparative MP-LC method 2, 2 Machine type: Reveleris (trademark) prep MPLC; Column: Phenomenex LUNA C18(3) (150×25mm, 10μ); Flow rate: 40mL / 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, 285nm.

[0252] Preparative LC-MS method 1, 3 MS machine type: Agilent Technologies G6130B quadrupole; HPLC machine type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect (trademark) CSH (C18, 100×30mm, 10μ); Flow rate: 25ml / min; Column temp: Room temperature; Eluent A: 100% acetonitrile; Eluent B: 10mM ammonium bicarbonate in water pH = 9.0; Linear gradient depending on product polarity: A t0 = 20%A, t2min = 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 Type of MS instrument: Agilent Technologies G6130B quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Shield (C18, 150×19 mm, 5 μm); Flow rate: 25 ml / min; Column temp: Room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; Linear 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 (Registered Trademark) C-815 Flash; Solvent Delivery System: 3-piston pump, auto-priming, 4 independent channels in one run, up to 4 solvents, automatic line switching when solvent runs out; Maximum pump flow rate 250 mL / min; Maximum pressure 50 bar (725 psi); Detection: UV 200 - 400 nm, combinations of up to 4 UV signals, scanning of the entire UV range, ELSD; Column size: 4 - 330 g on the device, luer type, 750 g to 3000 g with optional holder.

[0254] SO1861-EMCH Synthesis (Figure 13) To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol), methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol) were added. The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative MP-LC. 1 . The 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. Purity based on LC-MS 96%. LRMS (m / z): 2069 [M - 1] 1- LC-MS r.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 minutes. Next, a solution (5.00 mL) of di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (1.82 g, 6.00 mmol) in DMF was added, and the reaction mixture was stirred at room temperature. After 5 hours, the reaction mixture was evaporated in vacuo, and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (2 × 50 mL), and brine (50 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (ethyl acetate-heptane gradient, rising from 10:90 to 100:0) to give the title compound (2.67 g, 100%) as a white solid. Purity based on LC-MS was 98%. LRMS (m / z): 287 / 343 / 465 [M-155 / M-99 / M+23] 1+ LC-MS r.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 (5-6 N, 20.0 mL, 110 mmol) in isopropanol, and the reaction mixture was stirred at room temperature. After 4 hours, 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 (((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) was added Boc-Lys(Boc)-ONp (3.69 g, 7.90 mmol), and the mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo, 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 vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, increasing from 100:0 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-MS r.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). Next, DIPEA (6.97 mL, 40.0 mmol) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (5 × 50 mL), and brine (50 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (DCM - methanol / DCM (1 / 9 v / v) gradient, increasing from 100:0 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 - MS r.t. (min): 1.94 2A

[0259] Intermediate 5: (S)-2,6 - Diamino - N-(2-(6 - azido - N-(2 - ((S)-2,6 - diaminohexanamido)ethyl)hexanamido)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 (5 - 6N, 50.0 ml, 275 mmol) in isopropanol and the reaction mixture was stirred overnight at room temperature. The reaction mixture was evaporated in vacuo and the resulting crude product was co - evaporated with DCM (3 × 20 mL) to give the crude title product as a white solid. LRMS (m / z): 250 [M + 2] 2 +, 500 [M + 1] 1+

[0260] Intermediate 6: (2S)-2,6-Bis[3-(acetylsulfanyl)propanamide]-N-[2-(6-azido-N-{2-[(2S)-2,6-bis[3-(acetylsulfanyl)propanamide]hexanamide]ethyl}hexanamide)ethyl]hexanamide To a solution of (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamide)ethyl)hexanamide)ethyl)hexanamide tetrahydrochloride (1.29 g, 2.00 mmol) in DMF (30 mL) and DIPEA (3.48 mL, 20.0 mmol) was added 4-nitrophenyl 3-(acetylthio)propanoate (2.26 g, 8.40 mmol), and the reaction mixture was stirred at room temperature over the weekend. The reaction mixture was evaporated in vacuo and the residue was dissolved in DCM / methanol (95:5 v / v, 100 mL). The resulting solution was washed with 1N potassium bisulfate solution (100 mL), 1N sodium hydroxide solution (3 × 100 mL), and brine (100 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, rising from 100:0 to 0:100) to give the title product (1.33 g, 65%) as a white solid. By LC-MS, an impurity (15%) with an m / z value corresponding to the product with one deprotected thioacetic acid group was found. The impurity was formed during or after the workup. LC-MS based purity 85%. LRMS (m / z): 510 [M+2] 2+ , 1019 / 1041 [M+1 / M+23] 1+ LC-MS r.t. (min): 1.86 2B

[0261] Intermediate 7: N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamide)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide) formate Skeleton 2 (102 mg, 0.100 mmol) was dissolved in methanol (1.00 mL). Next, freshly prepared 1N sodium hydroxide solution (0.440 mL, 0.440 mmol) was added and the reaction mixture was stirred at room temperature. After 30 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 co-evaporated with methanol (2 x 10 mL). The residue was dissolved in a methanol / water mixture (9:1 v / v, 1.00 mL) and the resulting solution was subjected to preparative MP-LC 2 and the fractions corresponding to the product were immediately pooled together, frozen and freeze-dried overnight to give the title compound (75.6 mg, 87%) as a colorless gummy oil. Purity 96% based on LC-MS. LRMS (m / z): 513 [M+2] 2+ , 825 [M+1] 1+ LC-MS r.t. (min): 1.42 2A

