Saponins conjugated to epitope binding proteins

Abstract

The present invention relates to a therapeutic combination comprising a first protein molecule comprising a first binding site for binding a first epitope of a first cell surface molecule and comprising a second pharmaceutical composition, the first pharmaceutical composition comprises a first protein molecule having at least one saponin covalently bound to an amino acid residue of the first protein molecule, and a second pharmaceutical composition comprising a second protein molecule different from the first protein molecule, the second protein molecule comprises a second binding site for binding a second epitope of a second cell surface molecule different from the first cell surface molecule, and comprises an effector moiety wherein the second epitope is different from the first epitope. Aspects of the invention are compositions comprising the first protein molecule and the second protein molecule of the invention. The invention also relates to antibody-drug conjugates comprising said first protein molecule of the invention and an effector moiety. Aspects of the invention relate to pharmaceutical compositions comprising said composition or said antibody-drug conjugate of the invention and optionally further comprising a pharmaceutically acceptable excipient. The invention also relates to the therapeutic combination or the composition or the antibody-drug conjugate or the pharmaceutical composition of the invention for use as a medicament. The invention also relates to said therapeutic combination of the invention for use in the treatment or prevention of cancer.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980092811.X. The application date of Chinese patent application No. 201980092811.X is December 9, 2019, and all its contents are incorporated herein. Technical Field

[0002] The present invention relates to a first protein molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first protein molecule having at least one saponin covalently bound to an amino acid residue of the first protein molecule via at least one linker and / or via an oligomeric or polymeric scaffold or directly covalently bound to an amino acid residue of the first protein molecule. The present invention also relates to a therapeutic combination, wherein the therapeutic combination comprises a first pharmaceutical composition comprising the first protein molecule of the present invention, and a second pharmaceutical composition comprising a second protein molecule different from the first protein molecule, the second protein 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, wherein the second epitope is different from the first epitope. In addition, the present invention relates to a therapeutic combination, wherein the therapeutic combination comprises (a) the first pharmaceutical composition of the present invention, which comprises the first protein molecule according to the present invention and comprises the first binding site for binding to the first epitope on the first cell surface molecule; and (b) a third pharmaceutical composition comprising a third protein molecule, the third protein molecule comprising the first binding site for binding to the first epitope on the cell surface molecule of (a) and an effector portion, wherein the first binding site of the first protein molecule is the same as the first binding site of the third protein molecule, and wherein the first cell surface molecule to which the first protein molecule can bind and the first epitope on the first cell surface molecule to which the third protein molecule can bind are the same. One aspect of the invention is a composition comprising the first protein molecule of the present invention and the second protein molecule of the present invention. One aspect of the invention relates to a composition comprising the first protein molecule of the present invention and the third protein molecule of the present invention. The present invention also relates to a composition comprising the first protein molecule of the present invention and any one or more of the following: an oligonucleotide, a nucleic acid, a heterologous nucleic acid, preferably selected from a vector, a gene, a cell suicide-inducing transgene, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a 2'-deoxy-2'-fluoroarabinoic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA) or any one or more of their derivatives.The present invention also relates to an antibody-drug conjugate or ligand-drug conjugate comprising the first protein 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, 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 the therapeutic combination of the present invention, the composition of the present invention, the antibody-drug conjugate of the present invention, the ligand-drug conjugate of the present invention, or the pharmaceutical composition of the present invention for use as a medicament. Background Art

[0003] Molecules with therapeutic biological activity are theoretically suitable for use as effective therapeutic drugs in many cases, for treating diseases (such as cancer) in human patients in need thereof. A typical example is a small molecule biologically active portion. However, many (if not all) potential drug-like molecules and therapeutic agents currently used in clinical practice have at least one of numerous drawbacks and shortcomings. When applied to the human body, in addition to the biological activity for the potential aspects of the disease to be treated or health problems, therapeutically active molecules can also exert off-target effects. Such off-target effects are undesirable and there is a risk of inducing health or even life-threatening side effects of the administered molecules. It is the occurrence of such adverse events that causes many drug-like compounds and therapeutic portions to fail to pass Phase III clinical trials or even Phase IV clinical trials (post-marketing follow-up). Therefore, it is highly desirable to provide drug molecules (e.g., small molecule therapeutics) whose therapeutic effects should, for example, (1) be highly specific to the biological factors or biological processes driving the disease, (2) be sufficiently safe, (3) be sufficiently effective, (4) be sufficiently targeted to the diseased cells with little or no off-target activity against non-diseased cells, (5) have a sufficiently timely mode of action (e.g., the administered drug molecule should reach the target site in the human patient within a specific timeframe and should remain at the target site for a specific timeframe), and / or (6) have a sufficiently long-lasting therapeutic activity in the patient, etc. Despite long and intensive research, and despite impressive progress in several areas that have individually addressed the difficulties and shortcomings encountered, unfortunately, to date, an "ideal" therapeutic agent possessing many or even all of the aforementioned beneficial characteristics has not been available to patients.

[0004] Chemotherapy is one of the most important treatment options in cancer treatment. However, it is often associated with a lower therapeutic window because it is less specific for cancer cells than for dividing cells in healthy tissues. The invention of monoclonal antibodies provides the possibility of using their specific binding properties as a mechanism for targeting cytotoxic agents to cancer cells without harming normal cells. This can be achieved by chemically conjugating cytotoxic effectors (also known as payloads or warheads) to antibodies to create antibody-drug conjugates (ADCs). Typically, very effective payloads (such as emtansine (DM1)) are used, and their non-conjugated forms have a limited therapeutic index (the ratio of the toxic dose to the effective dose). The conjugate of DM1 with trastuzumab (ado-trastuzumab-emtansine), also known as Kadcycla, increased the tolerated dose of DM1 by at least two times in monkeys. Over the past few decades, tremendous efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, introducing ADCs into the clinic remains challenging. The first ADC approved for clinical use in 2000 was gemtuzumab ozogamicin (Mylotarg, CD33-targeted, Pfizer / Wyeth) for the treatment of relapsed acute myeloid leukemia (AML). However, Mylotarg was withdrawn from the market at the request of the Federal Drug Administration (FDA) due to numerous concerns, including its safety profile. Patients treated with Mylotarg were more likely to die than those receiving conventional chemotherapy. In 2017, Mylotarg was allowed to enter the market again with a lower recommended dose, different regimens used in combination with chemotherapy or alone, and in new patient populations. To date, only five ADCs have been approved for clinical use, while clinical development of approximately 55 ADCs has been discontinued. However, interest remains high, and approximately 80 ADCs are currently in clinical development in nearly 600 clinical trials.

[0005] While it is possible to use toxic payloads that patients generally cannot tolerate, a poor therapeutic index (the ratio of the toxic dose to the effective dose) is a major issue that has led to the discontinuation of many ADCs in clinical development. This can be due to several mechanisms, such as off-target toxicity to normal cells, the development of resistance to cytotoxic agents, and premature drug release in the circulation. An FDA systematic review of ADCs found that the toxicity profiles of most ADCs could be categorized by the payload used, rather than the antibody used, suggesting that toxicity was primarily determined by premature payload release. Of the approximately 55 discontinued ADCs, at least 23 are estimated to be due to a poor therapeutic index. For example, development of the trastuzumab-tesirine conjugate (ADCT-502, a HER2-targeted ADC therapeutic) was recently discontinued due to a narrow therapeutic index, likely due to on-target and off-tissue effects in lung tissue, which expresses significant levels of HER2. Furthermore, several ADCs in Phase 3 trials have been discontinued due to the lack of a primary endpoint. For example, phase 3 trials of ado-trastuzumab-emtansine conjugate (ABT-414, EGFR-targeted, AbbVie) tested in patients with newly diagnosed glioblastoma and ado-trastuzumab-emtansine conjugate (IMGN853, folate receptor α (FRα)-targeted, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently discontinued without showing a survival benefit. It is important to note that the clinically used doses of some ADCs may not be sufficient to exert their full anticancer activity. For example, the MTD of ado-trastuzumab-emtansine in humans is 3.6 mg / kg. In preclinical models of breast cancer, ado-trastuzumab-emtansine induced tumor regression at dose levels of 3 mg / kg or above, but more potent efficacy was observed at 15 mg / kg. This suggests that ado-trastuzumab-emtansine may not exert its maximum potential antitumor effect at clinically administered doses.

[0006] ADCs are primarily composed of antibodies, cytotoxic moieties (such as payloads), and linkers. In the design and development of new ADCs, several new strategies have been proposed and implemented for each component of the ADC to overcome existing problems. For example, by identifying and validating sufficient antigenic targets for the antibody component, by selecting antigens that are highly expressed in tumors but rarely or not expressed in normal tissues, antigens present on the cell surface that are accessible to circulating ADCs, and antigens that allow the ADC to be internalized into the cell after binding; and alternative activity mechanisms; designing and optimizing linkers to improve the solubility and drug-to-antibody ratio (DAR) of the ADC and overcome protein-induced resistance that can transport chemotherapeutic agents out of cells; improving the DAR ratio by including more payloads, and selecting and optimizing antibodies to improve the homogeneity and developability of the antibody. In addition to the technological development of ADCs, new clinical and translational strategies are being deployed to maximize the therapeutic index, such as changing the dosing regimen by fractionated dosing; conducting biodistribution studies; including biomarkers for optimizing patient selection, capturing response signals early, monitoring the duration and depth of response, and providing information for combination studies.

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

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

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

[0010] This conjugation principle has attracted widespread attention and has been studied for decades. Most conjugates of small molecule drugs in preclinical or clinical development are for oncology indications. However, as of 2014, only one drug not related to cancer (Movantik, a PEG oligomer conjugate of the opioid antagonist naloxone, AstraZeneca) has been approved for the treatment of opioid-induced constipation in patients with chronic pain, a non-oncology indication. To date, there has been little clinical success in translating the application of drug-scaffold conjugates to human treatment. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has demonstrated potent anticancer activity in mouse models of solid tumors and leukemias and is undergoing clinical development for oncology indications. Although it demonstrated significantly reduced nonspecific toxicity and improved pharmacokinetics in humans, the improvement in anticancer efficacy in patients was minimal, leading to the discontinuation of further development of PK1.

[0011] The failure of scaffold-small molecule drug conjugates has been attributed, at least in part, to their poor accumulation at tumor sites. For example, although PK1 accumulates 45-250 times more in tumors than in healthy tissues (liver, kidney, lung, spleen, and heart) in mouse models, tumor accumulation has only been observed in a small subset of patients in clinical trials.

[0012] A potential solution to the above problems is to apply nanoparticle systems to drug delivery, such as liposomes. Liposomes are spherical vesicles composed of one or more layers of phospholipid bilayers that form spontaneously when phospholipids are dispersed in water. The amphiphilic nature of phospholipids provides them with self-assembly, emulsification and wetting properties, and these properties can be used for the design of new drugs and new drug delivery systems. Drugs encapsulated in liposome delivery systems have multiple advantages over directly administering drugs, such as improving and controlling pharmacokinetics and pharmacodynamics, tissue targeting properties, reduced toxicity and enhanced drug activity. Such successful examples are the small molecule chemotherapy drug doxorubicin in liposome encapsulation form (Doxil: PEGylated liposome encapsulation form of doxorubicin; Myocet: non-PEGylated liposomal doxorubicin), which has been approved for clinical use.

[0013] Therefore, there remains a need to find a solution that allows drug therapies (such as anti-tumor therapies) to be suitable for non-systemic use when desired, where the drug has, for example, an acceptable safety profile, very low off-target activity, sufficient efficacy, sufficiently low clearance from the patient's body, etc. SUMMARY OF THE INVENTION

[0015] For embodiments of the present invention, a first object is to provide improved bioactive compounds or compositions comprising such improved bioactive compounds.

[0016] One of the several objectives of embodiments of the present invention is to provide a solution to the problem of nonspecificity encountered when administering small molecule therapeutically active compounds to human patients in need. One of the several objectives of embodiments of the present invention is to provide a solution to the problem of drugs having non-optimal specificity for biological factors or biological processes that drive disease. One of the several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs have insufficient safety profiles when administered to human patients in need. One of the several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs are less effective than expected when administered to human patients in need. One of the several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs, when administered to human patients in need, are not sufficiently targeted to diseased cells and have little or no off-target activity against non-diseased cells. One of the several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs, when administered to human patients in need, do not have a sufficiently timely mode of action (e.g., the administered drug molecule should reach the target site in the human patient's body within a specific time frame and should remain at the target site for a specific time frame). One of the several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs, when administered to human patients in need, do not have sufficiently long-lasting therapeutic activity in the patient's body.

[0017] At least one of the above-mentioned objects of embodiments of the present invention is achieved by providing a first protein molecule of the present invention, comprising a cell targeting moiety and at least one saponin, the first protein molecule being further suitable for use as a medicament or for inclusion in a pharmaceutical combination according to the present invention, and suitable for use as a semi-finished product of the ADC or antibody-oligonucleotide conjugate (AOC) of the present invention manufactured according to the present invention. The therapeutic combination comprises the first protein molecule comprising a covalently bound saponin and a second protein molecule comprising an effector molecule (also referred to as an effector moiety), wherein the first and second protein molecules comprise different binding sites for different epitopes exposed on different cell surface molecules of a target cell, wherein the different cell surface molecules are expressed by and exposed on the surface of the same target cell.

[0018] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.The embodiments of the invention described herein can be combined and operate in conjunction with each other unless otherwise indicated.

[0019] One aspect of the present invention relates to a first protein molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first protein molecule having at least one saponin covalently bound to an amino acid residue of the first protein molecule or directly covalently bound to an amino acid residue of the first protein molecule via at least one linker and / or via an oligomeric or polymeric scaffold. According to the present invention, the first protein molecule is a finished product for use in, for example, a therapeutic combination comprising a first pharmaceutical composition comprising the first protein molecule having a saponin covalently coupled thereto (a first conjugate comprising the first protein molecule having a covalently coupled saponin). Secondly, the first protein molecule with a covalently linked saponin is also a semi-finished product. The first protein molecule can be linked to, for example, at least one effector moiety, such as an enzyme, a toxin (e.g., a protein toxin), an oligonucleotide (e.g., a BNA), thereby providing an ADC or AOC according to the present invention having one or more covalently linked saponins, optionally via a linker and / or an oligomeric or polymeric scaffold. Therefore, one aspect of the present invention relates to a conjugate comprising or consisting of said first protein molecule, said first protein 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 said first protein molecule via at least one linker and / or at least one saponin is covalently bound to an amino acid residue of said first protein molecule via an oligomeric or polymeric scaffold, or is directly covalently bound to an amino acid residue of said first protein molecule.

[0020] One embodiment is said first protein molecule of the invention, wherein said 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.

[0021] One embodiment is the first protein molecule of the invention, wherein at least one saponin is a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, and / or a saponin isolated from a species of the genus Gypsophila and / or a species of the genus Saponaria and / or a species of the genus Agrostemma and / or a species of the genus Quillaja, such as Quillaja saponaria.

[0022] One embodiment is the first protein molecule of the present invention, wherein the first epitope of the first cell surface molecule bound by the first binding site of the first protein molecule is a tumor cell-specific first epitope of a tumor cell-specific receptor, and the tumor cell-specific receptor is preferably selected from the group consisting of: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, vascular integrin α-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.

[0023] One embodiment is the first protein molecule of the present invention, wherein the tumor cell-specific first epitope, the first tumor cell surface molecule or the first tumor cell-specific receptor is the first epitope or the first molecule or the first receptor that is internalized by the tumor cell after binding of the first protein molecule of the present invention to the first epitope or the first molecule or the first receptor, and wherein preferably, when the first protein molecule binds to the cell surface molecule comprising the first epitope, the tumor cell surface molecule or the tumor cell-specific receptor, it undergoes tumor cell receptor-mediated internalization, e.g., by endocytosis, or tumor cell surface molecule-mediated internalization, e.g., by endocytosis.

[0024] One aspect of the invention relates to a therapeutic combination, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition comprising the first protein molecule of the invention and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second protein molecule different from the first protein molecule, the second protein molecule comprising a second binding site for binding to a second epitope of the second cell surface molecule different from the first cell surface molecule and comprising an effector moiety, the second pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, wherein the second epitope is different from the first epitope.

[0025] One aspect of the invention relates to a therapeutic combination, wherein the therapeutic combination comprises: (a) the first pharmaceutical composition of the invention, which comprises the first protein molecule according to the invention and comprises a first binding site for binding to a first epitope on the first cell surface molecule, the first pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient; (b) a third pharmaceutical composition comprising a third protein molecule, the third protein molecule comprising 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, wherein the first binding site of the first protein molecule and the first binding site of the third protein molecule are the same, and wherein the first cell surface molecule and the first epitope on the first cell surface molecule to which the first protein molecule can bind are the same as the first epitope on the first cell surface molecule and the first epitope on the first cell surface molecule to which the third protein molecule can bind.

[0026] One embodiment is the first protein molecule and / or the second protein molecule of the present invention, which is a semi-finished product for the manufacture of an ADC conjugated to at least one saponin, or which is a semi-finished product for the manufacture of an AOC conjugated to at least one saponin, said at least one saponin being coupled to said ADC or said AOC via a covalent bond, preferably via at least one linker, and preferably via an oligomeric or polymeric scaffold to which at least one saponin is covalently linked, preferably via a linker. Figure 91 、 Figure 92 ).

[0027] One embodiment is the first protein molecule of the invention and / or the therapeutic combination of the 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, and preferably comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mocetuzumab, potuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and the antibody of Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one cell receptor binding fragment and / or domain thereof, with the proviso that the first binding site of the first protein molecule is different from the second binding site of the second protein molecule.

[0028] One embodiment is the therapeutic combination of the present invention, wherein the effector portion comprised by the second protein molecule and / or the third protein molecule comprises or consists of any one or more of the following: an oligonucleotide, a nucleic acid, a heterologous nucleic acid.

[0029] One embodiment is a therapeutic combination of the present invention, wherein the effector portion comprised by the second protein molecule and / or the third protein molecule comprises at least one protein molecule or consists of at least one protein molecule, preferably the at least one protein molecule is selected from any one or more of the following: peptides, proteins, enzymes (such as urease and Cre-recombinase), ribosome-inactivating proteins, protein toxins.

[0030] One embodiment is a therapeutic combination of the present invention, wherein the effector portion comprised by the second protein molecule and / or the third protein molecule comprises or consists of at least one payload, preferably the at least one payload is selected from any one or more of the following: a toxin targeting a ribosome, a toxin targeting an elongation factor, a toxin targeting tubulin, a toxin targeting DNA and a toxin targeting RNA.

[0031] One embodiment is a therapeutic combination according to the invention, wherein the first protein molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins, or any number therebetween (e.g. 7, 9, 12 saponins), covalently bound directly to an amino acid residue of the first protein molecule, preferably to a cysteine ​​and / or lysine, and / or covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric scaffold (preferably 1-8 such scaffolds or 2-4 such scaffolds), wherein at least one scaffold is optionally based on a dendron, wherein 1-32 saponins (e.g. 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins) or any number therebetween (e.g. 7, 9, 12 saponins) of saponins are covalently bound to at least one scaffold.

[0032] One aspect of the present invention relates to a composition comprising said first protein molecule of the present invention and said second protein molecule of the present invention.

[0033] One aspect of the present invention relates to a composition comprising said first protein molecule of the present invention and said third protein molecule of the present invention.

[0034] One embodiment is a composition according to the present invention, comprising the second or third protein molecule together with the first protein molecule, wherein the effector moiety comprised by the second protein molecule or the third protein molecule is any one of the effector moieties according to the present invention and is preferably a BNA.

[0035] One embodiment is a composition comprising the first protein molecule described in the present invention and any one or more of the following: oligonucleotides, nucleic acids, heterologous nucleic acids, preferably selected from vectors, genes, cell suicide-inducing transgenes, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamers, RNA aptamers, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acids (LNA), bridged nucleic acids (BNA), 2'-deoxy-2'-fluoroarabinoic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNA), plasmids, glycol nucleic acids (GNA) and threose nucleic acids (TNA) or any one or more of their derivatives, more preferably BNA, such as BNA that silences HSP27 protein expression.

[0036] One aspect of the present invention relates to an antibody-drug conjugate or ligand-drug conjugate comprising a first protein molecule according to the present invention and an effector moiety.

[0037] One embodiment is the antibody-drug conjugate or ligand-drug conjugate of the present invention, wherein the antibody can bind to any one of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin α-V, CA 6, 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, HE R2, and / or wherein the antibody is or comprises any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mocetuzumab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell thereof and / or at least one tumor cell receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxifloxacin pasudotox and potuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3, or wherein the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule, such as EGF or a cytokine.

[0038] One embodiment is an antibody-drug conjugate or ligand-drug conjugate according to the invention, wherein the effector moiety is any one or more of the effector moieties according to the invention.

[0039] One aspect of the present invention relates to a pharmaceutical composition comprising the composition of the present invention, 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.

[0040] One embodiment is a therapeutic combination according to the invention or a composition according to the invention or an antibody-drug conjugate or a ligand-drug conjugate according to the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0041] One aspect of the present invention relates to any one of the following ADCs and AOCs and their semi-finished conjugates, which comprises the first protein molecule according to the present invention and / or the second protein molecule according to the present invention and / or the third protein molecule according to the present invention and at least one effector molecule according to the present invention or at least one saponin according to the present invention, or both:

[0042] anti-EGFR antibody-saponin;

[0043] Anti-EGFR antibodies – triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type with an aldehyde function in position C-23, containing a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0044] anti-EGFR antibody-SO1861;

[0045] anti-EGFR antibody-GE1741;

[0046] anti-EGFR antibody-SA1641;

[0047] Anti-EGFR antibody-Quil-A;

[0048] Anti-EGFR antibody-QS-21;

[0049] Anti-EGFR antibody - saponins from the water-soluble saponin fraction of Quillaja saponaria;

[0050] Cetuximab-saponin;

[0051] Cetuximab-triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0052] Cetuximab-SO1861;

[0053] Cetuximab-GE1741;

[0054] Cetuximab-SA1641;

[0055] Cetuximab-Quil-A;

[0056] Cetuximab-QS-21;

[0057] Cetuximab - saponins from the water-soluble saponin fraction of Quillaja saponaria;

[0058] anti-HER2 antibody-saponin;

[0059] anti-HER2 antibodies - triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0060] anti-HER2 antibody - SO1861;

[0061] Anti-HER2 antibody – GE1741;

[0062] anti-HER2 antibody-SA1641;

[0063] Anti-HER2 antibody - Quil-A;

[0064] Anti-HER2 antibody - QS-21;

[0065] Anti-HER2 antibody - saponins from the water-soluble saponin fraction of Quillaja saponaria;

[0066] trastuzumab-saponin;

[0067] Trastuzumab-triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0068] Trastuzumab-SO1861;

[0069] Trastuzumab-GE1741;

[0070] Trastuzumab-SA1641;

[0071] Trastuzumab-Quil-A;

[0072] Trastuzumab-QS-21;

[0073] Trastuzumab - saponins from the water-soluble saponin fraction of Quillaja saponaria;

[0074] anti-CD71 antibody-saponin;

[0075] anti-CD71 antibody - triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0076] anti-CD71 antibody-SO1861;

[0077] anti-CD71 antibody-GE1741;

[0078] anti-CD71 antibody-SA1641;

[0079] anti-CD71 antibody-Quil-A;

[0080] Anti-CD71 antibody-QS-21;

[0081] Anti-CD71 antibody - saponins from the water-soluble saponin fraction of Quillaja saponaria;

[0082] OKT-9-saponin;

[0083] OKT-9-triterpenoid saponins and / or disaccharide-chain triterpenoid saponins of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin;

[0084] OKT-9-SO1861;

[0085] OKT-9-GE1741;

[0086] OKT-9-SA1641;

[0087] OKT-9-Quil-A;

[0088] OKT-9-QS-21;

[0089] OKT-9 - saponin from the water-soluble saponin fraction of Quillaja saponaria;

[0090] anti-EGFR antibody-oligonucleotide;

[0091] anti-EGFR antibody-antisense oligonucleotide;

[0092] anti-EGFR antibody-siRNA;

[0093] Anti-EGFR antibody - antisense BNA;

[0094] Anti-EGFR antibody - antisense BNA (HSP27);

[0095] anti-EGFR antibody-protein toxin;

[0096] Anti-EGFR antibody-ribosome inactivating protein; Anti-EGFR antibody-oleanolic acid;

[0097] anti-EGFR antibody-saporin;

[0098] Cetuximab-oligonucleotide;

[0099] Cetuximab-antisense oligonucleotide;

[0100] Cetuximab-siRNA;

[0101] Cetuximab-antisense BNA;

[0102] Cetuximab-antisense BNA (HSP27); Cetuximab-protein toxin;

[0103] Cetuximab-ribosome-inactivating protein;

[0104] Cetuximab-dianthin;

[0105] Cetuximab-saporin;

[0106] anti-HER2 antibody-oligonucleotide;

[0107] anti-HER2 antibody-antisense oligonucleotide;

[0108] anti-HER2 antibody-siRNA;

[0109] Anti-HER2 antibody - antisense BNA;

[0110] Anti-HER2 antibody antisense BNA (HSP27); Anti-HER2 antibody-protein toxin;

[0111] Anti-HER2 antibody-ribosome inactivating protein; Anti-HER2 antibody-oleanolic acid;

[0112] anti-HER2 antibody-saporin;

[0113] trastuzumab-oligonucleotide;

[0114] trastuzumab-antisense oligonucleotide;

[0115] trastuzumab-siRNA;

[0116] Trastuzumab-antisense BNA;

[0117] Trastuzumab-antisense BNA (HSP27); Trastuzumab-protein toxin;

[0118] Trastuzumab-ribosome-inactivating protein;

[0119] trastuzumab-dianthin;

[0120] trastuzumab-saporin;

[0121] anti-CD71 antibody-oligonucleotide;

[0122] anti-CD71 antibody-antisense oligonucleotide;

[0123] anti-CD71 antibody-siRNA;

[0124] anti-CD71 antibody-antisense BNA;

[0125] anti-CD71 antibody - antisense BNA (HSP27);

[0126] anti-CD71 antibody-protein toxin;

[0127] Anti-CD71 antibody-ribosome inactivating protein;

[0128] anti-CD71 antibody-oleanolic acid;

[0129] anti-CD71 antibody-saporin;

[0130] OKT-9-oligonucleotide;

[0131] OKT-9-antisense oligonucleotide;

[0132] OKT-9-siRNA;

[0133] OKT-9-antisense BNA;

[0134] OKT-9-antisense BNA (HSP27);

[0135] OKT-9 - protein toxin;

[0136] OKT-9 - ribosome-inactivating protein;

[0137] OKT-9-caryophyllin;

[0138] OKT-9-saporin;

[0139] An anti-EGFR antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), wherein the saponin is any one or more of the following: a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-EGFR antibody is preferably cetuximab;

[0140] An anti-EGFR antibody (-protein toxin) (-saponin), wherein the protein toxin is any one or more of ribosome-inactivating protein, oleanolic acid and saporin, and wherein the saponin is any one or more of the following: a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group in position C-23 and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and a saponin in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-EGFR antibody is preferably cetuximab;

[0141] An anti-HER2 antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA and an antisense BNA (HSP27), and wherein the saponin is any one or more of the following: a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and a saponin in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-HER2 antibody is preferably trastuzumab;

[0142] An anti-HER2 antibody (-protein toxin) (-saponin), wherein the protein toxin is any one or more of ribosome-inactivating protein, oleanolic acid and saporin, and wherein the saponin is any one or more of the following: a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and a saponin in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-HER2 antibody is preferably trastuzumab;

[0143] Anti-CD71 antibodies (-oligonucleotides) (-saponins), wherein the oligonucleotides are any one or more of antisense oligonucleotides, siRNAs, antisense BNAs and antisense BNAs (HSP27), and wherein the saponins are any one or more of the following: triterpenoid saponins and / or disaccharide-chain triterpenoid saponins belonging to the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and saponins in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-CD71 antibody is preferably OKT-9; and

[0144] Anti-CD71 antibody (-protein toxin) (-saponin), wherein the protein toxin is any one or more of ribosome inactivating protein, oleanolic acid and saponin, and wherein the saponin is any one or more of the following: triterpenoid saponins and / or disaccharide-chain triterpenoid saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group in position C-23 and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and saponins in the water-soluble saponin fraction of Quillaja saponaria, wherein the anti-CD71 antibody is preferably OKT-9.

