Saponin conjugated to epitope binding proteins

Abstract

To provide a first proteinaceous molecule having covalently coupled saponin.SOLUTION: Provided is a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell-surface molecule, the first proteinaceous molecule is provided with at least one saponin that is covalently bound to an amino-acid residue of the first proteinaceous molecule via at least one linker and / or via an oligomeric or polymeric scaffold, or covalently bound directly to an amino-acid residue of the first proteinaceous molecule, where the first epitope of the first cell surface molecule to which the first binding site binds is CD71.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first proteinaceous molecule being provided with at least one saponin covalently bound to an amino acid residue of the first proteinaceous molecule via at least one linker and / or via an oligomer or polymer backbone or directly to an amino acid residue of the first proteinaceous molecule. The present invention also relates to a combination of therapeutic agents. The combination of therapeutic agents comprises a first pharmaceutical composition comprising the first proteinaceous molecule of the present invention and a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell surface molecule different from the first cell surface molecule and comprising an effector moiety, the second epitope being different from the first epitope. Furthermore, the present invention relates to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the present invention comprising a first proteinaceous molecule according to the present invention and comprising a first binding site for binding to a first epitope on a first cell surface molecule; and (b) a third pharmaceutical composition comprising a third proteinaceous molecule; the third proteinaceous molecule comprising a first binding site for binding to the first epitope on the cell surface molecule of (a) and an effector moiety. The first binding site of the first proteinaceous molecule and the first binding site of the third proteinaceous molecule are the same. And, the first epitope on the first cell surface molecule and the first cell surface molecule to which the first proteinaceous molecule can bind, and the first epitope on the first cell surface molecule and the first cell surface molecule to which the third proteinaceous molecule can bind are the same. One aspect of the present invention is a composition comprising the first proteinaceous molecule of the present invention and the second proteinaceous molecule of the present invention. One aspect of the present invention relates to a composition comprising the first proteinaceous molecule of the present invention and the third proteinaceous molecule of the present invention.The present invention also relates to a composition comprising one or more of an oligonucleotide, a nucleic acid, and a xeno nucleic acid, which is preferably selected from at least one of a vector, a gene, a transgene that induces apoptosis, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), a DNA aptamer, an RNA aptamer, mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA), and a threose nucleic acid (TNA), or a derivative thereof, and a first proteinaceous molecule of the present invention. The present invention also relates to an antibody-drug conjugate or a ligand-drug conjugate comprising a first proteinaceous molecule of the present invention and an effector portion. One aspect of the present invention relates to a pharmaceutical composition comprising the composition of the present invention or the antibody-drug conjugate of the present invention or the ligand-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient. The present invention also relates to a combination of therapeutic agents of the present invention or the composition of the present invention or the antibody-drug conjugate of the present invention or the ligand-drug conjugate of the present invention or the pharmaceutical composition of the present invention for use as a medicament. 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 exhibit off-target effects in addition to the bioactivity directed towards the 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, and the therapeutic effect of the drug molecule should, for example, be highly specific for the biological factor or biological process driving the disease, (2) be sufficiently safe, (3) be sufficiently effective, (4) sufficiently target diseased cells with little or no off-target activity against non-diseased cells, (5) have a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in a human patient within a certain time frame and remain at the target site for a certain time frame), and / or (6) have a therapeutic activity that persists sufficiently long in the patient's body. Despite extensive and ongoing research, as well as impressive progress made in some areas in addressing the individual difficulties 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 utilizing their specific binding properties as a mechanism for targeted delivery of cytotoxic agents to cancer cells while sparing normal cells. This can be achieved by chemical conjugation of cytotoxic effectors (also known as payloads or warheads) to antibodies to create antibody-drug conjugates (ADCs). Typically, very potent payloads such as 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 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 patients treated with conventional chemotherapy. Mylotarg was re-approved for marketing 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 an antigen that has a high expression level in tumors and no or little expression in normal tissues, an antigen that is present on the cell surface and accessible to the circulating ADC, and an antigen that permits internalization of the ADC into the cell 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, by varying 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 that have been evaluated as treatment options for lymphatic 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 were each combined with rituximab, a monoclonal antibody administered concomitantly that binds to CD20, and no payload was provided (Non-Patent Document 1). 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 features mentioned earlier for ADCs.

[0007] On the one hand, over 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 majority of clinical trials are being conducted in the field of gene therapy, with nearly 2,600 ongoing or completed trials globally, but only about 4% entering 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 ASO as a potential therapeutic agent 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 ASO has been demonstrated, inefficient cellular uptake both in vitro and in vivo limits the effectiveness of ASO and has been a barrier to therapeutic development. Cellular uptake can be <2% of the dose, resulting in an ASO concentration that is too low at the active site for effective and sustained results. 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, which contain one or more molecules of a small molecule drug conjugated 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, 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 so far provided little clinical success. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has shown significant anti-cancer activity against both solid tumors and leukemia in mouse models and was under clinical investigation for oncology indications. Although it demonstrated a significant reduction in non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer efficacy was found to be marginal in patients, and as a result, further development of PK1 was discontinued.

[0010] The failure of skeleton-small molecule drug conjugates can be attributed, at least in part, 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 observed only 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, which are formed spontaneously when phospholipids are dispersed in water. The amphiphilic properties of phospholipids endow them with 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 has been approved for clinical use.

[0012] Therefore, there is still a need to find a solution that allows 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, and a sufficiently low clearance rate from the patient's body.

Prior Art Documents

Non-Patent Documents

[0013]

Non-Patent Document 1

Summary of the Invention

[0014] In one embodiment of the present invention, the first goal is to provide a compound with improved biological activity, or a composition containing such a compound with improved biological activity.

[0015] One of several objectives of some embodiments of the present invention is to provide a solution to the problem of non-specificity encountered 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 body of a patient when administered to a human patient in need thereof.

[0016] At least one of the above objects of the embodiments of the present invention is achieved by providing a first proteinaceous molecule of the present invention comprising at least one saponin and a cell targeting moiety, the first proteinaceous molecule being also suitable for use as a medicament according to the present invention or also suitable for the significance in a pharmaceutical combination according to the present invention and also suitable for use as a semi-finished product for the production of an ADC or an antibody-oligonucleotide conjugate (AOC) of the present invention according to the present invention. The therapeutic combination comprises a first proteinaceous molecule comprising a covalently bound saponin and a second proteinaceous molecule comprising an effector molecule, also referred to as an effector moiety. The first and second proteinaceous molecules comprise different binding sites for different epitopes exposed on different cell surface molecules of the target cell. The different cell surface molecules are expressed by the same target cell and are exposed on the surface of the same target cell.

[0017] 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.

[0018] One aspect of the present invention relates to a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first proteinaceous molecule being provided with at least one saponin covalently bound to an amino acid residue of the first proteinaceous molecule via at least one linker and / or via an oligomer or polymer backbone or directly to an amino acid residue of the first proteinaceous molecule. According to the present invention, the first proteinaceous molecule is, for example, a finished product for use in a combination of therapeutic agents comprising a first pharmaceutical composition (a first conjugate comprising a first proteinaceous molecule having a saponin (s) covalently coupled thereto) comprising a first proteinaceous molecule having a saponin covalently coupled thereto. Secondly, a first proteinaceous molecule having a saponin covalently coupled thereto is also a semi-finished product. The first proteinaceous molecule can be linked, for example, to at least one effector moiety, such as an enzyme, a toxin, such as a protein toxin, an oligonucleotide, such as a BNA, thereby providing an ADC or AOC according to the present invention. The ADC or AOC is optionally provided with one or more covalently linked saponins via a linker and / or an oligomer or polymer backbone. Therefore, one aspect of the present invention relates to a conjugate comprising or consisting of a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein at least one saponin is covalently bound to the first proteinaceous molecule via at least one linker and / or at least one saponin is covalently bound to an amino acid residue of the first proteinaceous molecule via an oligomer or polymer backbone or directly covalently bound to an amino acid residue of the first proteinaceous molecule.

[0019] One embodiment is the first proteinaceous molecule of the present invention, wherein the first binding site comprises or consists of an immunoglobulin or at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin.

[0020] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one saponin is a triterpenoid saponin and / or a bisdesmoside-type triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, and / or a saponin isolated from Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, such as Quillaja saponaria.

[0021] One embodiment is the first proteinaceous molecule of the present invention, wherein the first epitope of the first cell surface molecule to which the first binding site of the first proteinaceous molecule binds is the first epitope specific to tumor cells of a tumor cell-specific receptor, preferably 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-alpha V, 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, more preferably selected from CD71, EGFR, HER2.

[0022] One embodiment is the first proteinaceous molecule of the present invention, wherein the tumor cell-specific first epitope, first tumor cell surface molecule, or first tumor cell-specific receptor is the first epitope or the first molecule or the first receptor that is internalized by tumor cells after binding of the first proteinaceous molecule of the present invention to the first epitope or the first molecule or the first receptor. Preferably, when the first proteinaceous molecule binds to a cell surface molecule containing the first epitope, a tumor cell surface molecule, or a tumor cell-specific receptor, it is subjected to internalization mediated by a tumor cell receptor via endocytosis, for example, or internalization mediated by a tumor cell surface molecule via endocytosis, for example.

[0023] One aspect of the present invention relates to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition comprising the first proteinaceous molecule of the present invention and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell surface molecule different from the first cell surface molecule and comprising an effector portion, the second pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, and the second epitope being different from the first epitope.

[0024] One aspect of the invention relates to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the invention comprising a first proteinaceous molecule of the invention comprising a first binding site for binding to a first epitope on a first cell surface molecule (the first pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient); (b) a third pharmaceutical composition comprising a third proteinaceous molecule; wherein the third proteinaceous molecule comprises a first binding site for binding to the first epitope on the cell surface molecule of (a) and an effector portion, the third pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, the first binding site of the first proteinaceous molecule and the first binding site of the third proteinaceous molecule being the same, and the first epitope on the first cell surface molecule and the first cell surface molecule to which the first proteinaceous molecule can bind, and the first epitope on the first cell surface molecule and the first cell surface molecule to which the third proteinaceous molecule can bind being the same.

[0025] An embodiment is the first proteinaceous molecule and / or the second proteinaceous molecule of the invention, which is an intermediate product for the manufacture of an ADC conjugated to at least one saponin, or which is an intermediate product for the manufacture of an AOC conjugated to at least one saponin, wherein at least one saponin is conjugated to the ADC or AOC via a covalent bond, preferably via at least one linker, preferably via an oligomeric or polymeric backbone to which at least one saponin is covalently coupled, preferably via a linker (Figs. 91, 92).

[0026] One embodiment is a combination of the first proteinaceous molecule of the present invention and / or a therapeutic agent of the present invention, wherein the first binding site and / or the second binding site is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment and / or domain thereof, preferably cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and the antibodies in Table A4, preferably cetuximab or trastuzumab or OKT-9, or any one of at least one cell surface molecule binding fragment or domain thereof, or consists of the same, provided that the first binding site of the first proteinaceous molecule is different from the second binding site of the second proteinaceous molecule.

[0027] One embodiment is a combination of therapeutic agents of the present invention, wherein the effector moiety contained in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of any one or more of oligonucleotides, nucleic acids, and xeno nucleic acids.

[0028] One embodiment is a combination of therapeutic agents of the present invention, wherein the effector moiety contained in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of peptides, proteins, enzymes such as urease and Cre recombinase, ribosome-inactivating proteins, and proteinaceous toxins. One embodiment is a combination of therapeutic agents of the present invention, wherein the effector moiety contained in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of at least one payload, preferably selected from 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.

[0029] One embodiment is a combination of therapeutic agents of the present invention, wherein the first proteinaceous molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1 to 100 saponins, or any number of saponins therebetween, such as 7, 9, 12 saponins, and is covalently attached directly to the amino acid residues of the first proteinaceous molecule, preferably to cysteine and / or lysine, and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymer or oligomer backbone, preferably 1 to 8 or 2 to 4 of such backbones, and at least one backbone is optionally dendron-based, and 1 to 32 saponins, such as 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins therebetween, such as 7, 9, 12 saponins, are covalently attached to at least one backbone.

[0030] One aspect of the present invention relates to a composition comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule of the present invention.

[0031] One aspect of the present invention relates to a composition comprising a first proteinaceous molecule of the present invention and a third proteinaceous molecule of the present invention.

[0032] One embodiment is a composition of the present invention, comprising either the second or the third proteinaceous molecule together with the first proteinaceous molecule, and the effector moiety comprised in the second proteinaceous molecule or the third proteinaceous molecule is any one of the effector moieties according to the present invention, preferably BNA.

[0033] One embodiment is a composition comprising the first proteinaceous molecule of the invention and preferably one or more selected from a vector, a gene, a 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, more preferably BNA, for example, an oligonucleotide, nucleic acid, and xeno nucleic acid selected from at least one of BNA for silencing HSP27 protein expression.

[0034] One aspect of the invention relates to an antibody-drug conjugate or ligand-drug conjugate comprising the first proteinaceous molecule of the invention and an effector moiety.

[0035] One embodiment is an antibody-drug conjugate or ligand-drug conjugate of the present invention, wherein the antibody can bind to 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, 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 any one of CD71, HER2, EGFR, and / or the antibody is cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, the antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one of its tumor cell receptor binding fragments and / or at least one of its tumor cell receptor binding domains, and / or the antibody-drug conjugate includes gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates of Table A2 and Table A3, or the ligand-drug conjugate includes at least one ligand for binding to cell surface molecules such as EGF or cytokine.

[0036] One embodiment is an antibody-drug conjugate or a ligand-drug conjugate of the present invention, and the effector moiety is any one or more of the effector moieties according to the present invention.

[0037] One aspect of the present invention relates to a pharmaceutical composition comprising a composition of the present invention or an antibody-drug conjugate or a ligand-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient.

[0038] One embodiment is a combination of therapeutic agents of the present invention, or a composition of the present invention, or an antibody-drug conjugate or a ligand-drug conjugate of the present invention, or a pharmaceutical composition of the present invention for use as a medicament.

[0039] One aspect of the present invention relates to any of the following ADCs and AOCs and their semi-finished conjugates, comprising a first proteinaceous molecule of the present invention and / or a second proteinaceous molecule of the present invention and / or a third proteinaceous molecule of the present invention, and comprising at least one effector molecule of the present invention or at least one saponin of the present invention or both: Anti-EGFR antibody-saponin; Anti-EGFR antibody-triterpenoid saponin, and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin; Anti-EGFR antibody-SO1861; Anti-EGFR antibody-GE1741; Anti-EGFR antibody-SA1641; Anti-EGFR antibody-Quil-A; Anti-EGFR antibody-QS-21; Saponin in the water-soluble saponin fraction of anti-EGFR antibody-Quillaja saponaria; Cetuximab-saponin; Cetuximab - triterpenoid saponin, and / or a bisdesmoside - type triterpenoid saponin belonging to the 12,13 - dehydrooleanane type having an aldehyde functional group at position C - 23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C - 3 beta - OH group of the saponin; Cetuximab - SO1861; Cetuximab - GE1741; Cetuximab - SA1641; Cetuximab - Quil - A; Cetuximab - QS - 21; Saponins in the water - soluble saponin fraction of Cetuximab - Quillaja saponaria; Anti - HER2 antibody - saponin; Anti - HER2 antibody - triterpenoid saponin, and / or a bisdesmoside - type triterpenoid saponin belonging to the 12,13 - dehydrooleanane type having an aldehyde functional group at position C - 23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C - 3 beta - OH group of the saponin; Anti - HER2 antibody - SO1861; Anti - HER2 antibody - GE1741; Anti - HER2 antibody - SA1641; Anti - HER2 antibody - Quil - A; Anti - HER2 antibody - QS - 21; Saponins in the water - soluble saponin fraction of Anti - HER2 antibody - Quillaja saponaria; Trastuzumab - saponin; Trastuzumab - triterpenoid saponin, and / or a bisdesmoside - type triterpenoid saponin belonging to the 12,13 - dehydrooleanane type having an aldehyde functional group at position C - 23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C - 3 beta - OH group of the saponin; Trastuzumab - SO1861; Trastuzumab - GE1741; Trastuzumab - SA1641; Trastuzumab - Quil - A; Trastuzumab - QS - 21; Saponins in the water-soluble saponin fraction of trastuzumab - Quillaja saponaria; Anti-CD71 antibody - saponin; Anti-CD71 antibody - triterpenoid saponin, and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin; Anti-CD71 antibody - SO1861; Anti-CD71 antibody - GE1741; Anti-CD71 antibody - SA1641; Anti-CD71 antibody - Quil-A; Anti-CD71 antibody - QS-21; Anti-CD71 antibody - saponins in the water-soluble saponin fraction of Quillaja saponaria; OKT-9 - saponin; OKT-9 - triterpenoid saponin, and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin; OKT-9 - SO1861; OKT-9 - GE1741; OKT-9 - SA1641; OKT-9 - Quil-A; OKT-9 - QS-21; OKT-9 - saponins in the water-soluble saponin fraction of Quillaja saponaria; Anti-EGFR antibody - oligonucleotide; Anti-EGFR antibody - antisense oligonucleotide; Anti-EGFR antibody - siRNA; Anti-EGFR antibody - antisense BNA; Anti-EGFR antibody - antisense BNA (HSP27); Anti-EGFR antibody - proteinaceous toxin; Anti-EGFR antibody - ribosome-inactivating protein; Anti-EGFR antibody-dianthin; Anti-EGFR antibody-saporin; Cetuximab-oligonucleotide; Cetuximab-antisense oligonucleotide; Cetuximab-siRNA; Cetuximab-antisense BNA; Cetuximab-antisense BNA (HSP27); Cetuximab-proteinaceous toxin; Cetuximab-ribosome-inactivating protein; Cetuximab-dianthin; Cetuximab-saporin; Anti-HER2 antibody-oligonucleotide; Anti-HER2 antibody-antisense oligonucleotide; Anti-HER2 antibody-siRNA; Anti-HER2 antibody-antisense BNA; Anti-HER2 antibody-antisense BNA (HSP27); Anti-HER2 antibody-proteinaceous toxin; Anti-HER2 antibody-ribosome-inactivating protein; Anti-HER2 antibody-dianthin; Anti-HER2 antibody-saporin; Trastuzumab-oligonucleotide; Trastuzumab-antisense oligonucleotide; Trastuzumab-siRNA; Trastuzumab-antisense BNA; Trastuzumab-antisense BNA (HSP27); Trastuzumab-proteinaceous toxin; Trastuzumab-ribosome-inactivating protein; Trastuzumab-dianthin; Trastuzumab-saporin; Anti-CD71 antibody-oligonucleotide; Anti-CD71 antibody-antisense oligonucleotide; Anti-CD71 antibody-siRNA; Anti-CD71 antibody-antisense BNA; Anti-CD71 antibody-antisense BNA (HSP27); Anti-CD71 antibody-proteinaceous toxin; Anti-CD71 antibody-ribosome-inactivating protein; Anti-CD71 antibody-dianthin; Anti-CD71 antibody-saporin; OKT-9-oligonucleotide; OKT-9-antisense oligonucleotide; OKT-9-siRNA; OKT-9-antisense BNA; OKT-9-antisense BNA (HSP27); OKT-9-proteinaceous toxin; OKT-9-ribosome-inactivating protein; OKT-9-dianthin; OKT-9-saporin; An anti-EGFR antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and the saponin is any one or more of a triterpenoid saponin, and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab; An anti-EGFR antibody (-proteinaceous toxin) (-saponin), wherein the proteinaceous toxin is any one or more of ribosome-inactivating proteins, dianthins, and saporins, the saponin is any one or more of triterpenoid saponins, and / or bisdesmoside-type triterpenoid saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab; An anti-HER2 antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of antisense oligonucleotides, siRNAs, antisense BNAs, and antisense BNA (HSP27), the saponin is any one or more of triterpenoid saponins, and / or bisdesmoside-type triterpenoid saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab; An anti-HER2 antibody (-proteinaceous toxin) (-saponin), wherein the proteinaceous toxin is any one or more of ribosome-inactivating proteins, dianthins, and saporins, the saponin is any one or more of triterpenoid saponins, and / or bisdesmoside-type triterpenoid saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab; An anti-CD71 antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and the saponin is a triterpenoid saponin and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9; An anti-CD71 antibody (-proteinaceous toxin) (-saponin), wherein the proteinaceous toxin is any one or more of a ribosome-inactivating protein, dianthin, and saporin, and the saponin is a triterpenoid saponin and / or a bisdesmoside-type triterpenoid saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid group on the carbohydrate substituent at the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9.