[0262] Intermediate 8: Dendron (-L-SO1861) 4 -amine N,N’-((9S,19S)-14-(6-Aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamide)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamide) formate (2.73 mg, 3.13 μmol) was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TCEP / acetonitrile (3:1 v / v, 3.00 mL). Next, 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 subjected to preparative LC-MS 3B and the fractions corresponding to the product were immediately pooled together, frozen and freeze-dried overnight to give the title compound (12.3 mg, 43%) as a white fluffy solid. Purity 97% based on LC-MS. LRMS (m / z): 1517[M-6]6- , 1821 [M - 5] 5- , 2276 [M - 4] 4- LC - MS r.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 - tetraoxapentadecane - 15 - oate (2.90 mg, 7.48 μmol) were dissolved in DMF (1.00 mL). Next, DIPEA (1.302 μL, 7.48 μmol) was added, the mixture was shaken for 1 min, and allowed to stand at room temperature. After 2 h, the reaction mixture was subjected to preparative LC - MS 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (5.86 mg, 84%) as a white fluffy solid. Purity based on LC - MS was 90%. LRMS(m / z): 2344 [M - 4] 4- LC - MS r.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 - tetraoxapentadecane - 15 - oate (3.94 mg, 8.91 μmol) were dissolved in DMF (1.00 mL). Next, DIPEA (1.55 μL, 8.91 μmol) was added, the mixture was shaken for 1 min, and allowed to stand at room temperature. After 3 h, the reaction mixture was subjected to preparative LC - MS 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (6.76 mg, 80%) as a white fluffy solid. Purity based on LC - MS was 66%. LRMS (m / z): 2358 [M - 4] 4- LC - MS retention time (min): 2.13 6C

[0265] Intermediate 11: Dendron (-L-SO1861) 4 -Maleimide 2 Skeleton 2 (5.10 mg, 5.00 μmol) was dissolved in methanol (100 μL). Next, freshly prepared 1N sodium hydroxide solution (22.0 μL, 22.0 μmol) was added, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (25.0 μL, 25.0 μmol) was added, and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and co-evaporated with methanol (2 × 5 mL). The resulting residue was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TCEP / acetonitrile (3:1 v / v, 3.242 mL). Directly from this solution, 1000 μL was added to SO1861-EMCH (14.4 mg, 6.94 μmol, 4.5 equivalents compared to the skeleton), and the mixture was shaken for 1 min and allowed to stand at room temperature. After 10 min, the reaction mixture was freeze-dried overnight. To the resulting residue, 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(3-(2,5-dioxo-2h-pyrrol-1(5h)-yl)propanamide)ethoxy)ethoxy)propanoate (5.84 mg, 0.014 mmol) and DMF (1.00 mL) were added. Next, DIPEA (2.39 μL, 0.014 mmol) was added, and the suspension was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was subjected to preparative LC-MS 3C The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to give the title compound (10.9 mg, 85%) as a white fluffy solid. Purity based on LC-MS: 80%. LRMS (m / z): 2354 [M - 4] 4- LC - MS retention time (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-Butoxy)Carbonyl]Amino})Hexanamide]Hexanamide]Ethyl}Hexanamide)Ethyl]Carbamoyl}-5-[(2S)-2,6-Bis({[(tert-Butoxy)Carbonyl]Amino})Hexanamide]Pentyl]Carbamoyl}-5-{[(tert-Butoxy)Carbonyl]Amino}Pentyl]Carbamate (S)-2-6-Diamino-N-(2-(6-Azido-N-(2-((S)-2,6-Diaminohexanamide)Ethyl)Hexanamide)Ethyl)Hexanamide Tetrahydrochloride (964 mg, 1.50 mmol) was dissolved in DMF (25.0 mL) and Triethylamine (2.08 mL, 15.0 mmol). Next, Boc-Lys(Boc)-ONp (3.36 g, 7.18 mmol) was added and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was purified by flash chromatography (DCM-Methanol / DCM (1 / 9 v / v) gradient, rising from 100:0 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-MS r.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-Tetraazaoctacosane-1,5-Diyl)Bis(2,6-Diaminohexanamide) Octahydrochloride Intermediate 1 (2.71 g, 1.50 mmol) was dissolved in HCl (5 - 6 N, 25.0 mL, 138 mmol) in isopropanol, and the reaction mixture was stirred at room temperature overnight. Next, the reaction mixture was evaporated in vacuo, and the resulting crude product was co-evaporated with DCM (3 × 20 mL) to afford 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)propanamide]‐N‐[(1S)‐1‐{[2‐(6‐azido‐N‐{2‐[(2S)‐2,6‐bis[(2S)‐2,6‐bis[3‐(acetylsulfanyl)propanamide]hexanamide]hexanamide]ethyl}hexanamide)ethyl]carbamoyl}‐5‐[(2S)‐2,6‐bis[3‐(acetylsulfanyl)propanamide]hexanamide]pentyl]hexanamide (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-tetraazaoctacosan-1,5-diyl)bis(2,6-diaminohexanamide) octahydrochloride (300 mg, 0.230 mmol) was added to 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 stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the residue was first subjected to flash chromatography (DCM - methanol / DCM (1 / 9 v / v) gradient, rising from 100:0 to 0:100), and then preparative MP-LC 2Purification by [specific method] gave the title product (70 mg, 15%) as a white solid. Purity based on LC-MS was 100%. LRMS (m / z): 685 [M+3] 3+ LC-MS r.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-sulfanylpropanamide)hexanamide]hexanamide]ethyl}hexanamide)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamide)hexanamide]pentyl]-2,6-bis(3-sulfanylpropanamide)hexanamide formate backbone 4 (10.0 mg, 4.87 μmol) was dissolved in methanol (200 μL). Next, freshly prepared 1N sodium hydroxide solution (42.9 μL, 0.043 mmol) was 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 subjected directly to 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-MS r.t. (min): 1.54 2C