[0145] One embodiment is the first protein molecule according to the invention, the intermediate conjugate according to the invention or the conjugate according to the invention, wherein the first binding site is selected from the group consisting of: cetuximab, trastuzumab, OKT-9, and / or wherein the effector molecule is selected from the group consisting of: oleanolic acid, saporin and antisense BNA (HSP27), and / or wherein the saponin is selected from the group consisting of: SO1861, GE1741, SA1641, Quil-A, QS-21 and saponins from the water-soluble saponin fraction of Quillaja saponaria.

[0146] One embodiment is a conjugate according to the invention, wherein the protein molecule is selected from the group consisting of cetuximab, trastuzumab, OKT-9, and / or wherein the molecule is selected from the group consisting of oleanolic acid, saporin and antisense BNA (HSP27), and / or wherein the saponin is selected from the group consisting of SO1861, GE1741, SA1641, Quil-A, QS-21 and saponins from the water-soluble saponin fraction of Quillaja saponaria.

[0147] One aspect of the present invention relates to an ADC or an AOC or a semi-finished ADC conjugate or a semi-finished AOC conjugate comprising a protein molecule according to the present invention and at least one molecule according to the present invention and / or at least one saponin of structure C according to the present invention:

[0148] A(–S)b(–E)c

[0149] Structure C,

[0150] Where A is the first binding site;

[0151] S is saponin;

[0152] E is the effector molecule;

[0153] b=0-64, preferably: 0, 1, 2, 3, 4, 8, 16, 32, 64 or any integer or part thereof;

[0154] c=0-8, preferably: 0, 1, 2, 3, 4, 6, 8 or any integer or part thereof,

[0155] wherein S is coupled to A and / or E, and E is coupled to A and / or S, preferably S is coupled to A and E is coupled to A.

[0156] One embodiment is structure C of the 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 wherein S is any one or more of the following: a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21 and a saponin from the water-soluble saponin fraction of Quillaja saponaria, and / or wherein E is any one or more of the following: an oligonucleotide, an antisense oligonucleotide, an siRNA, an antisense BNA and an antisense BNA (HSP27), and / or any one or more of the following: a protein toxin, a ribosome-inactivating protein, dianthin and saporin.

[0157] One embodiment is structure C of the invention, the conjugate according to the invention or the intermediate conjugate according to the invention or the first protein molecule according to the invention, wherein the saponin (if present) and / or the effector molecule (if present) are linked to the saponin via at least one linker (such as a cleavable linker), and / or via at least one oligomeric or polymeric scaffold (such as a scaffold based on N-ε-maleimidocaproic acid hydrazide (EMCH) succinimidyl 3-(2-pyridyldithio) propionate or N-hydroxysuccinimide 3-(2-pyridyldithio) propionate (SPDP) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexahydrate). The invention relates to a method for preparing a saponin-containing protein, wherein the saponin-containing protein is covalently coupled to ...

[0158] One aspect of the present invention relates to the use of any of the above-mentioned conjugates of the present invention, the intermediate conjugates of the present invention, or the first protein molecule of the present invention as a medicament.

[0159] One aspect of the present invention relates to the use of any conjugate according to the present invention or the semi-finished conjugate according to the present invention or the first protein molecule according to the present invention for treating or preventing cancer or autoimmune diseases.

[0160] Figure 91 and Figure 92 Examples of ADCs according to the invention with covalently coupled saponins and OACs according to the invention with covalently coupled saponins are shown.

[0161] definition

[0162] The term "linker" has its conventional scientific meaning and refers herein to a linear segment of a chemical moiety or amino acid residue that is compounded by a peptide bond, which connects a molecule or atom to another molecule (e.g., a ligand or effector molecule or scaffold). Typically, a linker comprises a chain of atoms connected by chemical bonds. Any linker molecule or linker technology known in the art can be used in the present disclosure. Where specified, a linker is a linker for covalently binding a molecule via a chemical group on such a molecule that is suitable for forming a covalent bond or bond with the linker. The linker can be a non-cleavable linker, e.g., a linker that is stable under physiological conditions. The linker can be a cleavable linker, e.g., a linker that is cleavable in the presence of an enzyme or at a specific pH range or value, or under physiological conditions, such as intracellular conditions within endosomes (e.g., late endosomes) and in lysosomes of mammalian cells (e.g., human cells). Exemplary linkers useful in the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate or N-hydroxysuccinimide 3-(2-pyridyldithio)propionate (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidohexafluorophosphate (HATU).

[0163] The term "trifunctional linker" has its conventional scientific meaning and refers herein to a linker that connects three molecules by a chemical group on each of the three molecules. Technicians can design such trifunctional linkers based on the present disclosure and common knowledge. Such trifunctional linkers can show, for example, a maleimide group, which can be used to conjugate with a targeting ligand showing a thiol group to carry out a thiol-ene reaction. In addition, the trifunctional linker can show a dibenzocyclooctyne (DBCO) group to carry out a so-called strain-promoted alkyne-azide cycloaddition reaction (SPAAC, click chemistry) with a saponin with an azide group. Finally, the trifunctional linker can obtain a third functional group (such as a trans-cyclooctene (TCO) group) to carry out a so-called inverse electron demand Diels-Alder (IEDDA) reaction with an effector molecule with tetrazine (Tz). Technicians will understand that the chemical groups of the trifunctional linker can be all three identical or different, or that the linker can include two identical chemical groups for connecting a molecule to the trifunctional linker. The key formed between the trifunctional linker can be a covalent bond or a non-covalent bond, preferably a covalent bond. The key formed between the trifunctional linker and one or two or three binding molecules by respective chemical groups can be a cleavable (unstable) key, such as cleavable under the acidic conditions of intracellular (mammalian endosomes and lysosomes such as human cells), or can be a non-cleavable key. Of course, the trifunctional linker can include one or two chemical groups for forming a covalent bond, and other two or one chemical group are respectively used to form a non-covalent bond. Of course, the trifunctional linker can include one or two chemical groups for forming a cleavable key, and other two or one chemical group are respectively used to form a non-cleavable key.

[0164] The term "cleavable" (as used in the term "cleavable linker" or "cleavable bond") has its conventional scientific meaning and refers herein to a bond that undergoes cleavage under acidic conditions, reducing conditions, enzymatic conditions, or light-induced conditions. For example, a cleavable linker can undergo cleavage under acidic conditions, preferably, the cleavable linker undergoes cleavage in vivo under acidic conditions (preferably at pH 4.0-6.5, more preferably pH ≤ 5.5) as found in endosomes and / or lysosomes of mammalian cells, preferably human cells. As another example, a cleavable linker can undergo enzymatic cleavage (by cathepsins). Furthermore, an example of a covalent bond that is cleavable under reducing conditions is a disulfide bond.

[0165] In the context of oligomeric or polymeric scaffolds, the terms "oligomer" and "polymer" have their conventional scientific meanings. A polymer, as used herein, refers to a substance having a molecular structure composed primarily or entirely of a large number of identical or similar units bonded together; an oligomer, as used herein, refers to a polymer whose molecules are composed of a relatively small number of repeating units. For example, a structure comprising 5-10 or fewer identical or similar units may be referred to as an oligomeric structure, while a structure comprising 10-50 or more monomeric units may be referred to as a polymeric structure, and a structure comprising 10 monomeric units may be referred to as an oligomeric or polymeric structure.

[0166] The term "binding site" has its conventional scientific meaning and refers herein to a region or epitope on a molecule, such as a protein, DNA, or RNA, to which another molecule can bind.

[0167] The term "scaffold" has its conventional scientific meaning and refers herein to an oligomeric or polymeric template or carrier or matrix (basic molecule or basic structure) to which one or more molecules (e.g., ligand molecules, effector molecules) can be covalently bound directly or through a linker (e.g., a cleavable linker). The scaffold can have a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendrimer, or dendrimer-oligomer, or have an assembled polymeric structure (e.g., a hydrogel, microgel, nanogel, stable polymeric micelle, or liposome), but excludes structures composed of non-covalent assemblies of monomers (e.g., cholesterol / phospholipid mixtures). The scaffold may comprise a polymeric or oligomeric structure, such as a poly- or oligomeric (amine), for example polyethyleneimine and poly- (amide); or a structure, such as polyethylene glycol; a poly- or oligomeric (ester), such as poly- (lactide), poly- (lactam), poly- lactide-co-glycolide copolymer; or a poly- (dextrin), a poly- or oligosaccharide, such as cyclodextrin or polydextrose; or a structure, such as a natural and / or artificial poly- or oligoamino acid, such as polylysine or a peptide or protein, a DNA oligomer or polymer, a stable RNA polymer or a PNA (peptide nucleic acid) polymer. Preferably, the polymeric or oligomeric structure is biocompatible, wherein biocompatible means that the polymeric or oligomeric structure does not show significant acute or chronic toxicity in an organism and can be excreted intact or completely degraded into compounds that are excretable and / or physiological by the body's metabolism. Metabolism.

[0168] The term "ligand" has its conventional scientific meaning and refers herein to a target cell surface molecule or target cell surface receptor that can selectively bind to a target cell (e.g., a target cancer cell or a target autoimmune cell). A ligand can bind to an epitope contained by a receptor or other antigen on a target cell. Preferably, the cell-binding ligand is an antibody.

[0169] As used herein, the term "antibody" is used in the broadest sense and refers to an immunoglobulin (Ig) that is defined as a protein belonging to the IgG, IgM, IgE, IgA or IgD class (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 binding domain" of an immunoglobulin is defined as an antigen-binding fragment or domain or other derivative of a parent immunoglobulin that substantially retains the antigen-binding activity of such parent immunoglobulin. Functional fragments and functional domains are antibodies within the meaning of the present invention, even if their affinity for antigen is lower than that of the parent immunoglobulin. According to the present invention, "functional fragments and domains" include but are not limited to F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single domain antibodies (sdAb), monovalent IgG, scFv-Fc, reduced IgG (rIgG), miniantibodies, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanoantibodies, variable V domains (such as VHH, Vh) and other types of antigen-recognizing immunoglobulin fragments and domains. Fragments and domains can be engineered to minimize or completely remove the intermolecular disulfide bond interactions that occur between the CH1 domain and the CL domain. Functional fragments and domains have greater tumor penetration advantages due to their smaller size. In addition, compared to intact immunoglobulins, functional fragments or domains can be more evenly distributed throughout the tumor mass.

[0170] The antibodies (immunoglobulins) of the present invention can be bifunctional or multifunctional. For example, one arm of a bifunctional antibody has specificity for one receptor or antigen, while the other arm recognizes a different receptor or antigen. Alternatively, each arm of a bifunctional antibody can have specificity for a different epitope of the same receptor or antigen on a target cell.

[0171] The antibodies (immunoglobulins) of the present invention can be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, surface-reshaped antibodies, anti-idiotypic antibodies, mouse antibodies, rat antibodies, rat / mouse hybrid antibodies, llama antibodies, llama heavy chain antibodies only, heavy chain antibodies only, and veterinary antibodies. Preferably, the antibodies (immunoglobulins) of the present invention are monoclonal antibodies. Surface-reshaped, chimeric, humanized, and fully human antibodies are also more preferred because they are less likely to cause immunogenicity in humans. The antibodies of the ADC of the present invention preferably specifically bind to antigens expressed on the surface of cancer cells, autoimmune cells, diseased cells, and abnormal cells, while leaving any healthy cells substantially unchanged (e.g., by not binding to such normal cells, or by binding to such healthy cells at a lower degree in quantity and / or affinity).

[0172] Specific antibodies that can be used in the ADC of the present invention include, but are not limited to, anti-HER2 monoclonal antibodies (such as trastuzumab and pertuzumab), anti-CD20 monoclonal antibodies (such as rituximab, ofatumumab, tositumomab and ibritumomab tiuxetan), anti-CA125 monoclonal antibodies (such as ogavuzumab), anti-EpCAM (17-1A) monoclonal antibodies (such as edrecolomab), anti-EGFR monoclonal antibodies (such as cetuximab, panitumumab and nimotuzumab), anti-CD30 monoclonal antibodies (such as brentuximab), anti-CD33 monoclonal antibodies (such as gemtuzumab and huMy9-6), anti-vascular integrin α-vβ-3 monoclonal antibodies (such as irrituximab), and anti-CD30 monoclonal antibodies. monoclonal antibodies (e.g., daratumumab), anti-CD52 monoclonal antibodies (e.g., alemtuzumab), anti-CD22 monoclonal antibodies (e.g., epratuzumab), anti-CEA monoclonal antibodies (e.g., labetuzumab), anti-CD44v6 monoclonal antibodies (bivalizumab), anti-FAP monoclonal antibodies (e.g., sirolimus), anti-CD19 monoclonal antibodies (e.g., huB4), anti-CanAg monoclonal antibodies (huC242), anti-CD56 monoclonal antibodies (e.g., huN901), anti-CD38 monoclonal antibodies (e.g., daratumumab), anti-CA6 monoclonal antibodies (e.g., DS6), anti-IGF-IR monoclonal antibodies (e.g., cilutumumab and 3B7), anti-integrin monoclonal antibodies (CNTO 95), and anti-syndecan-1 monoclonal antibodies (e.g., B-B4).

[0173] Any other 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 for the ligand-drug conjugates of the present invention and provided with a ligand covalently bound to a saponin according to the present invention. Such ligands include, but are not limited to, proteins, polypeptides, peptides, small molecules. Examples of such non-antibody ligands are interferons (e.g., IFN-α, IFN-β, and IFN-γ), transferrin, lectins, epidermal growth factor (EGF) and EGF-like domains, gastrin-releasing peptide (GRP), platelet-derived growth factor (PDGF), transforming growth factor (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factor (IGF, e.g., IGF-1 and IGF-2), other suitable hormones (e.g., thyrotropin-releasing hormone (TRH), melanocyte stimulating hormone (MSC), thyrotropin-releasing hormone (MTH ... hormones (MSH), steroid hormones (such as estrogen and androgen)), somatostatin, lymphokines (such as IL-2, IL-3, IL-4 and IL-6), colony stimulating factors (CSF such as G-CSF, M-CSF and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (such as AS-1411, GBI-10, RNA aptamers to HIV glycoprotein), small molecules (such as folic acid, anisamide phenylboronic acid), vitamins (such as vitamin D), carbohydrates (such as hyaluronic acid, galactose).

[0174] "Effector molecule" or "effector moiety" or "payload" has its conventional scientific meaning and, in the context of the present invention, is any substance that affects cellular metabolism by interacting with an intracellular effector molecule target, wherein the effector molecule target is any molecule or structure within the cell, excluding but including the membranes of compartments and vesicles of the endocytic and recycling pathways. Thus, such structures within the cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytoplasm.

[0175] Effector molecules or moieties are pharmaceutically active substances, such as toxins (e.g., protein toxins), drugs, polypeptides, or polynucleotides. The pharmaceutically active substances in the present invention are effector molecules or moieties that are used to achieve beneficial results in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / subject. Benefits include diagnosis, prognosis, treatment, cure, and prevention (prevention / prophylaxis) of a disease and / or symptom and / or health problem. Pharmaceutically active substances may also cause undesirable, sometimes even harmful, side effects (e.g., adverse events observed in clinical trials). In this case, the pros and cons must be weighed to determine whether the pharmaceutically active substance is suitable for a particular situation. If the effect of the pharmaceutically active substance in a cell is primarily beneficial to the entire organism, the cell is referred to as a target cell. If the effect in the cell is primarily harmful to the entire organism, the cell is referred to as an off-target cell. In artificial systems (e.g., cell culture and bioreactors), target cells and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules and moieties are drugs, toxins, polypeptides (e.g., enzymes), polynucleotides (including polypeptides and polynucleotides comprising non-natural amino acids or nucleic acids), and any combination thereof.

[0176] Effector molecules or effector moieties as drugs may include, but are not limited to, anticancer agents, anti-inflammatory agents, and anti-infective agents (e.g., antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents). Preferably, the drug molecules of the present invention are anticancer agents or anti-autoimmune agents. Suitable anticancer agents include, but are not limited to, alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. Also included within the definition of "anticancer agent" are, for example, (i) antihormonal agents used to modulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands; (iii) antiandrogens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways associated with abnormal cell proliferation; (vii) ribozymes, such as inhibitors of VEGF expression and inhibitors of HER2 expression; (viii) vaccines, such as gene therapy vaccines; topoisomerase 1 inhibitors; (ix) antiangiogenic agents; and pharmaceutically acceptable salts, acids, solvates, and derivatives of any of the foregoing.

[0177] Effector molecules or moieties as toxins can include, but are not limited to, protein toxins (e.g., bacteria-derived toxins and plant-derived toxins), toxins targeting microtubule filaments, toxins targeting DNA, toxins targeting RNA. Examples of protein toxins are saporin, dianthin, ricin, modeccin, abrin, volkensin, viscumin, Shiga toxin, Shiga-like toxins, Pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of toxins targeting microtubule filaments are maytansinoids (e.g., DM1 and DM4), auristatins (e.g., monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF)), toxoids, tubulysin, nostoc, rhizoxin. Examples of toxins that target DNA are calicheamicins: N-acetyl-gamma calicheamicin, CC-1065 analogs, duocarmycins, doxorubicin, methotrexate, benzodiazepines, camptothecin analogs, and anthracyclines. Examples of toxins that target DNA are amanitin, spliceostatins, and tylanstatins. Toxins as used in the present invention are defined as pharmaceutically active substances that are capable of killing or inactivating cells. Preferably, the targeted toxin is a toxin that is toxic only or at least primarily to the target cells but not to off-target cells. The net effect of the targeted toxin is preferably beneficial to the entire organism.

[0178] The effector molecule or moiety as a polypeptide can be, for example, a polypeptide or toxin that restores a lost function (such as enzyme replacement, gene regulatory function). Examples of polypeptides as effector molecules are, for example, Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)bouganin, agrobacterin, ricin (toxin A chain); American pokeweed antiviral protein, apoptosis protein, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (e.g., argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; cell signaling enzymes; cell cycle regulators; gene regulators (transcription factors such as NF-κB or gene repressors such as methionine repressors).

[0179] The effector molecule or effector portion as a polynucleotide can be, for example, a polynucleotide comprising coding information (such as a gene or open reading frame encoding a protein). It can also include 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 acids (PNA), morpholinos and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA) and threose nucleic acids (TNA). Each of these is distinguished from naturally occurring DNA or RNA by changes in the molecular backbone. Examples of nucleotides as effector molecules are, but are not limited to, DNA: single-stranded DNA (e.g., DNA of adenine phosphoribosyltransferase); linear double-stranded DNA (e.g., coagulation factor IX gene); circular double-stranded DNA (e.g., plasmid); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA; antisense oligonucleotides (ASO, AON, such as PNA, PMO, LNA, and BNA).

[0180] The term "protein" (as used, for example, for "protein molecules" and "protein toxins") refers to molecules and toxins comprising at least one string of amino acid residues that can be obtained as the expression product of a single mRNA. Such molecules or toxins may also comprise any post-translational modifications, carbohydrates (such as N- or O-linked carbohydrates), disulfide bonds, phosphorylation, sulfation, etc., as a result of any post-translational modifications, and / or may also comprise any other modifications (such as those resulting from chemical modifications (e.g., the addition of effector moieties, saponins, scaffolds, ligands, etc.), either directly to, for example, amino acid side chains, or chemically bound (covalently) to protein molecules via at least one linker that is bound to a molecule for chemically modifying the protein molecule. The term "protein" also encompasses and includes assemblies of such molecules, such as homodimers, heterotrimers, heterohexamers, or complex assemblies (such as ribosomes).

[0181] In the context of, for example, "specific binding" and "a receptor or molecular target that is specifically present or expressed on the surface of a tumor cell," the terms "specific" and "specifically" have their normal scientific meaning as known in the art and refer herein to a binding interaction of a first molecule with a second molecule that occurs with a higher affinity or is expressed, or, for example, to a higher degree of expression, relative to any putative binding of the first molecule to another molecule that is different from the second molecule, e.g., considering the number of cell surface receptors or molecular targets on the surface of a first type of cell (e.g., a tumor cell, an autoimmune cell, a diseased cell, an abnormal cell) relative to the degree of expression of the same receptor or molecular target on a second type of cell (e.g., a healthy cell, etc.), wherein expression on the second type of cell may be completely absent or very low relative to any degree of expression on a tumor cell, etc. Furthermore, the term "specific," as in "specifically binds," has its normal scientific meaning as known in the art and refers herein to a molecule that can interact with another molecule with a higher binding affinity than background interactions between the molecules. Similarly, the term "specific" refers to an interaction, e.g., between two molecules or between a cell and a molecule, that has a higher binding affinity than background interactions between molecules. Binding molecules (e.g., immunoglobulins) bind to binding sites on molecules (e.g., epitopes, cell surface receptors, etc.) through their binding sites (e.g., immunoglobulin variable regions of immunoglobulins) with a higher binding affinity than background interactions between molecules. In the context of the present invention, background interactions typically have a K of as low as 10E-4 M. D Similarly, a "specific binding domain" is a domain that preferentially binds to a binding site on a molecule (e.g., an epitope, a cell surface receptor, etc.) with a higher binding affinity than background interactions between molecules. In the context of the present invention, "background interactions" are typically interactions with an affinity lower than 10E-4 M. D Preferably, the specific binding domain binds to the molecule with a K of greater than about 10E-5M. D affinity binding.

[0182] The term "binding" is defined as an interaction between molecules that can be distinguished from background interactions.

[0183] Throughout the specification, the term "fragment" refers to an amino acid sequence that is a portion of a protein domain or that constitutes an entire protein domain. A binding fragment according to the present invention must have binding specificity for the corresponding target, such as a cell surface receptor on the surface of a diseased cell (e.g., a tumor cell).

[0184] The term "ADC" or "antibody-drug conjugate" has the conventional scientific meaning known to those skilled in the art and refers herein to a class of biopharmaceutical drugs designed as targeted therapies for treating, for example, cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADCs consist of antibodies linked to a biologically active cytotoxic (anti-cancer) payload or drug. ADCs combine the targeting ability of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. They are designed to distinguish between healthy cells and diseased tissues (such as tumor cells in a tumor).

[0185] The term "saponin collection" has its normal meaning and refers herein to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, including SA1657 and predominantly SA1641.

[0186] The term "Quillaja saponin" has its normal meaning and refers herein to the saponin fraction of Quillaja saponin, and therefore the source of all other QS saponins, primarily comprising QS-18 and QS-21.

[0187] "QS-21" or "QS21" has its conventional scientific meaning and refers herein to a mixture of QS-21A-apio (-63%), QS-21A-xylo (-32%), QS-21B-apio (-3.3%), and QS-21B-xylo (-1.7%).

[0188] Similarly, "QS-21A" has its conventional scientific meaning and refers herein to a mixture of QS-21A-apio (-65%) and QS-21A-xylo (-35%).

[0189] Similarly, "QS-21B" has its conventional scientific meaning and refers herein to a mixture of QS-21B-apio (-65%) and QS-21B-xylo (-35%).

[0190] The term "Quil-A" refers to a commercially available semi-purified extract from the Quillaja saponaria tree and contains varying amounts of over 50 different saponins, many of which contain the triterpene-trisaccharide substructure Gal-(1-hydroxy-2-oxo-1 ... → 2)-[Xyl-(1 →3)]-GlcA-. The saponins found in Quil-A are listed in van Setten (1995), Table 2 [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon FAKersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 660-666 (1995)]. Quil-A and Quillaja saponins are saponin fractions from the Quillaja saponaria Molina tree, and both contain a large number of different saponins, with largely overlapping contents. The specific compositions of these two fractions differ because they were obtained through different purification procedures.

[0191] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. The molecular weight of QS1861 is 1861 Daltons, and the molecular weight of QS1862 is 1862 Daltons. QS186 is described in Fleck et al. (2019), Table 1, row 28 [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira, and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171]. The structure depicted is the api variant of QS-7, QS1862. The molecular mass is 1862 Daltons, as this is the formal mass including the proton at the glucuronic acid site. At neutral pH, the molecule is deprotonated. When measured in a mass spectrometer in negative ion mode, the measured mass is 1861 Daltons.

[0192] The terms first, second, third, etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. Where appropriate, these terms are interchangeable. The embodiments of the present invention can be operated in other sequences than those described or illustrated herein.

[0193] Furthermore, various embodiments, although referred to as "preferred" or "eg, for example" or "particularly" are to be construed as illustrative ways in which the invention may be practiced rather than limiting the scope of the invention.

[0194] The term "comprising" used in the claims should not be interpreted as being limited to the elements or steps listed thereafter; it does not exclude other elements or steps. It needs to be interpreted as specifying the presence of the stated features, integers, steps or components mentioned, but does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof. Thus, the scope of the expression "a pharmaceutical composition comprising A and B" should not be limited to pharmaceutical compositions consisting only of components A and B, but rather, for the purposes of the present invention, the only listed components of the pharmaceutical composition are A and B, and the claims should also be interpreted to include 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 step A and step B, but rather, for the purposes of the present invention, the only listed steps of the method are A and B, and the claims should also be interpreted to include equivalents of these steps.

[0195] Furthermore, the indefinite article "a / an" when referring to a feature does not exclude the possibility that more than one feature (e.g., component, excipient, saponin, etc.) is present, unless the context clearly requires one and only one feature. Thus, the indefinite article "a / an" generally means "at least one." BRIEF DESCRIPTION OF THE DRAWINGS

[0197] Figure 1 In vivo study of antibody-protein toxin + unconjugated SO1861 BT474 tumor-bearing mice were treated with different concentrations of trastuzumab-saporin (iv) + 1.5 mg / kg of unconjugated SO1861 (subQ injection 1 hour before trastuzumab-saporin treatment).