[0040] One embodiment is the first proteinaceous molecule of the present invention, the semi-finished conjugate of the present invention, or the conjugate of the present invention, wherein the first binding site is selected from cetuximab, trastuzumab, OKT-9, and / or the effector molecule is selected from dianthin, saporin, and antisense BNA (HSP27), and / or the saponin is selected from the saponins in the water-soluble saponin fraction of SO1861, GE1741, SA1641, Quil-A, QS-21, and Quillaja saponaria.

[0041] One embodiment is a conjugate according to the present invention, wherein the first proteinaceous molecule is selected from cetuximab, trastuzumab, OKT-9, and / or the effector molecule is selected from dianthin, saporin, and antisense BNA (HSP27), and / or the saponin is selected from SO1861, GE1741, SA1641, Quil-A, QS-21, and the saponins in the water-soluble saponin fraction of Quillaja saponaria.

[0042] One aspect of the present invention relates to an ADC or AOC or semi-finished ADC conjugate or semi-finished AOC conjugate of structure C, comprising the first proteinaceous molecule of the present invention, comprising at least one effector molecule of the present invention, and / or comprising at least one saponin of the present invention: A(-S)b(-E)c Structure C Wherein A is the first binding site; S is a saponin; E is an effector molecule; b = 0 to 64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64, or any integer or fraction therebetween; c = 0 to 8, preferably 0, 1, 2, 3, 4, 6, 8, or any integer or fraction therebetween, S is coupled to A and / or E, E is coupled to A and / or S, preferably, S is coupled to A, and E is coupled to A.

[0043] One embodiment is the structure C of the present invention, wherein A is an anti-EGFR antibody, such as cetuximab, an anti-HER2 antibody, such as trastuzumab, an anti-CD71 antibody, such as OKT-9, and / or S is saponin, triterpenoid saponin, and / or belongs 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 the carbohydrate substituent at the C-3 beta-OH group of saponin, any one or more of bisdesmoside-type triterpenoid saponins, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and / or E is any one or more of oligonucleotide, antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and / or any one or more of proteinaceous toxins, ribosome-inactivating proteins, dianthin, and saporin.

[0044] One embodiment is the structural C of the present invention, the conjugate of the present invention, or the semi-conjugate of the present invention, or the first proteinaceous molecule of the present invention, and when present, saponin and / or when present, the effector molecule is through at least one linker such as a cleavable linker, and / or at least one oligomer or polymer backbone, for example 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)-based linker, and dendron-based backbones such as G4-dendron, or trifunctional linkers such as the trifunctional linker of Scheme II, and are covalently coupled through. And / or, at least one lysine side chain and / or cysteine side chain of the first binding site of the first proteinaceous molecule, preferably a monoclonal antibody or a fragment or domain thereof, is involved in the covalent bond with the saponin and / or effector molecule and / or linker and / or cleavable linker and / or backbone, preferably, the saponin and / or effector molecule is covalently linked to the first binding site of the first proteinaceous molecule, preferably an antibody, and the covalent linkage comprises or consists of an amide bond, a hydrazone bond, a disulfide bond.

[0045] One aspect of the present invention relates to the use of any of the aforementioned conjugates of the present invention or semi-conjugates of the present invention or the first proteinaceous molecule of the present invention as a medicament.

[0046] One aspect of the present invention relates to the use of any of the conjugates of the present invention or semi-conjugates of the present invention or the first proteinaceous molecule of the present invention for use in the treatment or prevention of cancer or autoimmune diseases.

[0047] Figures 91 and 92 show examples of the ADCs of the present invention having covalently coupled saponin(s) and the OACs of the present invention having covalently coupled saponin(s). Definition

[0048] The term "linker" has its ordinary scientific meaning and herein 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, such as to a ligand or an effector molecule or 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 may be a non-cleavable linker. For example, the linker is stable under physiological conditions. The linker may 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 may be used in the context of the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate, or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU).

[0049] The term "trifunctional linker" has its usual scientific meaning and here refers to a linker that attaches to three molecules via chemical groups of each of the three molecules. Those skilled in the art can design such trifunctional linkers based on the present disclosure and ordinary general knowledge. Such trifunctional linkers can exhibit, for example, maleimide groups that can be used for conjugation to a targeting ligand that exhibits a thiol group for performing a thiol-ene reaction. In addition, trifunctional linkers can exhibit dibenzocyclooctyne (DBCO) groups for performing a so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with a saponin having an azide. Finally, trifunctional linkers 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). Those 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 groups for linking molecules 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 a 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 within cells such as endosomes and lysosomes of mammalian cells such as human cells, or non-cleavable bonds. Of course, a trifunctional linker can include one or two chemical groups for forming covalent bonds, and the further two or one chemical group(s) are for forming non-covalent bonds respectively. Of course, a trifunctional linker can include one or two chemical groups for forming cleavable bonds, and the further two or one chemical group(s) (singular or plural) are for forming non-cleavable bonds respectively.

[0050] 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 acidic conditions, reductive conditions, enzymatic conditions, or photoinduced 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, for example, by cathepsin. Furthermore, an example of a covalent bond cleavable under reductive conditions is a disulfide bond.

[0051] 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.

[0052] 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.

[0053] 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 attached 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 acids, 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.

[0054] 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.

[0055] 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 subclass thereof), 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 a parental immunoglobulin or other derivative that essentially maintains the antigen-binding activity of such parental immunoglobulin. Functional fragments and functional domains are antibodies in the meaning of the present invention even if their affinity for an antigen is lower than that of the parental 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 a tumor mass compared to the whole immunoglobulin.

[0056] 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.

[0057] The antibodies (immunoglobulins) of the present invention can be 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, llama antibodies consisting only of heavy chains, antibodies consisting only of heavy chains, and veterinary antibodies, but are not limited thereto. 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, autoreactive 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).

[0058] Specific antibodies that can be used in the ADCs 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.

[0059] 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, anisamidophenylboronic acid), vitamins (e.g., vitamin D), carbohydrates (e.g., hyaluronic acid, galactose).

[0060] "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.

[0061] 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 beneficial results in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / human subject. The benefits include 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 overwhelmingly beneficial to the organism as a whole, the cell is called a target cell. If the effect in a certain cell is overwhelmingly harmful to the organism as a whole, 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.

[0062] 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 in signal transduction pathways involved in abnormal cell proliferation; (vii) ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines; topoisomerase 1 inhibitors; (ix) angiogenesis inhibitors; and also includes any pharmaceutically acceptable salts, acids, solvates, and derivatives of the above.

[0063] 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 analog, duocarmycin, doxorubicin, methotrexate, benzodiazepine, camptothecin analog, and anthracycline. 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, rather than to off-target cells. The net effect of the targeted toxin is preferably beneficial to the organism as a whole.

[0064] The effector molecule or portion that is a polypeptide can be, for example, a polypeptide that restores a lost function such as, for example, enzyme supplementation, gene regulatory function, or a toxin. Examples of polypeptides as effector molecules are, for example, Cas9; toxins (such as saporin, dianthin, gelonin, (de)buganine, agrostin, ricin (toxin A chain)); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (such as argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; enzymes of cell signaling; cell cycle regulators; gene regulators (transcription factors such as NF-κB or gene repressors such as methionine repressor).

[0065] The effector molecule or effector portion 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 (such as DNA of adenine phosphoribosyltransferase); linear double-stranded DNA (such as the coagulation factor IX gene); circular double-stranded DNA (such as a plasmid); RNA: mRNA (such as TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA; antisense oligonucleotides (ASO, AON, such as PNA, PMO, LNA, and BNA), but are not limited thereto.

[0066] For example, the term "proteinaceous" as used in "proteinaceous molecule" and "proteinaceous toxin" refers to a molecule and a toxin 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 contain 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 contain that which results from any other modification, such as a chemical modification (e.g., directly to an amino acid side chain or to the molecule (covalently) attached to and (covalently) chemically bound to the proteinaceous molecule via at least one linker, which linker is either an effector moiety, a saponin, a backbone, a ligand, etc.). The term "proteinaceous" also encompasses and includes aggregates of such molecules, such as homodimers, heterotrimers, heterohexamers, or complex aggregates, such as ribosomes.

[0067] 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 a 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, the expression or a higher degree of expression of a cell surface receptor or molecular target on the surface of a first type of cell, such as a tumor cell, an autoimmune cell, a diseased cell, an abnormal cell, etc., relative to the degree of expression of the same receptor or molecular target in a second type of cell, such as a healthy cell. 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., with a binding affinity higher than the background interaction between molecules via their binding sites, for example, the immunoglobulin variable region of the immunoglobulin. 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 preferably 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.

[0068] The term "binding" is defined as an intermolecular interaction that can be distinguished from background interactions.

[0069] 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.

[0070] 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 treatment for treating cancer. Unlike 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 distinguishing between healthy cells and diseased tissues such as tumor cells in a tumor.

[0071] 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 and containing SA1657 and mainly SA1641.

[0072] 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 and mainly contains QS-18 and QS-21.

[0073] "QS-21" or "QS21" has its usual scientific meaning and here refers to a mixture of QS-21A-apio (~63%), QS-21A-xylo (~32%), QS-21 B-apio (~3.3%), and QS-21 B-xylo (~1.7%).

[0074] Similarly, "QS-21A" has its usual scientific meaning and here refers to a mixture of QS-21A-apio (~65%) and QS-21A-xylo (~35%).

[0075] 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%).

[0076] 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 saponins. Many of these incorporate the triterpenoid-trisaccharide partial structure 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 Table 2 of van Setten (1995) (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 QuilA, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.9, 660-666 (1995)). Quil-A, and also the quillaja saponins, are also fractions of saponins from Quillaja saponaria, both contain a large variety of different saponins and have largely overlapping contents. Since the two fractions are obtained by different purification procedures, the two fractions differ in their specific compositions.

[0077] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7api. QS1861 has a molecular mass of 1861 daltons, and QS1862 has a molecular mass of 1862 daltons. QS1862 is described in line 28 of Table 1 in Fleck et al. (2019) (Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira And Simone Gasparin Verza, Saponins from Quillaja saponaria And Quillaja brasiliensis: Particular Chemical Characteristics And Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171). The described structure is the api variant QS1862 of QS-7. The molecular mass is 1862 daltons 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.

[0078] 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 sequences other than those described or illustrated herein.

[0079] Furthermore, various embodiments are said to be "preferred" or "e.g." or "for example" or "specifically", but should be construed as exemplary manners in which the invention may be implemented rather than limiting the scope of the invention.

[0080] The term "comprising" as used in the claims is not to be interpreted as being limited to the elements or steps recited thereafter; it does not exclude other elements or steps. It is to be construed as identifying the presence of the recited features, integers, steps, or components but not precluding 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; rather, with respect to the present invention, the recited components of the pharmaceutical composition are only A and B, and further, the claims should be construed to encompass 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; rather, with respect to the present invention, the recited steps of the method are only A and B, and further, the claims should be construed to encompass equivalents of those steps.

[0081] In addition, unless the context clearly requires that there be only one of a feature, a reference to a feature by the indefinite article "a" or "an" does not exclude the possibility that more than one of the feature, for example, components, excipients, saponins, etc. are present. Accordingly, the indefinite article "a" or "an" usually means "at least one".

Brief Description of the Drawings

[0082]

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[0083] 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 modulating effect. In many constellations, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound outside the cell, (2) is recycled to the cell surface after internalization, or (3) is transported to the endosome where it is degraded. These root issues have been known for decades and over 500 targeted toxins have been investigated in the past decades, but the problem remains unsolved and only the protein toxin moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP), targeted by one antibody, has been approved by the FDA to date for relapsed or refractory hairy cell leukemia.

[0084] Numerous strategies have been described to overcome these problems, including approaches to transfer the toxin to endogenous cell membrane transport complexes in the biosynthetic pathway in the endoplasmic reticulum and techniques to block or weaken the membrane integrity of the endosome, i.e., the compartment of the endocytosis pathway in the cell, and thus facilitate 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, as well as photoinductive techniques. The efficacy of targeted toxins has typically increased by 100-fold or 1000-fold in cell culture and in exceptional cases by 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.

[0085] All strategies involving physicochemical techniques interact more or less directly with the membrane and require molecular improvement factors that include essentially small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not by themselves target cell-specific and are distributed in a non-targeted manner other than for targeted toxins. This is one of the major drawbacks of current approaches.

[0086] 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 may work in combination and synergistically unless otherwise specified.

[0087] Although the present invention is 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. Changes can be made without departing from the scope defined by the appended claims.

[0088] One aspect of the present invention relates to a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first proteinaceous molecule having at least one saponin covalently attached to an amino acid residue of the first proteinaceous molecule via at least one linker and / or via an oligomeric or polymeric backbone or directly covalently attached to an amino acid residue of the first proteinaceous molecule. Therefore, the present invention relates to the provision of a conjugate, the conjugate comprising or consisting of a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein at least one saponin is covalently attached to the first proteinaceous molecule via at least one linker and / or is attached to an amino acid residue of the first proteinaceous molecule via an oligomeric or polymeric backbone or is directly covalently attached to an amino acid residue of the first proteinaceous molecule.

[0089] One embodiment is the first proteinaceous molecule of the present invention, wherein the first binding site comprises or consists of an immunoglobulin, or at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, such as an antibody, IgG, Vhh domain or Vh domain, a molecule comprising or consisting of Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, and / or comprises or consists of at least one ligand for binding to a cell surface molecule such as EGF or cytokine.

[0090] One embodiment is the first proteinaceous molecule of the present invention, wherein the first epitope of the first cell surface molecule is the first tumor cell-specific epitope of the first tumor cell surface molecule, more preferably the first tumor cell-specific epitope of the first tumor cell surface receptor specifically present on tumor cells.

[0091] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one saponin is a triterpenoid saponin, and / or a bisdesmoside-type triterpenoid 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 as a carbohydrate substituent of the C-3 beta-OH group of the saponin, and / or a saponin isolated from Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, such as Quillaja saponaria.

[0092] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one saponin is a single specific saponin or a mixture of two or more different saponins, for example, the saponins of Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-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, one or more of them, 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 saponin having 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.

[0093] One embodiment is a first proteinaceous molecule according to the invention, wherein at least one saponin is a bidesmoside-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, which first branched carbohydrate side chain optionally contains glucuronic acid, and the saponin contains an ester group having a second branched carbohydrate side chain at the C-28 position, which second branched carbohydrate chain preferably contains at least 4 carbohydrate units, optionally contains at least one acetyl residue, such as 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 a carbohydrate via an ester bond, or at least one saponin is any one or more of QS-21, or QS-21A, QS-21A-api, QS-21A-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.

[0094] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one saponin is a bisdesmoside-type triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23, and at least one saponin is covalently coupled to an amino acid residue of the first proteinaceous molecule via the aldehyde functional group on the saponin, preferably the aldehyde functional group at position C-23, preferably via at least one linker, more preferably via at least one cleavable linker, and the amino acid residue is preferably selected from cysteine and lysine.

[0095] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one saponin is a bisdesmoside-type triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, and at least one saponin is covalently coupled to an amino acid residue of the first proteinaceous molecule via the glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, preferably via at least one linker, and the amino acid residue is preferably selected from cysteine and lysine.

[0096] One embodiment is the first proteinaceous molecule of 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-ether bond to a sulfhydryl group on the first proteinaceous molecule, such as the sulfhydryl group of cysteine.

[0097] One embodiment is a first proteinaceous molecule according to the present invention, wherein a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of at least one saponin is covalently coupled to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate of a linker, and this linker is covalently coupled via an amide bond to an amine group on the first proteinaceous molecule, such as a lysine or N-terminal amine group of the first proteinaceous molecule.

[0098] One embodiment is a first proteinaceous molecule of the present invention, wherein a first epitope of a first cell surface molecule to which a first binding site of the first proteinaceous molecule binds is a first epitope specific to tumor cells of a tumor cell-specific receptor, preferably 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, more preferably selected from CD71, EGFR, HER2.

[0099] One embodiment is the first proteinaceous molecule of the present invention, wherein the first epitope specific to tumor cells, the first tumor cell surface molecule, or the first tumor cell-specific receptor is internalized by tumor cells after binding of the first proteinaceous molecule according to any one of claims 1 to 11 to the first epitope or the first molecule or the first receptor, preferably, when the first proteinaceous molecule binds to a cell surface molecule containing the first epitope, a tumor cell surface molecule, or a tumor cell-specific receptor, it is subjected to internalization mediated by a tumor cell receptor, such as via endocytosis, or internalization mediated by a tumor cell surface molecule, such as via endocytosis.