[0270] Intermediate 5: Dendron (-L-SO1861) 8 -amine Skeleton 5 (0.52 mg, 0.299 μmol) and SO1861-EMCH (29.2 mg, 0.014 mmol) were dissolved in a mixture of 0.5 mM TCEP / acetonitrile and 20 mM NH4HCO3 (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. Next, the mixture was subjected directly to 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. Purity based on LC-MS: 97%. LRMS (m / z): 2282 [M-8] 8- , 2607 [M-7] 7- LC-MS r.t. (min): 4.41 5A

[0271] Dendron (NEM) 4 Synthesis (Figure 18) To benzylbis(2-((S)-2,6-bis(3-mercaptopropanamide)hexanamide)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 purified using preparative LC-MS 3A to give the title compound (1.53 mg, 57%) as a white fluffy solid. Purity based on LC-MS: 98%. LRMS (m / z): 1346 [M+1] 1+ LC-MS r.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 linker Ordered trifunctional linkers (DBCO, TCO, maleimide) from Bio-Synthesis Inc. (Lewisville, Texas). HSP27 BNA oligo Ordered HSP27 BNA (-thiol) oligo (sequence 5’-GGCacagccagtgGCG-3’) (Zhang et al., 2011) from Bio-synthesis Inc. (Lewisville, Texas).

[0273] Intermediate 1: SO1861-azide To 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. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was subjected to 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. Purity 100% based on LC-MS. LRMS (m / z): 2150 [M-1] 1- LC-MS r.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). The resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was subjected to preparative LC-MS 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity 89% based on LC-MS. LRMS (m / z): 1677 [M - 2] 2- LC - MS retention time (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-formylbenzamide)-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) were 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 purified 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. Purity 92% based on LC-MS LRMS (m / z): 394 [M + 2] 2+ , 789 [M + 1] 1+ LC - MS retention time (min): 1.28 7A

[0276] Intermediate 4: Methyltetrazine - BNA oligo HSP27 BNA oligosulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). Next, TCEP (14.3 mg, 0.050 mmol) was added, and 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 with a molecular weight cut-off of 3000 Da (5000×g, 30 min). Next, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residue solution, and the resulting mixture was filtered again under the same conditions described above. The residue 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)benzamide)-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. A 20 mM NH4HCO3 solution (20.0 mL) was added to the crude product, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter under the same conditions described above. Next, again, a solution of 20 mM NH4HCO3 (20.0 mL) was added to the residue solution, and the resulting mixture was filtered again using a centrifugal filter under the same conditions described above. The residue 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 based on LC-MS was 91%. LRMS (m / z): 1631 [M - 4] 4- , 2174 [M - 3] 3- LC-MS r.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 subjected to preparative LC - MS 4A . The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. Purity based on LC - MS was 92% (two peaks, both m / z values corresponding to the title compound). LRMS (m / z): 1641 [M - 6] 6- , 1970 [M - 5] 5- LC - MS r.t. (min): 3.24 and 3.40 6B

[0278] Intermediate 1: SO1861 - azide To 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. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was subjected to 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. Purity based on LC - MS was 100%. LRMS (m / z): 2150 [M - 1] 1- LC - MS r.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 subjected to preparative LC-MS 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS was 89%. LRMS (m / z): 1677 [M - 2] 2- LC-MS r.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-formylbenzamide)-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), methanol (extra dry, 2.00 mL) and TFA (11.1 μL, 0.145 mmol) were added, and the reaction mixture was stirred at 50 °C. After 30 min, the reaction mixture was evaporated in vacuo, and the resulting residue was purified 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. Purity based on LC-MS was 92%. LRMS (m / z): 394 [M + 2] 2+ , 789 [M + 1] 1+ LC-MS r.t. (min): 1.28 7A

[0281] Intermediate 4: Methyltetrazine-BNA oligo HSP27 BNA oligosulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). Next, TCEP (14.3 mg, 0.050 mmol) was added, and 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 with a molecular weight cut-off of 3000 Da (5000×g, 30 min). Next, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residue solution, and the resulting mixture was filtered again under the same conditions described above. The residue 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)benzamide)-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 freeze-dried over the weekend to give the crude title product as a pink, fluffy solid. 20 mM NH4HCO3 solution (20.0 mL) was added to the crude product, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter under the same conditions described above. Next, again, a solution of 20 mM NH4HCO3 (20.0 mL) was added to the residue solution, and the resulting mixture was filtered again using a centrifugal filter under the same conditions described above. The residue solution was diluted with 20 mM NH4HCO3 (20.0 mL), and the resulting mixture was freeze-dried overnight to give the title product (90.0 mg, 115%) as a pink, fluffy solid. Purity based on LC-MS was 91%. LRMS (m / z): 1631 [M - 4] 4- , 2174 [M - 3] 3- LC-MS r.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 subjected to preparative LC - MS 4A . The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. Purity based on LC - MS was 92% (two peaks, both m / z values corresponding to the title compound). LRMS (m / z): 1641 [M - 6] 6- , 1970 [M - 5] 5- LC - MS r.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 a gel. To prepare cDNA, first, 100 ng of total RNA was mixed with random hexamers (Qiagen; final concentration 2 μM) in NFW at a final volume of 12.5 μl, denatured at 70 °C for 5 min, and immediately ice-cooled. Next, 7.5 μl of cDNA synthesis mix consisting of 4 μl of 5× RT buffer (Promega), 0.4 μl of 25 mM dNTP (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 was added. 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) ∞ at 4 °C. For a single qPCR reaction, the following mix was prepared: 1 μL of cDNA, 0.05 μL of forward primer (250 μM), 0.05 μL of reverse primer (250 μM), 8.9 μl of LNFW, 10 μl of SYBR Green (Bio-Rad). The following qPCR protocol was used: 1 cycle: 5 min at 95 °C, 40 cycles: 15 s at 95 °C + 30 s at 60 °C.