[0198] Figure 2(1)-(3). Unconjugated saponin-mediated enhancement of endosomal escape and target cell killing. Figure 2(1), A) HeLa cells (EGFR +) cell viability analysis; Figure 2 (2), B) HeLa cells (EGFR + ) Cell viability analysis. The axes and legends of A) and B) are the same. Figure 2(3), C) HeLa cells (EGFR) treated with SO1861 or GE1741 in the presence or absence of 1.5 pM EGF oleanolic acid. + Figure 2(3), D) HeLa cells (EGFR) treated with various QSmix (saponin mixture from Quillaja saponaria) in the presence or absence of 1.5 pM EGF oleanolic acid. + ). The Y-axes of C) and D) are the same.

[0199] Figure 3 (1)-(3). Comparison of the activities of unconjugated SO1861 and SO1861-EMCH. According to the present invention, EGFR-targeted antisense BNA oligonucleotides are delivered and gene silencing is performed in cancer cells. Figure 3(1)-Figure 3(2) , A, B, C) A431 (EGFR ++ )、HeLa(EGFR + ) or A2058(EGFR - ) Cell viability analysis of cells. Figure 3(2)-Figure 3(3) , D, E) A431 (EGFR ++ ) or HeLa(EGFR + ) Cell viability analysis of cells. The axes and legends for A), B), C), D), and E) are the same. That is, the legends for Figures 3 A to C are displayed next to Figure 3 C; the legends for Figures 3 D and 3 E are displayed next to Figure 3 E.

[0200] Figure 4 Comparison of unconjugated SO1861 with SO1861-EMCH (unstable) and SO1861-S (stable). HeLa cells (EGFR + ) for cell viability analysis.

[0201] Figure 5(1)-(4). EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing. Using Cetuximab-(Cys-L-SO1861)3,9 or cetuximab-(Cys-L-SO1861) 3,9 +100nM HSP27BNA treated A431 (EGFR ++ ) and A2058 (EGFR - ) cells. The axes and legends of Figure 5(1), A) and Figure 5(2), B) are the same, and the legend is shown next to B in Figure 5(2). The Y axes of Figure 5(3), C) and Figure 5(4), D) are the same.

[0202] Figure 6 Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. Compared with the control group, HSP27BNA + cetuximab-(Cys-L-SO1861) in A431 tumor-bearing mice 3,9 Treated mice showed effective tumor-targeted gene silencing.

[0203] Figure 7 In vivo activity of 1T2C. Compared with the control, 50 mg / kg of cetuximab-(Cys-L-SO1861) 4 + 25 mg / kg of cetuximab-(-L-HSP27BNA) 4 The 1T2C combination showed strong tumor-targeted gene silencing.

[0204] Figure 8 In vivo activity of 1T2C. In a PDX tumor mouse model (high HER2 expression), 40 mg / kg of trastuzumab-(CYS-L-SO1861) 4 The 1T2C combination of trastuzumab-saporin + 0.02 / 0.03 mg / kg showed effective tumor growth inhibition.

[0205] Figure 9 (1)-(2). 1-Target 2-Component. By the therapeutic combination according to the present invention, in A431 cells (EGFR ++ )( Figure 9(1)-Figure 9(2) , A, C) and CaSKi cells (EGFR + )( Figure 9(1)-Figure 9(2) , B, D) EGFR-targeted cell killing. A, B) Cetuximab-(CYS-L-SO1861) on A431 (A) and CaSKi (B) cells 3,7 Titration + fixed concentration of 10 pM cetuximab-saporin and control. C, D) Titration of cetuximab-saporin + fixed concentration of 75 nM cetuximab-CYS-L-SO1861 on A431 (C) and CaSKi (D) cells. 3,7 and controls.

[0206] Figure 10 (1)-(2). 1-Target 2-Component. + / - )( Figure 10(1)-Figure 10(2) , A, C) and A2058 cells (EGFR - )( Figure 10(1)-Figure 10(2) , B, D) EGFR-targeted cell killing. A, B) Cetuximab-(CYS-L-SO1861) on HeLa (A) and CaSKi (B) cells 3,7 Titration + fixed concentration of 10 pM cetuximab-saporin and control. C, D) Titration of cetuximab-saporin + fixed concentration of 75 nM cetuximab-CYS-L-SO1861 on HeLa (C) and A2058 (D) cells. 3,7 and controls.

[0207] Figure 11 :1-target 2-component. By the therapeutic combination according to the present invention, in SKBR3 cells (HER2 ++ HER2-targeted cell killing in SKBR3 cells (A, B). A) Trastuzumab (CYS-L-SO1861) on SKBR3 cells 4 Titration + fixed concentration of 50 pM Trastuzumab-Saporin and control. B) Titration of Trastuzumab-Saporin + fixed concentration of 2.5 nM Trastuzumab-(Cys-L-SO1861) on SKBR3 cells 4 and controls.

[0208] Figure 12 (1)-(2). 1-Target 2-Component. By the therapeutic combination according to the present invention, in JIMT-1 cells (HER2 + / - )( Figure 12(1)-Figure 12(2) , A, C) and MDA-MB-468 cells (HER2 - )( Figure 12(1)-Figure 12(2) , B, D) HER2 cell killing. A, B) Trastuzumab Cys-L-SO1861 on JIMT-1 (A) and MDA-MB-468 (B) cells 4 Titration + fixed concentration of 50 pM trastuzumab-saporin and control. C, D) Titration of trastuzumab-saporin + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) on JIMT-1 (C) and MDA-MB-468 (D) cells. 4 and controls.

[0209] Figure 13: Chloroquine inhibits 1-target 2-component. By the therapeutic combination according to the present invention + chloroquine, in SK-BR-3 (HER2 ++ ) and A431 cells (EGFR ++ ) HER2 and EGFR-targeted cell killing in SK-BR-3 cells. A) Trastuzumab-saporin titration + fixed concentration of 5 nM Trastuzumab-Cys-L-SO1861) 4 +0.5 μM chloroquine and control. B) Cetuximab-saporin titration on A431 cells + fixed concentration of 5 nM cetuximab-(Cys-L-SO1861) 3,8 +0.5 μM chloroquine and control.

[0210] Figure 14 (1)-(2): 1-target 2-component. ++ ) and A2058 cells (EGFR - Figure 14 (1), A, B) Cetuximab-(Cys-L-SO1861) on A431 cells (A) and A2058 cells (B) 3,8 Titration + fixed concentration of 100 nM cetuximab-(Lys-L-HSP27BNA) 4 Figure 14 (2), C, D) Cetuximab-(LYS-L-HSP27BNA) on A431 cells (C) and A2058 cells (D). 4 Titration + fixed concentration of 77nM cetuximab-(CYS-L-SO1861) 3,8 and controls.

[0211] Figure 15 (1)-(2): 2-target 2-component. ++ ) and HeLa cells (EGFR + / - ) and EGFR- and HER2-targeted cell killing in SK-BR-3 cells (HER2 ++ ) and JIMT-1 cells (HER2 + / - Figure 15(1), A, B) Cetuximab-Cys-(dendron (-L-SO1861)) on MDA-MB-468 cells (Figure 15(1), A) and HeLa cells (Figure 15(1), B) 4 ) 3,9Titration + fixed concentration of 10 pM cetuximab-saporin and control. Figure 15 (2), C, D) Trastuzumab (Cys-(dendron (-L-SO1861)) on SK-BR-3 cells (C) and JIMT-1 cells (D) 4 ) 4 Titration + fixed concentration of 50 pM Trastuzumab-Saporin and control.

[0212] Figure 16 .2-Target 2-Component. The therapeutic combination according to the present invention, trastuzumab-saporin + 2.5nM trastuzumab (CYS-L-SO1861) can be used 4 Effectively kill SK-BR-3 cells (HER2 + / - ), whereas T-DM1+2.5nM trastuzumab-(Cys-L-SO1861) 4 Titration is ineffective at such low toxin concentrations. T-DM1 is trastuzumab-emtansine carrying ~3.5 emtansine (DM1) toxin molecules per antibody (DAR3.5).

[0213] Figure 17 (1)-(2). 1-Target 2-Component. By the therapeutic combination according to the present invention, in A431 cells (EGFR ++ )(Figure 17(1), A) and CaSKi cells (EGFR + )(Figure 17(1), B) and A2058 cells (EGFR - ) EGFR-targeted cell killing in A431 cells (A), CaSKi cells (B), and A2058 cells (Figure 17 (2), C) Cetuximab-(Cys-L-QSmix) 4,1 Titration + fixed concentration of 10 pM Cetuximab-Saporin or 10 pM Cetuximab-Oleanin and control. QSmix is ​​a mixture of saponins from Quillaja saponaria extract.

[0214] Figure 18 1-Target 2-Component Concept: mAb1-SO1861 + mAb1-Protein Toxin. SO1861 and a toxin (ribosome-inactivating protein) are each independently conjugated to an antibody (mAb1) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb1-Protein Toxin bind to cell surface receptors, 2) Receptor-mediated endocytosis of both conjugates occurs, 3) At low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) Toxin release into the cytoplasm occurs, and 5) the toxin induces cell death.

[0215] Figure 19 1-Target 2-Component Concept: mAb1-SO1861 + mAb2-BNA Oligonucleotide. SO1861 and the antisense BNA oligonucleotide are each independently conjugated to an antibody (mAb1) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb1-BNA oligonucleotide bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) BNA oligonucleotide release into the cytoplasm occurs, and 5) target gene silencing occurs.

[0216] Figure 20 :1-Target 2-Component Concept: mAb1-(Scaffold (-SO1861)n)n+mAb1-Protein Toxin. Dendrimer (-SO1861) n and protein toxin (ribosome inactivating protein) are each independently conjugated to an antibody (mAb1) for delivery and internalization into target cells. 1) mAb1-dendron (-SO1861) 4 and mAb1-protein toxin binds to cell surface receptors, 2) receptor-mediated endocytosis of the two conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active allowing endolysosomal escape, 4) toxin release into the cytoplasm occurs and 5) toxin induces cell death.

[0217] Figure 21 (1)-(2): Antibody-(-L-SO1861) 4 with antibody-(-L-SO1861) 2 By the therapeutic combination according to the present invention, in A431 cells (EGFR ++ ) and SK-BR-3(HER2 ++ ) in HER2- and EGFR-targeted cell killing. Figure 21 (1), A) Cetuximab-(-L-SO1861) in A431 cells 4 +10pM Cetuximab-Saporin and Cetuximab-(-L-SO1861) 2 Comparison of Cetuximab-Saporin at +10 pM. Figure 21 (2), B) Trastuzumab-(-L-SO1861) in SK-BR-3 cells 4 +50pM of trastuzumab-saporin and trastuzumab-(-L-SO1861) 2 Comparison of Trastuzumab-Saporin at +50 pM.

[0218] Figure 22 :Antibody-(-L-SO1861)4 with antibody-(-S-SO1861) 4 By the therapeutic combination according to the present invention, in SK-BR-3 (HER2 ++ ) HER2-targeted cell killing in SK-BR-3 cells. B) Trastuzumab-(-L-SO1861) in SK-BR-3 cells 4 +50pM of trastuzumab-saporin and trastuzumab-(-S-SO1861) 4 Comparison of Trastuzumab-Saporin at +50 pM.

[0219] Figure 23 The 2T2 component system demonstrated tumor regression when tested in an A431 tumor-bearing mouse model.

[0220] Figure 24 The 2T2 component system demonstrated tumor regression and eradication when tested in an A431 tumor-bearing mouse model.

[0221] Figure 25 (1)-(2): 2-target 2-component. ++ / HER2 + / - )( Figure 25(1)-Figure 25(2) ,A,C) and CaSKi cells (EGFR ++ / HER2 + / - )( Figure 25(1)-Figure 25(2) , B, D) EGFR / HER2-targeted cell killing. A, B) Cetuximab-(Cys-L-SO1861) on A431 cells 3,7 Titration + fixed concentration of 50 pM trastuzumab-saporin and control. C, D) Titration of trastuzumab-saporin + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) on Caski cells 3,7 The axes and legends for Figures A) and B) and C) and D) are the same. That is, the legends for Figures A and B are displayed next to Figure B; the legends for Figures C and D are displayed next to Figure D.

[0222] Figure 26 (1)-(2). 2-target 2-component. By the therapeutic combination according to the present invention, in HeLa cells (EGFR + / - / HER2 + / - )( Figure 26(1)-Figure 26(2) ,A,C) and A2058 cells (EGFR - / HER2 + / - )( Figure 26(1)-Figure 26(2)EGFR / HER2-targeted cell killing in (A, B) and (D) HeLa cells. 3,7 Titration + fixed concentration of 50 pM trastuzumab-saporin and control. C, D) Titration of trastuzumab-saporin + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) on A2058 cells 3,7 The axes and legends for A) and B) are the same. That is, the legends for Figures 26A and 26B are displayed next to Figure 26B.

[0223] Figure 27 :2-target 2-component. By the therapeutic combination according to the present invention, in SKBR3 cells (HER2 ++ / EGFR + / - HER2 / EGFR targeted cell killing in (A, B). A) Trastuzumab-(Cys-L-SO1861) on SKBR3 cells 4 Titration + fixed concentration of 1.5 pM EGF-oleanolic acid and control. B) Titration of EGF-oleanolic acid + fixed concentration of 2.5 nM Trastuzumab-(Cys-L-SO1861) on SKBR3 cells 4 and controls.

[0224] Figure 28 (1)-(2). 2-Target 2-Component. By the therapeutic combination according to the present invention, in JIMT-1 cells (HER2 + / - EGFR + / - )( Figure 28(1)-Figure 28(2) ,A,C) and MDA-MB-468 cells (HER2 - / EGFR ++ )( Figure 28(1)-Figure 28(2) HER2 / EGFR-targeted cell killing in (A, B) and (B) JIMT-1 cells. 4 Titration + fixed concentration of 1.5 pM EGF-oleanolic acid and control. C, D) Titration of EGF-oleanolic acid + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) on MDA-MB-468 cells 4 The axes and legends for A) and B) and C) and D) are the same. That is, the legends for Figures 28A and 28B are displayed next to Figure 28B; the legends for Figures 28C and 28D are displayed next to Figure 28D.

[0225] Figure 29 :2-target 2-component. By the therapeutic combination according to the present invention, in SKBR3 cells (HER2 ++ / EGFR+ / - HER2 / EGFR targeted cell killing in (A, B). A) Trastuzumab-(Cys-L-SO1861) on SKBR3 cells 4 Titration + fixed concentration of 10 pM cetuximab saporin and control. B) Titration of cetuximab-saporin + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) on SKBR3 cells 4 and controls.

[0226] Figure 30 (1)-(2). 2-Target 2-Component. By the therapeutic combination according to the present invention, in JIMT-1 cells (HER2 + / - EGFR + / - )( Figure 30(1)-Figure 30(2) ,A,C) and MDA-MB-468 cells (HER2 - / EGFR ++ )( Figure 30(1)-Figure 30(2) HER2 / EGFR-targeted cell killing in (A, B) and (B) JIMT-1 cells. 4 Titration + fixed concentration of 10 pM cetuximab-saporin and control. C, D) Titration of cetuximab-saporin + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) on MDA-MB-468 cells 4 The axes and legends for A) and B) and C) and D) are the same. That is, the legends for Figures 30 A and 30 B are displayed next to Figure 30 B; the legends for Figures 30 C and 30 D are displayed next to Figure 30 D.

[0227] Figure 31 (1)-(2): Chloroquine inhibits 2-target 2-component. By the therapeutic combination according to the present invention + chloroquine, in A431 cells (EGFR ++ / HER2 + / - / CD71 + )( Figure 31 (1), A, B), MDA-MB-468 cells (EGFR ++ / HER2 - / CD71 + )( Figure 31 (2), C) or SK-BR-3 (HER2 ++ / EGFR + / - / CD71 + )( Figure 31(2), EGFR / HER2, EGFR / CD71, or HER2 / CD71 targeted cell killing in D). A) Trastuzumab-oleanol or trastuzumab-saporin titration on A431 cells + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,9 +800nM chloroquine and control. B) CD71mab-saporin titration on A431 cells + fixed concentration of 10.5nM cetuximab-(Cys-L-SO1861) 3,9 +500nM chloroquine and control. C) CD71mab-saporin titration on MDA-MB-468 cells + fixed concentration of 10.5nM cetuximab-(Cys-L-SO1861) 3,9 +500nM chloroquine and control. D) CD71mab-saporin titration on SK-BR-3 cells + fixed concentration of 5nM trastuzumab-(Cys-L-SO1861) 3,9 +500 nM chloroquine and control.

[0228] Figure 32 (1)-(2): 2-target 2-component. By the therapeutic combination according to the present invention, in A431 cells (EGFR ++ / HER2 + / - )( Figure 32 (1), A) and A2058 cells (EGFR - / HER2 + / - )( Figure 32 (1), B) EGFR / HER2 targeted gene silencing. A) Cetuximab-(Cys-L-SO1861) on A431 cells (A) and A2058 cells (B) 3,9 Titration + fixed concentration of 100 nM Trastuzumab-(Lys-L-HSP27BNA) 4,4 and controls. Figure 32 (2), C, D) Trastuzumab-(Lys-L-HSP27BNA) on A431 cells (A) and A2058 cells (B) 4,4 Titration + fixed concentration of 77nM Cetuximab-(Cys-L-SO1861) 3,9 and control. The axes and legends of A) and B) and C) and D) are the same. That is, Figure 32 The legend of A and B is shown in Figure 32 Next to B; Figure 32 C and Figure 32 The legend of D is shown in Figure 32 Next to the D.

[0229] Figure 33(1)-(2): 2-target 2-component. By the therapeutic combination according to the present invention, in MDA-MB-468 cells (EGFR ++ / CD71 + )( Figure 33 (1), A) HeLa cells (EGFR + / - / CD71 + ), SK-BR-3 cells (HER2 ++ / CD71 + )( Figure 33 (1), B) and JIMT-1 cells (HER2 + / - / CD71 + ) EGFR / CD71 or HER2 / CD71 targeted cell killing in MDA-MB-468 cells. A) Cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9 Titration + fixed concentration of 10 pM CD71mab-saporin and control. B) A) Cetuximab-Cys-(Dendron (-L-SO1861) on HeLa cells 4 ) 3,9 Titration + fixed concentration of 10 pM CD71mab-saporin and control. Figure 33 (2), C) Trastuzumab-Cys-(dendron (-L-SO1861) on SK-BR-3 cells 4 ) 4 Titration + fixed concentration of 10 pM CD71mab-saporin and control. Figure 33 (2), D) Trastuzumab-Cys-(dendron (-L-SO1861) on JIMT-1 cells 4 ) 4 Titration + fixed concentration of 10 pM CD71mab-saporin and control.

[0230] Figure 34 .2- Target 2-component comparison with T-DM1. The therapeutic combination according to the present invention can effectively kill trastuzumab-saporin + 75nM cetuximab-(Cys-L-SO1861) 3,9 Killing A431 cells (EGFR ++ / HER2 + / - ), but T-DM1+75nM cetuximab-(Cys-L-SO1861) 3,9 Titration of trastuzumab-emtansine is ineffective at such low toxin concentrations. T-DM1 is a trastuzumab-emtansine toxin with ~3.5 emtansine (DM1) molecules per antibody.

[0231] Figure 35 (1)-(2): Control treatment for all cell lines. AD) Cell viability when treated with trastuzumab (Figure 35 (1), A), cetuximab (Figure 35 (1), B), T-DM1 (Figure 35 (2), C) free toxins: saporin and oleanolic acid (Figure 35 (2), D) or saporin conjugated to non-cell-bound IgG (D) with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474.

[0232] Figure 36 (1)-(2). 2-Target 2-Component. By the therapeutic combination according to the present invention, in A431 cells (EGFR +++ / HER2 + / - )(Figure 36(1), A) and CaSKi cells (EGFR ++ / HER2 + / - )(Figure 36(1), B) and A2058 cells (EGFR - / HER2 + / - ) EGFR / CD71 and EGFR / HER2 targeted cell killing in A431 cells (A), CaSKi cells (B) and A2058 cells (Figure 36 (2), C) Cetuximab-(Cys-L-QSmix) 4,1 Titration + fixed concentration of 10 pM trastuzumab-saporin or 10 pM CD71mab-saporin and control. QSmix is ​​a mixture of saponins from Quillaja saponaria extract. The legends for Figure 36A and Figure 36B are presented next to Figure 36B.

[0233] Figure 37 : 2-Target 2-Component Concept: mAb1-SO1861 + mAb2-Protein Toxin. SO1861 and the toxin (ribosome-inactivating protein) are each conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb2-Protein Toxin bind to their corresponding cell surface receptors, 2) Receptor-mediated endocytosis of both conjugates occurs, 3) At low endolysosomal pH and appropriate concentrations, SO1861 becomes active, allowing endolysosomal escape, 4) Toxin release into the cytoplasm occurs, and 5) the toxin induces cell death.

[0234] Figure 38: 2-Target 2-Component Concept: mAb1-SO1861 + mAb2-BNA Oligonucleotide. SO1861 and the antisense BNA oligonucleotide are each conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb2-BNA oligonucleotide bind to their corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) BNA oligonucleotide release into the cytoplasm occurs, and 5) target gene silencing occurs.

[0235] Figure 39 :2-target 2-component concept: mAb1-(scaffold (-SO1861) n ) n +mAb2-Protein toxin.Dendron (-SO1861) n and protein toxins (ribosome inactivating proteins) were each conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb1-(dendron (-SO1861) 4 ) 1 ) and mAb2-protein toxins are conjugated to their corresponding cell surface receptors, 2) receptor-mediated endocytosis of the two conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active to enable endolysosomal escape, 4) toxin release into the cytoplasm occurs, and 5) toxin induces cell death.

[0236] Figure 40 (1)-(2). Tumor-targeted protein toxin delivery results in tumor volume reduction and tumor growth inhibition in tumor-bearing mice. Figure 40 (1), A) Cetuximab-(Cys-L-SO1861) in A431 tumor-bearing mice compared to controls. 3,9 (Lys-S-caryophyllene) 2 Dose escalation (intraperitoneal, ip) showed a decrease in tumor volume. Figure 40 (2), B, C) Cetuximab-(Cys-L-SO1861) in A431 tumor-bearing mice compared with controls. 3,9 (Lys-L-caryophyllene) 2 Dose escalation (intraperitoneal, ip (B) or intravenous, iv (C)) in A431 tumor-bearing mice showed reduced tumor growth.

[0237] Figure 41 Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg of cetuximab-(Cys-L-SO1861) in A431 tumor-bearing mice compared to controls. 3,9 (Lys-L-HSP27BNA) 1,8Shown to induce potent tumor-targeted gene silencing.

[0238] Figure 42 Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg of cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27 BNA) in A431 tumor-bearing mice showed significant differences compared to the control group. 3,7 Shown to induce potent tumor-targeted gene silencing.

[0239] Figure 43 (1)-(4): HER2 or EGFR targeted protein toxin delivery and cell killing in cancer cells according to the present invention. Figure 43(1)-Figure 43(2) , A, B) in SK-BR-3 cells (HER2 ++ ) and MDA-MB-468 cells (HER2 - ) on trastuzumab-(Cys-L-SO1861) 3,8 (Lys-L-caryophyllene) 1,7 or trastuzumab-(Cys-L-SO1861) 3,8 (Lys-S-caryophyllene) 1,7 treatment and control. Figure 43(3)-Figure 43(4) , C, D) in A431 cells (EGFR ++ ) and A2058 cells (EGFR - ) on Cetuximab-(Cys-L-SO1861) 3,8 (Lys-L-caryophyllene) 1,7 or cetuximab-(Cys-L-SO1861) 3,8 (Lys-S-caryophyllene) 1,7 Figures 43 A and B have the same legend and are next to Figure 43 B. Figures 43 C and D have the same legend and are next to Figure 43 D.

[0240] Figure 44(1)-(2): EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing in cancer cells according to the present invention. Figure 44(1)-Figure 44(2) , A, B) in A431 cells (EGFR ++ ) and A2058 cells (EGFR - ) on Cetuximab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 1,7 Figure 44A and B have the same legend, next to Figure 44B.

[0241] Figure 45: According to the present invention, HER2-targeted antisense BNA oligonucleotide delivery and gene silencing in cancer cells. Trastuzumab-(Cys-L-SO1861) on SK-BR-3 cells (HER2++) 3,8 (Lys-L-HSP27BNA) 3,5 treatment and control.

[0242] Figure 46 : According to the present invention, EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing in cancer cells. A, B) In A431 cells (EGFR ++ ) and A2058 cells (EGFR - ) on Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 3,7 treatment and control.

[0243] Figure 47 :(S)n–(L)(E) Concept:mAb-(SO1861) n (Protein toxins) n SO1861 at a cysteine ​​residue (Cys) and a protein toxin (ribosome-inactivating protein) at a lysine residue are both conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-L-SO1861) 4 (Lys-protein toxins) 2 Upon binding to its corresponding cell surface receptor, 2) receptor-mediated endocytosis occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active to allow endolysosomal escape, 4) toxin release into the cytoplasm occurs, and 5) the toxin induces cell death.

[0244] Figure 48 :(S)n–(L)(E) Concept:mAb-(SO1861) n (Antisense BNA Oligonucleotide) n SO1861 on a cysteine ​​residue (Cys) and antisense BNA oligonucleotides on a lysine residue were both conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-SO1861)4(Lys-BNA oligonucleotide) 2 1) conjugation to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of the two conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active to enable endolysosomal escape, 4) release of the BNA oligonucleotide into the cytoplasm occurs, and 5) target gene silencing is induced.

[0245] Figure 49:(S)n–(L)(E) Concept: mAb-(SO1861-Scaffold-Antisense BNA Oligonucleotide) n (SO1861-Trifunctional Linker-BNA Oligonucleotide) n Conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb-(SO1861-trifunctional linker-BNA oligonucleotide) 4 1) binding to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of the two conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active allowing endolysosomal escape, 4) release of the BNA oligonucleotide into the cytoplasm occurs, and 5) target gene silencing is induced.

[0246] Figure 50 .Antibody-SO1861 conjugation procedure. Shown is a connection reaction in which four parts of the plant-derived saponin SO1861 are connected to four cysteines in the antibody light chain. First, the disulfide bonds in IgG are destroyed under the influence of exposure to TCEP (tris(2-carboxyethyl)phosphine); second, the saponin SO1861 containing a chemical linker bound thereto is added together with trifluoroacetic acid, and the four saponin parts are connected to IgG. In order to produce a cleavable "ready-to-use conjugate" saponin, the aldehyde group of SO1861 reacts with the EMCH (ε-maleimidocaproic acid hydrazide) linker. The hydrazide group of EMCH forms an acid-cleavable hydrazone bond with the aldehyde group of SO1861. At the same time, the EMCH linker presents a maleimide group with thiol (sulfhydryl) reactivity and can therefore be conjugated to the thiol (i.e., ligand portion) of IgG. Therefore, an endosomal escape enhancing conjugate of the present invention is provided, and / or a first binding molecule of the present invention is provided.