[0100] One embodiment is the first proteinaceous molecule of the present invention, wherein the first binding site of the first proteinaceous molecule comprises cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody in Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor-binding fragment and / or at least one tumor cell receptor-binding domain thereof, preferably at least one tumor cell-specific receptor-binding fragment and / or at least one tumor cell-specific receptor-binding domain thereof, and comprises or consists of any one of them.

[0101] One aspect of the present invention relates to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition comprising a first proteinaceous molecule of the present invention and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell surface molecule different from the first cell surface molecule and comprising an effector portion, the second pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, and the second epitope being different from the first epitope.

[0102] One embodiment is a combination of therapeutic agents of the present invention, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the present invention comprising a first proteinaceous molecule of the present invention (the first epitope on the first cell surface molecule is a first tumor cell-specific epitope on a first tumor cell-specific surface molecule, preferably a first tumor cell-specific epitope on a first cell surface receptor specifically present on tumor cells); and (b) a second pharmaceutical composition of the present invention; the second cell surface molecule is a second tumor cell-specific surface molecule different from the first tumor cell-specific surface molecule, preferably a second cell surface receptor specifically present on tumor cells different from the first cell surface receptor specifically present on said tumor cells, and the second epitope is a second tumor cell-specific epitope.

[0103] One aspect of the present invention relates to a combination of therapeutic agents of the present invention, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the present invention comprising a first proteinaceous molecule of the present invention comprising a first binding site for binding to a first epitope on a first cell surface molecule (the first pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient); (b) a third pharmaceutical composition comprising a third proteinaceous molecule; wherein the third proteinaceous molecule comprises a first binding site for binding to the first epitope on the cell surface molecule of (a) and an effector portion, the third pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, the first binding site of the first proteinaceous molecule and the first binding site of the third proteinaceous molecule being the same, and the first epitope on the first cell surface molecule to which the first proteinaceous molecule can bind and the first cell surface molecule, and the first epitope on the first cell surface molecule to which the third proteinaceous molecule can bind and the first cell surface molecule being the same.

[0104] One embodiment is a combination of therapeutic agents of the present invention, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the present invention; and (b) a third pharmaceutical composition of the present invention; wherein the first cell surface molecule is expressed on the tumor cell surface, preferably the first cell surface molecule is a tumor cell-specific surface molecule, and preferably the first epitope is a first tumor cell-specific epitope.

[0105] One embodiment is the first proteinaceous molecule of the present invention or a combination of therapeutic agents of the present invention, wherein the first binding site for binding to the first epitope on the first cell surface molecule is the binding site of a first tumor cell-specific epitope on a first cell surface receptor that is specifically present on tumor cells.

[0106] The inventors have established that when the first pharmaceutical composition is also administered to a mammal (mouse) having a tumor to which it is administered, the therapeutic window of an antibody-drug conjugate, such as the second and third proteinaceous molecules in the second or third pharmaceutical composition of the present invention respectively, is increased. The first proteinaceous protein has at least one glycoside, such as saponin, bound thereto, preferably covalently, more preferably via a cleavable linker. Saponin presumably increases the therapeutic effectiveness of the effector moiety bound to the second and third proteinaceous molecules by improving the endosomal escape of the effector moiety to the cytoplasmic matrix where the activity of the effector moiety is desired. In this way, a therapeutic effect is established in, near, and / or within target cells under the influence of the presence of the first proteinaceous molecule containing saponin at a dose already lower than the conventional dose of the second or third proteinaceous molecule, i.e., the ADC. Target cells are, for example, diseased cells, such as tumor cells or autoimmune cells or B cells associated with B cell diseases. According to the present invention, the effector moiety is, for example, a toxin as part of an ADC or an oligonucleotide such as BNA as part of an AOC.

[0107] One embodiment is a combination of the therapeutic agents of the present invention, wherein the second binding site of the second proteinaceous molecule and / or the first binding site of the third proteinaceous molecule comprises, consists of, or contains at least one binding domain of an immunoglobulin, 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, and / or comprises or consists of at least one ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0108] One embodiment is a combination of the therapeutic agents of the present invention comprising a second pharmaceutical composition, wherein the second binding site of the second proteinaceous molecule for binding to the second epitope is the second binding site of the tumor cell-specific second epitope on the second cell surface receptor that is specifically present on tumor cells, and the second binding site is different from the first binding site.

[0109] By targeting two different cell surface molecules with a first and a second proteinaceous molecule, delivery of saponin and effector molecules into and within the cytoplasmic matrix of exactly the same target cells that expose both different cell surface molecules on the cell surface is improved and more specific compared to such exposure of the cells to only a second proteinaceous molecule such as an ADC or AOC in the absence of the cell-targeted saponin (first proteinaceous molecule). The abnormal cells selected for separate targeting by the binding site of the first proteinaceous molecule and by the binding site of the second proteinaceous molecule (the binding sites are different, the epitopes to which the first and second proteinaceous molecules bind are different, and are located on different types and classes of cell surface molecules such as two different receptors), ideally have a high degree of the first epitope and the second epitope on the first cell surface molecule and the second cell surface molecule respectively (i.e., relatively high expression of two separate and different cell surface molecules on target cells such as tumor cells or auto-immune cells compared to expression on non-target cells such as healthy cells, for example), and / or specifically expose the first and second cell surface molecules when healthy (adjacent) cells of the patient are considered. Preferably, both cell surface molecules targeted by the first and second binding sites are expressed at a relatively high level and / or specifically in target (diseased, tumor) cells compared to healthy cells. One embodiment is a pharmaceutical combination, wherein at least one of the first and second binding sites, and thus at least one of the first and second cell surface molecules such as the first and second tumor cell receptors, is expressed specifically or to a relatively high degree when compared to the expression of the first cell surface molecule and / or the second cell surface molecule on the surface of healthy (adjacent) cells. Therefore, the first epitope or the second epitope on the target cell surface molecule, preferably the first epitope and the second epitope, are ideally unique to the diseased target cells and are present and exposed specifically at least on the surface of the target cells. Binding of the first and second proteinaceous molecules to their respective first and second epitopes on the target cells is followed by endocytosis of the complex of the first proteinaceous molecule and the first target cell surface molecule and of the second proteinaceous molecule and the second target cell surface molecule.The first and second proteinaceous molecules must enter the same target cell via binding interactions with two different cell surface molecules that are both expressed to a sufficient degree or uniquely on the target cell when compared to healthy cells that are not to be targeted. Thus, accumulation of a therapeutically active amount of the first and second proteinaceous molecules within the target cell will occur only if the expression levels of the two distinct target cell surface molecules are both above a certain minimum expression threshold. At the same time, when both the first and second proteinaceous molecules are able to enter the target cell in sufficient amounts by binding to the sufficiently exposed and expressed first and second cell surface molecules, the fact that the effector moiety conjugated to the second proteinaceous molecule can exert its intracellular (e.g., cytotoxic or gene silencing) activity only in the presence of the first proteinaceous molecule having a covalently bound saponin also provides a safeguard against unwanted side effects of the effector moiety on healthy cells and healthy tissues that are not meant to be targeted and affected by the effector moiety, e.g., when the expression of at least one of the first and second cell surface molecules is sufficiently low in healthy cells, preferably when the expression of both the first and second target cell surface molecules is sufficiently low in healthy cells. That is, for the first and second cell surface molecules bound by the first and second binding sites of the first and second proteinaceous molecules, respectively, or for at least either the first cell surface molecule or the second cell surface molecule, a sufficiently low expression or even absence of the exposed first and second cell surface molecules ideally does not permit entry of both the first and second proteinaceous molecules into (non-target) healthy cells to an amount that would cooperate to effect endosomal escape of the effector moiety under the influence of the saponin bound to the first proteinaceous molecule. Since the ADC or AOC can be used at a lower dose compared to when the first proteinaceous molecule was not added to the treatment regimen, the lower level of entry of the ADC or AOC into healthy cells already presents a lower risk of occurrence of unwanted side effects when targeting and killing target diseased cells such as tumor cells and autoimmune cells is contemplated.

[0110] One embodiment is a combination of a therapeutic agent of the present invention comprising a first proteinaceous molecule of the present invention or a second pharmaceutical composition, wherein the first and second proteinaceous molecules each comprise a first and a second binding site for binding to a first and a second tumor cell-specific epitope on a first and a second tumor cell-specific receptor, respectively, the receptors are different and present on the same tumor cell, the first and second binding sites are different, and the first and second tumor cell-specific epitopes are different.

[0111] One embodiment is a combination of a therapeutic agent of the present invention comprising a first proteinaceous molecule of the present invention or a third pharmaceutical composition, wherein the first and third proteinaceous molecules comprise the same first binding site for binding to a first tumor cell-specific epitope on a first tumor cell-specific receptor.

[0112] One embodiment is a combination of a therapeutic agent of the present invention comprising a first proteinaceous molecule of the present invention or a second pharmaceutical composition, wherein the first receptor and / or the second receptor is 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, and HER2.

[0113] One embodiment is a combination of a therapeutic agent of the present invention comprising the first proteinaceous molecule of the present invention or the second pharmaceutical composition, wherein the first and second tumor cell-specific receptors are internalized by tumor cells after binding to the second proteinaceous molecule of the present invention when the first proteinaceous molecule and / or combination of therapeutic agents of the present invention comprises the second pharmaceutical composition, and preferably, the binding of the first proteinaceous molecule and / or the second proteinaceous molecule to the first and second tumor cell-specific receptors respectively results in internalization mediated by the tumor cell receptor, for example, via endocytosis of the complex of the first proteinaceous molecule and the first tumor cell-specific receptor and the complex of the second proteinaceous molecule and the second tumor cell-specific receptor.

[0114] One embodiment is a combination of a therapeutic agent comprising the third pharmaceutical composition of the present invention or the first pharmaceutical composition according to the present invention, wherein the first tumor cell receptor, preferably the first tumor cell-specific receptor, is internalized by tumor cells after binding to the first proteinaceous molecule of the present invention and / or after binding to the third proteinaceous molecule of the present invention, and preferably, the binding of the first proteinaceous molecule and / or the third proteinaceous molecule to the first tumor cell receptor such as the first tumor cell-specific receptor is followed by internalization mediated by the tumor cell receptor, for example, via endocytosis of the complex of the first proteinaceous molecule and the first tumor cell receptor and the complex of the third proteinaceous molecule and the first tumor cell receptor.

[0115] Synchronization is the missing link between a 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 1 dose of free saponin and 1 dose of an ADC (a second or third proteinaceous molecule according to the invention) separately to mice does not result in any desired anti-tumor activity, such as delayed tumor growth, tumor regression, reduced 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 of 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 the free saponin did not enable effective anti-tumor activity. The referenced ADCs 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 in various in vivo animal tumor models can be achieved by treating the animals with a conjugate according to the invention optionally containing a backbone according to the invention, i.e., a combination of the first and second or first and third proteinaceous molecules of the invention. 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, and / or an effector moiety (e.g., diantin, silencing BNA (HSP27)) covalently linked via a non-cleavable or cleavable bond, and / or 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, the dendron containing chemical groups for (covalent) coupling to a ligand or antibody or fragment or domain thereof. Reference is made to the section of examples which illustrate various ones of these backbones according to the invention, which show anti-tumour cell activity in vivo and / or in vitro, for example when cytotoxicity exerted by a proteinaceous toxin is contemplated or when gene silencing in tumour cells is contemplated.

[0116] Judging from the failures observed when considering the treatment of animals bearing tumors with an ADC together with free saponin without being constrained by any theory, it is preferred to synchronize the presence of at least one saponin and an effector moiety, preferably a toxin or an oligonucleotide, in compartments or vesicles of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. For ADCs and free saponins, synchronizing the presence of molecules in late endosomes to obtain an in vivo synergistic effect could not be beneficially obtained according to the attempts of the inventors. In one aspect, the present invention preferably solves at least the following problems for combinations of an effector moiety contained in a second proteinaceous molecule and a saponin contained in a first proteinaceous molecule: without being constrained by any theory, for example, the only reasonable chemical group on a saponin that can be used for (covalent) particularly single and cleavable retainable coupling is required for endosomal escape activity. Most probably, the known limitation is that, for example, the significant endosomal escape enhancing effect of saponins listed in Table A1 and Scheme I has been known for more than 10 years, but the reason why saponins have not been used in combination with a prodrug in clinical studies other than the application of saponins in vaccination regimens involving the use of immune enhancing adjuvant substances. For example, providing the first proteinaceous molecule of the present invention having a covalently conjugated backbone solves these difficulties at least in part. Surprisingly, saponins previously applied by virtue of their immune enhancing activity in the context of vaccination involving saponin as an adjuvant component are also suitable here for (covalent) coupling to the first proteinaceous molecule of the present invention by virtue of their in vitro and in vivo anti-tumor activity.

[0117] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effect. For example, some effectors such as Pseudomonas exotoxin are rerouted to other organelles before the "late endosomal stage" and will therefore usually not benefit from coupling to a second proteinaceous molecule according to the present invention. However, such toxins can be adapted for use in the present invention, for example, by deleting the rerouting carried out by the signal peptide. 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 second proteinaceous molecule of the present invention preferably comprises a covalently conjugated functionalized backbone, i.e., a backbone comprising a conjugated effector moiety(ies), for targeting the backbone comprising the conjugated effector moiety(ies) to target cells such as tumor cells or autoimmune cells. Furthermore, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules compared to cell membrane-permeable toxins.

[0118] As used herein, the term "ligand" has its ordinary meaning and preferably refers to 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, preferably is 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 the target cell because it interferes with a process that occurs only in the target cell can also be considered a targeted toxin (as in off-target cells, it cannot exert its toxic effect. For example, apoptin). Preferably, a targeted toxin is a toxin combined with a ligand or, for example, a monoclonal antibody because it is active in the target cell and not in off-target cells (because it is bound and endocytosed only by the target cell). In a functionalized scaffold containing a carrier molecule (i.e., a second or third proteinaceous molecule) comprising a ligand and an effector moiety, the ligand or monoclonal antibody guides the effector moiety and the scaffold to the target cell. After internalization, at least one glycoside, preferably a saponin contained in a conjugate of a first proteinaceous molecule and saponin, mediates endosomal escape of the effector moiety. The saponin is typically a saponin listed in Table A1 and Scheme I, and preferably, the saponin is SO1861 and / or QS-21 and / or SA1641 and / or GE1741.

[0119] Preferably, the effector moiety conjugated to the second or third proteinaceous molecule, whose effect is enhanced by saponin conjugated to the first proteinaceous molecule, is removed from the second or third proteinaceous molecule, such as an antibody, when endocytosed. This can be achieved, for example, by a cleavable bond that breaks under acidic, reducing, enzymatic, or photoinductive conditions.

[0120] One embodiment is a combination of a therapeutic agent of the invention comprising the first proteinaceous molecule of the invention and / or the second pharmaceutical composition, wherein the first binding site and / or the second binding site is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment and / or domain thereof, preferably cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and the antibodies of Table A4, preferably cetuximab or trastuzumab or OKT-9, or any one of at least one cell surface molecule binding fragment or domain thereof, provided that the first binding site of the first proteinaceous molecule is different from the second binding site of the second proteinaceous molecule.

[0121] One embodiment is a combination of therapeutic agents comprising a third pharmaceutical composition of the present invention, or a first pharmaceutical composition according to the present invention when included in a combination of therapeutic agents comprising a third pharmaceutical composition, wherein the first binding site of the first proteinaceous molecule and the third proteinaceous molecule comprises a monoclonal antibody, or at least one cell surface molecule binding domain and / or fragment thereof, preferably 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 any one of at least one cell surface molecule binding fragment and / or domain thereof, or consisting of the same, provided that the first binding site of the first proteinaceous molecule is the same as the first binding site of the third proteinaceous molecule.

[0122] One embodiment is a combination of therapeutic agents comprising a second or third pharmaceutical composition of the present invention, wherein the second binding site of the second proteinaceous molecule and / or the first binding site of the third proteinaceous molecule is a monoclonal antibody, or at least one cell surface molecule binding fragment or domain thereof, or comprises the same, preferably gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates of Tables A2 and A3, or consisting of the same.

[0123] The inventor has established that such immunoglobulins, their domains, ligands, etc. are particularly suitable for application as the first binding site of a first proteinaceous molecule containing a first binding site (and the same binding site of a third proteinaceous molecule). Similarly, the inventor has established that such immunoglobulins, their domains, ligands, etc. are particularly suitable for application as the second binding site of a second proteinaceous molecule containing a second binding site. For example, antibodies and antibody binding domains are suitable for targeting epitopes on the exposed surface of selected cell surface molecules, resulting in targeting of the first and third (and separately the second) proteinaceous molecules to cells expressing the cell surface molecules targeted by the first and third proteinaceous molecules. And / or also targeting cells expressing a second cell surface molecule targeted by the second proteinaceous molecule. These cells also express the first and third cell surface molecules (which are the same cell surface molecule) and have said cell surface molecule on their cell surface. Similarly, ligands such as EGF that target EGFR on target cells are suitable for application as a binding site on the first and third proteinaceous molecules or as a second binding site on the second proteinaceous molecule. However, the second binding site is different from both the first and third binding sites, and the first and third binding sites are the same. Binding sites for the first and third epitopes or the second epitope that are specific for the binding of the first and third proteinaceous molecules to the first cell surface molecule and / or for the binding of the second proteinaceous molecule to the second cell surface molecule are preferred. The first and second cell surface molecules are exposed on exactly the same target cell. For example, binding sites based on an antibody or its domain or binding fragment provide such desired specificity for selected first, second, third epitopes on selected first or second cell surface molecules of selected cells, such as diseased cells, tumor cells, autoimmune cells, etc. Thus, the first, second, and third binding sites based on an antibody or binding molecule (fragment, domain) are preferred for the first, second, and third proteinaceous molecules.