[0284] HSP27 gene expression was determined using 2-( Ct HSP27 - GEOMEAN ( Ct ref1 ; Ct ref2 ; Ct ref3 ; Ct ref4)(()) was used for the calculation. Here, ref1, ref2, ref3, and ref4 are the reference genes IMMT, EIF2S2, GUSB, and UBC for tumor analysis. To select the most ideal and stable reference genes for this tumor sample, two reference genes were selected from among the nine reference genes tested based on the performance of the GeNORM analysis. To do so, the qPCR results were imported into the Qbase+ software program. Thereby, two quality metrics are calculated: the coefficient of variation (V) of the normalized reference gene expression levels; 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 a technical replicate 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 Column (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris Protein Gel (Thermo-Fisher), NuPAGE™ MES SDS Running Buffer (Thermo-Fisher), Novex™ Sharp Pre-Stained Protein Standard (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 Anhydrous (99%, Sigma-Aldrich), Polyethylene Glycol Sorbitan Monolaurate (TWEEN 20. Sigma-Aldrich, Dulbecco's Phosphate Buffered Saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetate dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filter 0.2 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), diantin-Cys (Dia-Cys, a diantin mutant with a single C-terminal cysteine functional group, Proteogenix), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200 PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio) propionate (PEG4-SPDP, Thermo-Fisher), HSP27 oligonucleotide (Biosynthesis).

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

[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 =64,500 M -1 cm -1 ;Rz 280:495 =0.232 Ellman's (TNB) OD 412 =14,150 M -1 cm -1 Size exclusion chromatography Size exclusion chromatography (SEC) was performed using an AKTA purification apparatus. Samples were analyzed by SEC using either a Biosep SEC-S3000 column or a Sephadex G50M column (10 × 40 cm). Elution was carried out with a TBS / isopropyl alcohol solution (85:15 v / v). Sample purity was determined by integration of the antibody sample peak relative to the trace aggregate peak.

[0290] Ellman assay Antibody-dendron (-L-SO1861) 4 (Cys and Lys) Synthesis of Trastuzumab-(L-d(SO1861)4)4 and Cetuximab-(L-d(SO1861)4)4 by DAR4 (FBR706 STB22 / 9-10) Trastuzumab and cetuximab are hereinafter referred to 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 exemplified as follows: Cetuximab was desalted in DPBS pH 7.5 buffer and then normalized to 2.50 mg / ml. An aliquot of Ab (9.19 mg, 61 nmol) was added to an aliquot of freshly prepared PEG4-SPDP solution (5.0 mg / ml, 6.70 molar equivalents, 411 nmol), the mixture was briefly vortexed, and then incubated at 20 °C for 60 min by 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). This mixture was roller mixed at 20 °C for 15 min by 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 the Ellman assay (SH to Ab ratio = 5.4). To the bulk Ab-SH (7.41 mg, 1.93 mg / ml, 49 nmol), an aliquot of freshly prepared d(SO1861)4-EMCH solution in DMSO was added (10 mg / ml, 8.0 molar equivalents per Ab, 0.4 μmol, 3.16 mg, 0.32 ml), the mixture was briefly vortexed, and then incubated overnight at 20 °C. In addition to the conjugation reaction, two aliquots of desalted Ab-SH (0.25 mg, 1.67 nmol) 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) at 20 °C overnight as positive and negative controls, respectively. After 18 h incubation (before addition of NEM), the crude conjugate mixture was briefly centrifuged, a 100 μl aliquot was removed for analysis by UV-vis, and characterized by the Ellman assay alongside the positive and negative controls to obtain the incorporation of d(SO1861)4.To the bulk Ab-d(SO1861)4 mixture, an aliquot (2.5 mg / ml, 5.0 molar equivalents, 0.25 μmol) of freshly prepared NEM solution was added and the mixture was purified by a 1.6 × 30 cm Sephadex G50 column eluting with DPBS pH 7.5 to give the purified cetuximab-(L-d(SO1861)4)4 conjugate. The product was filtered through a 0.2 μm filter to clarify and then carefully concentrated to ca. 3 mg / ml using a vivaspin T15 concentrator (3,000 g, 5 minute intervals, 5 °C) to give the final cetuximab-(L-d(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) Trastuzumab-(L-HSP27)4, cetuximab-(L-HSP27)4, PEG4-SPDP, and cetuximab-(L-HSP27)2 synthesized via PEG4-SPDP with DAR2, trastuzumab, cetuximab, trastuzumab-L-SO1861, cetuximab-L-SO1861 with DAR4 will hereafter be 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 minutes at 20 °C by roller mixing. Thereafter, oligo-SH was purified by a PD10 G25 desalting column eluting 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 of freshly prepared PEG4-SPDP solution in DMSO (1 mg / ml) (9.26 molar equivalents, 80.3 nmol, 45 μmol) by roller mixing at 20 °C for 60 minutes. Subsequently, the reaction was 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 eluting with TBS pH 7.5. An aliquot of the resulting Tras-(L-SO1861)-(L-PEG4-SPDP) was taken out and tested by UV-Vis analysis. SPDP incorporation was determined using TCEP to liberate 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 by roller mixing overnight at 20 °C. After 17 hours, the conjugate was analyzed by UV-vis analysis to confirm HSP27 incorporation by the release of pyridyl-2-thione (PDT) at 343 nm. The crude conjugate was purified using a 1.6 × 33 cm Sephadex G50 column eluting 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: Required for STB17 / 1-8

[0295]