[0247] Figure 51 .Synthesis of SO1861-EMCH.

[0248] Figure 52 (1)-(6).Dendrite-(-L-SO1861) 4 synthesis.

[0249] Figure 53 (1)-(5).Dendrite-(-L-SO1861) 8 synthesis.

[0250] Figure 54 (1)-(4). Synthesis of SO181-L-trifunctional linker-L-HSP27BNA.

[0251] Figure 55 .HSP27BNA-Dendron-(-L-SO1861) 4 synthesis.

[0252] Figure 56 Dendron primitive (NEM) 4 synthesis.

[0253] Figure 57 : A scaffold precursor with four amino groups for saponin attachment and an azide group for click chemistry.

[0254] Figure 58 Evidence of saponin conjugation to the model scaffold. The inset shows the theoretically expected peak and intensity distribution of the conjugated saponin. Experimental data obtained by LC-MS / ESI-MS show a near-identical peak at m / z 758-760 Da, confirming successful saponin conjugation.

[0255] Figure 59 :Use the cytotoxicity assay of the toxin oleanolic acid-epidermal growth factor (oleanolic acid-EGF) of targeting.Untreated cells are normalized to 1.Polymeric structure (Pentrimer) has no influence on cell viability, no matter in the presence or absence of oleanolic acid-EGF and saponin (SA1641), this shows that polymeric structure does not have intrinsic cytotoxicity.The activity of clickable targeted toxin (oleanolic acid-EGF-alkyne) significantly reduces, and this is the result of toxin modification, but does not have any relationship with support.Functionalized polymeric structure has the same activity as the targeted toxin that does not click, and this shows that the functionalization of support can not damage the activity of effector molecule.In the presence and absence of polymeric structure, the effect of saponin is identical, and this shows that polymeric structure can not damage the efficacy of saponin in the two-component system.

[0256] Figure 60 (1)-(2): H-NMR spectra of (A) SO1861 and (B) SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide). (Figure 60 (1), A) 9.43 ppm (H a ) corresponds to the aldehyde proton of SO1861. (Figure 60(2), B) 6.79 ppm (H c ) corresponds to the maleimide protons of SO1861-EMCH, while the peak at 7.68 ppm (H b The peak at 9.43 ppm corresponds to the hydrazone proton. The absence of a signal at 9.43 ppm indicates that quantitative conversion of the aldehyde group has occurred.

[0257] Figure 61 :MALDI-TOF-MS spectra of (A) SO1861-EMCH and (B) SO1861-EMCH-mercaptoethanol. (A) RP mode: m / z 2124 Da ([M+K] + , saponin-EMCH), m / z 2109Da ([M+K] +, SO1861-EMCH), m / z is 2094Da ([M+Na] + , SO1861-EMCH). (B) RP mode: m / z 2193 Da ([M+K] + , saponin-EMCH-mercaptoethanol), m / z 2185Da ([M+K] + , SO1861-EMCH-mercaptoethanol), m / z 2170Da ([M+Na] + , SO1861-EMCH-mercaptoethanol).

[0258] Figure 62 The structure of SO1861 features prominent chemical groups used to conjugate saponins that enhance endosomal escape into a multimeric structure. The highlighted groups are aldehydes (black circles), carboxylic acids (dashed circles), olefins (dashed pentagons), and alcohols (dashed boxes). The aldehyde group (arrow) is the most suitable group for chemoselective and reversible conjugation reactions.

[0259] Figure 63 : Strategies to generate (A) stable and (B) cleavable “ready-to-use conjugates” of the endosomal escape enhancer saponin.

[0260] Figure 64 :Hydrolysis of SO1861-EMCH hydrazone bond under acidic conditions.

[0261] Figure 65 : Structure of SO1861-EMCH. (A) Standard molecular structure, and (B) 3D model. The maleimide group is marked with a circle.

[0262] Figures 66 (1)-(3): (Figure 66 (1), A) Synthesis scheme of SO1861-EMCH. (Figure 66 (2), B) MALDI-TOF-MS spectra of SO1861 (m / z of 1861 Da) and (Figure 66 (3), C) SO1861-EMCH (m / z of 2068 Da) in negative reflectance mode. TFA: trifluoroacetic acid, rt: room temperature, h: hours and MW: molecular weight.

[0263] Figure 67 (1)-(2): MALDI-TOF-MS spectra of SO1861-EMCH before (Figure 67 (1), A) and after (Figure 67 (2), B) hydrolysis in pH 3 HCl solution.

[0264] Figure 68 : Reaction scheme for conjugation of SO1861-EMCH to any amine-containing polymer structure.

[0265] Figure 69: MALDI-TOF-MS spectra of (A) BSA-SO1861 (m / z 70.0 kDa, 72.1 kDa, 74.2 kDa) and (B) BSA (m / z 66.6 kDa).

[0266] Figure 70 : (A) SO1861-EMCH and (B) SO1861-HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) conjugated to cyanine 3 dye-labeled polyamidoamine (PAMAM) G5 dendrimers.

[0267] Figure 71 (1)-(2): (Figure 71 (1), A) Cy3-PAMAM, ( Figure 71(1)-Figure 71(2) , B-D) MALDI-TOF-MS spectra of Cy3-PAMAM-SO1861 with increasing SO1861-EMCH feed equivalents from (B) top to (D) bottom. (B) corresponds to Cy3-PAMAM-SO1861 with 5 SO1861 attached per PAMAM, (C) corresponds to Cy3-PAMAM-SO1861 with 13 SO1861 attached per PAMAM, and (D) corresponds to Cy3-PAMAM-SO1861 with 51 SO1861 attached per PAMAM.

[0268] Figure 72 : MALDI-TOF-MS spectra of (A) Cy3-PAMAM-SO1861 with 5 equivalents of feed SO1861-EMCH and (B) Cy3-PAMAM-SO1861 with 30 equivalents of feed SO1861-EMCH.

[0269] Figure 73 : MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 (NC=stable bond (“non-cleavable”)).

[0270] Figure 74 (1)-(2): (Figure 74 (1), A) Reaction scheme and (Figure 74 (2), B) Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO), (Figure 74 (2), C) Cy3-PAMAM-(SO1861)5-DBCO and (Figure 74 (2), D) Cy3-PAMAM-(SO1861) 27 MALDI-TOF-MS spectrum of -DBCO.

[0271] Figure 75 (1)-(2): Reaction schemes of (Figure 75 (1), A) oleanolic acid-EGF-Alexa488 and (Figure 75 (1), B) oleanolic acid-EGF-Alexa488-SS-PEG-N3. (Figure 75 (2), C) oleanolic acid-EGF, (Figure 75 (2), D) oleanolic acid-EGF-Alexa488 and (Figure 75 (2), E) oleanolic acid-EGF-Alexa488-SS-PEG-N3; Alexa488: MALDI-TOF-MS spectra of Alexa Fluor 488 dye.

[0272] Figure 76 (1)-(2): Reaction schemes of (Figure 76 (1), A) oleanolic acid-Alexa488 and (Figure 76 (1), B) oleanolic acid-Alexa488-SS-PEG-N3. (Figure 76 (2), C) oleanolic acid, (Figure 76 (2), D) oleanolic acid-Alexa488 and (Figure 76 (2), E) oleanolic acid-Alexa488-SS-PEG-N3; Alexa488: MALDI-TOF-MS spectra of Alexa Fluor 488 dye.

[0273] Figure 77 : Fluorescence image of SDS-PAGE gel performed on the VersaDoc imaging system. M = marker, P = Cy3-PAMAM-(SO1861) 27 -DBCO, D = oleanolic acid-EGF-Alexa488-SS-PEG-N3, C1 = Cy3-PAMAM-(SO1861)5-oleanolic acid-EGF-Alexa488, C2 = Cy3-PAMAM-NC-SO1861-oleanolic acid-EGF-Alexa488, and C3 = Cy3-PAMAM-(SO1861) 27 -Caryophyllin-EGF-Alexa488.

[0274] Figure 78 : (A) Synthesis scheme of Cy3-PAMAM-NC-SO1861 via reductive amination. (B and C) Respective MALDI-TOF-MS spectra.

[0275] Figure 79 : Reaction scheme for the formation of poly(SO1861) using SO1861-EMCH as monomer, APS / TMEDA system as polymerization initiator and aminopropanethiol as free radical quencher.

[0276] Figure 80: MALDI-TOF-MS spectra of poly (SO1861) reaction batches. (A) SO1861-EMCH at 60 ° C, (B) SO1861-EMCH + 11 at 60 ° C -3 Equivalent APS, (C) SO1861-EMCH+11 at 60℃ -3 Equivalent APS / TMEDA.

[0277] Figure 81 DNA method. DNA origami principles were used to generate a DNA-based scaffold capable of conjugating and releasing glycoside molecules. Furthermore, one of the DNA strands acquired a click chemistry moiety, which can be used to conjugate a targeted toxin to form a functionalized scaffold. bp: base pair.

[0278] Figure 82 : Poly (peptide-SO1861) method. Use a peptide sequence that can conjugate and release glycoside molecules and can react with itself to form a poly (peptide-SO1861) construct. The poly (peptide) chain end can be further modified with a click chemistry moiety (e.g., BCN-NHS linker) that can be used for conjugation to a toxin.

[0279] Figure 83 . MALDI-TOF-MS spectra of (A) native peptide, (B) peptide-SO1861 conjugate.

[0280] Figure 84 .Molecular structure of amino-protected G4-dendron.

[0281] Figure 85 .Synthetic schemes for generating dendron-based scaffolds and functional scaffolds.

[0282] Figure 86 (1)-(2). (Figure 86 (1), A) Reaction scheme for partial dye labeling and deprotection of G4-dendron. (Figure 86 (2), B) MALDI-TOF-MS spectra of deprotected and partially dye-labeled G4-dendron.

[0283] Figure 87 MALDI-TOF-MS spectra of G4-dendron-SO1861 scaffolds with (A) 22 feed equivalents of SO1861-EMCH, (B) 10 feed equivalents of SO1861-EMCH, and (C) 3 feed equivalents of SO1861-EMCH.

[0284] Figure 88 (1)-(2). Cell viability curves of HeLa cells treated with (Figure 88 (1), A) EGFR cell surface expression as determined by FACS analysis of HeLa cells (Figure 88 (1), B, see Table 19), SO1861 + oleanolic acid-EGF (Dia-EGF), SO1861 + oleanolic acid-EGF + 500nM chloroquine, SO1861 + oleanolic acid-EGF + 500nM The cell viability of HeLa cells treated with PAMAM, SO1861 + oleanol-EGF + 667nM dendron (Figure 88 (2), C) was significantly decreased after treatment with SO1861 + oleanol-EFG, SO1861 + oleanol-EGF + 500nM chloroquine, SO1861 + oleanol-EGF + 500nM PAMAM, SO1861 + oleanol-EGF + 500nM PAMAM-(SH) 16 、SO1861+oleanolic acid-EGF+500nM PAMAM-(SH) 65 、SO1861+oleanolic acid-EGF+500nM PAMAM-(SH) 108 The cell viability of HeLa cells treated with SO1861+oleanol-EGF, SO1861+oleanol-EGF+500nM chloroquine, SO1861+oleanol-EGF+500nM PAMAM, SO1861+oleanol-EGF+500nM PAMAM-(mPEG)3, SO1861+oleanol-EGF+500nM PAMAM-(mPEG)8, SO1861+oleanol-EGF+500nM PAMAM-(mPEG) 18 Cell viability of treated HeLa cells.

[0285] Figure 89 (1)-(2). (Figure 89 (1), A) Reaction scheme for the thiolation of PAMAM using the thiolation reagent 2-iminothiol. (Figure 89 (1), B) Native PAMAM, (Figure 89 (1), C) Thiolated PAMAM-(SH) 16 、(Figure 89(2), D) Thiolated PAMAM-(SH) 65 and (Figure 89(2), E) thiolated PAMAM-(SH) 108 MALDI-TOF-MS spectrum.

[0286] Figure 90 (1)-(3). (Figure 90 (1), A) Using PEGylation reagent mPEG 2k -NHS PEGylation reaction scheme of PAMAM. (Figure 90(2), B) native PAMAM, (Figure 90(2), C) PEGylated PAMAM-(mPEG2k )3、(Figure 90(3), D)PEGylated PAMAM-(mPEG 2k )8 and (Figure 90 (3), E) PEGylated PAMAM-(mPEG 2k ) 18 MALDI-TOF-MS spectrum.

[0287] Figure 91 : A basic scaffold with click chemistry functionality for attaching any desired effector molecule. The user determines the location of the click chemistry sites in the effector molecule as well as all other properties of the effector molecule, such as the choice and location of the optional ligand.

[0288] Figure 92 : Functionalized scaffolds with pre-bound effector molecules and click chemistry functionality for attachment of any desired ligand. Optionally, a pH sensitive link can be provided to release the effector molecule from the scaffold after reaching the endosome. Detailed Description of the Invention

[0290] In order for a bioactive molecule to be effective, the molecule must be able to bind to its target, for example in serum, externally on the cell surface, or internally within a cell or organelle. For example, the active portion of almost all protein-based targeted toxins must enter the cytoplasm of the target cell to mediate its target-modulating effects. In many cases, the toxins remain ineffective because (1) the targeting portion is poorly internalized and remains bound to the exterior of the cell, (2) after internalization it is recycled back to the cell surface, or (3) it is transported to the endolysosomes where it is degraded. Although these fundamental issues have been known for decades and more than 500 targeted toxins have been studied over the past few decades, these issues remain unresolved and to date, only one antibody targeting the protein toxin, pasudotox-tdfk ( AstraZeneca Pharmaceuticals LP) has been approved by the FDA for the treatment of relapsed or refractory hairy cell leukemia.

[0291] To overcome these problems, a number of strategies have been described, including methods to redirect toxins to endogenous cell membrane transport complexes of the biosynthetic pathway in the endoplasmic reticulum, and techniques to disrupt or weaken the integrity of the endosome membrane (i.e., the compartment of the endocytic pathway) to promote endosomal escape. This includes the use of lysosomal amines, carboxylate ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human, and synthetic origin, other organic molecules, and light-induced technologies. Although the efficacy of targeted toxins is generally increased by hundreds or thousands of times in cell culture, and in special cases by more than a million times, the need to co-administer endosomal escape enhancers with other substances brings new problems, including additional side effects, loss of target specificity, difficulties in determining the therapeutic window, and cell type-dependent variations.

[0292] All strategies (including physicochemical techniques) require enhancer molecules that interact more or less directly with the membrane and essentially include small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not themselves targeted to specific cells but are distributed with different kinetics than the target toxin. This is one of the main drawbacks of current methods.

[0293] The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.The embodiments of the invention described herein can be combined and operate in conjunction with each other unless expressly stated otherwise.

[0294] Although the present invention has been described in terms of several embodiments, it is expected that substitutions, modifications, permutations, and equivalents will become apparent to one of ordinary skill in the art upon reading the specification and studying the accompanying drawings and diagrams. The present invention is not limited in any way to the illustrated embodiments. Changes may be made without departing from the scope of the appended claims.

[0295] One aspect of the present invention relates to a first protein molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, the first protein molecule having at least one saponin covalently bound to an amino acid residue of the first protein molecule via at least one linker and / or via an oligomeric or multimeric scaffold or directly covalently bound to an amino acid residue of the first protein molecule. Accordingly, the present invention provides a conjugate comprising or consisting of a first protein molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein the at least one saponin is covalently bound to the first protein molecule via at least one linker and / or via an oligomeric or multimeric scaffold or directly covalently bound to an amino acid residue of the first protein molecule.

[0296] One embodiment is a first protein molecule of the invention, wherein the first binding site comprises or consists of an immunoglobulin such as an antibody, IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, a scFv, Fv, dAb, F(ab)2, an Fcab fragment, or at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, and / or comprises at least one ligand that binds to a cell surface molecule, such as EGF or a cytokine.

[0297] One embodiment is the first protein molecule of the present invention, wherein the first epitope of the first cell surface molecule is a tumor cell specific first epitope of a first tumor cell surface molecule, more preferably a tumor cell specific first epitope of a first tumor cell surface receptor specifically present on tumor cells.

[0298] One embodiment is the first protein molecule of the invention, wherein the at least one saponin is a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, and / or a saponin isolated from a species of the genus Gypsophila and / or a species of the genus Saponaria and / or a species of the genus Agrostemma and / or a species of the genus Quillaja, such as Quillaja saponaria.

[0299] One embodiment is the first protein molecule of the invention, wherein the at least one saponin is a single specific saponin or a mixture of two or more different saponins, such as one or more of the following: the saponins in Table A1 or Scheme 1, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Saponinum sylvestris saponins, Saponinum truncatum var. album), QS-18, Quil-A, Gyp1, gypsophila saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any one of their stereoisomers and / or any combination thereof, preferably the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or a saponin having a glycoside core, a Gal-(1-hydroxy-2-hydroxy-1-oxo-1-yl)-4-nitropropanecarboxylate residue at the C-3β-OH group.→ 2)-[Xyl-(1 → 3)]-GlcA carbohydrate substituent and Glc-(1 → 3)-Xyl-(1 → 4)-Rha-(1 → 2)-[Xyl-(1 → 3)-4-OAc-Qui-(1 → 4)]-Fuc carbohydrate substituted saponins, and / or 3-O-β-D-galactopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)]-β-D-glucopyranosyl salpingic acid 28-O-β-D-glucopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-[β-D-xylopyranosyl-(1 → 3)-4OAc-β-D-pyranoquinolyl-(1 → 4)]-β-D-fucopyranoside, more preferably the saponin is SO1861 and / or QS-21.

[0300] One embodiment is a first protein molecule of the invention, wherein the at least one saponin is a disaccharide chain saponin having a molecular weight of at least 1,500 Daltons and comprising an oleanolic 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, the first branched carbohydrate side chain optionally containing glucuronic acid, wherein the saponin contains an ester group and a second branched carbohydrate side chain at the C-28 position, the second branched carbohydrate chain preferably containing glucuronic acid. comprising at least four carbohydrate units, which optionally contain at least one acetyl residue (e.g., two acetyl residues), and / or optionally comprise deoxy carbohydrates and / or optionally comprise isorhamnose and / or optionally comprise glucose and / or optionally comprise 4-methoxycinnamic acid and / or optionally comprise 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprise 5-O-[5-O-Rha-(1 →2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyloctanoic acid, or wherein the at least one saponin is QS-21 or any one or more of the following: QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A

[0301] One embodiment is a first protein molecule according to the invention, wherein said at least one saponin is a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23, wherein said at least one saponin is covalently coupled to an amino acid residue of said first protein molecule via the aldehyde function in said saponin, preferably said aldehyde function in position C-23, preferably via at least one linker, more preferably via at least one cleavable linker, wherein said amino acid residue is preferably selected from cysteine ​​and lysine.

[0302] One embodiment is a first protein molecule according to the invention, wherein said at least one saponin is a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of said saponin, wherein said at least one saponin is covalently coupled to an amino acid residue of said first protein molecule via the glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of said saponin, preferably via at least one linker, wherein said amino acid residue is preferably chosen from cysteine ​​and lysine.

[0303] One embodiment is a first protein molecule of the invention, wherein the aldehyde functional group in position C-23 of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled to a sulfhydryl group (e.g., a sulfhydryl group of cysteine) in the first protein molecule via a thioether bond.

[0304] One embodiment is a first protein molecule of the invention, wherein the glucuronic acid functionality in the carbohydrate substituent at the C-3β-OH group of the at least one saponin is covalently coupled to a linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidohexafluorophosphate, which is covalently coupled to an amine group in the first protein molecule (such as an amine group of lysine or the N-terminus of the first protein molecule) via an amide bond.

[0305] One embodiment is the first protein molecule of the present invention, wherein the first epitope of the first cell surface molecule bound by the first binding site of the first protein molecule is a tumor cell-specific first epitope of the tumor cell-specific receptor, and the tumor cell-specific receptor is preferably selected from the group consisting of: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, vascular integrin α-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.

[0306] One embodiment is the first protein molecule of the present invention, wherein the tumor cell-specific first epitope, the first tumor cell surface molecule or the first tumor cell-specific receptor is the first epitope or the first molecule or the first receptor that is internalized by the tumor cell after the first protein molecule of any one of claims 1 to 11 binds to the first epitope or the first molecule or the first receptor, and wherein preferably, when the first protein molecule binds to the cell surface molecule comprising the first epitope, the tumor cell surface molecule or the tumor cell-specific receptor, it undergoes tumor cell receptor-mediated internalization (e.g., by endocytosis) or tumor cell surface molecule-mediated internalization (e.g., by endocytosis).

[0307] One embodiment is a first protein molecule of the present invention, wherein the first binding site of the first protein molecule comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mocetuzumab, potuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof, preferably at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof.

[0308] One aspect of the invention relates to a therapeutic combination, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition comprising a first protein molecule described herein and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second protein molecule different from the first protein molecule, the second protein molecule comprising a second binding site for binding to a second epitope of the second cell surface molecule different from the first cell surface molecule, and comprising an effector moiety, the second pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, wherein the second epitope is different from the first epitope.

[0309] One embodiment is a therapeutic combination of the present invention, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition of the present invention, which comprises a first protein molecule of the present invention, wherein the first epitope on the first cell surface molecule is a tumor cell-specific first epitope on a first tumor cell-specific surface molecule, preferably a tumor cell-specific first epitope specifically present on a first cell surface receptor of a tumor cell; and (b) a second pharmaceutical composition of the present invention, wherein the second cell surface molecule is a second tumor cell-specific surface molecule different from the first tumor cell-specific surface molecule, preferably the second cell surface receptor specifically present on a tumor cell is different from the first cell surface receptor specifically present on the tumor cell, and wherein the second epitope is a tumor cell-specific second epitope.

[0310] One embodiment is a therapeutic combination of the invention, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition of the invention, comprising a first protein molecule of the invention and comprising a first binding site for binding to a first epitope on a first cell surface molecule, optionally further comprising a pharmaceutically acceptable excipient; and (b) a third pharmaceutical composition comprising a third protein molecule, said third protein molecule comprising said first binding site for binding to a first epitope on a cell surface molecule of (a) and an effector portion, optionally further comprising a pharmaceutically acceptable excipient, wherein the first binding site of the first protein molecule and the first binding site of the third protein molecule are the same, and wherein the first cell surface molecule and the first epitope on the first cell surface molecule to which the first protein molecule can bind are the same as the first epitope on the first cell surface molecule and the first epitope on the first cell surface molecule to which the third protein molecule can bind.

[0311] One embodiment is a therapeutic combination of the present invention, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition as described herein; and (b) a third pharmaceutical composition as described herein, wherein the first cell surface molecule is expressed on the surface of a tumor cell, and preferably the first cell surface molecule is a tumor cell-specific surface molecule, and wherein preferably the first epitope is a first tumor cell-specific epitope.

[0312] One embodiment is a first protein molecule of the invention or a therapeutic combination of the invention, wherein said first binding site for binding to said first epitope on said first cell surface molecule is a binding site for a tumor cell-specific first epitope on a first cell surface receptor specifically present on tumor cells.

[0313] The inventors of the present invention have determined that the therapeutic window of an antibody drug conjugate (e.g., the second or third protein molecule in the second or third pharmaceutical composition of the present invention) is increased when administered to a tumor-bearing mammal (mouse) that has also been administered a first pharmaceutical composition. The first protein molecule has at least one glycoside (e.g., a saponin) bound thereto, preferably covalently, and more preferably bound thereto via a cleavable linker. The saponin enhances the efficacy of the effector moiety bound to the second and third protein molecules by enhancing the endosomal escape of the effector moiety into the cytoplasm where the desired effector moiety is active. This approach has established a therapeutic effect at lower doses than conventional doses of the ADC (i.e., the second or third protein molecule) by bringing the saponin to, at, and / or within the target cell, under the influence of the presence of the first protein molecule comprising the saponin. Target cells are, for example, diseased cells (e.g., tumor cells) or autoimmune cells or B cells associated with B cell diseases. The effector moiety is, for example, a toxin as part of an ADC according to the present invention or an oligonucleotide (BNA) as part of an AOC according to the present invention.

[0314] One embodiment is a therapeutic combination according to the invention, wherein the second binding site of the second protein molecule and / or the first binding site of the third protein molecule comprises or consists of an immunoglobulin such as an antibody, IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, a scFv, Fv, dAb, F(ab)2, an Fcab fragment, or at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, and / or comprises at least one ligand that binds to a cell surface molecule, such as EGF or a cytokine.

[0315] One embodiment is a therapeutic combination of the invention comprising a second pharmaceutical composition, wherein the second binding site of the second protein molecule for binding to the second epitope is a second binding site for a tumor cell-specific second epitope specifically present on a second cell surface receptor of the tumor cell, wherein the second binding site is different from the first binding site.

[0316] By targeting (two) different cell surface molecules with the first and second protein molecules, delivery of the saponin and effector moiety to and within the cytoplasm of the very same target cell exposed to two different cell surface molecules on the cell surface is improved and more specific than such exposure to only the second protein molecule (e.g., ADC or AOC) in the absence of the cell-targeting saponin (the first protein molecule). When considering (adjacent) healthy cells of the patient, the abnormal cells are selected for isolation targeting by the binding site of the first protein molecule and the binding site of the second protein molecule, wherein the binding sites are different and wherein the epitopes bound by the first and second protein molecules (e.g., two different receptors, ideally having a first epitope and a second epitope on the first cell surface molecule and the second cell surface molecule, respectively) are different and located in or on different kinds and types of cell surface molecules to a high degree (i.e., higher expression of the two distinct and different cell surface molecules on the targeted cells (e.g., tumor cells or autoimmune cells) than on non-targeted cells (e.g., healthy cells), and / or the first and second cell surface molecules are specifically exposed. Preferably, the two cell surface molecules targeted by the first and second binding sites are relatively highly and / or specifically expressed on the targeted (disease, tumor) cells compared to healthy cells. One embodiment is a pharmaceutical composition wherein at least one of the first and second binding sites, and therefore at least one of the first and second cell surface molecules (e.g., first and second tumor cell receptors), is specifically expressed or expressed to a relatively higher degree when compared to the expression of the first cell surface molecule and / or second cell surface molecule on the surface of healthy (adjacent) cells. Thus, the first epitope or the second epitope, preferably the first epitope and the second epitope on the targeted cell surface molecule, is ideally unique to the targeted diseased cell and is at least specifically present and exposed on the surface of the targeted cell. The first and second protein molecules, after binding to their respective first and second epitopes on the targeted cell, are internalized by the complex of the first protein molecule and the first target cell surface molecule and the second protein molecule and the second target cell surface molecule. Because the first and second protein molecules must enter the same target cell via binding interactions with two different cell surface molecules that are both expressed to a sufficient extent or uniquely expressed on the targeted cell, accumulation of therapeutically effective amounts of the first and second protein molecules within the target cell is only possible and will occur if both of the two distinct targeted cell surface molecules are above a certain minimum expression threshold, when compared to healthy cells that should not be targeted.At the same time, the fact that the effector moiety bound to the second protein molecule is only able to exert its intracellular activity (e.g., cytotoxicity or gene silencing) in the presence of the first protein molecule having the covalently bound saponin, also provides protection of the effector moiety against negative and undesirable side effects on healthy cells and healthy tissues that are not targeted and affected by the effector moiety when both the first and second protein 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, when at least one of the first and second cell surface molecules is expressed at sufficiently low levels in healthy cells, and preferably when both the first and second targeted cell surface molecules are expressed at sufficiently low levels in healthy cells. That is, with respect to the first and second cell surface molecules, in the case of sufficiently low expression or even absence of exposed first and second cell surface molecules, and in the case of at least the first or second cell surface molecule to which the first and second binding sites of the first and second protein molecules, respectively, bind, does not ideally allow both the first and second protein molecules to enter (non-targeted) healthy cells in amounts that would conceptually lead to endosomal escape of the effector moiety under the influence of the saponin bound to the first protein molecule. Because the ADC or AOC can be used at a lower dose than when the first protein molecule is not added to the therapeutic regimen, it is contemplated that the ADC or AOC can target and kill target diseased cells (such as tumor cells and autoimmune cells) at a low level with a lower risk of undesirable side effects occurring in healthy cells.