[0124] By targeting the same cell surface molecule with the first and third proteinaceous molecules, delivery of saponin and effector moiety into and within the cytoplasmic matrix of exactly the same target cells is improved and more specific. The abnormal cells selected for targeting by the binding sites of the first and third proteinaceous molecules preferably have a high degree and / or specifically have cell surface molecules when the patient's (adjacent) healthy cells are considered. Therefore, the epitope on the target cell surface molecule is preferably unique to the diseased target cells, present and exposed at least specifically on the surface of the target cells. Binding of the first and third proteinaceous molecules is followed by endocytosis of the complex of the first proteinaceous molecule and the target cell surface molecule and of the third proteinaceous molecule and the target cell surface molecule. Since the first and third proteinaceous molecules must enter the same target cells by binding interactions with exactly the same cell surface molecule, accumulation of a therapeutically active amount of the first and third proteinaceous molecules within the target cells is only possible and will occur when the expression level of the target cell surface molecule is above a certain minimum expression threshold. At the same time, when both the first and third proteinaceous molecules were able to enter the target cells in sufficient amounts by binding to sufficiently exposed and expressed cell surface molecules, the effector moiety bound to the third proteinaceous molecule was only able to exert its intracellular (e.g., cytotoxic or gene silencing) activity in the presence of the first proteinaceous molecule carrying the covalently linked saponin. This fact also provides a safeguard against negative and unwanted side effects of the effector moiety on, for example, healthy cells and healthy tissues, which are not meant to be targeted and affected by the effector moiety when the expression of the target cell surface molecule is sufficiently low in healthy cells. That is, low expression of the cell surface molecule bound by the binding sites of the first and third proteinaceous molecules does not allow entry of both the first and third proteinaceous molecules to an amount that would cooperate to effect endosomal escape of the effector moiety under the influence of the saponin bound to the first proteinaceous molecule.Since the ADC or AOC can be used at a lower dose compared to when the first proteinaceous molecule was not added to the treatment regimen, entry of the ADC or AOC into healthy cells to a low extent already has a lower risk of unwanted side effects when targeting and killing target diseased cells such as, for example, tumor cells and autoimmune cells is contemplated.

[0125] In this specification and the claims (the whole of the present application), when the first and third epitopes, the first and third binding sites, and the first and third cell surface molecules are contemplated, the terms "first" and "third" have the same meaning. That is, for the first and third proteinaceous molecules, the target epitope is the same, the binding site is the same, and the target cell surface molecule, for example, the tumor cell (specific) receptor is the same.

[0126] Tables A2, A3, and A4 list preferred examples of a first cell surface molecule that includes a first epitope for a first binding site of a first and a third proteinaceous molecule. In addition, Tables A2, A3, and A4 also list preferred examples of a second cell surface molecule that includes a second epitope for a second binding site of a second proteinaceous molecule. When the first and / or the second cell surface molecule, preferably both the first and the second cell surface molecules, are specifically expressed on a target cell, and when the first and second epitopes on the first and second cell surface molecules to which the first and second binding sites can bind are specifically present on the first and / or the second cell surface molecule, specific targeting of the first, third, and / or second proteinaceous molecule to the same desired target cells, such as tumor cells that expose the first and second tumor cell surface molecules, is facilitated. On the other hand, other cells, such as healthy cells, are not targeted or are targeted to a lesser extent by the first, third, and second proteinaceous molecules. These do not express the first and / or the second cell surface molecule or express the first and / or the second cell surface molecule to a lesser extent. Preferably, these do not express the first and second cell surface molecules or express the first and second cell surface molecules to a lesser extent compared to the expression of the cell surface molecule(s) on the target (abnormal) cell.

[0127] One embodiment is a combination of therapeutic agents comprising the second or third pharmaceutical composition of the invention, wherein the effector moiety contained in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of any one or more of an oligonucleotide, nucleic acid, xeno nucleic acid, preferably 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 derivatives thereof, more preferably BNA, for example any one or more selected from BNA for silencing HSP27 protein expression.

[0128] One embodiment is a combination of therapeutic agents comprising the second or third pharmaceutical composition of the invention, wherein the effector moiety comprised in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of a peptide, a protein, an enzyme such as urease and Cre recombinase, a ribosome-inactivating protein, a proteinaceous toxin, more preferably 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 plant toxin comprising the A chain of a ribosome-inactivating protein and 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, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumine, viscumine A chain; or an animal or human toxin such as frog RNase, or granzyme B or angiogenin from human, or any one or more of any fragment or derivative thereof; preferably, the protein toxin is dianthin and / or saporin.

[0129] One embodiment is a combination of therapeutic agents comprising the second or third pharmaceutical composition of the present invention, wherein the effector molecule(s) comprised in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of at least one payload, preferably any one or more of a toxin targeting ribosome, a toxin targeting elongation factor, a toxin targeting tubulin, a toxin targeting DNA, and a toxin targeting RNA, more preferably, emtansine, pasudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mavodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepine, AZ13599185, cryptophycin, lysocine, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, a shortened form of Pseudomonas Aeruginosa exotoxin (PE38), duocarmycin derivative, amanitin, α-amanitin, spliceostatin, tyranstatin, ozogamicin, tesirine, ambrastatin 269, and solabubtansin, or any one or more of derivatives thereof.

[0130] The active ingredient in the present invention is an effector moiety, which is used to achieve beneficial results in organisms, preferably vertebrates, more preferably humans, such as cancer patients or autoimmune patients. The benefits include the diagnosis, prognosis prediction, treatment, cure, and / or prevention of diseases and / or symptoms. The active ingredient can also lead to unwanted harmful side effects. In this case, a balance must be struck to determine whether the active ingredient is suitable for a particular case. If the effect of the active ingredient 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 cultures and bioreactors, the target cells and off-target cells depend on the purpose and are defined by the user.

[0131] The effector moiety, which is a polypeptide, can be, for example, a polypeptide that restores a lost function such as enzyme supplementation, gene regulatory function, or a toxin.

[0132] One embodiment is the first proteinaceous molecule of the present invention, wherein the first proteinaceous molecule contains more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1 to 100 saponins, or any number in between, such as 7, 9, 12 saponins, and is covalently bound directly to the amino acid residues of the first proteinaceous molecule, preferably to cysteine and / or lysine, and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymer or oligomer backbone, preferably 1 to 8 or 2 to 4 of such backbones, and at least one backbone is optionally dendron-based and 1 to 32 saponins, such as 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number in between, such as 7, 9, 12 saponins, are covalently bound to at least one backbone.

[0133] 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, particularly human tumor cells, in free form together with a second molecule (e.g., an effector moiety or effector molecule such as a toxin, oligonucleotide). Indeed, in cell-based bioassays using human tumor cells, for the saponins tabulated in Table A1 described herein, as well as those of Scheme I and the various embodiments of the present invention, in the presence of these saponins when bound to a first proteinaceous molecule, a second molecule (effector moiety) bound to a second or third proteinaceous molecule, such as a nucleic acid and / or a toxin, such as a protein toxin (e.g., one or more of the protein toxins listed in Table A5), is delivered to the cytoplasmic matrix with increased efficiency and / or effectiveness, presumably by intracellular release from (late) endosomes and lysosomes. That is, the endosomal and / or lysosomal escape of such a second molecule (effector moiety bound to the second or third proteinaceous molecule of the present invention), such as a nucleic acid and / or a toxin, is less efficient in the absence of saponin.

[0134] Surprisingly, the inventors have now demonstrated that water-soluble saponin fractions from Quillaja saponaria, including QS-21 and its family members QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18, and Quil-A, 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 (examples of the second and / or third proteinaceous molecules of the invention comprising a payload such as a covalently linked oligonucleotide or (protein) toxin). At this time, a monoclonal antibody (the first proteinaceous molecule of the invention) is administered to tumor cells of mammalian species (human) in the form of a covalent conjugate comprising a second and / or third proteinaceous molecule (the second and / or third proteinaceous molecule mentioned above) comprising an effector moiety and at least one glycoside comprised in the first proteinaceous molecule as a covalent conjugate, such as QS-21 and its family member saponins (e.g., the water-soluble fraction of Quillaja saponaria) encapsulated by such QS-21 preparations. The effector molecule and the glycoside, such as the saponin fraction of Quillaja saponaria, QS-21, SO1861, SA1641, GE1741, are covalently bound to the proteinaceous molecule either directly or via a linker, or either directly or via at least one linker via a polymeric or oligomeric structure. Without wishing to be bound by any theory, the observed stimulation or enhancement of, for example, the reduction mediated by antisense BNA of tumor cell HSP27 expression (HSP27 gene silencing) in the presence of saponins derived from Quillaja saponaria in vitro may (also) be related to the activation of the inflammasome in tumor cells by the saponin, resulting in, for example, tumor cell pyroptosis.The inventor has established that, for example, second and third proteinaceous molecules conjugated to antisense BNA or dianthin or saporin exhibit, when contacting cells in a bio-based cell assay in the presence of the first proteinaceous molecule of the invention, which is targeted to the same (tumor) cells as the cell surface molecule targeted by the second and / or third proteinaceous molecules and which comprises saponin, any anti-tumor cell activity or improved anti-tumor cell activity in vitro. On the other hand, in the absence of the first proteinaceous molecule, and hence in the absence of saponin, such activity against tumor cells was not observed.

[0135] Water-soluble saponin fractions containing QS-21, and QS-21 from Quillaja saponaria, have also long been known and have been previously intensively applied, for example, by virtue of their immunopotentiating ability as an adjuvant for subunit vaccines. For example, QS-21 is being 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. This is specifically expressed and presented by tumor cells. The anti-tumor vaccination enhanced by QS-21 aimed at prolonging 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 vaccine treatments, 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 surface of cancer cells under the influence of QS-21 saponin containing an adjuvant (AS15; GSK). Surprisingly to the inventors, the saponin fraction of Quillaja saponaria, and thus presumably QS-21 (as part of the water-soluble saponin fraction of Quillaja saponaria), enhances the anti-tumor cell activity of a payload such as a protein toxin (dianthin) conjugated to a second proteinaceous molecule (e.g., the ligand EGF).

[0136] The inventors provide a covalently coupled antisense BNA, such as BNA(HSP27), and show that a tumor cell-targeting monoclonal antibody contacted with tumor cells together with a first proteinaceous molecule of the invention having a covalently coupled saponin (e.g., SO1861, QS-21) can silence in vivo HSP27 of the tumor, compared to a control and compared to an AOC (a third proteinaceous molecule) without the presence of the first proteinaceous molecule having the coupled saponin. Both BNA and saponin are coupled via a cleavable bond to respective antibodies (e.g., cetuximab) of the first and third proteinaceous molecules. Therefore, co-administering an ADC or an antibody-oligonucleotide conjugate (AOC), such as an antibody-BNA conjugate, with a first proteinaceous molecule having a saponin confers anti-tumor cell activity to the ADC or AOC that is not seen with the same dose of the ADC alone or the AOC alone. Notably, an AOC (a second or third proteinaceous molecule) and a monoclonal antibody having a covalently coupled saponin (a first proteinaceous molecule), when administered separately to mice bearing tumors in separate mouse groups, increase HSP27 expression of the tumor cells compared to a control group (administered vehicle only). Only the co-administration of an AOC (a second or third proteinaceous molecule) containing an effector portion of the invention and a first proteinaceous molecule having a covalently coupled saponin shows reduced HSP27 expression when compared to a control.Antisense BNA (HSP27) was a BNA having the oligonucleotide sequence 5'-GGCacagccagtgGCG-3' according to Zhang et al. (2011) (Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic Acid (LNA)-based Antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333). Notably, to the best of the inventors' knowledge, the BNA was 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 a manner such that the gene silencing activity is retained in tumor cells of animals bearing tumors in vitro and more importantly in vivo. This approach of providing BNA-based AOCs opens up a new avenue for administering the targeted BNA to human (cancer) patients in need thereof.

[0137] Here, the inventors disclose that coupling a saponin, such as the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, Table A1, the saponin of Scheme I, to a first proteinaceous molecule via a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B, or via an oligomeric or polymeric structure of the backbone, and comprising the covalently coupled saponin, results in improved cytotoxicity exerted by effector moieties, such as toxins, contained in a second and / or third proteinaceous molecule under the influence of the covalently coupled saponin on the first proteinaceous molecule.

[0138] One embodiment is a first proteinaceous molecule of the invention, comprising a saponin that includes one or some or all of the indicated structural features of the saponin of Structure A of Scheme I (the saponin of Structure A is said to be a saponin having an “ideal” structure when there is endosome escape improvement activity for the effector moiety in the endosomes of cells contacted with the first proteinaceous molecule), and / or a saponin selected from any one or more of the further saponins of Scheme I:

[0139]

Chemical formula

[0140]

Chemical formula

[0141]

Chemical formula

[0142]

Chemical formula

[0143]

Chemical formula

[0144] According to the invention, a glycoside such as a saponin according to the invention, which has a "desirable" structure for the purpose of improving the endosomal escape of an effector molecule bound to a second or third proteinaceous molecule of the invention, is a bidesmosidic saponin according to structure A of Scheme I, has a molecular mass of at least 1,500 daltons, contains an oleanane-type triterpene having an aldehyde group at C-23 and optionally a hydroxyl group at C-16, has 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 and 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.

[0145] SO1861 differs from the "ideal structure" represented by Scheme I Structure A in that it has only one acetyl residue on the quinovose and only has additional xylose. The "ideal structure" of a saponin for improving endosomal escape of an effector molecule or effector moiety 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 represented by Structure A of Scheme I. Without wishing to be bound by any theory, the inventors believe that Structure A of Scheme I corresponds to an "ideal saponin" (but not a minimum requirement saponin) for improved endosomal escape activity. This means that not all of the structures (chemical groups) have to be present or must be present on each saponin that has at least sufficient improved endosomal escape activity to promote the accumulation of the effector moiety in the cytoplasmic matrix. And this means that some saponins can have other structural elements such as acyl chains, and / or for other saponins showing improved endosomal escape activity, the sugars can be different from the sugars represented by Scheme I. For example, when considering the ideal structure of Structure A of Scheme I, some of the saponins in the QS-21 saponin and the water-soluble fraction of Quillaja saponaria (Quillaja 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.

[0146] One embodiment is the first proteinaceous molecule of the present invention, wherein at least one linker is a non-cleavable linker or a cleavable linker, and the cleavable linker undergoes cleavage when bound to saponin, for example, under acidic conditions, reducing conditions, enzymatic conditions, or photoinductive conditions. Preferably, the cleavable linker contains a hydrazone bond or a hydrazide bond that undergoes cleavage under acidic conditions when bound to saponin, and / or contains a bond sensitive to proteolysis, for example, proteolysis by cathepsin B, and / or contains a bond sensitive to cleavage under reducing conditions, such as a disulfide bond.

[0147] One embodiment is the first proteinaceous molecule of the present invention, wherein when the cleavable linker is bound to saponin, the cleavable linker undergoes in vivo cleavage under 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.

[0148] One embodiment is the first proteinaceous molecule of the present invention, wherein the oligomer or polymer backbone contains a polymer or oligomer structure and contains chemical groups for covalent coupling of the backbone to amino acid residues of the first proteinaceous molecule.

[0149] According to the present invention, typically, the saponin is the saponin listed in Table A1, Scheme I. When the entry of the effector moiety covalently coupled to the second or third proteinaceous molecule into the cell and its accumulation in the cytoplasmic matrix are considered, for the activity of the saponin, such as the activity of improving endosomal escape in the cell, it has been proven beneficial when the saponin is covalently coupled to the first proteinaceous molecule and hydrazone bonds and / or hydrazide bonds and / or disulfide bonds are involved. Such types of bonds are readily cleaved under acidic conditions in mammalian cells, such as human cells, in (late) endosomes and lysosomes, and / or under reducing conditions. Alternatively, the inventor has also demonstrated that the covalent coupling of saponin to the first proteinaceous molecule via a bond that is not readily cleavable under physiological conditions in cells, such as (late) endosomes, lysosomes, and cytoplasmic matrix, is also beneficial for the enhancing activity of saponin on the biological effects of effector moieties such as proteinaceous toxins such as nucleic acids (e.g., BNA that silences HSP27) and saporin. In the present application including the claims, when referring to the conjugate of the present invention, for example, the first proteinaceous molecule optionally including a backbone having a saponin coupled to the first proteinaceous molecule via a linker and / or a backbone via a hydrazone bond or a disulfide bond, the terms "cleavable linker", "cleavable bond", etc. are also referred to as "labile linker" ("L") and "labile bond" in the context of cleavage of such bonds or linkers in (late) endosomes and / or lysosomes. For example, FIGS. 6 and 7 show in vivo HSP27 gene silencing in human tumors in mice. Mice bearing tumors were treated with a first proteinaceous molecule consisting of a monoclonal antibody having a saponin coupled thereto via a labile linker (hydrazone bond) according to the present invention, and the third proteinaceous molecule included a conjugated antisense BNA for silencing the HSP27 gene of tumor cells covalently coupled to the monoclonal antibody (the same type as the first monoclonal antibody) via a disulfide bond.That is, without being bound by any theory, once the combination of the therapeutic agents of the present invention is internalized, for example, by endocytosis, the hydrazone bond and the disulfide bond are cleaved in the (late) endosome and / or lysosome of the target tumor cell that expresses the epitope on the target cell surface molecule, here EGFR, on the cell surface. When entry of BNA from the endosome and / or lysosome into the cytoplasmic matrix is considered, cleavage of the bond will probably contribute to the endosome escape enhancing activity of saponin, but such cleavage is not essential for observing the gene silencing effect of the combination of the cetuximab-SO1861 conjugate and the cetuximab-BNA conjugate of the present invention.

[0150] Those skilled in the art will understand that the trifunctional linker is a backbone of the present invention suitable for covalently coupling one, two, or three saponin moieties. For the trifunctional linker, covalent coupling of one or two saponin moieties is preferred. The second and / or third binding sites are suitable for covalently binding a proteinaceous ligand, such as a first proteinaceous molecule. Typical proteinaceous ligands are EGF for targeting (tumor) cells expressing EGFR on the cell surface, and cytokines for targeting tumor cells or autoimmune cells. Moreover, the second or third binding site of the trifunctional linker is an immunoglobulin, such as a monoclonal antibody, for binding to a cell surface molecule, such as a tumor cell surface molecule, preferably a tumor cell-specific molecule, more preferably a tumor cell receptor specifically (over)expressed on the surface of tumor cells, i.e., suitable for covalent coupling of a first proteinaceous molecule. Similarly, an immunoglobulin, or any fragment(s) and / or domain(s) thereof encompassing the binding specificity of the immunoglobulin is suitable for binding to a cell surface molecule, such as a receptor expressed on the surface of an autoimmune cell. Therefore, in one embodiment, the first proteinaceous molecule comprises a trifunctional linker, said linker comprising a covalently bound saponin, such as QS-21, SO1861, and a covalently bound binding site, such as a cell targeting moiety, such as a ligand or antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, non-healthy cells, B cell diseases.