Table 41

[0296] Example 8 Materials and methods In our current study, we examined a model skeleton consisting of four molecular arms for saponin binding via Schiff base (imine) and one arm for click chemistry. The polymer structure (Figure 19) is a dendrimer based on pentavalent polyethylene glycol of the first generation (i.e., the number of repeating branching cycles). This was purchased from Iris Biotech GmbH (Marktredwitz, Germany). Saponin (SA1641 in this example) was purified from a complex crude extract of saponins from the Gypsophila species called saponinum album obtained from Merck (Darmstadt, Germany). The powdered crude 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 pH 5.0 was reached. To remove tannins, the solution was shaken with 30 mL of butanol in a separating funnel. The aqueous phase was re-acquired and the butanol extraction was repeated twice. Anhydrous sodium sulfate was added to the butanol phase, filtered, and pooled. 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, different saponins were separated by high performance liquid chromatography (HPLC). The tube (column exclusion) was rinsed with warm water (40 °C) at a flow rate of 1.5 mL / min and then with isopropanol (100%) including a Eurospher RP-C18 column (5 μm, 250×8 mm). The saponin was applied to the column and eluted with a methanol gradient (from 20% methanol to 70% methanol within 30 min at 1.5 mL / min in water with 0.01% trifluoroacetic acid added, then 70% methanol for a further 60 min) (Sama et al, 2018). Aliquots of the fractions were analyzed for their SA1641 content by electrospray ionization mass spectrometry (ESI-MS). Fractions containing pure SA1641 were pooled and methanol was evaporated. The aqueous solution was frozen as a thin film in a rotary round bottom flask by using dry ice. After storage at -80 °C for 16 h, the sample was freeze-dried.To generate the backbone defined in the present invention, the polymer structure (0.2 mM) and SA1641 (3.2 mM) were dissolved in water (approx. pH 8), equal volumes were mixed, and shaken at 26 °C for 24 h. Then, sodium cyanoborohydride (NaCNBH3; 0.1 M) was added at a 4-fold molar excess relative to SA1641, and the sample was incubated for an additional 24 h. The structure was then verified by ultra-high performance liquid chromatography (UPLC) / ESI-MS. The sample was applied to an RP-C4 column and eluted with a methanol gradient (from 25% to 80% methanol within 15 min, then 80% methanol for an additional 10 min in water with 0.01% trifluoroacetic acid). Fractions were analyzed using a LockSpray™ ion source, which is designed specifically for accurate mass measurements by electrospray ionization using an LC-time of flight (LC-TOF) mass spectrometer from Waters Corporation.

[0297] Results The inset in Figure 20 shows the theoretically predicted mass spectrum obtained from calculations by 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 the reason why more than one peak is expected for a single substance. The experimental data obtained by UPLC / ESI-MS (Figure 20) shows almost exactly the same peaks at m / z 758 - 760 with the same intensities as predicted, thus demonstrating successful SA1641 coupling to the polymer structure.

[0298] Example 9 Materials and Methods As an example of a prodrug, we used the targeted toxin, dianthin-epidermal growth factor (dianthin-EGF). The plasmid His-dianthin-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). Subsequently, 300 μL of lysogenic medium (LB) was added and the suspension was incubated at 37 °C for 1 h with shaking at 200 rpm. 100 μl of the bacterial suspension was inoculated onto a pre-warmed lysogenic medium agar plate containing 50 μg / mL ampicillin and the plate was incubated overnight at 37 °C. Colonies from the plate were inoculated into lysogenic medium (3 mL) containing 50 μg / mL ampicillin and the bacteria were incubated at 37 °C and 200 rpm for 8 h. The suspension (50 μL) was added to 500 mL of lysogenic medium containing 50 μg / mL ampicillin and incubated overnight at 37 °C and 200 rpm. Subsequently, the volume was scaled up to 2.0 L and the bacteria were grown under the same conditions until an optical density at a wavelength of 600 nm of 0.9 was reached. Subsequently, protein expression was 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 5,000 g and 4 °C for 5 min, resuspended in 20 mL of 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 (15,800 g, 4 °C, 30 min) and imidazole was added to a final concentration of 20 mM. The supernatant was incubated with 2 mL of Ni-nitrilo triacetic acid agarose for 30 min at 4 °C with continuous shaking in the presence of 20 mM imidazole. Subsequently, the material was poured into a 20 mL column and washed three times with 10 mL of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole), and diantin-EGF was eluted with increasing concentrations of imidazole (31, 65, 125, and 250 mM) in 10 mL aliquots of wash buffer.The eluate fraction (2 mL) was dialyzed against 2.0 L of PBS at 4 °C overnight. Desalted diantin-EGF was concentrated by Amicon® Ultra-15 (10 kDa) and the protein concentration was quantified.

[0299] To introduce a suitable click chemistry group into diantin-EGF, an 8-fold molar excess of alkyne-PEG5-N-hydroxysuccinimidyl ester relative to diantin-EGF was dissolved in dimethyl sulfoxide and added to 9 volumes of diantin-EGF (1 mg in 0.2 M NaH2PO4 / Na2HPO4, pH 8). After incubation at room temperature for 4 h, unbound alkyne was separated by use of a PD10 column (GE-Healthcare, Freiburg, Germany). Click chemistry with the polymer structure was carried out by copper(I)-catalyzed alkyne-azide cycloaddition. Alkyne-diantin-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 in 0.1 M NaH2PO4 / Na2HPO4, pH 8, at room temperature for 1 h under gentle agitation. Then, low molecular mass substances were separated using a PD10 column.