[0317] One embodiment is a first protein molecule of the invention or a therapeutic combination of the invention comprising a second pharmaceutical composition, wherein the first and second protein molecules comprise first and second binding sites, respectively, for binding to first and second tumor cell-specific epitopes on first and second tumor cell-specific receptors, respectively, said receptors being different and present on the same tumor cell, wherein the first and second binding sites are different, and the first and second tumor cell-specific epitopes are different.

[0318] One embodiment is a first protein molecule of the invention or a therapeutic combination of the invention comprising a third pharmaceutical composition, wherein the first and third protein molecules comprise the same first binding site for binding to a first tumor cell specific epitope on a first tumor cell specific receptor.

[0319] One embodiment is a first protein molecule of the invention or a therapeutic combination of the invention comprising a second pharmaceutical composition, wherein the first receptor and / or the second receptor is selected from the group consisting of: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, vascular integrin αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected from: CD71, EGFR, HER2.

[0320] One embodiment is a first protein molecule of the invention or a therapeutic combination of the invention comprising a second pharmaceutical composition, wherein the first and second tumor cell-specific receptors are internalized by the tumor cell upon binding to the first protein molecule of the invention and / or upon binding to the second protein molecule of the invention when the therapeutic combination comprises the second pharmaceutical composition, and wherein preferably the binding of the first protein molecule and / or the second protein molecule to the first and second tumor cell-specific receptors results in tumor cell receptor-mediated internalization (e.g., by endocytosis) of the complex of the first protein molecule and the first tumor cell-specific receptor and the complex of the second protein molecule and the second tumor cell-specific receptor, respectively.

[0321] One embodiment is a therapeutic combination comprising a third medicament according to the invention or a first pharmaceutical composition according to the invention, wherein a first tumor cell receptor, preferably a first tumor cell-specific receptor, is internalized by the tumor cell after binding to the first protein molecule according to the invention and / or after binding to the third protein molecule according to the invention, and wherein preferably, binding of the first protein molecule and / or the third protein molecule to the first tumor cell receptor (such as the first tumor cell-specific receptor) is followed by tumor cell receptor-mediated internalization (e.g., by endocytosis) of the complex of the first protein molecule and the first tumor cell receptor and of the complex of the third protein molecule and the first tumor cell receptor.

[0322] Synchronization is the missing link between successful mouse delivery strategies and their application in humans. In fact, the inventors of the present invention have established in a series of in vivo mouse tumor models that, compared with control animals not treated with ADC and free saponin, administering a certain dose of free saponin and a certain dose of ADC (according to the second or third protein molecule of the present invention) to mice, respectively, did not produce any desired anti-tumor effect (such as tumor growth delay, tumor regression, reduction and slowing of tumor growth). Free saponin was administered using various routes of administration and at different time points using free saponin administration compared to the moment of administration of the ADC (administering free saponin before, during and after administration of the ADC). The ADC tested in the in vivo tumor model was cetuximab-oleanol (containing free SO1861) or trastuzumab-saporin (containing free SO1861). Changing the dose of free saponin did not provide effective anti-tumor activity. The dose of the ADC mentioned by itself did not produce any beneficial anti-tumor effect on tumor-bearing animals. Surprisingly, the present inventors have now determined that beneficial anti-tumor activity in various in vitro mammalian cell-based bioassays and / or various in vivo animal tumor models can be achieved by treating animals with a conjugate according to the invention, optionally comprising a scaffold according to the invention, i.e., a combination of a first and a second or a first and a third protein molecule according to the invention. For example, the scaffold is a trifunctional linker that covalently binds a saponin (e.g., SO1861, QS-21) via a cleavable or non-cleavable bond and / or covalently binds an effector moiety (e.g., oleanolic acid, silencing BNA (HSP27)) via a non-cleavable or cleavable bond, and / or covalently binds a monoclonal antibody (e.g., cetuximab, trastuzumab, OKT-9), or the scaffold is a dendron, such as, for example, a dendron to which four moieties (e.g., four saponin molecules) can be bound, or a dendron for binding, for example, two saponins and two effector molecules, said dendron comprising chemical groups for (covalent) coupling to a ligand or antibody or fragment or domain thereof. Reference is made to the Examples section, which illustrates that various of these scaffolds according to the invention show anti-tumor cell activity in vivo and / or in vitro when considering cytotoxicity exerted by, for example, protein toxins or when considering gene silencing in tumor cells.

[0323] Without wishing to be bound by any theory, in view of the observed failures when treating tumor-bearing animals with ADCs and free saponins, it is preferred that at least one saponin and an effector moiety (preferably a toxin or oligonucleotide) be present simultaneously in a compartment or vesicle of the endocytic pathway of the target cell (e.g., a tumor cell or an autoimmune cell). According to the inventors' efforts, it has not been beneficial to synchronize the presence of the molecules in the late endosome for ADCs and free saponins to achieve a synergistic effect in vivo. In one aspect, the present invention preferably addresses at least the following issues regarding combining an effector moiety contained in a second protein molecule with a saponin contained in a first protein molecule: without wishing to be bound by any theory, for example, endosomal escape activity requires a single rational chemical group within the saponin that can be used for (covalent), particularly single and cleavable, retainable coupling. In addition to the use of saponins in vaccination protocols, this known limitation is likely why saponins have not been used in combination with pharmaceutically active substances in clinical studies, where the use of immunopotentiating adjuvant substances is implied, despite the fact that the significant endosomal escape enhancer effects of the saponins listed in Table A1 and Scheme I have been known for over 10 years. For example, providing a first protein molecule of the present invention with a covalent conjugation scaffold at least partially addresses these difficulties. Surprisingly, saponins, which have previously been used for their immunopotentiating activity in the context of vaccination involving saponins as adjuvant components, are now also suitable for (covalent) coupling to the first protein molecule of the present invention for in vitro and in vivo anti-tumor activity.

[0324] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effects. Some effectors (such as, for example, Pseudomonas exotoxin) are redirected to other organelles before the "late endosomal stage" and therefore generally do not benefit from coupling to a second protein molecule according to the present invention. However, such toxins may be suitable for use in the present invention, for example, by deleting the signal peptide responsible for rerouting. In particular, toxins that are very toxic and require only a single molecule to escape the endosome to kill the cell may be modified to be less effective. Cell-killing toxins are preferably used if at least 2, more preferably at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and most preferably at least 100 toxin molecules escape the endosome. It is further preferred that the second protein molecule of the present invention comprises a covalently conjugated functionalized scaffold, i.e., a scaffold comprising a covalently bound effector moiety, for targeting the scaffold comprising the bound effector moiety to a target cell (e.g., a tumor cell or autoimmune cell). In addition, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0325] The term "ligand" as used in the present invention has its general meaning and preferably means a molecule or structure that is capable of binding to another molecule or structure on the cell surface of a target cell, wherein the molecule or structure on the cell surface can be endocytosed and preferably is not present 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. For example, this is the case with 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 in a carrier molecule is one possibility for producing a targeted toxin. A toxin that is toxic only in target cells, because it only interferes with processes occurring in target cells, can also be considered a targeted toxin (because in off-target cells, it cannot exert its toxic effect, such as apoptin). Preferably, the targeted toxin is a toxin combined with a ligand or, for example, a monoclonal antibody so that it is active in target cells rather than in off-target cells (because it only binds to and is endocytosed by the target cells). In a functionalized scaffold comprising a carrier molecule comprising a ligand and an effector moiety (i.e., a second or third protein molecule), 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 comprised by a conjugate of the first protein molecule and a saponin, mediates the endosome escape of the effector moiety. The saponin is typically a saponin listed in Table A1 and Scheme 1, and preferably the saponin is SO1861 and / or QS-21, and / or SA1641 and / or GE1741.

[0326] Preferably, upon endocytosis, the effector moiety bound to the second or third protein molecule, for example, is separated from the second or third protein molecule (e.g., an antibody), which is enhanced by the saponin binding to the first protein molecule. This can be achieved by breaking a cleavable bond, for example, under acidic, reducing, enzymatic, or light-induced conditions.

[0327] One embodiment is a first protein molecule of the invention and / or a therapeutic combination of the invention comprising a 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, and preferably comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mocetuzumab, potuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and the antibody of Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is different from the second binding site of the second protein molecule.

[0328] One embodiment is a therapeutic combination comprising the third pharmaceutical composition of the present invention or the first pharmaceutical composition according to the present invention, when included in a therapeutic combination comprising the third pharmaceutical composition, wherein said first binding site of said first protein molecule and said third protein molecule comprises at least one of a monoclonal antibody or a cell surface molecule binding domain and / or fragment thereof, and preferably comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, , moxifloxacin, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is the same as the first binding site of the third protein molecule.

[0329] One embodiment is a therapeutic combination comprising the second or third pharmaceutical composition of the present invention, wherein the second binding site of the second protein molecule and / or the first binding site of the third protein molecule is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment thereof or a domain thereof, and preferably comprises or consists of any one of the following: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxifloxacin pasudotox and potuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3.

[0330] The present inventors have determined that such immunoglobulins, their domains, ligands, etc. are particularly suitable for use as the first binding site of the first protein molecule comprising the first binding site (and the same binding site of the third protein molecule). Similarly, the present inventors have determined that such immunoglobulins, their domains, ligands, etc. are particularly suitable for use as the second binding site of the second protein molecule comprising the second binding site. For example, antibodies and the binding domains of antibodies are suitable for targeting the epitope on the exposed surface of a selected cell surface molecule, which results in the first and third (and respectively second) protein molecules being targeted to express target cells of the cell surface molecules targeted by the first and third protein molecules and / or also to express cells of the second cell surface molecule targeted by the second protein molecule, these cells also expressing the first and third cell surface molecules (which are the same cell surface molecules) and having the cell surface molecules on their cell surfaces. Similarly, a ligand (such as EGF) targeting the EGFR on the target cell is suitable for use as the binding site in the first and third protein molecules, or as the second binding site in the second protein molecule, provided that the second binding site is different from the first and third binding sites, wherein the first and third binding sites are identical. Preferred are binding sites for the first and third epitopes or the second epitope that are specific for binding of the first and third protein molecules to the first cell surface molecule and / or binding of the second protein molecule to the second cell surface molecule, the first and second cell surface molecules being exposed on the same target cell. For example, binding sites based on antibodies or their domains or binding fragments provide such desired specificity for selected first, second, and third epitopes on selected first or second cell surface molecules of selected cells, for targeting, for example, diseased cells, tumor cells, autoimmune cells, etc. Thus, the first, second, and third protein molecules are preferably based on the first, second, and third binding sites of antibodies or binding molecules (fragments, domains).

[0331] By targeting the same cell surface molecule with the first and third protein molecules, the delivery of the saponin and effector moiety to and within the cell cytoplasm of the same target cell is improved and more specific. When considering the patient's (adjacent) healthy cells, the abnormal cells selected for targeting by the binding sites of the first and third protein molecules ideally have a high and / or specific cell surface molecule. Therefore, the epitope on the targeted cell surface molecule is ideally unique to the targeted diseased cells and is at least specifically present and exposed on the targeted cell surface. The binding of the first and third protein molecules is followed by endocytosis of the complex of the first protein molecule and the target cell surface molecule and the complex of the third protein molecule and the target cell surface molecule. Since the first and third protein molecules must enter the same target cell through binding interactions with the very same cell surface molecule, the accumulation of therapeutically active amounts of the first and third protein molecules within the target cell is only possible and occurs when the expression level of the targeted cell surface molecule is above a certain minimum expression threshold. At the same time, the fact that the effector moiety bound to the third protein molecule is only able to exert its intracellular (e.g., cytotoxic or gene silencing) activity in the presence of the first protein molecule having a covalently bound saponin, when both the first and third protein molecules are able to enter the target cell in sufficient amounts by binding to sufficiently exposed and expressed surface molecules, and when the targeted cell surface molecules are expressed at sufficiently low levels in healthy cells, also provides protection for the effector moiety against negative and undesirable side effects, for example, on healthy cells and healthy tissues that are not targeted and affected by the effector moiety. That is, low expression of the cell surface molecules bound by the binding sites of the first and third protein molecules does not allow the first and third protein molecules to enter simultaneously, which in turn leads to an amount of endosomal escape of the effector moiety under the influence of the saponin bound to the first protein molecule. Because the ADC or AOC can be used at a lower dose than when the first protein molecule is not added to the therapeutic regimen, it is considered that when, for example, the ADC or AOC targets and kills target diseased cells (such as tumor cells and autoimmune cells) at a low level, there is a lower risk of undesirable side effects occurring in healthy cells.

[0332] Throughout the specification and claims (the entire application), the terms "first" and "third" have the same meaning when referring to the first and third epitopes, the first and third binding sites, and the first and third cell surface molecules. That is, for the first and third protein molecules, the targeted epitope is the same, the binding site is the same, and the targeted cell surface molecule (e.g., tumor cell (specific) receptor) is the same.

[0333] Tables A2, A3, and A4 list preferred examples of first cell surface molecules comprising a first epitope of a first binding site for a first and third protein molecule. In addition, Tables A2, A3, and A4 also list preferred examples of second cell surface molecules comprising a second epitope of a second binding site for a second protein molecule. When the first and / or second cell surface molecules are specifically expressed on target cells, preferably both the first and second cell surface molecules, and when the first binding site and / or the second binding site bind to the first and second epitopes on the first and second cell surface molecules, respectively, they can bind, specifically present in the first and / or second cell surface molecules, and specific targeting of the first, third, and / or second protein molecules to the same desired target cells (e.g., tumor cells that expose the first and second tumor cell surface molecules) is promoted, while other cells are not targeted or are targeted to a lesser extent by the first, third, and second protein molecules, such as healthy cells that do not express the first and / or second cell surface molecules or that do express the first and / or second cell surface molecules to a lesser extent, preferably those that do not express the first and / or second cell surface molecules or express the first and / or second cell surface molecules to a lesser extent than the expression of the cell surface molecules on the targeted (abnormal) cells.

[0334] One embodiment is a therapeutic combination comprising the second or third pharmaceutical composition of the present invention, wherein the effector portion comprised by the second protein molecule and / or by the third protein molecule comprises any one or more of the following, or consists of any one or more of the following: an oligonucleotide, a nucleic acid, a heterologous nucleic acid, preferably selected from a vector, a gene, a cell suicide-inducing transgene, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, Any one or more of a ligand, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinoic 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, such as BNA that silences HSP27 protein expression.

[0335] One embodiment is a therapeutic combination comprising the second or third pharmaceutical composition of the present invention, wherein the effector moiety comprised by the second protein molecule and / or by the third protein molecule comprises or consists of at least one protein molecule, preferably selected from any one or more of the following: peptides, proteins, enzymes such as urease and Cre-recombinase, ribosome inactivating proteins, protein toxins, more preferably selected from any one or more of the following: any one or more of the protein toxins selected from Table A5 and / or viral toxins such as apoptin; bacterial toxins such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins such as α-sarcin ; plant toxins comprising ribosome-inactivating proteins and the A chain of type 2 ribosome-inactivating proteins, such as oleanolic acid (e.g., oleanolic acid-30 or oleanolic acid-32), saporin (e.g., saporin-S3 or saporin-S6), bouganin or a deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modicin, modicin A chain, abrin, abrin A chain, modeccin, modeccin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog ribonuclease, or granzyme B or angiogenin from humans, or any fragment or derivative thereof; preferably, the protein toxin is oleanolic acid and / or saporin.

[0336] One embodiment is a therapeutic combination comprising the second or third pharmaceutical composition of the present invention, wherein the effector portion comprised by the second protein molecule and / or the third protein molecule comprises or consists of at least one payload, preferably selected from any one or more of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, pasudotox, a maytansine derivative DM1, a maytansine derivative DM4, a monomethyl auristatin E(M) MAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-gamma calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analogs, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolebenzodiazepine, AZ13599185, sanguinimidyl, rhizoctonia solani, methotrexate, anthracycline, camptothecin analogs, SN 38. DX 8951f, exitecan mesylate, a truncated form of Pseudomonas aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, spliceostatin, talanstatin, ozogamicin, tesirine, amberstatin 269, and soravtansine, or derivatives thereof.

[0337] The pharmaceutical active substance in the present invention is an effector moiety that is used to achieve a beneficial result in an organism, preferably a vertebrate, more preferably a human (such as a cancer patient or an autoimmune patient). Benefits include diagnosis, prognosis, treatment, cure and / or prevention of diseases and / or symptoms. Pharmaceutical active substances may also cause unwanted side effects. In this case, the pros and cons must be weighed to determine whether the pharmaceutical active substance is suitable for a particular situation. If the effect of the pharmaceutical active substance in a cell is mainly beneficial to the entire organism, the cell is called a target cell. If the effect in the cell is mainly harmful to the entire organism, the cell is called an off-target cell. In artificial systems (cell culture and bioreactors), target cells and off-target cells depend on the purpose and are defined by the user.

[0338] The effector moiety being a polypeptide may be, for example, a polypeptide or a toxin that restores a lost function (such as, for example, enzyme replacement, gene regulatory function).

[0339] One embodiment is a first protein molecule according to the invention, wherein said first protein molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins, or any number therebetween (e.g. 7, 9, 12 saponins), covalently bound directly to an amino acid residue of said first protein molecule, preferably to a cysteine ​​and / or lysine, and / or covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric scaffold (preferably 1-8 such scaffolds or 2-4 such scaffolds), wherein said at least one scaffold is optionally based on a dendron, wherein 1-32 saponins (e.g. 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number therebetween such as 7, 9, 12 saponins) are covalently bound to at least one scaffold.

[0340] Table A1 and Scheme 1 and the above-mentioned embodiments summarize a series of saponins that, when in free form with a second molecule (e.g., effector moiety or effector molecule, such as a toxin, an oligonucleotide), contact with mammalian cells (particularly human tumor cells), identified their endosome escape enhancing activity. In fact, in a cell-based bioassay using human tumor cells, for the saponins listed in Table A1 and the saponins in Scheme 1 and the saponins in the various embodiments of the invention as described herein, it was determined that under the influence of these saponins, when combined with a first protein molecule, a second molecule (effector moiety) (e.g., nucleic acid and / or toxin (e.g., one or more protein toxins listed in Table A5)), when combined with a second or third protein molecule, the saponin was delivered into the cytoplasm with increased efficiency and / or efficacy, presumably by intracellular release from (late) endosomes and lysosomes. That is, in the absence of saponins, the endosome and / or lysosomal escape of such second molecule (effector moiety combined with a second or third protein molecule of the present invention) (e.g., nucleic acid and / or toxin) was less efficient.

[0341] Surprisingly, the inventors of the present invention have now demonstrated that a water-soluble saponin fraction from Quillaja saponaria (comprising QS-21 and its family members QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18 and Quil-A), when combined with a monoclonal antibody (the first protein molecule of the present invention) and a second and / or third protein molecule comprising an effector moiety (the second and / or third protein molecule described above) and at least one of the first protein molecule as a covalent conjugate. The ability to enhance the in vitro biological effects of nucleic acids bound to monoclonal antibodies or protein toxins bound to monoclonal antibodies (examples of second and / or third protein molecules of the present invention, including covalently bound oligonucleotides or payloads such as (protein) toxins) when administered to tumor cells of mammalian species (humans) in the form of covalent conjugates of a glycoside such as QS-21 and its family member saponins contained in such QS-21 preparations (e.g., water-soluble fractions of Quillaja sylvestris)) was also demonstrated, wherein the effector molecule is covalently bound to the glycoside (e.g., saponin fractions of Quillaja sylvestris, QS-21, SO1861, SA1641, GE1741) directly or through a linker or through a polymeric or oligomeric scaffold or directly or through at least one linker to, for example, a protein molecule. Without wishing to be bound by any theory, the stimulation or enhancement of, for example, antisense BNA-mediated reduction of HSP27 expression (HSP27 gene silencing) of tumor cells observed in vitro in the presence of saponins derived from Quillaja saponaria may (also) be related to activation of inflammasomes in tumor cells by activating saponins (e.g., leading to pyroptosis of tumor cells). When in the presence of a first protein molecule of the invention comprising a saponin and targeting the same cell surface molecules as those targeted by the second and / or third protein molecule, the second and third protein molecules conjugated to, for example, antisense BNAs or oleanolic acid or saponin exhibit all or any improved anti-tumor cell activity, whereas in the absence of the first protein molecule and therefore of the saponin, no such activity against tumor cells is observed.

[0342] QS-21 and the water-soluble saponin fraction containing QS-21 from the Quillaja sapodilla tree are already known and have been widely used for their immunopotentiating abilities, for example as adjuvants for subunit vaccines. For example, QS-21 was used in two Phase III clinical trials in human patients who received a subunit vaccine adjuvanted with QS-21 (Glaxo-Smith-Kline, MAGRIT trial, DERMA study). The subunit was the MAGE-A3 protein, specifically expressed and presented by tumor cells. QS-21-enhanced anti-tumor vaccination was intended to prolong disease-free survival in patients with cancer (melanoma and non-small cell lung cancer). Additionally, QS-21 has been tested as an adjuvant in clinical trials for the development of anti-cancer vaccine therapies, vaccines for HIV-1 infection, vaccines against hepatitis B, and anti-malarial vaccines using QS-21 with Glaxo-Smith-Kline's adjuvants AS01 and AS02. Previous studies have shown that QS-21 saponin, in combination with an adjuvant (AS15; GSK), elicits an immune response against the MAGE-A3 peptide displayed on the surface of cancer cells. Surprisingly, the inventors discovered that the saponin fraction of Quillaja saponin, and therefore likely QS-21 (as part of the water-soluble saponin fraction of Quillaja saponin), enhances the anti-tumor cell activity of, for example, a second protein molecule, such as the ligand EGF.

[0343] The inventors of the present invention have shown that a tumor cell-targeting monoclonal antibody having a covalently coupled antisense BNA (e.g., BNA (HSP27)) and contacting tumor cells with a first protein molecule of the present invention having a covalently coupled saponin (e.g., SO1861, QS-21) can silence HSP27 in tumors in vivo, compared to a control and an AOC (third protein molecule) alone in the absence of a first protein molecule having a coupled saponin, wherein the BNA and saponin are coupled to the respective antibodies (e.g., cetuximab) of the first and third protein molecules via cleavable bonds. Co-administration of an ADC or antibody-oligonucleotide conjugate (AOC) (e.g., an antibody-BNA conjugate) with a first protein molecule having a saponin imparts anti-tumor cell activity to the ADC or AOC, whereas no anti-tumor cell activity was observed with the ADC or AOC alone at the same dose. It is noteworthy that when AOC (second or third protein molecule) and a monoclonal antibody with a covalently coupled saponin (first protein molecule) were administered to tumor-bearing mice in different mouse groups, respectively, the expression of HSP27 in tumor cells was increased compared to the control group (only vehicle was administered). When compared to the control, only the co-administration of an AOC comprising an effector portion (second or third protein molecule) of the present invention and a first protein molecule with a covalently coupled saponin showed reduced HSP27 expression. 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], antisense BNA (HSP27) is a BNA with an oligonucleotide sequence 5'-GGCacagccagtgGCG-3'. Notably, to the best of the inventors' knowledge, BNAs are designed for use as free nucleic acids. The inventors are now the first to demonstrate that antisense BNAs can be covalently coupled to ligands or antibodies via (non-)cleavable linkers in a manner that retains gene silencing activity in vitro and, more importantly, in vivo in tumor cells of tumor-bearing animals. This approach to delivering BNA-based anticancer agents (AOCs) opens up new avenues for administering targeted BNAs to human (cancer) patients in need.

[0344] The inventors herein disclose that a saponin (e.g., saponins in the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, Table A1, Scheme I) is covalently coupled to a first protein molecule, such as via a trifunctional linker (e.g., the trifunctional linker of Scheme II and Structure B), or via an oligomeric or polymeric structure comprising a scaffold to which the saponin is covalently bound, resulting in improved cytotoxicity exerted by an effector moiety (e.g., a toxin) comprised by a second and / or third protein molecule under the influence of the covalently coupled saponin in the first protein molecule.

[0345] One embodiment is a first protein molecule of the invention comprising a saponin comprising one or several or all of the indicated structural features of a saponin of structure A in Scheme 1, wherein the saponin of structure A is referred to as a saponin having an 'ideal' structure, where endosomal escape enhances activity against an effector moiety present in an endosome of a cell in contact with the first protein molecule and / or any one or more other saponins selected from Scheme 1: Scheme 1:

[0346]

[0347] Plan I (continued)

[0348]

[0349] Plan I (continued)

[0350]

[0351] Plan I (continued)

[0352]

[0353]

[0354] Plan I (continued)

[0355]

[0356] According to the present invention, the glycoside bound to the first protein molecule of the present invention (such as a saponin according to the present invention) having an 'ideal' structure for the purpose of enhancing the endosomal escape of an effector molecule bound to the second or third protein molecule of the present invention is a disaccharide chain saponin of structure A according to Scheme 1 having a molecular weight of at least 1,500 Daltons and comprising an oleanolic triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, wherein the saponin contains a first branched carbohydrate side chain at the C-3 position, the first branched carbohydrate side chain optionally comprising glucuronic acid, wherein the saponin contains a second branched carbohydrate side chain at the C-28 position. The ester group of a branched carbohydrate side chain, the second branched carbohydrate chain preferably comprises at least four carbohydrate units, optionally comprising at least one acetyl residue (such as two acetyl residues) and / or optionally comprising a deoxy carbohydrate and / or optionally comprising quinovose and / or optionally comprising glucose and / or optionally comprising 4-methoxycinnamic acid and / or optionally comprising 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprising 5-O-[5-O-Rha-(1 → 2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyloctanoic acid.