[0151] One embodiment is a first proteinaceous molecule of the present invention and comprises an oligomeric trifunctional linker as a backbone core structure according to Scheme II:

[0152]

Chemical Structure

[0153] In the formula, the saponin is covalently bonded to the trifunctional linker backbone via an unstable cleavable hydrazone linker (acid-sensitive) and / or via a bond containing maleimide. On the other hand, the binding of the backbone to the binding site such as an antibody is established via a bond containing maleimide with cysteine at the binding site such as 1, 2, 3, or 4 cysteines and / or via an unstable cleavable hydrazone linker (acid-sensitive), thereby forming Structure B:

[0154]

Chemical formula

[0155] As a result, 1 to 4 backbones are covalently bonded to a single, for example, antibody, such as a monoclonal antibody.

[0156] One embodiment is the first proteinaceous molecule of the present invention, the glycoside molecule is a saponin, and the linkage between the saponin and the first proteinaceous molecule preferably occurs by an acid-labile bond that is stable at pH 7.4 and preferably releases the saponin below pH 6.5, more preferably between pH 6.5 and 5.0. This is achieved, for example, by an imine formed by the amino group of the linker linking the saponin and the first proteinaceous molecule and the aldehyde group of the saponin. Other chemical bonds that meet the pH conditions can also be used for aldehyde coupling. For example, specific hydrazones or acetals, each requiring hydrazide and hydroxyl groups as functional groups of the linker. 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 the aldehyde functional group, more preferably via the aldehyde functional group, preferably the aldehyde functional group at position 23. Alternatively, the saponin is preferably attached to the first proteinaceous molecule either via the aldehyde functional group or via the carboxylic acid functional group of the glycoside molecule, i.e., saponin, via the polymer or oligomer structure of the backbone and via a linker connecting the polymer or oligomer structure of the backbone.

[0157] One embodiment is the first proteinaceous molecule of the present invention, and at least one saponin is bound to the first proteinaceous molecule via a stable bond. In a more preferred embodiment, the stable bond between the saponin and the first proteinaceous molecule preferably occurs via amide coupling or amine formation. This is achieved, for example, by amide bond formation mediated by carbodiimide between the amino group of the polymer or oligomer backbone structure that links the saponin and the first proteinaceous molecule together 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 of the linker or backbone. When the bond is a stable bond, the saponin is preferably attached via one of the carboxyl groups of the saponin to the linker or backbone, and the linker or backbone is further linked to the first proteinaceous molecule.

[0158] One embodiment is the first proteinaceous molecule of the present invention, wherein the saponin is coupled to the binding site via a backbone according to the present invention, and the chemical group for covalent coupling of the backbone to the binding site is a click chemistry group.

[0159] One embodiment is the first proteinaceous molecule of the invention, wherein the saponin is coupled to the binding site via a backbone according to the 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. The click chemistry group is a chemical functional group 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 solvent-free or mild (e.g., water) or easily removable solvents, and simple product isolation. The click chemistry group for coupling the saponin to the binding site on the first proteinaceous molecule, optionally via a backbone or linker, is preferably a tetrazine, azide, alkene, or alkyne, or a reactive derivative thereof, such as methyl-tetrazine or maleimide (alkene), more preferably an alkyne, or a cyclic derivative of these groups, such as cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]nona-4-yne, dibenzocyclooctyne).

[0160] Therefore, the first proteinaceous molecule according to the present invention comprises at least one saponin. In this context, "at least one" means that the first proteinaceous molecule contains one saponin molecule, but may also contain several (e.g., 2, 3, or 4) saponins or a large number (e.g., 10, 20, or 100) of saponins. Depending on the application, the first proteinaceous molecule may comprise a covalently bound backbone having covalently bound saponins. The backbone can be designed to contain a defined number of saponins. Preferably, the first proteinaceous molecule according to the present invention contains a defined number or range of saponins rather than a random number. This is particularly advantageous for drug development with respect to marketing approval. In this regard, a defined number means that the first proteinaceous molecule preferably contains a predetermined number of saponins. This is achieved, for example, by designing a backbone comprising a polymer structure having a certain number of possible moieties to which the saponin(s) can attach. In an ideal situation, all of these moieties are coupled to saponins, and then the backbone contains a predetermined number of saponins. It is envisioned to provide a standard set of backbones containing, for example, 2, 4, 8, 16, 32, 64 saponins, etc., so that the optimal number can be easily tested by the user according to his needs. One embodiment is the first proteinaceous molecule of the present invention comprising the backbone of the present invention, and in a non-ideal situation, for example, not all moieties present on the polymer structure bind saponins, and the saponins are present within a defined range. Such ranges can be, for example, 2 to 4 saponin molecules per backbone, 3 to 6 saponin molecules per backbone, 4 to 8 saponin molecules per backbone, 6 to 8 saponin molecules per backbone, 6 to 12 saponin molecules per backbone, etc. Therefore, in such a case, the first proteinaceous molecule comprising the backbone according to the present invention contains 2, 3, or 4 saponins when the range is defined as 2 to 4.

[0161] The backbone is, in essence, independent of the type of saponin covalently attached to the backbone, and the backbone is subsequently (in sequential order) covalently coupled to a first proteinaceous molecule. Therefore, the first proteinaceous molecule containing the backbone is the basic product of a new platform technology. Since at least one covalently attached saponin mediates the intracellular delivery of an effector moiety attached to a second proteinaceous molecule, the backbone technology according to the invention is the first known system to mediate controlled intracellular effector moiety delivery by saponin. The backbone provides an optimized and functionally active unit, which can be linked at a single and defined position to the binding sites contained in the saponin(s) and the first proteinaceous molecule, such as a ligand, an antibody, etc.

[0162] One embodiment is a first proteinaceous molecule comprising a backbone according to the present invention, wherein 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 either 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 degraded to excretable and / or physiological compounds by the body's metabolism. The aggregates can be assembled by covalent cross-linking or non-covalent 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 strictly 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.

[0163] 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. Dendrimers are the connection of two or more dendrons at their focal points. Dendronized polymers are the connection of the focal points of one or more dendrons to a polymer. In preferred embodiments, a backbone according to the present invention is provided, and the polymer or oligomer structure comprises, 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 or DNA polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. Preferably, the polymer or oligomer structure is biocompatible.

[0164] One embodiment is a combination of therapeutic agents of the invention, or a combination of therapeutic agents for use according to the invention, wherein the first proteinaceous molecule comprises more than one covalently attached saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128, or from 1 to 100 saponins, or any number in between, such as 7, 9, 12 saponins.

[0165] One embodiment is the first proteinaceous molecule of the invention, wherein at least one saponin is covalently attached to the polymer or oligomer structure of the oligomer or polymer backbone via at least one cleavable linker according to the invention.

[0166] One embodiment is the first proteinaceous molecule of the invention, wherein the chemical group of the oligomer or polymer backbone for covalent coupling to the amino acid residues of the first proteinaceous molecule is a click chemistry group, preferably selected from tetrazine, azide, alkene, or alkyne, or a cyclic derivative of these groups, more preferably, the chemical group is an azide.

[0167] One embodiment is the first proteinaceous molecule of the invention, wherein the polymer or oligomer structure of the oligomer or polymer backbone 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, and this assembly is preferably assembled by covalent cross-linking.

[0168] The present inventors have now established that covalent coupling of saponin, preferably via a cleavable bond or linker, to a first proteinaceous molecule according to any of the above embodiments provides efficient and cell-targeted enhancement of the activity of effector moieties coupled to second and third proteinaceous molecules. The first and third proteinaceous molecules comprise the same first binding site, and the first and second proteinaceous molecules comprise different first and second binding sites. When a third proteinaceous molecule is contemplated it comprises the same first binding site as the first proteinaceous molecule, and when first and second proteinaceous molecules are contemplated they each comprise different first and second binding sites. By using second and / or third proteinaceous molecules such that when the effector moieties are delivered to the same target cell, coupling of saponin directly or via a linker to a cysteine side chain or lysine side chain of a first proteinaceous molecule such as a monoclonal antibody has been found to be a useful way of specifically and efficiently delivering effector moiety enhancing activity into target cells.

[0169] To describe the present invention in more detail, the process of cellular uptake of substances (although the inventor does 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 vesicle transport and substance 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 physiologically irreversible biochemical changes. 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 even 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 with a pH of approximately 6.5. Most of the internalized cargo and membrane are recycled back 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 it is often difficult to distinguish between them. Degradation of endocytosed molecules occurs within the endolysosomes or lysosomes. Endosomal escape is the active or passive release of substances from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably from clathrin-mediated endocytosis or recycling pathways 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.

[0170] Unless specifically indicated otherwise, and particularly when referring to the endosomal escape mechanism of glycoside molecules such as saponins of the present invention, whenever the term "endosome" or "endosomal escape" is used herein, it also 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.

[0171] In formal terms, a glycoside is any molecule in which a sugar group is attached via a glycosidic bond through its anomeric carbon to another group. Without being bound by any theory, glycoside molecules such as saponins in the context of the present invention are molecules that can further enhance the effect of an effector moiety, particularly by facilitating endosomal escape of the effector moiety. Without being bound by any theory, glycoside molecules (saponins, such as those listed in Table A1) interact with the membranes of compartments and vesicles of the endocytosis pathway and recycling pathway, making them leaky for said effector molecules and resulting in increased endosomal escape. The term "scaffold is capable of increasing endosomal escape of an effector moiety" means that at least one saponin (glycoside molecule) coupled to the polymeric or oligomeric structure of the scaffold is capable of improving endosomal escape of the effector moiety. At this time, both molecules are within an endosome, for example a late endosome, and optionally and preferably, at least one glycoside such as a saponin is released from a first proteinaceous molecule, for example from a linker or polymeric or oligomeric structure contained in said first proteinaceous molecule, for example by cleavage of a cleavable bond between at least one glycoside (saponin) and the first proteinaceous molecule (for example, via the polymeric or oligomeric structure of the scaffold and / or via a linker). The bond between at least one glycoside such as a saponin according to the invention and a first proteinaceous molecule, optionally via a linker or scaffold, can be a "stable bond", but this does not mean that such a bond cannot be cleaved by, for example, an enzyme in an endosome. For example, a glycoside or saponin, optionally together with a part of an oligomer or polymer of a linker or scaffold, can be cleaved from the remaining linker fragment or oligomer or polymeric structure. For example, it can happen that a protease cleaves a (proteinaceous) linker or proteinaceous polymeric structure, such as albumin, thereby releasing at least one glycoside, saponin.However, it is preferred that the glycoside molecule (preferably a saponin) is released in its original form in which it is in an active form, preferably before it is optionally coupled (prepared to be coupled) to a first proteinaceous molecule via a linker and / or an oligomer or polymer backbone; thus, the glycoside (saponin) has its native structure after such cleavage, or the glycoside (saponin) has a chemical group or linker (part thereof) attached thereto after such cleavage. On the other hand, the glycoside bioactivity (saponin bioactivity), for example, the endosome / lysosome escape improvement activity against an effector moiety present in the same endosome or lysosome, is maintained or restored by the cleavage of the bond between the carrier molecule optionally containing the linker and / or backbone of the present invention, i.e., the first proteinaceous molecule and the glycoside (saponin). In the present invention, the term "stable" means, for example, with respect to the bond between a saponin and an amino acid residue, linker, polymer or oligomer structure (of the backbone), ligand, (monoclonal) immunoglobulin or its binding domain or fragment, and / or effector (effector moiety, effector molecule) of a first proteinaceous molecule, that the bond is not readily broken, or at least is not designed to be readily broken by, for example, pH differences, salt concentrations, or UV light, reducing conditions. In the present invention, the term "cleavable" means, for example, with respect to the bond between a saponin and a first proteinaceous molecule, linker, amino acid residue, polymer or oligomer structure of the backbone, ligand, antibody, and / or effector, that the bond is designed to be readily cleaved by, for example, pH differences, salt concentrations, reducing conditions, etc. Those skilled in the art are well aware of such cleavable bonds and how to prepare them.

[0172] Prior to the present invention, one of the major hurdles to introducing ADCs and AOCs to the market was the small therapeutic window: the therapeutically effective dose of an ADC or AOC was associated with (unacceptable) side effects, inhibiting the development and significance of the treatment of patients with ADCs. By applying the first proteinaceous molecule of the present invention, it has now become possible to direct one or more glycoside molecules (saponins), together with an ADC bearing a payload or together with a (monoclonal) antibody conjugated to an oligonucleotide such as a BNA according to the present invention (i.e., a specific second or third proteinaceous molecule of the present invention), to (target) cells. In particular, it has not been possible previously to specifically direct simultaneously the effector part of the second or third proteinaceous molecule and a specific number or range of glycoside molecules (saponins) per effector part, for example, to the cytoplasmic matrix of cells by way of the endocytosis pathway of the cells.

[0173] The solution provided by the present invention involves the covalent attachment of at least one saponin to a first proteinaceous molecule. A further solution provided by the present invention involves (firstly) polymerizing a glycoside molecule (saponin) using an oligomeric or polymeric backbone and providing a cluster of covalently attached saponins to the first proteinaceous molecule, for example enabling the remonomerization of one or more saponins at an intracellular location where the mechanism of action of the saponin is desired after endocytosis. In this context, "polymerizing" refers to the reversible and / or irreversible multiple conjugation of saponin molecules to either a first proteinaceous molecule via a linker, directly, or via a polymer or oligomer structure for forming a backbone, or the reversible and / or irreversible multiple conjugation of (modified) saponins that form a polymer or oligomer structure for forming a backbone thereby. In this context, "remonomerization" means, for example after endocytosis, the cleavage of saponin(s) from a first proteinaceous molecule, from a linker that links the saponin(s) to the first proteinaceous molecule, or from a backbone, and the reacquisition of the (native) chemical state of the unbound saponin. These unbound saponins may or may not contain additional chemical groups, such as chemical groups for linking the saponin to a linker, an amino acid residue of a first proteinaceous molecule, or a backbone, and / or a (chemical) linker attached to a chemical group of the saponin such as an aldehyde group or a carboxylic acid group. Due to the complex chemistry of saponins, for example, the "polymerization" of saponins in a backbone or other linking linker and their "remonomerization" at a desired location, for example intracellularly, after endocytosis, has been a difficult task. In particular, the chemical reactions (polymerization of glycosides) used to provide a covalently linked glycoside, such as a linker and backbone containing a triterpenoid saponin, for covalent attachment to a first proteinaceous molecule typically occur in a water-free organic solvent, while saponins and, for example, biocompatible polymers applied as a backbone for carrying the attached saponins are water-soluble molecules.The chemical properties of the unmodified saponin further prevented its polymerization. Also, one other solution for (directly) binding multiple saponins to an effector molecule was estimated to be less promising. This is because effector molecules (drugs, toxins, polypeptides, or polynucleotides) typically do not provide sufficient binding sites. And because the coupling products become highly heterogeneous and / or coupling a bioactive molecule such as saponin with, for example, a peptide, toxin, or nucleic acid carries the risk of affecting and interfering with the activity of one or even both of the molecules bound together in the conjugate containing such saponin. Furthermore, there was a significant risk that the effector moiety contained in the second or third proteinaceous molecule would lose its function after coupling of the saponin to, for example, an ADC or an antibody-oligonucleotide conjugate (AOC). Embodiments of the present invention address at least one of these drawbacks.

[0174] One aspect of the present invention relates to a composition comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule of the present invention.

[0175] One aspect of the present invention relates to a composition comprising a first proteinaceous molecule of the present invention and a third proteinaceous molecule of the present invention.

[0176] One embodiment is a composition comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule of the present invention, or a composition comprising a first proteinaceous molecule of the present invention and a third proteinaceous molecule of the present invention, wherein the effector moiety contained in the second proteinaceous molecule or the third proteinaceous molecule is any one of the effector moieties according to the present invention, preferably BNA.

[0177] One aspect of the present invention relates to a composition, which comprises the first proteinaceous molecule of the present invention and, preferably, any one or more of a vector, a gene, a transgene that induces cell death, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), an 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, more preferably BNA, for example, an oligonucleotide, nucleic acid, and any one or more of xeno nucleic acids selected from at least one of BNA (antisense BNA (HSP27)) for silencing HSP27 protein expression.

[0178] In the context of the present invention, an effector molecule or effector moiety is any substance that affects the metabolism of a cell by interacting with an effector molecule target within the cell, where the effector molecule target is any molecule or structure within the cell and excludes the compartments of the endocytosis and recycling pathways and the lumen of vesicles, but includes the membranes of these compartments and vesicles. Thus, said structures within the cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytoplasmic matrix. Delivery of an effector moiety to the cytoplasmic matrix in the context of the present invention preferably means that the effector moiety can escape from endosomes (and / or lysosomes) (which also includes escaping from endolysosomes and lysosomes as defined above), and preferably can reach the effector moiety target described herein. The present invention also encompasses a new type of molecule referred to as a scaffold. This serves to simultaneously bring into endosomes both an effector moiety and at least one glycoside molecule such as the saponin of the present invention in a predetermined ratio when the effector moiety is included in a second or third proteinaceous molecule of the present invention and the saponin is included in a first proteinaceous molecule. In the context of the present invention, the polymeric or oligomeric structure of the scaffold 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, but excludes 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 joined together, and an oligomer is a polymer whose molecules consist of a relatively small number of repeating units. There is no one specific cutoff consensus 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 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, such as the saponin of the present invention. The backbone preferably includes a polymer or oligomer structure, such as poly or oligo(amine), such as polyethyleneimine and poly(amidoamine), and biocompatible structures, 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 (and thus includes at least one glycoside molecule, such as the saponin of the present invention), (b) a functionalized backbone (and thus includes at least one glycoside molecule, such as saponin, and a ligand, antibody, etc. as a first proteinaceous molecule), (c) a polymer or oligomer structure without a glycoside molecule such as the saponin of the present invention (see, for example, Table A1) without a ligand, antibody, etc. as a first proteinaceous 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 containing at least one ligand, antibody, etc. as a first proteinaceous molecule.Polymers or oligomeric structures on an aggregated polymer structure that do not contain any of the molecules mentioned above (i.e., no glycosides such as saponins, no first proteinaceous molecules such as ligands, antibodies) are added, in particular, as structural components of the aggregated structure. This helps to assemble or stabilize the aggregated structure ("glue-like"). Without being bound by any theory, the acidic environment appears to be a prerequisite for the synergistic action between the glycoside (saponin) and the effector moiety.