[0300] To test the effectiveness of the present invention, we performed a viability assay with HER14 cells. These cells are fibroblasts stably transfected with the human epidermal growth factor receptor and are thus 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 at 37 °C, 5% CO2, and 98% humidity for 24 h with DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Then, different test substances (see Results and Figure 21) were added in triplicate at a volume of 25 μL, and an 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. Thereafter, 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. The solubilized formazan was photometrically quantified at 570 nM using a microplate reader (Spectra MAX 340PC, Molecular Devices, Sunnyvale, CA, USA). Untreated cells were normalized to 1, and all samples were referenced to the untreated control. Significance was determined by an unpaired two-sample t-test.

[0301] Results The exemplary polymer structure, the pentamer dendrimer (pentrimer), has no cytotoxic effect on target cells, either in the absence or presence of SA1641 (Figure 21, columns 2 and 3). In the absence of the backbone, the targeted toxin (dianthin-EGF) shows half of the maximum 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 endosomal escape enhancer (column 5). The presence of the polymer structure does not affect the toxicity of dianthin-EGF, either in the presence or absence of SA1641 (columns 6 and 7), indicating that the backbone does not affect the toxicity of dianthin-EGF. To couple the model polymer structure to the exemplary drug substance dianthin-EGF by click chemistry, the substance had to be previously coupled to an alkyne group. The drug substance manufacturer can directly introduce the click position into the substance during synthesis at his chosen position where the activity of the substance remains unaffected. When the alkyne-modified drug substance was clicked with the polymer structure, there was no additional loss of activity, indicating that the polymer structure itself is not toxic.

[0302] Example 10 Materials 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%, Carl Roth), 2-mercaptoethanol (98%, Sigma-Aldrich), poly(amidoamine) (PAMAM dendrimer, ethylenediamine core, 5.0 generation 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), 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 15 mL centrifugal filter (10 kDa MWCO, Sigma).

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

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

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

[0306] Size exclusion chromatography Size exclusion chromatography (SEC) was performed using Sephadex G25 Superfine from GE Healthcare and a prepacked PD10 column (GE Healthcare, Sephadex G25M). Before performing chromatography, the materials were activated by swelling them in their respective eluents.

[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 carried out for 24 h with 1 L of solvent, which was replaced after the first 6 h of the process.

[0308] Lyophilization Lyophilization was performed using an Alpha 1-2 LD plus (Martin Christ Gefriertrocknungsanlagen GmbH). Typically, the samples were frozen in liquid nitrogen and placed in the lyophilizer under high vacuum.

[0309] SO1861-EMCH synthesis SO1861 (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) from Saponaria officinalis L were placed in a round flask with a stirrer and dissolved in 13 mL of methanol. TFA (400 μL, cat.) was added to the solution, and the reaction mixture was stirred at 800 rpm and room temperature for 3 h with an RCT B magnetic stirrer (IKA Labortechnik). After stirring for 3 h, the mix was diluted either with MilliQ water or PBS and dialyzed against either MilliQ water or PBS for 24 h with great care. A regenerated cellulose membrane tube (Spectra / Por 7) with a MWCO of 1 kDa was used. After dialysis, the solution was lyophilized to obtain a white powder. Yield 62.4 mg (95%). The dried aliquot was further 1 used for characterization by 1H NMR and MALDI-TOF-MS.

[0310] 1 1H NMR (400 MHz, methanol-d4) (Figure 22A, SO1861): δ = 0.50 - 5.50 (m, sapogenin triterpenoid and sugar backbone protons), 9.43 (1H, s, aldehyde proton of saponin, H a ).

[0311] 1 1H NMR (400 MHz, methanol-d4) (Figure 22B. SO1861-EMCH, PBS workup): δ = 0.50 - 5.50 (m, sapogenin triterpenoid and sugar backbone protons), 6.79 (2H, s, maleimide protons, H c ), 7.62 - 7.68 (1H, 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 ([M-H] - , saponin-EMCH conjugate), 2244 Da ([M-H] - , saponin-EMCH conjugate), 2222 Da ([M-H] - , saponin-EMCH conjugate), 2178 Da ([M-H] - , saponin-EMCH conjugate), 2144 Da ([M-H] - , saponin-EMCH conjugate), 2122 Da ([M-H] - , saponin-EMCH conjugate), 2092 Da ([M-H] - , saponin-EMCH conjugate), 2070 Da ([M-H] - , SO1861-EMCH), 2038 Da ([M-H] - , SO1832-EMCH), 1936 Da ([M-H] - , SO1730-EMCH), 1861 Da ([M-H] - , SO1861).

[0313] SO1861-EMCH-mercaptoethanol To SO1861-EMCH (0.1 mg, 48 nmol), 200 μL of mercaptoethanol (18 mg, 230 μmol) was added, and the solution was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 1 h. After 1 h of shaking, the solution was diluted with methanol and dialyzed against methanol carefully for 4 h using a regenerated cellulose membrane tube (Spectra / Por 7) with a MWCO of 1 kDa. After dialysis, an aliquot was taken out 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 47 μL of 2-iminothiolane (231 μg, 1.1 μmol) dissolved in PBS was added to 200 μL of BSA-RhodB solution (10 mg, 0.15 μmol) in PBS, and the mixture was shaken at 800 rpm at room temperature for 40 min on a ThermoMixer C (Eppendorf). After 40 min of shaking, the reaction mixture 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 at 800 rpm at room temperature for 12 h on a ThermoMixer C (Eppendorf). After 12 h of shaking, BSA-SO1861 was concentrated using centrifugal filtration at 4,000 rpm (15 °C) with an Amicon Ultra 15 filter having a MWCO of 3 kDa. The conjugate was stored in the refrigerator as a solution, and aliquots were 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 attached), 35.9 kDa ([M+H] 2+ , BSA-SO1861 with three SO1861 attached), 34.7 kDa ([M+H] 2+, two SO1861s were attached (BSA - SO1861).