[0357] SO1861 differs from the "ideal structure" shown in Scheme 1, Structure A, only in having only one acetyl residue on the quinovose and having an additional xylose. The "ideal structure" of a saponin for enhancing endosomal escape of an effector molecule or effector moiety is preferably a saponin having Structure A of Scheme 1, and a saponin that exhibits endosomal escape enhancing activity and has one or more of the structural features shown in Structure A of Scheme 1. Without wishing to be bound by any theory, the inventors believe that Structure A of Scheme 1 represents an "ideal saponin" (rather than a minimal saponin) for endosomal escape enhancing activity, meaning that not all structures (chemical groups) can or must be present in every saponin to have at least sufficient endosomal escape enhancing activity to promote accumulation of the effector moiety in the cytoplasm, and that some saponins may have other structural elements (such as acyl chains), and / or for other saponins that exhibit endosomal escape enhancing activity, the sugar may be different from the sugar shown in Scheme 1. For example, when considering the ideal structure of Structure A in Scheme 1, QS-21 saponin and some saponins in the water-soluble fraction of Quillaja saponaria (Quil-A) differ in the carbohydrate modification at C-28: for example, the presence of an acyl chain in QS-21. In the water-soluble fraction of Quillaja saponaria, saponins (QS-7, QS1862) are similar to the ideal structure A and are similar to QS1861.

[0358] One embodiment is a first protein molecule of the invention, wherein at least one linker is a non-cleavable linker or a cleavable linker, wherein the cleavable linker is cleaved, for example, under acidic conditions, reducing conditions, enzymatic conditions or light-induced conditions, preferably the cleavable linker comprises a hydrazone bond or a hydrazide bond, which is cleaved under acidic conditions when bound to a saponin, and / or comprises a bond that is susceptible to proteolysis (e.g., proteolysis by cathepsin B), and / or is a bond that is susceptible to cleavage under reducing conditions (e.g., a disulfide bond) when bound to a saponin.

[0359] One embodiment is the first protein molecule of the invention, wherein when the cleavable linker is conjugated to a saponin, the cleavable linker undergoes cleavage in vivo under acidic conditions as found in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0-6.5, more preferably at pH ≤ 5.5.

[0360] One embodiment is a first protein molecule of the invention, wherein the oligomeric or multimeric scaffold comprises a multimeric or oligomeric structure and comprises a chemical group for covalently coupling the scaffold to an amino acid residue of the first protein molecule.

[0361] According to the present invention, saponins are typically those listed in Table A1 of Scheme 1. The activity of saponins has been shown to be beneficial, for example, when the effector moiety covalently coupled to the second or third protein molecule enters the cell and accumulates in the cytoplasm, when the saponin is covalently coupled to the first protein molecule involving a hydrazone bond and / or a hydrazide bond, and / or a disulfide bond, to enhance endosomal escape. Such bond types are easily cleaved under acidic conditions within (late) endosomes and lysosomes of mammalian cells (e.g., human cells) and / or under reducing conditions. Alternatively, the inventors have also demonstrated that covalent coupling of saponins to the first protein molecule via a bond that is not easily cleaved under physiological conditions within the cell (e.g., (late) endosomes, lysosomes, cytoplasm) is also beneficial for enhancing the activity of saponins on the biological effects of, for example, effector moieties (e.g., nucleic acids, e.g., BNAs that silence HSP27) and protein toxins (e.g., saporin). Throughout the application, including the claims, the terms 'cleavable linker', 'cleavable bond' and the like, also referred to as 'labile linker' ('L') and 'labile bond', refer to the context of cleavage of such bonds or linkers, e.g. in (late) endosomes and / or lysosomes, when the conjugate of the invention is, e.g., optionally comprising a scaffold with a saponin coupled to a first protein molecule via a linker and / or a first protein molecule of the scaffold via a hydrazone bond or a disulfide bond. For example, Figure 6 and Figure 7In vivo HSP27 gene silencing in human tumors in mice was demonstrated. Tumor-bearing mice were treated with a first protein molecule according to the present invention, consisting of a monoclonal antibody and a saponin bound thereto via an unstable linker (hydrazone bond), while a third protein molecule comprised an antisense BNA covalently coupled to a monoclonal antibody (of the same type as the first monoclonal antibody) via a disulfide bond for silencing the binding of the HSP27 gene in tumor cells. That is, without wishing to be bound by any theory, the hydrazone and disulfide bonds are cleaved in the (late) endosomes and / or lysosomes of target tumor cells expressing an epitope on a targeted cell surface molecule (here, EGFR) at the cell surface, once the therapeutic combination of the present invention is internalized, for example, by endocytosis. Considering that BNAs enter the cytoplasm from endosomes and / or lysosomes, the cleavage of the bond may contribute to the endosomal escape-enhancing activity of the saponin, although such cleavage is not a necessary condition for observing the gene silencing effect of the combination of the cetuximab-SO1861 conjugate and the cetuximab-BNA conjugate of the present invention.

[0362] The skilled person will appreciate that the trifunctional linker is a scaffold suitable for covalently coupling one, two or three saponin moieties of the present invention. It is preferred for the trifunctional linker of covalently coupling one or two saponin moieties. The second and / or third binding site is for example applicable to covalently coupling protein ligands (such as the first protein molecule). Typical protein ligands are EGF for targeting (tumor) cells expressing EGFR on the cell surface, and cytokines for targeting tumor cells or autoimmune cells. In addition, the second or third binding site of the trifunctional linker is applicable to the covalent coupling of immunoglobulins (such as monoclonal antibodies), i.e., for combining the first protein molecule of cell surface molecules, cell surface molecules are for example tumor cell surface molecules, preferably tumor cell specific molecules, more preferably tumor cell receptors specifically (over) expressed on tumor cell surfaces. Similarly, immunoglobulins comprising immunoglobulin binding specificity or any fragment and / or domain thereof are applicable to combining on autoimmune cell surface expression cell surface molecules (such as receptors). Thus, in an embodiment, the first protein molecule comprises a trifunctional linker comprising or consisting of a covalently bound saponin (e.g. QS-21, SO1861) and the covalently bound binding site is, for example, a cell targeting moiety, such as a ligand or antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, healthy lung cells or diseased B cells.

[0363] One embodiment is the first protein molecule of the present invention, comprising an oligomeric trifunctional linker according to Scheme II as a scaffold core structure:

[0364]

[0365] wherein the saponin is covalently bound to the trifunctional linker scaffold via a labile, cleavable hydrazone linker (acid-labile) and / or via a bond comprising a maleimide, and the binding of the scaffold to the binding site (e.g., antibody) is established via a labile, cleavable hydrazone linker (acid-labile) and / or via a bond comprising a maleimide bonded to a cysteine ​​(e.g., 1, 2, 3, or 4 cysteines) in the binding site, thereby forming structure B:

[0366]

[0367] One to four scaffolds are covalently bound to a single antibody (eg, a monoclonal antibody).

[0368] One embodiment is a first protein molecule of the present invention, wherein the glycoside molecule is a saponin, and the connection between the saponin and the first protein molecule is preferably via an acid-labile bond that is stable at pH 7.4 and preferably releases the saponin at pH 6.5 or below, more preferably at pH 6.5 to 5.0. This is achieved, for example, by an imine formed by an amino group of a linker connecting the saponin to the first protein molecule and an aldehyde group of the saponin. Other chemical bonds that meet the pH conditions can also be used for aldehyde coupling, such as certain hydrazones or acetals, which require hydrazide and hydroxyl groups as linker functional groups, respectively. If the bond is a cleavable bond, the saponin is preferably linked to the polymeric or oligomeric structure of the scaffold via the aldehyde functional group or via one of the carboxyl groups in the saponin, more preferably via the aldehyde functional group, preferably the aldehyde functional group at the 23 position. Alternatively, the saponin is preferably linked to the first protein molecule via the polymeric or oligomeric structure of the scaffold, via a linker that is linked to the polymeric or oligomeric structure of the scaffold via the aldehyde functional group or the carboxylic acid functional group of the glycoside molecule (i.e., saponin).

[0369] One embodiment is a first protein molecule of the present invention, wherein at least one saponin is bound to the first protein molecule via a stable bond. In a more preferred embodiment, the stable bond between the saponin and the first protein molecule preferably occurs via amide coupling or amine formation. This is achieved, for example, by carbodiimide-mediated amide bond formation, which is achieved by an amino group of the polymeric or oligomeric scaffold structure that connects the saponin and the first protein molecule and an activated glucuronic acid group of the saponin. A chemical bond that meets the definition of a stable bond can also be used for aldehyde coupling (e.g., specific amines derived after reductive amination) where a primary amino group is required as a functional group of a polymeric or oligomeric structure of a scaffold or linker. If the bond is a stable bond, the saponin is preferably connected to the linker or scaffold via one of the carboxyl groups of the saponin, which is also connected to the first protein molecule.

[0370] One embodiment is the first protein molecule of the invention, wherein the saponin is coupled to the binding site via a scaffold according to the invention, wherein the chemical group used to covalently couple the scaffold to the binding site is a click chemistry group.

[0371] One embodiment is a first protein molecule of the invention wherein a saponin is coupled to a binding site via a scaffold according to the invention, wherein the click chemistry group is a tetrazine, an azide, an alkene, or an alkyne, or a derivative of any of these groups, preferably an azide. Click chemistry groups are functional chemical groups suitable for click chemistry that are defined as modular, broad, very high yielding, producing only harmless by-products, highly selective and highly tolerant to different functional groups, and stereospecific. Desirable process characteristics include simple reaction conditions, readily available starting materials and reagents, the absence of solvents or the use of mild solvents (e.g., water) or easily removable solvents, and simple product isolation. The click chemistry group used to couple the saponin to the binding site in the first protein molecule, optionally via a scaffold or a linker, is preferably a tetrazine, an azide, an alkene or an alkyne, or a reactive derivative thereof such as methyltetrazine or maleimide (alkene), more preferably alkyne, or a cyclic derivative of these groups such as cyclooctyne (e.g. aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]non-4-yne, dibenzocyclooctyne).

[0372] Therefore, the first protein molecule according to the present invention comprises at least one saponin. In this article, "at least one" means that the first protein molecule comprises a saponin molecule, but can also comprise a pair (for example two, three or four) saponins or a plurality of (for example 10, 20 or 100) saponins. Depending on the application, the first protein molecule can comprise a covalently bound scaffold with a covalently bound saponin, wherein the scaffold can be designed so that it comprises a limited number of saponins. Preferably, the first protein molecule according to the present invention comprises a limited number or range of saponins, rather than a random number. This is particularly advantageous for the drug development relevant to a marketing authorization. The quantity limited in this respect means that the first protein molecule preferably comprises a previously limited number of saponins. This is for example achieved by designing a scaffold comprising a multimeric structure with a certain number of possible parts for saponin connection. Ideally, all of these parts are coupled with saponin and scaffold, rather than comprising a previously limited number of saponins. It is envisaged that a standard set of scaffolds is provided, which comprises for example two, four, eight, sixteen, thirty-two, sixty-four, etc. saponins, so that the user can easily test the optimal number according to his needs. One embodiment is a first protein molecule according to the invention comprising a scaffold according to the invention, wherein the saponin is present within a defined range, e.g., in non-ideal situations, not all moieties present in the multimeric structure are bound to saponin. For example, such a range may be 2-4 saponin molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules per scaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules per scaffold, etc. In this case, if the range is defined as 2-4, then the first protein molecule comprising the scaffold according to the invention thus comprises 2, 3, or 4 saponins.

[0373] The scaffold is essentially independent of the type of saponin to which the scaffold is covalently bound, and the scaffold is subsequently (in sequence) covalently coupled to the first protein molecule. Thus, the first protein molecule comprising the scaffold is the base product of the new platform technology. Since at least one covalently bound saponin mediates the intracellular delivery of the effector moiety bound to the second protein molecule, the scaffold technology according to the present invention is the first system known for the controlled intracellular delivery of effector moieties mediated by saponins. The scaffold provides an optimized functionally active unit that can be connected to a saponin and a binding site contained in a single and defined position by the first protein molecule (ligand, antibody, etc.).

[0374] One embodiment is a first protein molecule comprising a scaffold according to the invention, wherein the number of monomers in the polymeric or oligomeric structure is a precisely defined number or range. Preferably, the polymeric or oligomeric structure comprises a structure such as a poly(amine) (e.g., polyethyleneimine and poly(amidoamine)), or a structure such as polyethylene glycol, a poly(ester) (e.g., poly(lactide)), a poly(lactamide), a poly(lactide-co-glycolide copolymer, a poly(dextrin), or a peptide or protein, or a structure such as a natural and / or artificial polyamino acid (e.g., polylysine, a DNA polymer, a stable RNA polymer, or a PNA (peptide nucleic acid) polymer), and can be a linear, branched, or cyclic polymer, oligomer, dendrimer, dendrimer, dendrimer-oligomer, or an assembly of these structures, whether pure or mixed. Preferably, the polymeric or oligomeric structure is biocompatible, wherein biocompatible means that the polymeric or oligomeric structure does not exhibit significant acute or chronic toxicity in an organism and can be excreted intact or completely degraded into excretable and / or physiological compounds. Assembly can be established by covalent cross-linking or non-covalent bonds and / or attractive forces. Therefore, they can also form nanogels, microgels or hydrogels, or they can be attached to a carrier (such as inorganic nanoparticles, colloids, liposomes, micelles or granular structures comprising cholesterol and / or phospholipids). The polymeric or oligomeric structure preferably has a precisely defined number or range of coupling moieties for coupling glycoside molecules (and / or effector molecules and / or carrier molecules, such as ligands, monoclonal antibodies or fragments thereof). Preferably, at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% of the precisely defined number or range of coupling moieties in the polymeric or oligomeric structure are occupied by the glycoside molecules in the scaffold according to the present invention.

[0375] Preferably, the dendron is a branched, well-defined dendrimer with a single chemically addressable group at the origin of the tree, referred to as the focal point. A dendrimer is a macromolecule formed by the connection of two or more dendrons at their focal point. A dendrimer is a multimer formed by the connection of one or more dendron focal points to a multimer. In a preferred embodiment, a scaffold according to the invention is provided, wherein the polymeric or oligomeric structure comprises a linear, branched or cyclic polymer, oligomer, dendrimer, dendron, dendron polymer, dendritic oligomer or an assembly of these structures, whether pure or mixed, wherein the assembly can be established by covalent crosslinking or non-covalent attraction and can form a nanogel, microgel or hydrogel, and wherein 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), poly(lactide-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, stable RNA polymers or PNA (peptide nucleic acid) polymers. Preferably, the polymeric or oligomeric structure is biocompatible.

[0376] One embodiment is a therapeutic combination according to the invention or for use according to the invention, wherein the first protein molecule comprises more than one covalently bound saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponins, or any number therebetween (e.g. 7, 9, 12 saponins).

[0377] One embodiment is the first protein molecule of the invention, wherein at least one saponin is covalently bound to the polymeric or oligomeric structure of the oligomeric or multimeric scaffold via at least one cleavable linker according to the invention.

[0378] One embodiment is a first protein molecule of the invention, wherein the chemical group of the oligomeric or polymeric scaffold used to covalently couple the oligomeric or polymeric scaffold to the amino acid residues of the first protein molecule is a click chemistry group, preferably selected from tetrazines, azides, alkenes or alkynes, or cyclic derivatives of these groups, more preferably, said chemical group is an azide.

[0379] One embodiment is a first protein molecule according to the invention, wherein the polymeric or oligomeric structure of the oligomeric or multimeric scaffold comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendron, dendrimer, dendri-oligomer, DNA, a polypeptide, polylysine, polyethylene glycol or an assembly of these polymeric or oligomeric structures, said assembly preferably being established by covalent cross-linking.

[0380] The inventors have determined that, according to any of the above embodiments, covalent conjugation of a saponin to a first protein molecule, preferably via a cleavable bond or linker, provides for an effective and cell-targeted enhancement of the activity of an effector moiety bound to a second protein molecule as well as a third protein molecule, wherein the first and third protein molecules comprise the same first binding site, and wherein the first and second protein molecules comprise different first and second binding sites. When the effector moiety is also delivered to the same target cell by using a second and / or third protein molecule comprising the same first binding site as the first protein molecule, when the third protein molecule is considered and comprises different first and second binding sites, respectively, when the first and second protein molecules are considered, conjugating the saponin to a cysteine ​​side chain or a lysine side chain of the first protein molecule (e.g., a monoclonal antibody) directly or via a linker has proven to be a beneficial means for specific and effective delivery of the effector moiety enhancing activity within the target cell.

[0381] To explain the present invention in more detail, the processes of cellular uptake of substances in the present invention are described (although the inventors do not wish to be bound by any theory) and the terminology used. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. The vesicle budding is characterized by (1) receptor-dependent ligand uptake mediated by the cytosolic protein clathrin, (2) lipid raft uptake mediated by the cholesterol binding protein caveolin, (3) non-specific fluid uptake (pinocytosis), or (4) non-specific particle uptake (phagocytosis). All types of endocytosis enter the cellular process of vesicle transport and substance sorting called the endocytic pathway. The endocytic pathway is complex and not yet fully understood. Without wishing to be bound by any theory, organelles can be formed from scratch and mature into the next organelle along the endocytic pathway. However, it is currently assumed that the endocytic pathway involves stable compartments connected by vesicle transport. A compartment is a complex, multifunctional membrane organelle that is specialized for a specific set of basic functions of the cell. Vesicles are considered to be transient organelles with a simpler composition and are defined as membrane-enclosed containers that are formed from scratch by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early endosomes and late endosomes represent stable compartments in the endocytic pathway, while primary endocytic vesicles, phagosomes, multivesicular bodies (also known as endosomal carrier vesicles), secretory granules, and even lysosomes represent vesicles. Endocytic vesicles emerge from the plasma membrane, most prominently from clathrin-coated pits, and initially fuse with early endosomes, a major sorting compartment with a pH of approximately 6.5. Most internalized cargo and membranes are recycled back to the plasma membrane via recycling vesicles (the recycling pathway). Components requiring degradation are transported to acidic late endosomes (pH below 6) via multivesicular bodies. Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to the late endosomal compartment when needed. The resulting organelle is called a hybrid organelle or endolysosome. Lysosomes bud from the hybrid organelle in a process called lysosomal reorganization. Late endosomes, lysosomes, and hybrid organelles are extremely dynamic, making it often difficult to distinguish them. The degradation of endocytosed molecules occurs within endolysosomes or lysosomes. Endosomal escape is the active or passive release of substances from the lumen of any type of compartment or vesicle into the cytoplasm via the endocytic pathway, preferably from clathrin-mediated endocytosis or recycling pathways. Thus, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediate and hybrid organelles.

[0382] Unless otherwise specified, especially when referring to the endosomal escape mechanism of glycoside molecules (such as the saponins of the present invention), whenever the term "endosome" or "endosomal escape" is used herein, it also includes endolysosomes and lysosomes, and escapes from endolysosomes and lysosomes, respectively. After entering the cytoplasm, the substance may move to other cellular units (such as the nucleus).

[0383] In formal terms, a glycoside is any molecule in which a sugar group is bound to another group via a glycosidic bond through its anomeric carbon. In the context of the present invention, glycoside molecules (such as saponins) are molecules that, without wishing to be bound by any theory, are able to further enhance the action of the effector moiety, in particular by promoting endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (saponins, such as those listed in Table A1) interact with the membranes of compartments and vesicles of the endocytic and recycling pathways and render them leaky for the effector moiety, leading to enhanced endosomal escape. The term "scaffold capable of enhancing 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 enhancing endosomal escape of the effector moiety when both molecules are within an endosome (e.g., a late endosome), optionally and preferably after the at least one glycoside (e.g., saponin) is released from a first protein molecule (e.g., from a linker or polymeric or oligomeric structure comprised by the first protein molecule), for example, by cleaving a cleavable bond between the at least one glycoside (e.g., saponin) and the first protein molecule (e.g., via the polymeric or oligomeric structure of the scaffold and / or via the linker). Even if the bond between the at least one glycoside (e.g., saponin) and the first protein molecule according to the present invention, optionally via the linker or scaffold, can be a "stable bond," this does not mean that such a bond cannot be cleaved enzymatically within the endosome. For example, the glycoside or saponin, optionally together with a portion of the oligomeric or polymeric structure of the linker or scaffold, can be cleaved from the remaining linker fragment or oligomeric or polymeric structure. For example, a protease cleaves the (protein) linker or protein polymeric structure (e.g., albumin) to release the at least one glycoside or saponin. However, preferably, the glycoside molecule (preferably a saponin) is released in an active form, preferably in its original form before being coupled to the first protein molecule, optionally via a linker and / or an oligomeric or polymeric scaffold (to be coupled); thus, the glycoside (saponin) has its native structure after such cleavage, or the glycoside (saponin) has (part of) a chemical group or a linker attached thereto, after such cleavage, whereas the glycoside biological activity (saponin biological activity), e.g. an endosomal / lysosomal escape enhancing activity towards an effector moiety present in the same endosome or lysosome, is maintained or restored after said cleavage of the bond between the glycoside (saponin) and the carrier molecule, i.e. the first protein molecule, optionally comprising a linker and / or a scaffold of the invention. In connection with the present invention, the term "stable" with respect to the bond between, for example, a saponin and an amino acid residue of a first protein molecule, a linker, a polymeric or oligomeric structure (of a scaffold), a ligand, a (monoclonal) immunoglobulin or a binding domain or fragment thereof, and / or an effector (effector moiety, effector molecule) means that the bond is not easily cleaved or is at least designed to be not easily cleaved by, for example, differences in pH, salt concentration or UV-light, reducing conditions.In connection with the present invention, the term "cleavable" refers to a bond between, for example, a saponin and a first protein molecule, a linker, an amino acid residue, a polymeric or oligomeric structure of a scaffold, a ligand, an antibody and / or an effector, which is designed to be easily broken by, for example, pH differences, salt concentrations, under reducing conditions, etc. The skilled person is well aware of such cleavable bonds and how to prepare them.

[0384] Prior to the present invention, one of the major obstacles to the introduction of ADCs and AOCs on the market was the narrow therapeutic window: therapeutically effective doses of ADCs or AOCs were accompanied by (unacceptable) side effects, hindering the development and impact of ADC-based patient treatment. By applying the first protein molecule of the present invention, it is now possible to direct one or more glycoside molecules (saponins) together with a payload-carrying ADC 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 protein molecule of the present invention), to (target) cells. In particular, it was previously impossible to simultaneously direct the effector portion of a second or third protein molecule and a (predefined, controllable) specific number or range of glycoside molecules (saponins) per effector portion specifically to the cytoplasm of a cell, for example via the cell's endocytic pathway.

[0385] The solution provided by the present invention comprises the covalent binding of at least one saponin to a first protein molecule. Another solution provided by the present invention comprises (first) polymerizing glycoside molecules (saponins) using an oligomeric or polymeric scaffold and providing a first protein molecule with a covalently bound saponin cluster, so that one or more saponins can be re-monomerized at the intracellular site of the desired saponin mode of action, for example after endocytosis. "Polymerization" in this context means reversible and / or irreversible multiconjugation of saponin molecules to a first protein molecule, either directly or through a polymeric or oligomeric structure to form a scaffold or reversible and / or irreversible multiconjugation (modified) saponins, via a linker, to form a polymeric or oligomeric structure to form a scaffold or a reversible and / or irreversible multiconjugation (modified) saponin. In the present context, "re-monomerization" means that the saponin is cleaved from the first protein molecule, from the linker that connects the saponin to the first protein molecule, or from the scaffold, for example after endocytosis, and regains the chemical state of the (natural) unbound saponin, which may or may not contain additional chemical groups, such as chemical groups for connecting the saponin to the linker head, the amino acid residue of the first protein molecule or the scaffold, and / or (chemical) linkers that bind to chemical groups of the saponin (such as aldehyde or carboxylic acid groups). Due to the complex chemical properties of saponins, for example, the "polymerization" of saponins at the scaffold or other linker and their "re-monomerization" at the desired location (such as within the cell) (for example after endocytosis) is a challenging task. In particular, the chemical reactions used to provide the linker and the scaffold include covalently linked glycosides for covalently binding to the first protein molecule, for example, triterpenoid saponins (polymerization of glycosides), which are usually present in anhydrous organic solvents, but the saponin and, for example, the biocompatible polymer used as a scaffold to carry the bound saponin are water-soluble molecules. The chemical properties of unmodified saponins further prevent them from polymerizing themselves. Another possible solution is to (directly) conjugate multiple saponins to effector molecules, which is not very promising as effector molecules (drugs, toxins, polypeptides or polynucleotides) generally do not provide enough binding sites and because the conjugated products can become very heterogeneous and / or conjugating bioactive molecules such as saponins and, for example, peptides, toxins, nucleic acids together carries the risk of affecting and hindering the activity of one or even two molecules combined in such saponin-containing conjugates. In addition, there is a considerable risk that the effector portion contained in the second or third protein molecule loses its function after the saponin is conjugated to, for example, an ADC or an antibody-oligonucleotide conjugate (AOC). Embodiments of the present invention address at least one of these drawbacks.

[0386] One aspect of the invention relates to a composition comprising a first protein molecule of the invention and a second protein molecule of the invention.

[0387] One aspect of the invention relates to a composition comprising a first protein molecule of the invention and a third protein molecule of the invention.

[0388] One embodiment is a composition comprising a first protein molecule of the present invention and a second protein molecule of the present invention, or a composition comprising a first protein molecule of the present invention and a third protein molecule of the present invention, wherein the effector portion comprised by the second protein molecule or the third protein molecule is any effector portion according to the present invention, preferably a BNA.

[0389] One aspect of the present invention relates to a composition comprising a first protein molecule of the present invention and one or more of an oligonucleotide, a nucleic acid and a heterologous nucleic acid, wherein the one or more of the oligonucleotide, the nucleic acid and the heterologous nucleic acid is preferably selected from the group consisting of a vector, a gene, a cell suicide-inducing transgene, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a 2'-deoxy-2'-fluoroarabinoic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA) or derivatives thereof, more preferably a BNA, such as a BNA that silences HSP27 protein expression (antisense BNA (HSP27)).