[0179] Whether a first proteinaceous molecule containing one or more (cleavable) linkers and / or optionally a backbone and / or either containing or not containing a saponin can disrupt the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be readily determined by assays as described in Example 3 and as known in the art. Inhibition is described as "an increase in the amount of glycoside ~ times that required for induced 50% cell killing". The backbone preferably does not result in an increase that is at least the increase in the amount of glycoside molecules (saponin) required to obtain 50% cell killing as observed when using chloroquine as a positive control. Alternatively and preferably, a first proteinaceous molecule containing one or more (cleavable) linkers and / or optionally a backbone and / or either containing or not containing a saponin does not result in an increase of at least 4-fold, more preferably at least 2-fold, in the amount of glycoside molecules required to induce 50% cell killing. The ~-fold increase should be measured essentially by an assay as described in Example 4. Chloroquine as a positive control induces an increase in the amount of glycoside, preferably saponin, by 2-fold required to observe 50% cell killing, and the saponin is any one or more of the saponins of the present invention (see Table A1, Scheme I, previous embodiments).

[0180] The term "improving or enhancing the effect of the effector moiety" means that a glycoside molecule, preferably a saponin of the present invention, increases the functional effectiveness of its effector moiety (e.g., the therapeutic index of a toxin or drug or oligonucleotide, such as 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 the effector moiety" can also mean that an effector moiety that could not be used due to lack of effect (and, for example, was not known to be an effector moiety) 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 a combination of an effector moiety with a second or third proteinaceous molecule is considered an "enhanced effect". In certain embodiments, the backbone contained in the first proteinaceous molecule and comprising the conjugated saponin(s) enhances the effect of the effector moiety contained in the second proteinaceous molecule whose effect is intended and / or desired. In the case of a first proteinaceous molecule comprising a saponin conjugated to a proteinaceous backbone, the proteinaceous polymer structure of the backbone itself can have an effect, for example, on the colloid osmotic pressure in the bloodstream. If such an effect is not the intended or desired effect of such a functionalized backbone contained in the first proteinaceous molecule, the proteinaceous structure of the backbone is not an effector moiety as defined in the present invention. Alternatively, for example, in the case of a backbone based on DNA or RNA that bears a conjugated saponin and is contained by the first proteinaceous molecule, the DNA or RNA part thereof 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 moiety as defined in the present invention.

[0181] Several favorable characteristics can be formulated for the endosome escape enhancing factor contained in the first proteinaceous molecule, i.e., glycoside or saponin, preferably the saponin 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 the effector moiety into off-target cells, (3) Preferably, their presence at the site of action is synchronized with the presence of the effector moiety, (4) Preferably, they are biodegradable or excretable, (5) Preferably, they do not substantially interfere with biological processes of organisms irrelevant to the biological activity of the effector molecule with which the endosome escape enhancing factor is combined, e.g., do not interact with hormones. Examples of glycoside molecules such as the saponins of the present invention that fulfill 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, and the saponins of Table A1, Scheme I.

[0182] One aspect of the present invention relates to an antibody-drug conjugate or an antibody-oligonucleotide conjugate or a ligand-drug conjugate, comprising the first proteinaceous molecule and the effector moiety of the present invention.

[0183] One embodiment is an antibody-drug conjugate or an antibody-oligonucleotide conjugate or a ligand-drug conjugate of the present invention, wherein the antibody can bind to 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, 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 any one of CD71, HER2, EGFR, and / or cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG-type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, the antibodies in Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor binding fragment and / or at least one tumor cell receptor binding domain thereof, and / or the antibody-drug conjugate includes gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates in Table A2 and Table A3, or the ligand-drug conjugate includes at least one ligand for binding to cell surface molecules such as EGF or cytokine.

[0184] One embodiment is an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate of the present invention, and the effector moiety is any one or more of the effector moieties according to the present invention.

[0185] One aspect of the present invention relates to a pharmaceutical composition, comprising the first proteinaceous molecule of the present invention and the second proteinaceous molecule of the present invention, or the first proteinaceous molecule of the present invention and the third proteinaceous molecule of the present invention, or an antibody-drug conjugate of the present invention, or an antibody-oligonucleotide conjugate of the present invention, or a ligand-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient.

[0186] One aspect of the present invention relates to a combination of therapeutic agents of the present invention for use as a medicament, comprising a second pharmaceutical composition, or a third pharmaceutical composition, or a composition comprising the first proteinaceous molecule of the present invention and the second proteinaceous molecule of the present invention or a composition comprising the first proteinaceous molecule of the present invention and the third proteinaceous molecule of the present invention, or an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate or a pharmaceutical composition of the present invention.

[0187] One aspect of the present invention relates to a combination of therapeutic agents of the present invention for use in the treatment or prevention of cancer or autoimmune diseases, comprising a second pharmaceutical composition, or a third pharmaceutical composition, or a composition comprising the first proteinaceous molecule of the present invention and the second proteinaceous molecule of the present invention or a composition comprising the first proteinaceous molecule of the present invention and the third proteinaceous molecule of the present invention, or an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate or a pharmaceutical composition of the present invention.

[0188] As mentioned before, at least one saponin comprised in the first proteinaceous molecule according to the invention increases the effectiveness of at least the current and new effector parts as defined in the present invention. Potential side effects will be reduced without reducing effectiveness, due to a reduction in the dosage of the effector part comprised in the second or third proteinaceous molecule. Thus, the present invention provides a first proteinaceous molecule according to the invention for use in medicine or for use as a medicament. Therefore, one aspect of the invention relates to a first proteinaceous molecule according to the invention for use as a medicament, the first proteinaceous molecule comprising at least saponin. Use of a first proteinaceous molecule according to the invention for manufacturing a medicament is also provided. In particular, cancer medicine, especially classical chemotherapeutic medicaments, are notorious for their side effects. Since the first and third proteinaceous molecules have the same binding site for the same epitope on the same cell surface molecule, or since the first and second proteinaceous molecules have different binding sites for different first and second epitopes on the first and second cell surface molecules respectively, the combination of therapeutic agents according to the invention is beneficial, in particular for use as a medicament, in particular for use in methods for treating cancer, due to the temporal and local synchronization and targeting of both the prodrug comprised in the second or third proteinaceous molecule and the saponin comprised in the first proteinaceous molecule. Therefore, the present invention provides a combination of therapeutic agents according to the invention or a first proteinaceous molecule of the invention for use in a method for treating cancer. The present invention also provides a combination of therapeutic agents according to the invention or a first proteinaceous molecule of the invention for use in a method for treating acquired or genetic disorders, in particular monogenic deficiency disorders. Therefore, the combination of therapeutic agents comprises the first and second proteinaceous molecules and / or the first and third proteinaceous molecules. Therefore, one aspect of the invention relates to a combination of therapeutic agents according to the invention for use in a method for treating cancer or an autoimmune disease, the second or third proteinaceous molecule comprising a covalently bound effector part.

[0189] Further applications of the first, second, and third proteinaceous molecules of the invention in 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 poor treatment options lead to a shortened lifespan of the patients concerned. An example of such a disease is phenylketonuria, which is an inborn metabolic disorder in which the metabolism of the amino acid phenylalanine, an amino acid of the amino acid phenylalanine, is reduced. The disease is characterized by mutations in the gene for the liver 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. The second or third proteinaceous molecule, preferably an antibody, having bound phenylalanine hydroxylase or a bound polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells and replace the defective enzyme in liver cells by the use of suitable specific antibodies. This is one example of the use of a combination of therapeutic agents of the invention for replacement or gene therapy, which, according to the invention, comprises a first proteinaceous molecule having a saponin bound thereto and a second or third proteinaceous molecule having an enzyme or oligonucleotide bound thereto. In a preferred embodiment, a combination of therapeutic agents according to the invention for use in a method of gene therapy or replacement therapy is provided.

[0190] The invention also provides a method for treating cancer, the method comprising administering to a patient in need thereof a medicament comprising a combination of therapeutic agents according to the invention, preferably administering an effective dose of said medicament to a patient in need thereof, preferably a human cancer patient.

[0191] Considerations regarding suitable forms for administration are known in the art and include maintaining toxic effects, solubility, route of administration, and activity. For example, a pharmacological composition to be injected into the bloodstream should be soluble.

[0192] Suitable dosage forms depend, for example, in part on the route of use or administration, whether percutaneous 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 that retard the compound or composition from exerting its effect and toxicity.

[0193] One embodiment is a combination of endosome escape enhancing conjugates according to the invention, comprising a first proteinaceous molecule comprising at least one covalently attached saponin and a binding moiety. The binding moiety comprises at least one effector moiety. The binding moiety is a second or third proteinaceous molecule comprising the attached effector moiety. The endosome escape enhancing conjugate and the binding moiety can each independently specifically bind to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of the complex of the endosome escape enhancing conjugate and the target cell-specific surface molecule and of the complex of the binding moiety and the target cell-specific surface molecule. The endosome escape enhancing conjugate and the binding moiety can bind to the same target cell-specific surface molecule via their same binding sites. Alternatively, the endosome escape enhancing conjugate and the binding moiety can bind to different target cell-specific surface molecules via their different binding sites. One embodiment is a combination according to the invention, wherein the endosome escape enhancing conjugate can compete with the binding moiety for binding to the target cell-specific surface molecule or structure. One embodiment is a combination according to the invention, wherein the endosome escape enhancing conjugate and the binding moiety can each independently specifically bind to the same epitope or to different epitopes. One embodiment is a combination for use in a method of treating an abnormality such as cancer according to the invention, wherein the endosome escape enhancing conjugate and the binding moiety should be administered simultaneously or sequentially, preferably simultaneously.

[0194] One aspect of the present invention relates to a kit, which includes a first container containing an endosome escape improvement conjugate (i.e., a first proteinaceous molecule) according to the present invention, and a second container containing a binding moiety (i.e., a second and / or third proteinaceous molecule) according to the present invention. The kit further includes instructions for using a binding molecule (i.e., a combination of therapeutic agents including a first and second or a first and third pharmaceutical composition).

[0195]

Table A1-1

[0196]

Table A1-2

[0197]

Table A1-3

[0198]

Table A1-4

[0199]

Table A1-5

[0200]

Table A1-6

[0201]

Table A2-1

[0202]

Table A2-2

[0203]

Table A2-3

[0204]

Table A2-4

[0205]

Table A2-5

[0206]

Table A2-6

[0207]

Table A2-7

[0208]

Table A2-8

[0209]

Table A2-9

[0210]

Table A2-10

[0211]

Table A2-11

[0212]

Table A2-12

[0213]

Table A3-1

[0214]

Table A3-2

[0215]

Table A4

[0216]

Table A5-1

[0217]

Table A5-2

[0218]

Table A5-3

[0219]

Table A5-4

[0220]

Table A5-5

[0221]

Table A5-6

[0222]

Table A5-7

[0223]

Table A5-8

[0224]

Table A5-9

[0225]

Table A5-10

[0226]

Table A5-11

[0227]

Table A5-12

[0228] The combination of the therapeutic agents of the present invention, the first pharmaceutical composition, the first proteinaceous molecule, the second or third pharmaceutical composition, or the second or third proteinaceous molecule may further be combined with a covalent conjugate (complex) of a binding molecule or moiety and a saponin, or may further be combined with a pharmaceutical compound, an antibody, etc., which is part of the present invention. Thereby, a composition comprising three or more enhancing factors, prodrugs, etc. of the present invention, such as a conjugate (e.g., the first proteinaceous molecule and / or the second or third proteinaceous molecule), which is combined with a binding moiety complexed with an effector molecule and further combined with a pharmaceutical, is provided. This is either linked to a saponin or not, and this is either coupled to a ligand, such as a targeting immunoglobulin, its domain or fragment, or not. Further, certain embodiments are the combination of the therapeutic agents of the present invention, the first pharmaceutical composition, the first proteinaceous molecule, the second or third pharmaceutical composition, or the second or third proteinaceous molecule, wherein the second or third proteinaceous molecule provides two or more effector moieties, such as toxins or immunotoxins, and the two or more effector moieties are the same or different.

[0229] Exemplary embodiments Certain embodiments are endosome escape enhancing conjugates of the present invention, wherein the saponin is a bidesmoside type triterpenoid saponin belonging to the type of 12,13 - dehydrooleanane having an aldehyde functional group at position 23, and the saponin is preferably a saponin that can be isolated from Gypsophila or Saponaria species, and more preferably the saponin is either saponin SO1861 or its diastereomer.

[0230] Certain embodiments are endosome escape enhancing conjugates of the present invention, wherein the binding site is at least a ligand, such as an immunoglobulin, to which at least an effector moiety is bound.

[0231] One embodiment is an endosome escape improvement conjugate of the present invention, and the binding site is an immunoglobulin or at least a binding domain thereof for binding to a cell surface molecule. Preferably, the cell surface molecule is selected from any of HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, Ephrin A4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD71.

[0232] One embodiment is an endosome escape improvement conjugate of the present invention, the linker is coupled to the glycoside via a cleavable bond, and the ligand is an immunoglobulin. Preferably, the cleavable bond is cleaved under acidic, reducing, enzymatic, or photoinductive conditions, preferably, the cleavable bond is a covalent bond, preferably an imine bond, a hydrazone bond, an oxime bond, a 1,3-dioxolane bond, or an ester bond, preferably, the cleavable bond is a disulfide bond or a peptide bond.

[0233] One embodiment is an endosome escape improvement conjugate of the present invention, the saponin moiety is a terminal saponin, preferably saponin SO1861, the linker is a chemical linker that covalently links the saponin to the binding site of the first proteinaceous molecule, and the same first binding site of the third and first proteinaceous molecules is an immunoglobulin, such as trastuzumab or cetuximab, and the linker preferably provides a cleavable bond between the terminal saponin moiety and the first binding site contained in the first and third proteinaceous molecules.

[0234] One embodiment is a combination of an endosome escape enhancing conjugate (i.e., a first proteinaceous molecule) according to the present invention and a binding moiety (i.e., a second or third proteinaceous molecule), the binding moiety comprising at least one effector moiety, wherein the endosome escape enhancing conjugate and the binding moiety can each independently specifically bind to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of a complex of the endosome escape enhancing conjugate and the target cell-specific surface molecule and of a complex of the binding moiety and the target cell-specific surface molecule.

[0235] One embodiment is a combination according to the present invention, wherein when the binding moiety is a third proteinaceous molecule, the endosome escape enhancing conjugate and the binding moiety can specifically bind to the same target cell-specific surface molecule or structure.

[0236] One embodiment is a combination according to the present invention, wherein when the binding moiety is a third proteinaceous molecule, the endosome escape enhancing conjugate can compete with the binding moiety for binding to the target cell-specific surface molecule or structure.

[0237] One embodiment is a combination according to the present invention, wherein the endosome escape enhancing conjugate and the binding moiety can each independently specifically bind to the same epitope.

[0238] One embodiment is a combination according to the present invention, wherein the endosome escape enhancing conjugate can specifically bind to a first epitope, which is the same as the first epitope to which the binding moiety can specifically bind when the binding moiety is a third proteinaceous molecule.

[0239] One embodiment is a combination according to the present invention, wherein when the binding moiety is a second proteinaceous molecule, the endosome escape enhancing conjugate and the binding moiety can specifically bind to different target cell-specific surface molecules or structures.

[0240] One embodiment is a combination according to the present invention, and the target cell-specific surface molecule or structure is selected from HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, Ephrin A4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD71.

[0241] One embodiment is a combination according to the present invention, and the glycoside molecule is a bisdesmoside-type triterpene, preferably a saponin.

[0242] One embodiment is a combination according to the present invention, and the glycoside molecule is a bisdesmoside-type triterpene saponin.

[0243] One embodiment is a combination according to the present invention, and the saponin is a bisdesmoside-type triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at the 23rd position.

[0244] One embodiment is a combination according to the present invention, and the saponin is a saponin that can be isolated from Gypsophila or Saponaria species.

[0245] One embodiment is a combination according to the present invention, and the saponin is either SO1861 or one of its diastereomers.

[0246] One embodiment is a combination according to the present invention, and at least one glycoside is bound via a cleavable bond to a ligand (the binding site for an epitope on the cell surface molecule). Preferably, the cleavable bond undergoes cleavage under acidic, reductive, enzymatic, or photoinductive conditions, and the cleavable bond is preferably a disulfide bond or a peptide bond.

[0247] One embodiment is a combination according to the invention, wherein the cleavable bond is a covalent bond, preferably an imine bond, a hydrazone bond, an oxime bond, a 1,3-dioxolane bond, or an ester bond.

[0248] One embodiment is a combination according to the invention, wherein the endosome escape enhancing conjugate comprises a defined number of glycosides or a defined range.

[0249] One embodiment is a combination according to the invention, wherein the defined range is from 1 to 30 glycosides, preferably from 1 to 20, more preferably from 1 to 10, more preferably from 1 to 6, more preferably from 2 to 6, more preferably from 2 to 5, more preferably from 3 to 5, more preferably from 3 to 4 glycosides.

[0250] One embodiment is a combination according to the invention, wherein the effector moiety is a prodrug, such as a toxin, such as a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide.

[0251] One embodiment is a combination according to the invention, wherein the target cell is a diseased cell or a disease-related cell, preferably a tumor cell or a tumor-related cell (e.g., tumor vascular cell), or an immune cell (e.g., regulatory T cell), or an autoimmune cell.

[0252] One embodiment is a combination according to the invention, wherein at least one effector moiety is coupled to a linking moiety (a second or third proteinaceous molecule) via a cleavable bond, preferably, the cleavable bond is cleaved under acidic, reducing, enzymatic, or photoinductive conditions, and / or the cleavable bond is a disulfide bond or a peptide bond.

[0253] One embodiment is a combination according to the invention, wherein the glycoside (saponin) can increase the endosome escape of the effector molecule.

[0254] One embodiment is a combination according to the present invention for use as a medicament.

[0255] One embodiment is a pharmaceutical composition comprising a combination according to the present invention (previous embodiment) and a pharmaceutically acceptable excipient.

[0256] One embodiment is a pharmaceutical composition according to the present invention, further comprising at least one additional active ingredient, such as an additional immunoglobulin.

[0257] One embodiment is a combination for use according to the present invention, or a pharmaceutical composition according to the present invention, for use in a method of treating cancer or an autoimmune disease.

[0258] One embodiment is a combination for use according to the present invention, wherein the endosome escape enhancing conjugate (first proteinaceous molecule) and the binding moiety (second or third proteinaceous molecule) should be administered simultaneously or sequentially, preferably simultaneously.

[0259] One embodiment is a method of treating cancer, the method comprising administering a combination according to the present invention to a patient in need thereof.