[0317] Cy3 - PAMAM 720 μL of PAMAM (30 mg, 1.04 μmol) dissolved in methanol was placed in a 250 mL round - bottom flask, and the methanol was removed by a rotary evaporator (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 a solution of Cy3 - COOH (0.6 mg, 1.2 μmol) in DMSO, and the mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 1 h. After 1 h of shaking, the HATU - Cy3 solution was added to the stirred PAMAM solution, and the reaction mixture was stirred at room temperature for 12 h. After 12 h of stirring, the reaction mixture was diluted with MilliQ water and dialyzed against MilliQ water using a regenerated cellulose membrane tube (Spectra / Por6) with a 2 kDa MWCO for 24 h. After dialysis, the volume of the conjugate solution was reduced by a rotary evaporator (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 obtain a viscous pink PAMAM - Cy3 conjugate. The formation of the PAMAM - Cy3 conjugate was confirmed by thin - layer chromatography (methanol / water, v / v 1:1) and the appearance of faster bands on a Sephadex G25 Superfine column. Yield 21.3 mg (63%). The molar ratio of dye per PAMAM measured by UV - Vis spectrophotometry was 0.43.

[0318] MALDI - TOF - MS (Figure 33 A) (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 exemplarily. 2-Iminothiolane (1 mg, 6.7 μmol) dissolved in 250 μL of MilliQ water was added to the PAMAM-Cy3 solution (0.5 mg, 17 nmol) in 125 μL of MilliQ water, and the mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 40 min. After 40 min of shaking, the reaction mixture 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 on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After 12 h of stirring, the reaction mixture was diluted with MilliQ water and dialyzed against MilliQ water using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12 - 14 kDa for 24 h with great care. After dialysis, the Cy3-PAMAM-SO1861 solution was concentrated using centrifugal filtration at 4000 rpm (15 °C) with an Amicon Ultra 15 filter having 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 FIGS. 33B - D and 34. MALDI-TOF-MS of Cy3-PAMAM-(SO1861)6 (FIG. 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 was carried out by the methodology described above, but the charged equivalents of the starting materials 2-iminothiolane and SO1861-EMCH were different. The charged equivalent of each starting material and the mass of each conjugate 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 separately dissolved in 200 μL DMSO. The SO1861 and HATU solutions were mixed and shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 20 min. After 20 min of shaking, the Cy3-PAMAM solution was added to the shaken SO1861-HATU solution, and the reaction mixture was allowed to shake 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 against MilliQ water for 24 h using a regenerated cellulose membrane tube (ZelluTrans, Carl Roth) with a MWCO of 12 - 14 kDa. After dialysis, the Cy3-PAMAM-NC-SO1861 solution was concentrated using centrifugal filtration at 4,000 rpm (15 °C) with an Amicon Ultra 15 filter having a MWCO of 3 kDa. Cy3-PAMAM-NC-(SO1861) 17 The conjugate was stored as a 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-azido-NH-BOC (G4-dendron) (9.75 mg, 2.11 μmol) was placed in a 2 mL reaction tube (Eppendorf) and dissolved in 200 μL of DMSO. 100 μL of a Cy5-DBCO solution (1.72 μmol * mL -1 / mL, 170 nmol) was added to the G4-dendron solution, and the mixture was shaken on a ThermoMixer C (Eppendorf) at room temperature and 800 rpm for 12 h. After 12 h of stirring, the reaction mixture was diluted with MilliQ water and dialyzed against MilliQ water for 24 h using a regenerated cellulose membrane tube (Spectra / Por 7) with a 1 kDa MWCO. After dialysis, the solution was lyophilized to obtain a blue powder. The crude product was used in the deprotection step as obtained from lyophilization.

[0326] The lyophilized G4-dendron that was partially Cy5-labeled was dissolved in 12 mL of CHCl3 in a 50 mL round-bottom flask equipped with a stir bar. 12 mL of TFA was added, and the reaction mixture was stirred on an RCT B magnetic stirrer (IKA Labortechnik) at 800 rpm and room temperature for 3 h. 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 formation of the G4-dendron conjugate was confirmed by chromatography on thin layer chromatography (methanol / water, v / v 1:1) and the appearance of a faster band by the PD10 column. The fractions obtained from size exclusion chromatography were lyophilized to obtain a blue powder.

[0327] Yield 5.7 mg (93%). The molar ratio of dye per G4-dendron measured 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-counter ion), 3820 Da ([M+Na] + , Cy5-G4-dendron - PF6-counter ion), 3617 Da ([M+H] + , G4-dendron impurity), 3017 ([M+H] + , G4-dendron).

[0329] Synthesis of G4-dendron-SO1861 The procedure for the lowest G4-dendron to SO1861-EMCH ratio is described illustratively. 2-Iminothiolane (2.65 mg, 19.2 μmol) dissolved in 300 μL of MilliQ water was added to a partially Cy5-labeled G4-dendron solution (0.577 mg, 192 nmol) in 252 μL of MilliQ water, and the mixture was shaken on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 40 min. After 40 min of shaking, the reaction mixture 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 on a ThermoMixer C (Eppendorf) at 800 rpm and room temperature for 12 h. After 12 h of shaking, the reaction mixture 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 spectra are illustrated in Figure 33. 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 charged equivalents of the starting material SO1861-EMCH. The charged equivalent of each starting material and the mass of each conjugate are highli...

Claims

1. The base is a) a polymeric or oligomeric structure, and b) at least one saponin SO1861 covalently attached to said polymeric or oligomeric structure; The base further comprises: c) a first chemical group for covalently coupling said base to a carrier molecule; ； d) 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; To DNA oligomers or polymers, RNA polymers, stabilized RNA polymers, and and PNA (peptide nucleic acid) polymers; e) said base is suitable for covalently binding said at least one saponin SO1861 to said carrier molecule, f) A 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 of claim 1, wherein at least one SO1861 is covalently attached to the polymer or oligomer structure via a non-cleavable bond or via a cleavable bond.