[0390] In the context of the present invention, an effector molecule or effector moiety is any substance that affects cellular metabolism by interacting with an intracellular effector molecule target, wherein the effector molecule target is any molecule or structure in the cell, excluding compartments and vesicles of endocytosis and recycling pathways, but including the membranes of these compartments and vesicles. Thus, the intracellular structures include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane and the cytoplasm. In the context of the present invention, cytoplasmic delivery of the effector moiety preferably refers to the ability of the effector moiety to escape endosomes (and / or lysosomes), as defined above, which also includes escape endosomes, lysosomes and lysosomes, and preferably is able to escape to reach the effector moiety target as described herein. The present invention also includes a novel molecule referred to as a scaffold, which is used to bring the effector moiety and at least one glycoside molecule (e.g., a saponin of the present invention) into the endosome at a predetermined ratio when the effector moiety is contained in a second or third protein molecule of the present invention and a saponin is contained in a first protein molecule. In the context of the present invention, a polymeric or oligomeric structure of a scaffold is a structurally ordered structure, such as a polymer, oligomer, dendrimer, dendrimer, or dendrimer-oligomer, or an assembled polymeric structure (such as a hydrogel, microgel, nanogel, stable polymer micelle, or liposome), but does not include structures composed of non-covalent assemblies of monomers (such as a cholesterol / phospholipid mixture). The terms "polymer, oligomer, dendrimer, dendrimer, or dendrimer-oligomer" have their ordinary meanings. In particular, a polymer is a substance having a molecular structure composed primarily or entirely of a large number of identical or similar units bonded together, while an oligomer is a polymer whose molecules are composed of a relatively small number of repeating units. There is no consensus on the specific thresholds for "many" and "few" as used in the above definitions of polymer and oligomer, respectively. However, since a scaffold can contain polymeric or oligomeric structures, or both, the full range of numbers of similar units bonded together applies to such structures, i.e., from 2 monomeric units to 100 monomeric units, 1000 monomeric units, and more. In some embodiments, the present invention provides the polynucleotide of the present invention.For example, the structure comprising 5 or less units can be referred to as oligomeric structure, and the structure comprising 50 monomeric units can be referred to as polymeric structure.The structure of 10 monomeric units can be referred to as oligomeric or polymeric.As defined herein, the support also comprises at least one glycoside molecule (such as saponin of the present invention).Support preferably includes polymeric or oligomeric structure, such as polymeric or oligomeric (amine), for example polyethyleneimine and poly (amidoamine), and biocompatible structure, such as polyethylene glycol, poly- or oligomeric (ester), such as poly (lactic acid), poly (lactam), polylactide-co-glycolide copolymer and poly (dextrin), polysaccharide or oligosaccharide, such as cyclodextrin or polydextrose, and polymeric or oligomeric amino acids, such as polylysine or peptide or protein, or DNA oligomer or polymer.Assembling polymeric structure as defined herein comprises at least one support and optional other independent polymeric or oligomeric structure.Other individual polymeric or oligomeric structures of the assembly can be (a) a scaffold (thus comprising at least one glycoside molecule, such as a saponin of the present invention), (b) a functionalized scaffold (thus comprising at least one glycoside molecule (e.g., a saponin) and a ligand, antibody, etc. as a first protein molecule), or (c) a polymeric or oligomeric structure without a glycoside molecule (e.g., a saponin of the present invention (see, for example, Table A1)) and a polymeric or oligomeric structure without a ligand, antibody, etc. as a first protein molecule. A functionalized assembled polymeric structure is an assembled polymeric structure comprising (a) at least one functionalized scaffold or (b) at least one scaffold comprising at least one ligand, antibody, etc. as a first protein molecule and at least one polymeric structure. The polymeric or oligomeric structure in the assembled polymeric structure does not contain any of the above molecules (i.e., no glycoside (e.g., saponin) and no first protein molecule (e.g., ligand, antibody)) that is specifically added as a structural component of the assembled structure, which helps to establish or stabilize the assembled structure ("gel-like"). Without wishing to be bound by any theory, an acidic environment appears to be a prerequisite for the synergistic action of the glycoside (saponin) and the effector moiety.

[0391] Whether the first protein molecule comprising a saponin or not further comprising one or more (cleavable) linkers and / or an optional scaffold is able to disrupt the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be easily determined using the assays described in Example 3 and known in the art. Such inhibition is described as the "fold increase in the number of glycosides required to induce 50% cell killing". Preferably, the scaffold does not result in an increase, which is at least the increase in the number of glycoside molecules (saponin) required to obtain 50% cell killing observed when using chloroquine as a positive control. Alternatively, and preferably, the first protein molecule comprising a saponin or not further comprising one or more (cleavable) linkers and / or an optional scaffold does not result in at least a 4-fold increase in glycoside molecules to induce 50% cell killing, more preferably does not result in at least a 2-fold increase. The fold increase will be measured in an assay essentially as described in Example 4, wherein chloroquine as a positive control induces a 2-fold increase in the amount of glycoside (preferably saponin), wherein the saponin is any one or more of the saponins of the present invention for which 50% cell killing is observed (see Table A1, Scheme I, previous embodiment).

[0392] The term "improving or enhancing the effect of an effector moiety" means that a glycoside molecule, preferably a saponin of the present invention, increases the functional efficacy of the effector moiety (e.g., the therapeutic index of a toxin or drug or oligonucleotide (e.g., BNA); the metabolic efficacy of a modifier in a biotechnological process; the transfection efficacy of a gene in a cell culture research experiment), preferably by enabling or improving its target engagement. Preferably, acceleration, prolongation, or enhancement of an antigen-specific immune response is not included. Therapeutic efficacy includes, but is not limited to, a stronger therapeutic effect, preferably with a lower dose and / or fewer side effects. "Improving the effect of an effector moiety" may also mean that an effector moiety that cannot be used due to lack of effect (and, for example, is not referred to as an effector moiety) becomes effective when used in combination with the present invention. Any other beneficial or desired effect provided by the present invention and attributable to the combination of an effector moiety and a second or third protein molecule is considered to be an "improved effect." In an embodiment, the scaffold comprising a saponin-bound scaffold comprised by a first protein molecule enhances the effect of the effector moiety comprised by the second protein molecule, which effect is expected and / or desired. In the case of a first protein molecule comprising a saponin bound to a protein scaffold, the protein polymeric structure of the scaffold itself may have, for example, an impact on the colloid osmotic pressure in the bloodstream. If this effect is not the expected or desired effect of the functionalized scaffold comprised by the first protein molecule, the protein structure of the scaffold is not the effector portion defined in the present invention. Alternatively, for example, in the case of a scaffold based on DNA or RNA carrying a bound saponin and contained in the first protein molecule, the portion of the DNA or RNA may have (unexpected) function, for example, by interfering with expression. If this interference is not the expected or desired effect of the final functionalized scaffold, the DNA or RNA polymeric structure of the scaffold is not the effector portion defined in the present invention.

[0393] The endosomal escape enhancers (i.e., glycosides or saponins, preferably saponins according to the present invention) contained in the first protein molecule can be formulated with a number of preferred features: (1) they are preferably non-toxic and do not elicit an immune response, (2) they preferably do not mediate cytoplasmic uptake of the effector moiety into off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector moiety, (4) they are preferably biodegradable or excretable, and (5) they preferably do not substantially interfere with biological processes of the organism unrelated to the biological activity of the effector molecule to which the endosomal escape enhancer is bound, such as, for example, hormone interactions. Examples of glycoside molecules (such as saponins according to the present invention) that meet the above criteria at least to some extent are disaccharide-chain triterpenes, preferably disaccharide-chain triterpenoid saponins, such as SO1861, SA1641, QS-21, GE1741 and the saponins in Table A1, Scheme I.

[0394] One aspect of the invention relates to an antibody-drug conjugate or an antibody-oligonucleotide conjugate or a ligand-drug conjugate comprising a first protein molecule of the invention and an effector moiety.

[0395] One embodiment is an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate of the present invention, wherein the antibody can bind to any one of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, Integrin α-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 CD71, EGFR FR, HER2, and / or or comprising any of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mocetuzumab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one of these tumor cells Receptor binding fragments and / or at least one tumor cell receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxifloxacin pasudotox and potuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3, or wherein the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule, such as EGF or a cytokine.

[0396] One embodiment is an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate of the invention, wherein the effector moiety is any one or more of the effector moieties according to the invention.

[0397] One aspect of the present invention relates to a pharmaceutical composition comprising a composition comprising a first protein molecule of the present invention and a second protein molecule of the present invention, or comprising a first protein molecule of the present invention and a third protein molecule of the present invention, or comprising an antibody-drug conjugate of the present invention, or comprising an antibody-oligonucleotide conjugate of the present invention, or comprising a ligand-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient.

[0398] One aspect of the invention relates to a therapeutic combination of the invention comprising a second pharmaceutical composition or comprising a third pharmaceutical composition, or comprising a composition comprising a first protein molecule of the invention and a second protein molecule of the invention or comprising a first protein molecule of the invention and a third protein molecule of the invention, or comprising an antibody-drug conjugate or an antibody-oligonucleotide conjugate or a ligand-drug conjugate or a pharmaceutical composition of the invention, for use as a medicament.

[0399] One aspect of the invention relates to a therapeutic combination of the invention comprising a second pharmaceutical composition or a third pharmaceutical composition, or a composition comprising a first protein molecule of the invention and a second protein molecule of the invention or a first protein molecule of the invention and a third protein molecule of the invention, or an antibody-drug conjugate or an antibody-oligonucleotide conjugate or a ligand-drug conjugate or a pharmaceutical composition of the invention for treating or preventing cancer or an autoimmune disease.

[0400] As previously mentioned, the at least one saponin contained in the first protein molecule according to the present invention at least increases the efficacy of existing and novel effector moieties defined herein. By reducing the dose of the effector moiety contained in the second or third protein molecule, potential side effects are reduced without compromising efficacy. Thus, the present invention provides a first protein molecule according to the present invention for use in medicine or as a medicament. Thus, one aspect of the present invention relates to a first protein molecule according to the present invention for use as a medicament, comprising at least one saponin. Also provided is the use of the first protein molecule according to the present invention for the manufacture of a medicament. Cancer drugs, in particular, classical chemotherapeutic drugs, are notorious for their side effects. Due to the temporally and spatially targeted and synchronized delivery of the pharmaceutically active substance contained in the second or third protein molecule and the saponin contained in the first protein molecule, because the first and third protein molecules have the same binding site for the same epitope on the same cell surface molecule, or because the first and second protein molecules have different binding sites for different first and second epitopes on first and second cell surface molecules, respectively, the therapeutic combination according to the present invention is particularly suitable for use as a medicament, in particular for use in a method for treating cancer. Thus, the present invention provides a therapeutic combination according to the present invention or a first protein molecule according to the present invention for use in a method for treating cancer. The present invention also provides a therapeutic combination according to the invention or a first protein molecule according to the invention for use in a method of treating an acquired or inherited disorder, in particular a monogenic disorder. Thus, the therapeutic combination comprises a first and a second protein molecule and / or comprises a first and a third protein molecule. Thus, one aspect of the present invention relates to a therapeutic combination according to the invention, wherein the second or third protein molecule comprises a covalently bound effector moiety, for use in a method of treating cancer or an autoimmune disease.

[0401] A further medical application of the first, second, and third protein molecules of the present invention is to replace intracellular enzymes in target cells that are produced in insufficient amounts or are not functioning properly. The resulting disease may be hereditary or acquired. In most cases, only symptomatic treatment is available, and for some rare diseases, the lack of treatment options can lead to a shortened lifespan for affected patients. An example of such a disease is phenylketonuria, an inborn error of metabolism that results in reduced metabolism of the amino acid phenylalanine. This disease is characterized by a genetic mutation in the liver enzyme phenylalanine hydroxylase. Phenylketonuria is currently incurable. The incidence rate is approximately 1:10,000, with the highest known incidence in Turkey being 1:2,600. A second or third protein molecule (preferably an antibody) with bound phenylalanine hydroxylase or a bound polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells using a suitable specific antibody and replace the defective enzyme in the liver cells. This is an example of a therapeutic combination using the present invention, comprising a first protein molecule bound to a saponin and a second or third protein molecule according to the present invention bound to an enzyme or oligonucleotide for replacement or gene therapy. In a preferred embodiment, there is provided a therapeutic combination according to the invention for use in a method of gene therapy or replacement therapy.

[0402] The present invention also provides a method for treating cancer, which comprises administering a medicament comprising the therapeutic combination according to the present invention to a patient in need thereof, preferably administering an effective dose of the medicament to a patient in need thereof (preferably a human cancer patient).

[0403] Considerations regarding a form suitable for administration are known in the art and include toxic effects, solubility, route of administration, and maintenance of activity.For example, a pharmaceutical composition injected into the bloodstream should be soluble.

[0404] The appropriate dosage form depends in part on the route of use or entry, e.g., transdermal or injection. Such dosage forms should allow the compound to reach the target cells, whether or not they are present in a multicellular host. Other factors are known in the art and include considerations such as toxicity and dosage form that may hinder the compound or composition from exerting its effect.

[0405] One embodiment is a combination of an endosomal escape-enhancing conjugate according to the present invention, the conjugate comprising a first protein molecule containing at least one covalently bound saponin and a binding moiety, wherein the binding moiety comprises at least one effector moiety, the binding moiety being a second or third protein molecule comprising a binding effector moiety, wherein the endosomal escape-enhancing conjugate and the binding moiety are independently capable of specifically binding to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of the complex of the endosomal escape-enhancing conjugate and the target cell-specific surface molecule and the complex of the binding moiety and the target cell-specific surface molecule, wherein the endosomal escape-enhancing conjugate and the binding moiety can bind to the same target cell-specific surface molecule via their same binding site, or wherein the endosomal escape-enhancing conjugate and the binding moiety can bind to different target cell-specific surface molecules via different binding sites. One embodiment is a combination according to the present invention, wherein the endosomal escape-enhancing conjugate is capable of competing with the binding moiety for binding to the target cell-specific surface molecule or structure. One embodiment is a combination according to the present invention, wherein the endosomal escape-enhancing conjugate and the binding moiety are independently capable of specifically binding to the same epitope or to different epitopes. One embodiment is a combination for use in a method according to the invention for treating an abnormality such as cancer, wherein the endosomal escape-enhancing conjugate and the binding moiety are administered simultaneously or sequentially, preferably simultaneously.

[0406] One aspect of the invention relates to a kit comprising a first container containing an endosomal escape-enhancing conjugate according to the invention (i.e., a first protein molecule) and a second container containing a binding moiety according to the invention (i.e., a second and / or third protein molecule), the kit further comprising instructions for using the binding molecules (i.e., a therapeutic combination comprising the first and second or the first and third pharmaceutical compositions).

[0407]

[0408]

[0409]

[0410]

[0411]

[0412] Table A2 - ADCs previously studied in a human clinical setting and subsequently withdrawn from further clinical studies

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421] Table A3 - ADCs that have reached Phase III clinical development

[0422]

[0423]

[0424]

[0425] Table A5: RIPs from plants*

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435] Part of the invention is a therapeutic combination, first pharmaceutical composition, first protein molecule, second or third pharmaceutical composition, or second or third protein molecule of the invention further combined with a covalent conjugate (complex) of a binding molecule or binding moiety and a saponin, or further combined with a drug compound, antibody, etc., thereby providing a composition comprising three or more enhancers, pharmaceutical active ingredients, etc., for example, a conjugate of the invention (e.g., a first protein molecule and / or a second or third protein molecule) combined with a binding moiety complexed with an effector molecule, further combined with a drug, said drug being linked to a saponin or not, and being coupled to a ligand (e.g., a targeting immunoglobulin, a domain thereof, or a fragment thereof) or not. In addition, one embodiment is a therapeutic combination, first pharmaceutical composition, first protein molecule, second or third pharmaceutical composition, or second or third protein molecule of the invention, wherein the second or third protein molecule has two or more effector moieties (e.g., a toxin or immunotoxin), wherein the two or more effector moieties are the same or different.

[0436] Exemplary embodiments

[0437] One embodiment is the endosomal escape-enhancing conjugate of the present invention, wherein the saponin is a disaccharide-chain triterpenoid saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group in position 23, and wherein the saponin is preferably a saponin that can be isolated from a Gypsophila or Saponaria species, more preferably the saponin is saponin SO1861 or any diastereomer thereof.

[0438] One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the binding site is at least one ligand (eg, immunoglobulin) to which at least one effector moiety is bound.

[0439] One embodiment is an endosomal escape-enhancing conjugate of the present invention, wherein the binding site is an immunoglobulin or at least a binding domain thereof that binds to a cell surface molecule, wherein preferably, the cell surface molecule is selected from any one of the following: HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, CD352, DLL3, ephrin A4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD71.

[0440] One embodiment is an endosomal escape-enhancing conjugate of the present invention, wherein the linker is coupled to the glycoside via a cleavable bond, and wherein the ligand is an immunoglobulin, wherein preferably, the cleavable bond undergoes cleavage under acidic, reducing, enzymatic or light-induced 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, wherein preferably the cleavable bond is a disulfide bond or a peptide bond.

[0441] One embodiment is an endosomal escape-enhancing conjugate of the present invention, wherein 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 protein molecule, and the same first binding site of the first and third protein 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 protein molecules.

[0442] One embodiment is a combination of an endosomal escape-enhancing conjugate according to the present invention (i.e., a first protein molecule) and a binding moiety (i.e., a second or third protein molecule), wherein the binding moiety comprises at least one effector moiety, wherein the endosomal escape-enhancing conjugate and the binding moiety are independently capable of specifically binding to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of the complex of the endosomal escape-enhancing conjugate and the target cell-specific surface molecule and the complex of the binding moiety and the target cell-specific surface molecule.

[0443] One embodiment is a combination according to the invention, wherein when the binding moiety is a third protein molecule, the endosomal escape-enhancing conjugate and the binding moiety are capable of specifically binding to the same target cell-specific surface molecule or structure.

[0444] One embodiment is a combination according to the invention, wherein when the binding moiety is a third protein molecule, the endosomal escape-enhancing conjugate is capable of competing with the binding moiety for binding to a target cell-specific surface molecule or structure.

[0445] One embodiment is a combination according to the invention, wherein the endosomal escape-enhancing conjugate and the binding moiety are capable of specifically binding to the same epitope independently of each other.

[0446] One embodiment is a combination according to the invention, wherein when the binding moiety is a third protein molecule, the endosomal escape-enhancing conjugate is capable of specifically binding to a first epitope that is the same as the first epitope to which the binding moiety is capable of specifically binding.

[0447] One embodiment is a combination according to the invention, wherein when the binding moiety is a second protein molecule, the endosomal escape-enhancing conjugate and the binding moiety are capable of specifically binding to different target cell-specific surface molecules or structures.

[0448] One embodiment is a combination according to the invention, wherein the target cell-specific surface molecule or structure is selected from the group consisting of: HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, F4GF2, FLT3, CD71.

[0449] One embodiment is a combination according to the invention, wherein the glycoside molecule is a disaccharide-chain triterpene, preferably a saponin.

[0450] One embodiment is a combination according to the invention, wherein the glycoside molecule is a disaccharide-chain triterpenoid saponin.

[0451] One embodiment is a combination according to the invention, wherein the saponin is a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position 23.

[0452] One embodiment is a combination according to the invention, wherein the saponin is a saponin isolatable from a species of the genus Gypsophila or Saponaria.

[0453] One embodiment is a combination according to the invention, wherein the saponin is SO1861 or any diastereoisomer thereof.

[0454] One embodiment is a combination according to the invention, wherein at least one glycoside is bound to the ligand (binding site for an epitope on a cell surface molecule) via a cleavable bond, wherein preferably said cleavable bond undergoes cleavage under acidic, reducing, enzymatic or light-induced conditions, and wherein the cleavable bond is preferably a disulfide bond or a peptide bond.

[0455] 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.

[0456] One embodiment is a combination according to the invention, wherein the endosomal escape-enhancing conjugate comprises a defined number or a defined range of glycosides.

[0457] One embodiment is a combination according to the invention, wherein the defined range is 1-30 glycosides, preferably 1-20, more preferably 1-10, more preferably 1-6, more preferably 2-6, more preferably 2-5, more preferably 3-5, more preferably 3-4 glycosides.

[0458] One embodiment is a combination according to the invention, wherein the effector moiety is a pharmaceutically active substance, such as a toxin (eg a protein toxin), a drug, a polypeptide or a polynucleotide.

[0459] One embodiment is a combination according to the invention, wherein the target cell is a diseased cell or a disease-associated cell, preferably a tumor cell or a tumor-associated cell (such as a tumor vascular cell), or an immune cell (such as a regulatory T cell) or an autoimmune cell.

[0460] One embodiment is a combination according to the invention, wherein at least one effector moiety is bound to the binding moiety (second or third protein molecule) via a cleavable bond, wherein preferably the cleavable bond undergoes cleavage under acidic, reducing, enzymatic or light-induced conditions, and / or wherein the cleavable bond is a disulfide bond or a peptide bond.

[0461] One embodiment is a combination according to the invention, wherein the glycoside (saponin) is capable of increasing the endosomal escape of the effector molecule.

[0462] One embodiment is a combination according to the invention for use as a medicament.

[0463] One embodiment is a pharmaceutical composition comprising the combination according to the invention (previous embodiment) and a pharmaceutically acceptable excipient.

[0464] One embodiment is a pharmaceutical composition according to the invention, which further comprises at least one additional active pharmaceutical ingredient (such as an additional immunoglobulin).

[0465] One embodiment is the combination for use according to the invention or the pharmaceutical composition according to the invention for use in a method for the treatment of cancer or an autoimmune disease.

[0466] One embodiment is a combination for use according to the invention wherein the endosomal escape-enhancing conjugate (first protein molecule) and the binding moiety (second or third protein molecule) are administered concomitantly or sequentially, preferably concomitantly.

[0467] One embodiment is a method of treating cancer comprising administering a combination according to the invention to a patient in need thereof.

[0468] One embodiment is a method of treating cancer comprising administering a pharmaceutical composition according to the present invention to a patient in need thereof.

[0469] One embodiment is a kit comprising a first container comprising an endosomal escape-enhancing conjugate according to the invention and a second container comprising a binding moiety according to the invention, the kit further comprising instructions for use of the binding molecules.

[0470] The first protein molecule is suitable for use as a semi-finished product for the manufacture of a functionalized ADC or functionalized OAC, wherein 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. One embodiment is a first protein molecule of the invention further comprising a payload or effector moiety of the invention (e.g., a toxin or oligonucleotide) covalently bound to the first protein molecule of the invention directly or via a linker of the invention, preferably a cleavable linker of the invention, and / or via an oligomeric or multimeric scaffold of the invention. For example, such a functionalized ADC or OAC comprises 2-4 saponins covalently coupled, for example, to cysteine ​​side chains in the first protein molecule (e.g., a ligand or antibody (fragment)) directly or via a (cleavable) linker, or comprises a dendron comprising, for example, 1-16 covalently coupled saponins bound thereto, said dendron being covalently coupled, for example, to cysteine ​​side chains and / or lysine side chains of the first protein molecule according to the invention.

[0471] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. Example

[0472] Example A - Treatment of tumor-bearing mammals with a combination of a conjugate of the invention and an ADC results in survival and tumor regression

[0473] A suspension of human A431 tumor cells was injected subcutaneously into female Balb / c nude mice. Human epidermal carcinoma was established under the skin of the mice in a xenograft animal tumor model. Following injection of the tumor cells, xenograft tumors were allowed to grow to approximately 170-180 mm. 3 A431 tumor cells have the following characteristics: high expression of EGFR, moderate expression of CD71, and low expression of HER2.

[0474] In Table A, the treatment results of control mice and tumor-bearing mice are presented. Tumor-bearing mice were treated with designated antibodies directed against human Her2 / neu, human EGFR or human CD71, cell surface receptors on xenograft tumors. Cetuximab was covalently conjugated to saponin SO1861. SO1861 first had a linker EMCH (N-ε-maleimidocaproic acid hydrazide), where EMCH is a maleimide-hydrazide cross-linker for covalently conjugating a sulfhydryl group (reduced cysteine ​​of the antibody) to a carbonyl group (aldehyde or ketone; here, the carbonyl group of the aldehyde at position C-23 of the saponin). Saponin-EMCH was covalently coupled to the reduced cysteine ​​of cetuximab, forming a covalent thioether bond between EMCH and the cysteine ​​side chain. ADC trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9, IgG)-saporin (covalent conjugate) were tested in mice for their tumor attack efficacy, measured as tumor volume over time after the start of ADC treatment. The ADC dose was suboptimal in the tumor model. That is, based on previous experiments, it was determined that at suboptimal doses of ADC, no tumor regression or tumor growth arrest was observed.

[0475]

[0476] These results indicate that, while ADC combination therapy at doses that were ineffective when used alone to treat tumor-bearing mice (tumor growth and mouse death were not prevented (euthanasia)), the covalent conjugates of the present invention, consisting of a tumor cell-specific receptor-targeting antibody covalently bound to a saponin (i.e., SO1861), provided an effective and efficacious treatment regimen when administered alone to cancer-bearing mice at doses that were ineffective (tumor growth and mouse death were not prevented (euthanasia)), as demonstrated by tumor regression and prolonged survival of treated animals (exceeding the duration of the experiment). Suboptimal doses of ADCs that lack anti-tumor activity when administered alone, combined with the covalently bound saponin-containing conjugates of the present invention, thus provide an effective treatment option for cancer patients, in which relatively low doses of ADC are effective. Lower doses of ADCs are expected to reduce the risk of adverse events, or even have no side effects at all. Furthermore, when considering the efficacy of the ADCs, the stimulatory effects of the saponin-containing conjugates of the present invention suggest that ADCs that have previously been shown to lack efficacy in treating tumor patients may regain interest and value because, as demonstrated in the current examples, their efficacy is improved in a combination therapy setting. With reference to Tables A2 and A3, a summary of ADCs previously studied in human clinical settings, but subsequently withdrawn from further clinical development, is provided. In particular, ADCs whose clinical development was terminated due to observed lack of efficacy and / or due to the occurrence of unacceptable adverse events are ADCs that, when combined with the covalently bound saponin-containing conjugates of the present invention (e.g., the tested cetuximab-saponin), may offer new value to cancer patients.

[0477] Example B - Quillaja saponin mixture containing QS-21 having endosomal / lysosomal escape enhancing activity

[0478] Scheme 1 shows a series of common molecular structures of QS-21 saponins (partially adapted from: Conrado Pedebos, Laércio Pol-Fachin, Ramon Pons, Cilaine V. Teixeira Hugo Verli, Atomic Model and Microle Dynamics of QS-21 Saponin, Molecules 2014, 19, 3744-3760). Mixtures of water-soluble saponins obtained from Quillaja saponin (Sigma-Aldrich, product number S4521; Roth, product number 6857; InvivoGen, product "Quil-A") can be used in the endosomal / lysosomal escape-enhancing conjugates and combinations of compositions of the present invention, based on the endosomal / lysosomal escape-enhancing properties of at least one individual saponin present in the mixture (e.g., QS-21), or based on a combination of two or more saponins included in the mixture (e.g., QS-21 and QS-7).

[0479] The inventors have demonstrated that a 2.5 μg / ml dose of a mixture of Quillaja saponins enhances the endosomal escape of oleanolic acid in a cell-based bioassay using mammalian tumor cells. The effector moiety exposed to the cells was oleanolic acid covalently conjugated to the ligand EGF: EGF-oleanolic acid. For the free saponins, the cells tested were the tumor cell lines HeLa, while A431, MDA-MB-468, CaSki, and A2058 were used to test the saponins when covalently conjugated to cetuximab.