[0260] One embodiment is a method of treating cancer, the method comprising administering a pharmaceutical composition according to the present invention to a patient in need thereof.

[0261] One embodiment is a kit comprising a first container containing an endosome escape enhancing conjugate according to the present invention and a second container containing a binding moiety according to the present invention, the kit further comprising instructions for using the binding molecule.

[0262] The first proteinaceous molecule is suitable for use as a semi-finished product for the manufacture of a functionalized ADC or a functionalized AOC, and the functionalized ADC or functionalized OAC comprises at least one covalently coupled saponin of the invention and at least one effector moiety of the invention. Certain embodiments are the first proteinaceous molecules of the invention and further comprise a payload or effector moiety of the invention, such as a toxin or oligonucleotide. The first proteinaceous molecule of the invention is covalently bound either directly or via a linker of the invention, preferably a cleavable linker of the invention, and / or via an oligomer or polymer backbone according to the invention. For example, such a functionalized ADC or OAC comprises 2 to 4 saponins covalently coupled directly or via a (cleavable) linker, for example to a cysteine side chain, on a first proteinaceous molecule such as a ligand or antibody (fragment). Alternatively, it comprises, for example, a dendron comprising 1 to 16 covalently coupled saponins attached thereto, the dendron being covalently coupled to, for example, a cysteine side chain and / or a lysine side chain of a first proteinaceous molecule according to the invention.

[0263] The invention is further illustrated by the following examples. These should in no way be construed as limiting the invention.

Examples

[0264] Treating animals with mammalian tumors with the conjugate of the 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 epidermoid carcinoma was developed as 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, low HER2 expressors.

[0265] 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 the xenograft tumors. Cetuximab was covalently conjugated to saponin SO1861. First, the linker EMCH (N-ε-maleimidocaproic acid hydrazide) was provided to SO1861. This EMCH is a maleimide and hydrazide cross-linking agent for covalently conjugating sulfhydryl (the reduced cysteine of the antibody) to a carbonyl (aldehyde or ketone; here, the carbonyl of the aldehyde at position C-23 of the saponin). The saponin-EMCH was covalently coupled to the reduced cysteine of cetuximab, forming a thio-ether covalent bond between EMCH and the cysteine side chain. The trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9, IgG)-saporin (covalent conjugate) of the ADCs 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 the previous experiments at which sub-optimal dose of the ADC would no tumor regression or arrest of tumor growth be observable.

[0266]

Table A

[0267] These results demonstrate that the combination therapy of the ADC with the conjugate of the present invention, which consists of an antibody targeting a tumor cell-specific receptor covalently bound to saponin, i.e., SO1861, provides an efficient and effective treatment regimen manifested as regressing tumors and prolonged survival of the treated animals (beyond the duration of the experiment), when treatment of mice bearing tumors with the ADC alone is ineffective (tumor growth and mouse death are not prevented (euthanasia)) at doses of the ADC. The covalent conjugate is administered to cancer-bearing mice at doses that are 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 bound saponin of the present invention, which has no anti-tumor activity when administered alone, provides an effective treatment option for cancer patients and is effective at a relatively low dose of the ADC. 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 stimulating effect of the conjugate with saponin of the present invention when the effectiveness of the ADC is considered indicates that ADCs, which have been previously found to lack effectiveness when treatment of tumor patients is concerned, may gain renewed attention and value. This is because the effectiveness of the ADC is improved under combination therapy conditions as demonstrated in this example. 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 investigation. In particular, ADCs that terminated 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 bound saponin of the present invention, such as the tested cetuximab-saponin.

[0268] Example B - Saponin mixture of Quillaja saponaria containing QS-21 having improved 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 improvement properties of at least one individual saponin present in the mixture, e.g., QS-21, or on a combination of two or more saponins contained by the mixture, e.g., QS-21 and QS-7, and can be applied to the endosome / lysosome escape improvement conjugates, compositions, combinations of the present invention.

[0269] 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 coupled to the ligand EGF: EGF-dianthin. The cells tested for dianthin 29 were the tumor cell line HeLa for free saponin, and A431, MDA-MB-468, CaSki, and A2058 for testing saponin when covalently coupled to cetuximab.

[0270] Example 1 Various concentrations of trastuzumab-saporin (HER2-targeted protein-toxin conjugate; intravenous) were tested in combination with 1.5 mg / kg SO1861 (subcutaneous, 1 hour before antibody-toxin injection) for improved efficacy in a BT474 (HER2 ++ ) xenograft mouse model. Tumors were ~150 mm 3Treatment was initiated on the 13th day when the size reached, and the tumor volume was determined after each treatment. Tumor growth inhibition was observed in mice treated with 1 mg / kg and 0.3 mg / kg of trastuzumab-saporin, but improved tumor growth inhibition was not observed in mice treated with the combination of trastuzumab-saporin + SO1861. This indicates that unconjugated SO1861 cannot improve the antibody-protein toxin under the current conditions and in the mouse model.

[0271] Example 2 Materials: QS Mix (1): S4521 (SigmaAldrich); QS Mix (2): 6857.1 (Carl Roth) QS Mix (3): Quil-A (registered trademark) Adjuvant: vac-quil (InvivoGen / Brenntag).

[0272] Previously, the efficacy of various saponins (SO1861, SO1642) has been co-administered to cells as "free" unconjugated molecules in combination with ligand-toxin fusions (e.g., EGF dianthin) or antibody-protein toxin conjugates, resulting in improved cytotoxic activity against target-expressing cells. Here, three different saponin molecules (SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904) isolated from the root extract of Saponaria officinalis were used in HeLa (EGFR +)Cells were titrated in the presence and absence of 1.5 pM EGF-diantin at a non-effective fixation concentration. This revealed a strong improvement in cytotoxic activity for all tested saponin variants compared to treatment without EGF-diantin (IC50 = 300 nM; Figure 2A). Next, EGF-diantin was titrated with a fixed concentration of saponin (~1000 nM), which revealed a strong improvement in killing target cells at low pM concentrations of EGF-diantin (IC50 = 0.4 pM; Figure 2B). Observation was made for all saponins SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904 used. EGF-diantin alone was able to induce cytotoxicity only at very high concentrations (IC50 = 10,000 pM). This indicates that these specific types of saponins all have an inherent ability to efficiently induce endosomal escape with only very low amounts of available targeted toxin.

[0273] To extend this assay, saponins from other sources were analyzed. Saponin (GE1741) purified from the root extract of Gypsophila elegans M.Bieb. was titrated against HeLa cells in the presence and absence of 1.5 pM EGF-dianthin and compared to purified SO1861. GE1741 also enhanced the killing of HeLa cells induced by EGF-dianthin but showed slightly lower efficacy compared to SO1861 (GE1741 IC50 = 800 nM; Figure 2C) and also showed higher general toxicity (IC50 = 5,000 nM in the absence of EGF-dianthin; Figure 1C). Similar assays were performed with different partially purified mixtures of Quillaja saponaria saponins (QS Mix 1–3) co-administered with 1.5 pM EGF-dianthin to HeLa cells, which revealed similar activity to SO1861 for two of the three (QS Mix 1 and QS Mix 3) (IC50 QS Mix / QS Mix 3 = 300 nM; Figure 1D). QS Mix (2) was less efficient in enhancing cell killing induced by 1.5 pM EGF-dianthin (IC50 = 2,000 nM; Figure 2D). However, no general toxicity was observed. This indicates that specific types of saponins are available from the QS extract that can also efficiently induce endosomal escape of the targeted ligand toxin EGF-dianthin.

[0274] Example 3 To conjugate the SO1861 molecule to an antibody, according to the present invention, a labile / acidsensitive linker (-EMCH or -N3) was conjugated to SO1861 via an aldehyde group to produce SO1861-EMCH or SO1861-N3 (Figs. 60 - 66). To verify the activity of SO1861-EMCH, the molecule was titrated in EGFR-expressing cells (A431, HeLa) and non-expressing cells (A2058) in the presence and absence of a fixed non-effective (1.5 pM) EGF diantin concentration. In all three cell lines, SO1861 alone showed strong cell viability reduction, while SO1861-EMCH as a single compound showed no toxicity up to 25,000 nM (Figs. 3A - C). When SO1861-EMCH was combined with 1.5 pM EGF diantin, strong target-specific cell viability reduction was observed in + A431 and HeLa cells (IC50 = 3,000 nM; Figs. 2A, B). On the other hand, - A2058 cells were not affected at all (Fig. 3C). Similar results were obtained for SO1861-N3. Co-administration of SO1861-N3 with 1.5 pM EGF diantin also showed efficient killing in A431 and HeLa cells (IC50 = 3,000 nM), but without EGF diantin, general toxicity was observed above 10,000 nM (Figs. 3D, 2E).

[0275] For stable conjugation of SO1861 to an antibody according to the present invention, a stable linker (HATU, Fig. 70) was conjugated to SO1861 via the carboxylic acid group of SO1861 to produce SO1861-(S). To determine the activity, different concentrations of SO1861-(S) were co-administered with 1.5 pM EGF diantin and tested for killing activity in EGFR-expressing HeLa cells. SO1861-(S) showed activity similar to SO1861, indicating that conjugation to the carboxylic acid did not affect the endosomal escape enhancing titer of the molecule as observed with SO1861-EMCH (Fig. 4).

[0276] 1 The 1-target 2-component system (1T2C) is a combination treatment of mAb1-protein toxin and mAb1-SO1861, as illustrated in Figure 19. In both cases, by DAR4, SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine residue (Cys), and HSP27BNA oligo was conjugated via a lysine residue, resulting in the production of two conjugates: cetuximab-(Cys-L-SO1861) 4 and cetuximab-(Lys-L-HSP27BNA) 4 . Cetuximab-(Cys-L-SO1861) 4 (intraperitoneal administration (i.p.)) and cetuximab-(Lys-L-HSP27BNA) 4 (intravenous administration (i.v.)) were combined and tested in an A431 xenograft mouse tumor model for EGFR tumor-targeted gene silencing activity. Treatment was initiated on day 12 when the tumors reached a size of ~150 mm 3 . Tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression compared to the control gene mRNA expression level (reference gene). This revealed that a single dose of 50 mg / kg cetuximab-(Cys-L-SO1861) 4 + 25 mg / kg cetuximab-(Lys-L-HSP27BNA) 4 resulted in a 50% reduction in HSP27 gene expression in A431 tumors compared to a single dose of either cetuximab-(Cys-L-SO1861) 4 or cetuximab-(Lys-L-HSP27BNA) 4 alone (Figure 7). A 40% reduction in HSP27 gene silencing was observed compared to vehicle control tumors. This indicates and enables that the combination of cetuximab-conjugated SO1861 + cetuximab-conjugated HSP27BNA oligo according to the 1T2C invention induces efficient targeted delivery of the therapeutic antisense oligonucleotide to the cytoplasm of solid tumor cells, thereby inducing in vivo tumor-targeted gene silencing.

[0277] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) with a DAR of 4 via a cysteine residue (Cys), resulting in trastuzumab-(Cys-L-SO1861). 4 The combination of trastuzumab-(Cys-L-SO1861) and trastuzumab-saporin (a trastuzumab protein toxin conjugate) was tested in a mouse tumor model (patient-derived xenograft tumor model PDX) with high HER2 expression levels and resistant to trastuzumab monotherapy. The combination according to the 1T2C invention of 40 mg / kg trastuzumab-(Cys-L-SO1861) (intraperitoneal administration (i.p.)) + 0.03 (days 1, 8) / 0.02 (days 15, 22, 30, 36, 43) mg / kg trastuzumab-saporin (intravenous administration (i.v.)) revealed strong tumor growth inhibition compared to vehicle control and monotherapy with 40 mg / kg trastuzumab-(Cys-L-SO1861) or 0.03 / 0.02 mg / kg trastuzumab-saporin alone (Figure 8). Additionally, no tumor growth inhibition activity was observed in mice with tumors treated with a lower dose combination (40 mg / kg trastuzumab-(Cys-L-SO1861) + 0.01 mg / kg trastuzumab-saporin) (Figure 8). This indicates and enables that the 1T2C combination of trastuzumab-conjugated SO1861 + trastuzumab-conjugated protein toxin induces efficient targeted delivery of the therapeutic protein toxin to the cytoplasm of solid tumor cells, thereby inducing in vivo tumor cell death and tumor growth inhibition. 4 Next, the combination of trastuzumab-(Cys-L-SO1861) and trastuzumab-saporin (a trastuzumab protein toxin conjugate) was tested in a mouse tumor model (patient-derived xenograft tumor model PDX) with high HER2 expression levels and resistant to trastuzumab monotherapy. 4 (intraperitoneal administration (i.p.)) + 0.03 (days 1, 8) / 0.02 (days 15, 22, 30, 36, 43) mg / kg trastuzumab-saporin (intravenous administration (i.v.)) The combination according to the 1T2C invention revealed strong tumor growth inhibition compared to vehicle control and monotherapy with 40 mg / kg trastuzumab-(Cys-L-SO1861) or 0.03 / 0.02 mg / kg trastuzumab-saporin alone (Figure 8). 4 In addition, no tumor growth inhibition activity was observed in mice with tumors treated with a lower dose combination (40 mg / kg trastuzumab-(Cys-L-SO1861) + 0.01 mg / kg trastuzumab-saporin) (Figure 8). This indicates and enables that the 1T2C combination of trastuzumab-conjugated SO1861 + trastuzumab-conjugated protein toxin induces efficient targeted delivery of the therapeutic protein toxin to the cytoplasm of solid tumor cells, thereby inducing in vivo tumor cell death and tumor growth inhibition. 4 The 1T2C system is a combination treatment of mAb1-SO1861 and mAb1-protein toxin (Figure 19).

[0278] The 1T2C system is a combination treatment of mAb1-SO1861 and mAb1-protein toxin (Figure 19). SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) with a DAR of 3,7 via a cysteine residue (Cys) (cetuximab-(Cys-L-SO1861) 3,7 ). Cetuximab-(Cys-L-SO1861) 3,7 was titrated with a fixed concentration of 10 pM cetuximab-saporin (cetuximab conjugated to the protein toxin saporin), and the killing mediated by the targeted protein toxin in EGFR-expressing cells (A431, EGFR ++ ; CaSki, EGFR + ) was determined. This revealed strong killing at low concentrations of cetuximab-(Cys-L-SO1861) 3,7 (A431: IC50 = 0.6 nM and Caski IC50 = 1 nM; Figures 9A, 9B), while cetuximab, cetuximab-(Cys-L-SO1861) 3,7 , or cetuximab + 10 pM cetuximab-saporin could not induce any killing activity in EGFR-expressing cells. This indicates that cetuximab-conjugated SO1861 efficiently improves the endosomal escape of cetuximab-conjugated protein toxin (at non-effective concentrations), thereby inducing killing of EGFR-expressing cells. The killing activity in A431 is more effective compared to CaSki and correlates with the EGFR expression levels in these cell lines. EGFR receptor binding competition between both conjugates of 1T2C is also observed. As the concentration of cetuximab-(Cys-L-SO1861) 3,7 increases, the killing activity drops due to the overriding receptor binding and internalization of cetuximab-saporin (Figures 9A, 9B).

[0279] Next, cetuximab-saporin was titrated with a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 , and the killing mediated by the targeted protein toxin in EGFR-expressing cells was determined. This was 75 nM cetuximab-(Cys-L-SO1861) 3,7has been shown to already induce efficient killing in EGFR-expressing cells by combination with low concentrations of cetuximab-saporin (A431: IC50 = 0.4 pM; and CaSKi: IC50 = 2 pM; FIGS. 9C and 9D), while cetuximab-saporin alone or cetuximab-saporin + 75 nM cetuximab showed killing only at high concentrations of cetuximab-saporin in both cell lines (IC50 = 40 pM and IC50 = 1000 pM, respectively) (FIGS. 9C, 9D). All of this indicates that relatively low concentrations of cetuximab-saporin may be effective and capable of inducing killing only in combination with low cetuximab-SO1861 concentrations in high EGFR-expressing cells. When comparing the killing activities of cetuximab-saporin with and without 75 nM cetuximab, receptor competition between both conjugates of the 1T2C system was also observed in the cetuximab-toxin titration treatment (FIGS. 9C, 9D).

[0280] Next, cetuximab-(Cys-L-SO1861) 3,7 was titrated with a fixed concentration of 10 pM cetuximab-saporin, and killing mediated by the targeted protein toxin in low EGFR-expressing cells or cells without EGFR expression (HeLa, EGFR + / - ; A2058, EGFR - ) was determined. Low (HeLa) EGFR-expressing or EGFR-negative (A2058) cells were not at all sensitive to any combination of cetuximab-(Cys-L-SO1861) 3,7 + 10 pM cetuximab-saporin (HeLa: IC50 > 1000 nM; A2058: IC50 > 1000 nM; FIGS. 10A, 10B). This indicates that in the absence of sufficient EGFR receptor expression, the effective intracellular delivery SO1861 concentration is not optimal (threshold) to induce escape of the endosomal protein toxin and killing mediated by the toxin. Next, cetuximab-saporin was combined with a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7Titrated and determined the cytotoxicity mediated by the targeted protein toxin in cells with low EGFR expression (HeLa) or no EGFR expression (A2058). Low EGFR-expressing cells (HeLa) showed cytotoxicity only at high cetuximab-saporin concentrations combined with 75 nM cetuximab-(Cys-L-SO1861) 3,7 (HeLa: IC50 = 60 pM, Figure 10C), while A2058 cells (EGFR - ) were not sensitive at any of the tested concentrations (A2058: IC50 > 10,000 pM; Figure 10D). All of this indicates that cells with low or no EGFR receptor expression are not sensitive to the combination of cetuximab-(Cys-L-SO1861) 3,7 + cetuximab-saporin. This is due to the lack of sufficient EGFR receptors to facilitate antibody-mediated delivery of SO1861 within the endosomal compartment and thus facilitate the escape of the protein toxin.