3. The base according to claim 1 or 2, wherein the cleavable bond undergoes cleavage under acidic, reductive, enzymatic or photoinduced conditions.

4. 4. The base according to any one of claims 1 to 3, wherein at least one SO1861 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 a mammalian cell.

5. 5. The base according to any one of claims 1 to 4, wherein at least one SO1861 is covalently bound to the polymeric or oligomeric structure of the base via an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thio-ethyl bond, an amide bond, a peptide bond or an ester bond.

6. 6. The base according to any one of claims 1 to 5, wherein at least one SO1861 is covalently bound to the polymeric or oligomeric structure of the base via a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thio-ethyl bond, an amide bond, a peptide bond or an ester bond or via at least one linker.

7. The base according to any one of claims 1 to 6, wherein at least one SO1861 is covalently bound to the polymeric or oligomeric structure of the base via a hydrazone bond.

8. 8. The base according to claim 1, wherein an aldehyde functional group at position C-23 of at least one SO1861 is involved in a covalent bond to a polymeric or oligomeric structure of the base and / or a glucuronic acid functional group on a carbohydrate substituent at C-3 beta-OH group of at least one SO1861 is involved in a covalent bond of said base to said polymeric or oligomeric structure, either via a direct bond or via at least one linker.

9. 9. The base according to any one of claims 1 to 8, wherein at least one aldehyde functional group at position C-23 of SO1861 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-ethyl bond.

10. 10. The base according to any one of claims 1 to 9, wherein a glucuronic acid function on the carbohydrate substituent at the C-3 beta-OH group of at least one SO1861 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 base, such as a lysine or N-terminal amine group of a proteinaceous molecule.

11. The base according to any one of claims 1 to 10, wherein the chemical group for the covalent coupling of the base to a carrier molecule is a click chemistry group.

12. A base according to any one of claims 1 to 11, wherein the click chemistry group is a tetrazine, an azide, an alkene or an alkyne, or a cyclic derivative of any of these groups.

13. 13. The base according to any one of claims 1 to 12, wherein the base is a trifunctional linker, comprising a second chemical group to which at least one SO1861 is covalently attached, comprising a third chemical group for covalent attachment to a molecule, and comprising a first chemical group for covalent attachment to a carrier.

14. 14. The base of any one of claims 1 to 13, wherein at least one SO1861 has a defined number of saponin molecules or a defined range of saponin molecules, or 1 to 128 or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 saponin molecules, or any number therebetween, for example 7, 9, 12 saponin molecules.

15. 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 14, which is selected from the group consisting of poly- or oligo(amino acids), such as proteins, peptides and polylysine.

16. 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.

17. 17. The base according to any one of claims 1 to 16, 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.

18. 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 18. The base according to any one of claims 1 to 17, comprising or consisting of an Fcab fragment.

19. 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 19. The base according to any one of claims 1 to 18, 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.

20. 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 20. The base according to any one of claims 1 to 19, 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.

21. 21. The base according to any one of claims 1 to 20, 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.

22. The base according to any one of claims 1 to 21, wherein the carrier molecule comprises or consists of at least one effector molecule or, when dependent on any one of claims 16 to 20, 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.

23. 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, Pseudomonas23. The base of claim 22, 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.

24. 24. The base according to claim 22 or 23, 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.

25. The base according to any one of claims 22 to 24, wherein the effector molecule comprises or consists of at least one payload.

26. The base according to any one of claims 1 to 25, wherein the carrier molecule comprises or consists of a covalently linked combination of an effector molecule and a monoclonal antibody.

27. 1. A method for producing a base suitable for covalently binding at least one saponin SO1861 belonging to the type of 12,13-dehydrooleanane having an aldehyde function in 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 said at least one SO1861 molecule belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 to said oligomeric or polymeric structure; b) covalently coupling, via said second chemical group(s), at least one saponin SO1861 belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 to said polymeric or oligomeric structure; 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; - 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; 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.

28. A method for producing a base covalently bound to a carrier molecule, said base comprising at least one covalently bound SO1861 of the type 12,13-dehydrooleanane having an aldehyde function at position C-23, said method comprising the steps of: ： a) providing a base comprising at least one SO1861 molecule belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23, covalently bonded to the polymeric or oligomeric structure of said base; b) covalently coupling the base of a) to a carrier molecule according to any one of claims 19 to 27, This provides said base covalently bound to a carrier molecule, said base comprising at least one covalently bound SO1861 of the type of 12,13-dehydrooleanane having an aldehyde function in 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; - 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; The method, wherein said aldehyde functionality at position C-23 is covalently coupled to said polymeric or oligomeric structure via a hydrazone bond.

29. the carrier molecule comprises or consists of at least one non-proteinaceous and / or at least one proteinaceous ligand for binding to a cell surface molecule, and optionally comprises or consists of one or more of a protein, a peptide, a nucleic acid, an oligonucleotide, a lipid, a fat, a fatty acid, a nanoparticle, a carbohydrate, optionally an immunoglobulin, at least one binding domain of an immunoglobulin, and / or at least one binding fragment of an immunoglobulin, or the carrier molecule comprises or consists of an effector molecule, which 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 A combination of such a ligand and such an effector molecule. A functional base comprising a base according to any one of claims 1 to 26 and a carrier molecule.

30. Carrier molecule one or more of proteins, peptides and carbohydrates, optionally an immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, and -oligonucleotides 30. The functional base of claim 29, comprising:

31. 31. The functional base according to claim 29 or 30, wherein at least one SO1861 is covalently bound to the polymeric or oligomeric structure of the base via a hydrazone bond.

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