[0281] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) via a cysteine residue (Cys) with DAR4 (trastuzumab-(Cys-L-SO1861) 4 ). Trastuzumab-(Cys-L-SO1861) 4 was titrated with a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin saporin), and the cytotoxicity mediated by the targeted protein toxin in HER2-expressing cells (SK-BR-3, HER2 ++ ) was determined. This revealed strong cytotoxicity at low concentrations of trastuzumab-(Cys-L-SO1861) 4 (SK-BR-3: IC50 = 0.8 nM; Figure 11A), while equivalent concentrations of trastuzumab, trastuzumab-(Cys-L-SO1861) 4、or trastuzumab + 50 pM trastuzumab - saporin failed to induce any cytotoxic activity in HER2-expressing cells. This indicates that trastuzumab-conjugated SO1861 efficiently enhances the endosomal escape of trastuzumab-conjugated protein toxin (at non-effective concentrations), thereby inducing the killing of HER2-expressing cells. Receptor competition between both conjugates of 1T2C was also observed. Trastuzumab-(Cys-L-SO1861) 4 As the concentration of trastuzumab-saporin increases, cytotoxic activity decreases due to overriding receptor binding and internalization of trastuzumab-saporin (Figure 11A).

[0282] Next, trastuzumab-saporin was titrated with a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) according to the present invention 4 to determine the targeting protein toxin-mediated killing in HER2-expressing cells. This revealed that 2.5 nM trastuzumab-(Cys-L-SO1861) 4 already induced efficient killing in HER2-expressing cells in combination with low concentrations of trastuzumab-saporin (SK-BR-3: IC50 = 2 pM; Figure 11B), whereas trastuzumab-saporin alone or trastuzumab-saporin + 2.5 nM trastuzumab showed killing only at high concentrations of trastuzumab-saporin (Figure 11B). All of this indicates that relatively low concentrations of trastuzumab-saporin may be effective and able to induce killing only in combination with low trastuzumab-(Cys-L-SO1861) 4 concentrations in high HER2-expressing cells.

[0283] Next, trastuzumab-(Cys-L-SO1861) 4 was titrated with a fixed concentration of 50 pM trastuzumab-saporin according to the present invention, and the targeting protein toxin-mediated killing was performed in low HER2-expressing cells (A431 HER2 + / -) or cells without HER2 expression (JIMT-1:HER2 + ;MDA-MB-468:HER2 - ) were determined. Cells with low or no HER2 expression were not at all sensitive to any combination of trastuzumab-(Cys-L-SO1861) 4 + 50 pM trastuzumab-saporin (JIMT-1: IC50 > 1000 nM; MDA-MB-468: IC50 > 1000 nM; Figures 13A, 13B). This indicates that in the absence of sufficient HER2 receptor expression, the effective intracellular delivery SO1861 concentration is not optimal (threshold) to induce escape of the endosomal protein toxin and toxin-mediated killing of cells. Next, trastuzumab-saporin was titrated with a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 and the target protein toxin-mediated killing in low HER2-expressing or HER2-negative cells was determined. Low HER2-expressing cells (JIMT-1) showed killing only at high trastuzumab-saporin concentrations in combination with 2.5 nM trastuzumab-(Cys-L-SO1861) 4 (JIMT-1: IC50 > 10,000 pM; Figure 13C), while MDA-MB-468 cells (HER2 - ) were not sensitive at any of the tested concentrations (MDA-MB-468: IC50 > 10,000 pM; Figure 13D).

[0284] All of this indicates that cells with low or no HER2 receptor expression are not sensitive to the combination of trastuzumab-(Cys-L-SO1861) 3,7 + trastuzumab-saporin. This is due to the lack of sufficient HER2 receptors to facilitate antibody-mediated delivery of SO1861 within the endosomal compartment and thus facilitate escape of the protein toxin.

[0285] To show that the activity of the 1T2C system is driven by the acidification of the endosomal compartment, the 1T2C system according to the present invention was tested in combination with the endosomal acidification inhibitor chloroquine. Trastuzumab-saporin was titrated in combination with chloroquine or without chloroquine in combination with 5 nM trastuzumab-(Cys-L-SO1861) 4 Trastuzumab-saporin + 5 nM trastuzumab-(Cys-L-SO1861) 4 showed strong cytotoxic activity in high HER2-expressing cells (SK-BR-3, HER2 ++ ; IC50 = 0.2 pM). However, trastuzumab-saporin + 5 nM trastuzumab-(Cys-L-SO1861) 4 + 0.5 μM chloroquine resulted in a strong inhibition of 1T2C cytotoxic activity in SK-BR-3 (HER2 ++ ) cells (IC50 = 40 pM). This indicates that when the acidification of the endosome is prevented, the activity of the antibody-conjugated SO1861 is reduced / blocked (Figure 14A). The same result was obtained in EGFR-expressing cells (A431, EGFR 3,8 ) with cetuximab-saporin + 5 nM cetuximab-(Cys-L-SO1861) 3,8 + 0.5 μM chloroquine (IC50 = 200 pM) compared to cetuximab-saporin + 5 nM cetuximab-(Cys-L-SO1861) ++ ; IC50 = 1 pM) with the 1T2C combination according to the present invention (Figure 14B).

[0286] 1 target 2 component system (1T2C) can also be a combined treatment of mAb1-SO1861 and mAb1-antisense BNA oligonucleotide, as illustrated in Figure 20. For this, we used an antisense BNA oligonucleotide against the mRNA of the cancer-specific target gene (upregulated in cancer cells) heat shock protein 27 (HSP27). By release into the cytoplasm, the antisense BNA recognizes and binds to the mRNA encoding HSP27, targets the mRNA for destruction, thereby depleting HSP27 mRNA expression in cancer cells. HSP27BNA was conjugated to cetuximab with DAR4 (cetuximab-(Lys-L-HSP27BNA) 4 ) and cetuximab-(Cys-L-SO1861) 3,8 were combined and tested according to the present invention for improved HSP27 gene silencing activity in EGFR-expressing cells (A431, EGFR ++ ) and non-expressing cells (A2058, EGFR) (Figure 20). Cetuximab-(Cys-L-SO1861) 3,8 + 100 nM cetuximab-(Lys-L-HSP27BNA) 4 showed strong HSP27 gene silencing in EGFR-expressing cells (A431: IC50 = nM, Figure 15A), but cetuximab-(Cys-L-SO1861) 3,8 alone showed no gene silencing activity. In A2058 cells (EGFR - ), gene silencing activity was not observed with the 1T2C combination (Figure 15B). Next, cetuximab-(Lys-L-HSP27BNA) 4 + 76.9 nM cetuximab-(Cys-L-SO1861) 3,8 showed strong HSP27 gene silencing activity in EGFR-expressing cells (A431: IC50 = 4 nM, Figure 15C), but cetuximab-(Lys-L-HSP27BNA) 4 or cetuximab-(Cys-L-SO1861) 3,8 , or cetuximab-(Lys-L-HSP27BNA) 4The combination of +77 nM cetuximab did not reveal any significant gene silencing activity (IC50 > 100 nM). When the experiment was conducted in EGFR non-expressing cells (A2058), gene silencing activity was not observed with the 1T2C combination (IC50 > 100 nM; Figure 15D). All of this indicates that the 1T2C system efficiently delivers the antisense BNA oligo to the cytoplasm of highly EGFR-expressing cells, thereby inducing mRNA degradation of the BNA target mRNA and resulting in target gene silencing.

[0287] The 1-target 2-component system (1T2C), as illustrated in Figure 21, may also be in the form of a combination treatment of mAb1-(scaffold (-SO1861) n ) n and mAb1-protein toxin. The dendron (-L-SO1861) 4 was conjugated to cetuximab via cysteine residue (Cys) conjugation with DAR3,9 to form cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9 and tested for enhanced 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 induced toxin-mediated killing in highly EGFR-expressing cells (IC50 = 0.4 nM; Figure 16A), which was not induced by cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9 or cetuximab + 10 pM cetuximab-saporin or cetuximab alone (Figure 16A). This indicates that, according to the 1T2C invention, the cetuximab-conjugated dendron (-L-SO1861) 4has been shown to efficiently improve (at non-effective concentrations) the endosomal escape of the cetuximab-conjugated protein toxin, thereby inducing the killing of high HER2-expressing cells. Cells expressing low levels of EGFR (HeLa, EGFR + / - ) were subjected to a similar 1T2C experiment. This revealed no killing activity (IC50 > 100 pM; Figure 16B) when using the 1T2C combination according to the present invention, indicating that in the absence of sufficient EGFR receptor expression, the effective intracellular SO1861 concentration is not optimal (threshold) to induce cytoplasmic delivery of the protein toxin that results in toxin-mediated killing of cells.

[0288] Next, the dendron (-L-SO1861) 4 was conjugated to the anti-HER2 antibody trastuzumab by cysteine conjugation (Cys) to DAR4, trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 and tested for improved killing activity in HER2-expressing cells (SK-BR-3, HER2 ++ ) in combination with an anti-HER2 antibody-protein toxin conjugate (trastuzumab-saporin). Trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 + 50 pM trastuzumab-saporin efficiently induced toxin-mediated killing of cells (IC50 = 2 nM, Figure 16C), which was not induced by trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 or trastuzumab + 50 nM trastuzumab-saporin or trastuzumab (Figure 16C). This indicates that the trastuzumab-conjugated dendron (-L-SO1861) 4 efficiently improves (at non-effective concentrations) the endosomal escape of the trastuzumab-conjugated protein toxin, thereby inducing the killing of high HER2-expressing cells. Cells expressing low levels of HER2 (JIMT-1, HER2 + / -) Similar exp...

Claims

**Claim 1** A first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein the first proteinaceous molecule has at least one saponin covalently bound to an amino acid residue of the first proteinaceous molecule via at least one linker and / or via an oligomer or polymer backbone, or directly covalently bound to an amino acid residue of the first proteinaceous molecule, and wherein the first epitope of the first cell surface molecule to which the first binding site binds is CD71, the first proteinaceous molecule. **Claim 2** The first binding site comprises or consists of an immunoglobulin, or at least one binding domain and / or at least one binding fragment of an immunoglobulin, such as an antibody, IgG, Vhh domain or Vh domain, a molecule comprising or consisting of the same, Fab, scFv, Fv, dAb, F(ab) ２ , Fcab fragment, and / or comprises or consists of at least one ligand for binding to a cell surface molecule, the first proteinaceous molecule according to claim 1. **Claim 3** The first proteinaceous molecule according to claim 1 or 2, wherein the at least one saponin is SO1861. **Claim 4** The at least one saponin is a bisdesmoside-type triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23, The first proteinaceous molecule according to any one of claims 1 to 3, wherein the at least one saponin is covalently coupled to the amino acid residue of the first proteinaceous molecule via an aldehyde functional group on the saponin. **Claim 5** The first proteinaceous molecule according to claim 4, wherein the aldehyde functional group at position C-23 of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, and this linker is covalently coupled to a sulfhydryl group on the first proteinaceous molecule via a thio-ether bond. **Claim 6** The at least one saponin is a bisdesmoside-type triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 and comprising a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, The first proteinaceous molecule according to claim 3 or 4, wherein the at least one saponin is covalently coupled to the amino acid residue of the first proteinaceous molecule via the glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin. **Claim 7** The glucuronic acid functional group as the carbohydrate substituent of the C-3 beta-OH group of the at least one saponin is covalently coupled to 1-[(bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate of a linker, and this linker is covalently coupled via an amide bond to an amine group on the first proteinaceous molecule, the first proteinaceous molecule according to claim 6.

8. The first proteinaceous molecule comprises more than one saponin, and the saponin is covalently bound directly to an amino acid residue of the first proteinaceous molecule, and / or covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one polymer or oligomer backbone, the first proteinaceous molecule according to any one of claims 1 to 7.

9. The at least one linker is a non-cleavable linker or a cleavable linker, the first proteinaceous molecule according to any one of claims 1 to 8.

10. The cleavable linker undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, the first proteinaceous molecule according to claim 8 or 9.

11. The oligomer or polymer backbone comprises a polymer or oligomer structure and comprises chemical groups for covalent coupling of the backbone to the amino acid residues of the first proteinaceous molecule, the first proteinaceous molecule according to any one of claims 1 to 10.

12. The oligomer or polymer backbone comprises a polymer or oligomer structure and comprises chemical groups, and the at least one saponin is covalently bound to the polymer or oligomer structure via at least one cleavable linker as defined in claim 8 or 9, the first proteinaceous molecule according to any one of claims 1 to 11.

13. The oligomer or polymer backbone includes a polymer or oligomer structure and a chemical group, and the chemical group for covalent coupling of the oligomer or polymer backbone to the amino acid residue of the first proteinaceous molecule is a click chemistry group, the first proteinaceous molecule according to any one of claims 1 to 12.

14. The oligomer or polymer backbone includes a polymer or oligomer structure and a chemical group, and the polymer or oligomer structure includes a linear, branched, and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an aggregate of these polymer or oligomer structures, the first proteinaceous molecule according to any one of claims 1 to 13.

15. The therapeutic composition comprises: (a) a first pharmaceutical composition comprising the first proteinaceous molecule according to any one of claims 1 to 14, and optionally further a pharmaceutically acceptable excipient; and (b) a third pharmaceutical composition comprising a third proteinaceous molecule and optionally further a pharmaceutically acceptable excipient; wherein the third proteinaceous molecule comprises: (a) a third binding site for binding to the first epitope on the cell surface molecule of (a); and an effector moiety, the first binding site and the third binding site are the same, and the first epitope and the first cell surface molecule on the first cell surface molecule to which the first proteinaceous molecule can bind, and the first epitope and the first cell surface molecule on the first cell surface molecule to which the third proteinaceous molecule can bind are the same, a therapeutic composition for co-administration.

16. (a) the therapeutic composition comprises the first pharmaceutical composition, and the first epitope on the first cell surface molecule is a tumor cell-specific first epitope on a first tumor cell-specific surface molecule; and (b) the therapeutic composition comprises the first pharmaceutical composition and the third pharmaceutical composition, and the first cell surface molecule is expressed on the tumor cell surface, the therapeutic composition according to claim 15.

17. The therapeutic composition according to claim 15 or 16, wherein the third binding site of the third proteinaceous 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.

18. The therapeutic composition according to any one of claims 15 to 17, wherein the same first binding site and the third binding site are binding sites for epitopes on CD71.

19. The therapeutic composition according to any one of claims 15 to 18, wherein the cell surface molecule is CD71, provided that when the first and third proteinaceous molecules are present in the therapeutic composition, the first and third binding sites are the same.

20. The therapeutic composition according to any one of claims 15 to 19, wherein the first binding site and the third binding site comprise a monoclonal antibody, or a cell surface molecule binding domain or fragment thereof, provided that the first binding site of the first proteinaceous molecule is the same as the third binding site of the third proteinaceous molecule.

21. The therapeutic composition according to any one of claims 15 to 20, wherein the effector portion contained in the third proteinaceous molecule comprises or consists of any one or more of an oligonucleotide, nucleic acid, xeno nucleic acid, or a derivative thereof.

22. The therapeutic composition according to any one of claims 15 to 21, wherein the effector portion contained in the third proteinaceous molecule is a vector, gene, transgene that induces cell suicide, 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 any one or more of a derivative thereof, or comprises or consists of the same.

23. The first proteinaceous molecule according to any one of claims 1 to 14, the third proteinaceous molecule defined in any one of claims 15 to 22, and optionally a pharmaceutically acceptable excipient, Optionally, a composition in which the effector moiety contained in the third proteinaceous molecule is any one of the effector moiety: oligonucleotide, nucleic acid, xeno nucleic acid, or a derivative thereof.

24. The first proteinaceous molecule according to any one of claims 1 to 14 or defined in any one of claims 15 to 20, Oligonucleotide, nucleic acid, and xeno nucleic acid, or a derivative thereof, vector, gene, transgene inducing cell suicide, 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 any one or more of derivatives thereof.

25. An antibody-drug conjugate or ligand-drug conjugate, comprising the first proteinaceous molecule and an effector moiety according to any one of claims 1 to 14, (i) the antibody or ligand can bind to CD71 and / or is or contains an IgG-type OKT-9 anti-CD71 monoclonal antibody, and / or, (ii) the effector moiety is any one or more of the effector moieties defined in claims 21 or 22. An antibody-drug conjugate or ligand-drug conjugate.

26. A pharmaceutical composition comprising the antibody-drug conjugate or ligand-drug conjugate according to claim 25 and optionally further comprising a pharmaceutically acceptable excipient.

27. The therapeutic composition according to any one of claims 15 to 22, or the composition according to claim 23 or 24, or the antibody-drug conjugate or ligand-drug conjugate according to claim 25, or the pharmaceutical composition according to claim 26, for use as a medicine or for use in the treatment or prevention of cancer or autoimmune diseases.

28. An endosome escape enhancing conjugate comprising at least one covalently attached saponin and comprising a first proteinaceous molecule as defined in any one of claims 1 to 14 or in any one of claims 15 to 20, wherein the endosome escape enhancing conjugate is capable of specifically binding to a target cell-specific surface molecule or structure.

29. The therapeutic composition comprises: a. The endosome escape enhancing conjugate according to claim 28; and b. A binding moiety comprising at least one effector moiety and comprising a third proteinaceous molecule as defined in any one of claims 15 to 23, the effector moiety being any one or more of the effector moieties as defined in claim 21 or 22; for simultaneous administration. The endosome escape enhancing conjugate and the binding moiety are capable of binding to the same target cell-specific surface molecule via their same binding sites.

30. The therapeutic composition comprises: a. The endosome escape enhancing conjugate according to claim 28; and b. A binding moiety comprising at least one effector moiety and comprising a third proteinaceous molecule as defined in any one of claims 15 to 23, the effector moiety being any one or more of the effector moieties as defined in claim 21 or 22; for simultaneous administration. The endosome escape enhancing conjugate is capable of specifically binding to the same first epitope to which the binding moiety is capable of specifically binding.

31. The endosome escape enhancing conjugate according to claim 28 or the therapeutic composition according to claim 29 or 30, wherein the target cell-specific surface molecule or structure is CD71.

32. The effector moiety contained in the third proteinaceous molecule is any one of an oligonucleotide, nucleic acid and xeno nucleic acid, or a derivative thereof, a vector, a gene, a transgene that induces cell suicide, 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, the endosome escape improvement conjugate according to claim 28 or 31, or the therapeutic composition according to any one of claims 29 to 31.

33. The saponin is a bisdesmoside-type triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position 23, the endosome escape improvement conjugate according to any one of claims 28 and 31 to 32, or the therapeutic composition according to any one of claims 29 to 32.

34. The saponin is any one of SO1861 or its diastereomer, the endosome escape improvement conjugate according to any one of claims 28 and 31 to 33, or the therapeutic composition according to any one of claims 29 to 33.

35. The endosome escape improvement conjugate according to any one of claims 28 and 31 to 34, or the therapeutic composition according to any one of claims 29 to 34, for use as a medicament or for use in the treatment or prevention of cancer or autoimmune diseases.

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