[0480] Example 1

[0481] In the BT474 (HER2++) xenograft mouse model, various concentrations of trastuzumab-saporin (a HER2-targeting protein-toxin conjugate; intravenous injection) were tested for their enhanced efficacy in combination with 1.5 mg / kg SO1861 (injected subcutaneously 1 hour before antibody-toxin injection). 3 Dosing began on day 13, and tumor volume was determined after each treatment. Although tumor growth inhibition was observed in mice treated with 1 mg / kg and 0.3 mg / kg of trastuzumab-saporin, no enhanced tumor growth inhibition was observed in mice treated with the trastuzumab-saporin + SO1861 combination. This suggests that unconjugated SO1861 is unable to enhance the antibody-protein toxin in the current setting and mouse model.

[0482] Example 2

[0483] Material:

[0484] QSmix(1): S4521 (Sigma Aldrich); QSmix(2): 6857.1 (Carl Roth) QSmix(3): Adjuvant: vac-quil (InvivoGen / Brenntag).

[0485] Previously, the efficacy of various saponins (SO1861, SO1642) as "free" unconjugated molecules co-administered to cells in combination with ligand-toxin fusions (e.g., EGF-oleanolic acid) or antibody-protein toxin conjugates enhanced the cell-killing activity of cells expressing the target. Here, in the presence and absence of a non-effective fixed concentration of 1.5 pM EGF-oleanolic acid, the cell-killing activity of HeLa (EGFR + ) cells. This revealed a strong enhancement of the cell-killing activity of all tested saponin variants (IC50 = 300 nM; Figure 2A) compared to the treatment without EGF-oleanol. Next, EGF-oleanol was titrated with a fixed concentration of saponin (~1000 nM). This showed a strong enhancement of targeted cell killing at low pM concentrations of EGF-oleanol (IC50 = 0.4 pM; Figure 2B), observed for all used saponins: SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904. EGF-oleanol alone could only induce cell killing at very high concentrations (IC50 = 10.000 pM). This suggests that these specific types of saponins all possess the intrinsic ability to effectively induce endosomal escape, with only very small amounts of targeted toxin available.

[0486] To expand this test, saponins from other sources were analyzed. A saponin purified from the root extract of Gypsophila elegans M. Bieb (GE1741) was titrated on HeLa cells in the presence and absence of 1.5 pM EGF-oleanolic acid and compared with purified SO1861. GE1741 also enhanced the killing of HeLa cells induced by EGFd-oleanolic acid, but with slightly lower efficacy than SO1861. (GE1741 IC50 = 800 nM; Figure 2 C) and also exhibited higher general toxicity (IC50 = 5.000 nM in the absence of EGF-oleanolic acid; Figure 2 C). Figure 1C). In a similar test, different partially purified Quillaja saponin mixtures (QSmix 1-3) were co-applied with 1.5 pM EGF oleanolic acid to HeLa cells, showing that two of the three (QSmix 1 and QSmix 3) had similar activity to SO1861 (IC50 QSmix / QSmix3 = 300 nM; Figure 1 QSmix (2) was less efficient in enhancing cell killing induced by 1.5 pM EGF-oleanolic acid (IC50 = 2000 nM; Figure 2D), however, no general toxicity was observed. This suggests that, also in the QS extract, a specific type of saponin can be used that effectively induces endosomal escape of the targeted ligand toxin EGF-oleanolic acid.

[0487] Example 3

[0488] According to the present invention, to conjugate the SO1861 molecule to an antibody, an unstable / acid-sensitive linker (-EMCH or -N3) was conjugated to SO1861 via an aldehyde group, yielding SO1861-EMCH or SO1861-N3 ( Figures 60-66 ). To validate the activity of SO1861-EMCH, the molecule was titrated in the presence and absence of a fixed, ineffective (1.5 pM) EGF-oleanolic acid concentration on EGFR-expressing (A431, HeLa) and non-expressing (A2058) cells. In all three cell lines, SO1861 alone showed a strong reduction in cell viability, while SO1861-EMCH as a single compound showed no toxicity up to 25.000 nM ( Figures 3 AC ). When SO1861-EMCH was combined with 1.5 pM EGF-oleanolic acid, the molecule was titrated in the presence and absence of a fixed, ineffective (1.5 pM) EGF-oleanolic acid concentration on EGFR-expressing (A431, HeLa) and non-expressing (A2058) cells. In all three cell lines, SO1861 alone showed a strong reduction in cell viability, while SO1861-EMCH as a single compound showed no toxicity up to 25.000 nM ( Figures 3 AC ). + Strong target-specific cell viability reduction was observed in A431 and HeLa cells (IC50 = 3.000 nM; Figure 2A, B), while EGFR-A2058 cells were not affected at all (Figure 3C). Similar results were obtained with SO1861-N3. SO1861-N3 also showed effective cell killing in A431 and HeLa cells with 1.5 pM of EGF-oleanolic acid (IC50 = 3.000 nM), but without EGF-oleanolic acid, general toxicity was observed above 10.000 nM (Figure 3D, Figure 2E).

[0489] For the stable conjugate of SO1861 and antibody according to the present invention, the stable linker (HATU, Figure 70) was conjugated to SO1861 via the carboxylic acid group of SO1861, generating SO1861-(S). To determine activity, varying concentrations of SO1861-(S) were co-administered with 1.5 pM of EGF oleanolic acid and tested for cell killing activity in HeLa cells expressing EGFR. SO1861-(S) exhibited similar activity to SO1861, suggesting that conjugation to the carboxylic acid did not affect the molecule's endosomal escape-enhancing potency, as observed with SO1861-EMCH ( Figure 4 ).

[0490] like Figure 19 As shown in , the 1 target 2-component system (1T2C) is a combined treatment of mAb1-protein toxin and mAb1-SO1861. SO1861-EMCH was conjugated via a cysteine ​​residue (Cys) using DAR 4, and the HSP27 BNA oligonucleotide was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) 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, (ip)) and cetuximab-(Lys-L-HSP27BNA) 4 The combination of (iv) was tested for EGFR tumor-targeted gene silencing activity in an A431 xenograft mouse tumor model. 3 When the drug was administered on day 12, tumor samples were collected 72 hours after the first dose and analyzed for HSP27 gene expression compared with the control gene mRNA expression level (reference gene). This showed that compared with a single dose of cetuximab-(Cys-L-SO1861) 4 or cetuximab-(Lys-L-HSP27BNA) 4 Compared with monotherapy, a single dose of 50 mg / kg of cetuximab-(Cys-L-SO1861) 4 + 25 mg / kg of cetuximab-(Lys-L-HSP27BNA) 4 Resulting in a 50% decrease in HSP27 gene expression in A431 tumors ( Figure 7Compared to vehicle-controlled tumors, a 40% reduction in HSP27 gene silencing was observed. This demonstrates that the combination of cetuximab-conjugated SO1861 and cetuximab-conjugated HSP27 BNA oligonucleotides, according to the 1T2C invention, induces effective targeted delivery of therapeutic antisense oligonucleotides to the cytoplasm of solid tumor cells, thereby inducing tumor-targeted gene silencing in vivo.

[0491] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) via a cysteine ​​residue (Cys), with DAR 4 resulting in the production of trastuzumab-(Cys-L-SO1861) 4 Trastuzumab-(Cys-L-SO1861) 4 The combination of trastuzumab-saporin (trastuzumab protein toxin conjugate) was tested in a mouse tumor model (patient-derived xenograft tumor model, PDX) with high HER2 expression levels and resistance to trastuzumab monotherapy. 4 or 0.03 / 0.02 mg / kg of trastuzumab-saporin monotherapy compared to 40 mg / kg of trastuzumab-(Cys-L-SO1861) according to the 1T2C invention 4 The combination of trastuzumab-saporin (administered intravenously, (iv)) + 0.03 (days 1, 8) / 0.02 (days 15, 22, 30, 36, 43) mg / kg showed strong tumor growth inhibition ( Figure 8 In addition, the lower dose combination (40 mg / kg trastuzumab-(Cys-L-SO1861) 4 No tumor growth inhibitory activity was observed in tumor-bearing mice treated with trastuzumab-saporin (+0.01 mg / kg). Figure 8 ). This demonstrates and demonstrates that the 1T2C combination of trastuzumab-conjugated SO1861 + trastuzumab-conjugated protein toxin induces efficient targeted delivery of therapeutic protein toxins in the cytoplasm of solid tumor cells, thereby inducing tumor cell death and tumor growth inhibition in vivo.

[0492] The 1 target 2 component system (1T2C) is a combination treatment of mAb1-SO1861 and mAb1-protein toxin ( Figure 19 )

[0493] SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR 3,7 (Cetuximab-(Cys-L-SO1861)3,7 At a fixed concentration of 10 pM cetuximab-saporin (cetuximab, conjugated to a protein toxin, saporin) versus cetuximab-(Cys-L-SO1861) 3,7 Titration was performed and the targeted protein toxin-mediated inhibition of EGFR-expressing cells (A431, EGFR ++ ;CaSKi,EGFR + ) cell killing. This indicates that at low concentrations of cetuximab-(Cys-L-SO1861) 3,7 (A431: IC50 = 0.6 nM and Caski IC50 = 1 nM; Figure 9 A, B) showed strong cell killing effects, while cetuximab and cetuximab-(Cys-L-SO1861) 3,7 or cetuximab + 10 pM cetuximab-saporin failed to induce any cell killing activity in cells expressing EGFR. This suggests that cetuximab-conjugated SO1861 effectively enhances the endosomal escape of cetuximab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell killing of cells expressing EGFR. Compared with CaSki, which is related to the EGFR expression level in these cell lines, the cell killing activity in A431 is more effective. When cetuximab-(Cys-L-SO1861) 3,7 Competition for EGFR receptor binding between the two conjugates within 1T2C was also observed as the concentration increased, and the cell killing activity decreased due to competition for receptor binding and internalization of cetuximab-saporin ( FIG. 9 , A and B).

[0494] Next, at a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 Cetuximab-saporin was titrated on the EGFR-expressing cells and the targeted protein toxin-mediated killing of cells was determined. This showed that 75 nM cetuximab-(Cys-L-SO1861) 3,7Combining with low concentrations of cetuximab-saporin already induced effective cell killing in cells expressing EGFR (A431: IC50 = 0.4 pM; and CaSKi: IC50 = 2 pM; Figure 9 C, D), while cetuximab-saporin alone or cetuximab-saporin + 75 nM cetuximab only showed cell killing in both cell lines at high concentrations of cetuximab-saporin (IC50 = 40 pM, IC50 = 1000 pM, respectively) (Figure 9 C, D). All of this indicates that in cells that highly express EGFR, relatively low concentrations of cetuximab-saporin can be effective and induce cell killing only in combination with low cetuximab-SO1861 concentrations. In the cetuximab-toxin titration treatment, receptor competition between the two conjugates within the 1T2C system was also observed when comparing the cell killing activity of cetuximab-saporin in the presence and absence of 75 nM cetuximab ( FIG. 9C , D ).

[0495] Next, the cetuximab-(Cys-L-SO1861) 3,7 Titration was performed and the targeted protein toxin-mediated inhibition of EGFR-low or no EGFR expression cells (HeLa, EGFR + / - ;A2058,EGFR - Cells with low (HeLa) or no (A2058) EGFR expression are resistant to cetuximab-(Cys-L-SO1861) 3,7 Any combination of cetuximab + 10 pM saporin was insensitive (HeLa: IC50>1000 nM; A2058: IC50>1000 nM; Figure 10 A, B). This suggests that in the absence of sufficient EGFR receptor expression, the effective intracellular delivery concentration of SO1861 is not the optimal (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing. Next, at a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 Cetuximab-saporin was titrated on the 20 μg / ml RT-PCR assay and the targeted protein toxin-mediated killing of cells with low (HeLa) or no (A2058) EGFR expression was determined. Low EGFR expressing cells (HeLa) were killed only at high cetuximab-saporin concentrations and 75 nM cetuximab-(Cys-L-SO1861). 3,7 When combined, it showed cell killing (HeLa: IC50=60pM), Figure 10C), while A2058 cells (EGFR -) at any tested concentration (A2058: IC50>10.000 pM; Figure 10D). All of these indicate that cells with low or no EGFR receptor expression are sensitive to cetuximab-(Cys-L-SO1861) 3,7 The cetuximab-saporin combination was insensitive due to the lack of sufficient EGFR receptors to promote antibody-mediated delivery of sufficient SO1861 in the endolysosomal compartment to facilitate the escape of the protein toxin.

[0496] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2 with a DAR of 4, (Trastuzumab-(Cys-L-SO1861)) via a cysteine ​​residue (Cys) 4 ) conjugated. Trastuzumab-(Cys-L-SO1861) was conjugated to a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab, conjugated to a protein toxin, saporin). 4 ) were titrated and the targeted protein toxin-mediated inhibition of HER2-expressing cells (SK-BR-3, HER2 ++ This suggests that at low concentrations of trastuzumab-(Cys-L-SO1861) 4 (SK-BR-3: IC50 = 0.8 nM; Figure 11 A) has a strong cell killing effect, while equivalent concentrations of trastuzumab, trastuzumab-(Cys-L-SO1861) 4 or trastuzumab + 50 pM trastuzumab-saporin could not induce any cell killing activity in cells expressing HER2. This indicates that trastuzumab conjugated with SO1861 effectively enhances the endosomal escape of trastuzumab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell killing of HER2-expressing cells. When trastuzumab-(Cys-L-SO1861) 4 As concentrations increased, receptor competition between the two conjugates within 1T2C was also observed, and cell killing activity decreased due to competitive receptor binding and internalization of trastuzumab-saporin ( Figure 11 A).

[0497] Next, according to the present invention, at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 Trastuzumab-saporin was titrated on the HER2-expressing cells and the targeted protein toxin mediated cell killing was determined. This showed that 2,5 nM trastuzumab-(Cys-L-SO1861) 4Combination with low concentrations of trastuzumab-saporin already induced potent cell killing in HER2-expressing cells (SK-BR-3: IC50 = 2 pM; Figure 11 ), while trastuzumab-saporin alone or trastuzumab-saporin + 2.5 nM trastuzumab showed cell killing only at high concentrations of trastuzumab-saporin ( Figure 11 B). All of this suggests that relatively low concentrations of trastuzumab-saporin can be effective and only in combination with low concentrations of trastuzumab-(Cys-L-SO1861) 4 The combination can induce cell killing only in cells that highly express HER2.

[0498] Next, according to the present invention, trastuzumab-(Cys-L-SO1861) was detected on a fixed concentration of 50 pM trastuzumab-saporin. 4 Titration was performed and the targeted protein toxin-mediated inhibition of HER2-low expressing cells (A431 HER2 + / - ) or cells without HER2 expression (JIMT-1: HER 2+ ;MDA-MB-468:HER2 - Cells with low or no HER2 expression are resistant to trastuzumab-(Cys-L-SO1861) 4 All combinations of trastuzumab and saporin at +50 pM were insensitive (JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM; Figure 13 A, B). This suggests that in the absence of sufficient HER2 receptor expression, the effective intracellular delivery concentration of SO1861 is not optimal (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing. Next, at a fixed concentration of 2,5 nM trastuzumab-(Cys-L-SO1861) 4 Trastuzumab-saporin was titrated on the HER2-low expressing or non-expressing cells and the targeted protein toxin mediated cell killing was determined. Cells expressing low HER2 (JIMT-1) were killed only at high trastuzumab-saporin concentrations and 2.5 nM trastuzumab-(Cys-L-SO1861). 4 The combination of JIMT-1 and JIMT-2 showed cell killing effect (JIMT-1: IC50>10.000pM; Figure 13 C), while MDA-MB-468 cells (HER2 - ) were not sensitive at any concentration tested (MDA-MB-468: IC50>10.000 pM; Figure 13 D).

[0499] All of this suggests that cells with low or no HER2 receptor expression are resistant to trastuzumab-(Cys-L-SO1861) due to the lack of sufficient HER2 receptors to facilitate antibody-mediated delivery of sufficient SO1861 in the endolysosomal compartment to promote the escape of the protein toxin. 3,7 +Trastuzumab-saporin insensitive.

[0500] To show that the activity of the 1T2C system is driven by acidification of the endolysosomal compartment, the 1T2C system according to the present invention was tested in combination with the endosomal acidification inhibitor chloroquine. Trastuzumab-saporin was combined with 5 nM trastuzumab-(Cys-L-SO1861) 4 and titrated with or without chloroquine. Trastuzumab-saporin 5nM Trastuzumab-(Cys-L-SO1861) 4 In cells that highly express HER2 (SK-BR-3, HER2 ++ ; IC50 = 0.2pM;) showed strong cell killing activity, however, trastuzumab-saporin + 5nM trastuzumab-(Cys-L-SO1861) 4 +0.5μM chloroquine resulted in the ++ ) cells (IC50 = 40 pM). This indicates that when the acidification of endolysosomes is inhibited, the activity of the antibody conjugated with SO1861 is reduced / blocked (Figure 14A). 3,8 +0.5μM chloroquine (IC50=200pM) in cells expressing EGFR (A431, EGFR ++ ; FIG14B) compared, using the 1T2C drive according to the present invention to achieve the same result.

[0501] like Figure 20 As shown in , the 1 target 2 component system (1T2C) can also be a combined treatment of mAb1-SO1861 and mAb1-antisense BNA oligonucleotides. To this end, we used antisense BNA oligonucleotides against cancer-specific target gene mRNA (upregulated in cancer cells), heat shock protein 27 (HSP27). After release into the cytoplasm, the antisense BNA recognizes and binds to the mRNA encoding HSP27, targeting the mRNA for destruction, thereby depleting the expression of HSP27 mRNA in cancer cells. According to the present invention ( Figure 20 ), HSP27BNA was conjugated to cetuximab using DAR4 (cetuximab-(Lys-L-HSP27BNA) 4 ), and with cetuximab-(Cys-L-SO1861)3,8 Combination testing to enhance the expression of EGFR in cells expressing EGFR (A431, EGFR ++ ) and HSP27 gene silencing activity in non-expressing cells (A2058, EGFR). Cetuximab-(Cys-L-SO1861) 3,8 +100nM Cetuximab-(Lys-L-HSP27BNA) 4 Strong HSP27 gene silencing was demonstrated in cells expressing EGFR (A431: IC50 = nM, FIG15A ), while cetuximab-(Cys-L-SO1861) 3,8 It did not show any gene silencing activity alone. - ), no gene silencing activity was observed in the 1T2C combination (Figure 15B). Next, cetuximab-(Lys-L-HSP27BNA) 4 +76.9nM Cetuximab-(Cys-L-SO1861) 3,8 A431 showed strong HSP27 gene silencing activity in cells expressing EGFR (IC50 = 4 nM, Figure 15C), while cetuximab-(Lys-L-HSP27BNA) 4 or cetuximab-(Cys-L-SO1861) 3,8 or cetuximab-(Lys-L-HSP27BNA) 4 The combination of 1T2C and 77nM cetuximab did not show any significant gene silencing activity (IC50>100nM). When the experiment was performed in cells that do not express EGFR (A2058), no gene silencing activity was observed in the 1T2C combination (IC50>100nM; Figure 15D). All of this indicates that the 1T2C system effectively delivers antisense BNA oligonucleotides to the cytoplasm of cells that highly express EGFR, thereby inducing mRNA degradation of the BNA target mRNA, leading to target gene silencing.

[0502] like Figure 21 As shown in , the 1 target 2 component system (1T2C) can also be mAb1-(scaffold (-SO1861) n ) n The dendron (-L-SO1861) was treated with DAR3,9 and mAb1-protein toxin combination. 4 The conjugation was conducted with cetuximab conjugated via a cysteine ​​residue (Cys) and the cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9Enhanced cell killing activity in EGFR-expressing cells (MDA-MB-468) in combination with an anti-EGFR antibody-protein toxin conjugate (cetuximab-saporin). Cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9 +10 pM cetuximab-saporin effectively induced toxin-mediated cell killing in cells overexpressing EGFR (IC50 = 0.4 nM; Figure 16 A), which was not detected by cetuximab-Cys-(dendron (-L-SO1861) 4 ) 3,9 or cetuximab + 10 pM cetuximab-saporin or cetuximab-induced ( Figure 16 This indicates that according to the 1T2C invention, the dendron (-L-SO1861) conjugated to cetuximab 4 The results showed that 1T2C effectively enhanced the endosomal escape of cetuximab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell killing of cells expressing high levels of HER2. + / - ), which showed that there was no cell killing activity (IC50>100pM; Figure 16 B), 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 leading to toxin-mediated cell killing.

[0503] Next, use DAR4 to convert the dendron primitive (-L-SO1861) 4 It is conjugated with the anti-HER2 antibody trastuzumab via cysteine ​​conjugation (Cys) to form trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 , and tested the efficacy of the drug in combination with an anti-HER2 antibody-protein toxin conjugate (trastuzumab-saporin) on HER2-expressing cells (SK-BR-3, HER2 ++ ) in the enhanced cell killing activity of trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 +50 pM of trastuzumab-saporin effectively induced toxin-mediated cell killing (IC50 = 2 nM, Figure 16 C), which is not composed of trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 or trastuzumab + 50nM trastuzumab-saporin or trastuzumab ( Figure 16 C) induced by trastuzumab conjugated dendron (-L-SO1861). 4 It effectively enhanced the endosomal escape of trastuzumab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell killing in cells expressing high levels of HER2. + / - Similar experiments in cells expressing trastuzumab-Cys-(dendron (-L-SO1861) 4 ) 4 +50 pM of trastuzumab-saporin was inactive (IC50>200 nM; Figure 16 D), indicating that in the absence of sufficient HER2 receptor expression, the effective intracellular SO1861 concentration is not optimal (threshold) for inducing endosomal protein toxin escape and toxin-mediated cell killing.

[0504] The clinically approved ADC, trastuzumab-emtansine (T-DM1), is a conjugate of the anti-Her2 antibody, trastuzumab, and the small molecule toxin emtansine (DAR3.5). According to the present invention, T-DM1 is conjugated with trastuzumab-(Cys-L-SO1861) 4 Combination titration and with antibody protein toxin conjugate trastuzumab-saporin + trastuzumab-(Cys-L-SO1861) 4 For comparison, trastuzumab-saporin + 2.5nM trastuzumab-(Cys-L-SO1861) 4 Compared with trastuzumab-saporin alone + 2.5 nM trastuzumab or trastuzumab-saporin, T-DM1 + ​​25.6 nM trastuzumab-(Cys-L-SO1861) showed enhanced activity (IC50 = 2 pM, Figure 17). 4 No enhanced cell killing activity was shown (IC50>100 pM; Figure 17). This indicates that the 1T2C system according to the present invention cannot enhance the delivery of antibody-small molecule conjugates, as small molecules can already passively cross the (endolysosomal) membrane.

[0505] The 1 target 2 component system (1T2C) can also be a combined treatment of mAb1-QSmix (a mixture of Quillaja saponins) and mAb1-protein toxin.

[0506] QSmix-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR 4.1 (Cetuximab-(Cys-L-QSmix) 4,1The cetuximab-(Cys-L-QSmix) was detected at a fixed concentration of 10 pM cetuximab-saporin or 10 pM cetuximab-oleanin. 4,1 Titration was performed and the targeted protein toxin-mediated inhibition of A431 (EGFR ++ )、CaSKi(EGFR + ) and A2058 (EGFR - ) cell killing effect. This indicates that in A431 (EGFR ++ ) and CaSKi(EGFR + ) cells, low concentrations of cetuximab-(Cys-L-QSmix) 4,1 +10 pM cetuximab-saporin or 10 pM cetuximab-oleanin had strong cell killing effect (A431: IC50=3 nM, Figure 18 A; CaSKi: IC 50 = 1 nM, Figure 18 B), while all control treatments could not induce any cell killing in cells expressing EGFR. - ) cells, no HSP27 gene silencing (IC50>1000nM; Figure 18 C). This indicates that cetuximab-conjugated QS21mix effectively enhances the endosomal escape of cetuximab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell killing only in cells expressing EGFR.

[0507] Example 4

[0508] The labile SO1861 was conjugated to the anti-EGFR antibody cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR3.9 (cetuximab-(Cys-L-SO1861) 3.9 ) and tested their enhanced antisense BNA oligonucleotides leading to enhanced target gene silencing. In this study, we used antisense BNA oligonucleotides against the cancer-specific target gene, heat shock protein 27 (HSP27) mRNA. Within the cytoplasm of the cell, HSP27 BNA binds to the mRNA encoding HSP27, targeting the mRNA for destruction, thereby reducing the expression of HSP27 in cancer cells. ++ (A431) and EGFR - (A2058) cells, at a fixed concentration of 100 nM HSP27BNA, to cetuximab-(Cys-L-SO1861) 3.9The combination according to the present invention showed effective HSP27 silencing on A431 (IC50 = 2 nM; Figure 5A), while cetuximab-(Cys-L-SO1861) alone 3.9 No silencing was observed. Cetuximab-(Cys-L-SO1861) 3.9 +100nM HSP27BNA in EGFR - No gene silencing activity was observed in cells (A2058) ( FIG5B ). This suggests that low concentrations of antibody-conjugated SO1861 can effectively enhance the cytoplasmic delivery and endolysosomal escape of antisense BNA oligonucleotides, thereby inducing effective gene silencing in cells expressing the target.

[0509] Next, the cells were treated with a fixed concentration of cetuximab-(Cys-L-SO1861) 3.9 Combined EGFR ++ (A431) and EGFR - (A2058) cells. This showed that HSP27BNA and 28.6nM cetuximab-(Cys-L-SO1861) 3.9 or 77nM cetuximab-(Cys-L-SO1861) 3.9 The combination was very effective in enhancing HSP27 gene silencing in A431 cells (IC50 = 10 nM; Figure 15C). HSP27BNA alone or in combination with a fixed equivalent of 77 nM cetuximab was less effective (IC50 = 1.000 nM; Figure 12C). HSP27BNA + 77 nM cetuximab-(Cys-L-SO1861) 3.9 The combined treatment was also tested on EGFR cells (A2058), and this showed no enhancement of HSP27 gene silencing (IC50 = 1.000 nM; Figure 5D). This suggests that cells with low or no EGFR receptor expression are resistant to cetuximab-(Cys-L-SO1861) 3,9 The combination of +HSP27BNA was insensitive, while cetuximab-targeted SO1861 could effectively enhance HSP27 gene silencing in high EGFR cells at low concentrations of non-targeted HS27BNA.

[0510] Next, SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR 3,8. The combination according to the invention was tested in the A431 xenograft 'nude' mouse tumor model with cetuximab-(Cys-L-SO1861) 3,8+HSP27BNA (antisense HSP27BNA oligonucleotides targeting and inducing degradation of tumor target hsp27 mRNA in cancer cells (gene silencing)) is used for EGFR-mediated tumor-targeted HSP27 gene silencing. When the tumor size reaches ∼150 mm 3 To determine HSP27 mRNA expression, dosing was initiated on day 12. For this purpose, tumor samples were collected 72 hours after the first dose and analyzed for HSP27 gene expression levels compared to cell control mRNA expression levels (reference gene). Tumor-bearing mice (n=3) received treatment (intraperitoneally; ip) on day 12 with 25 mg / kg of cetuximab-(Cys-L-SO1861). 3,8 + 25 mg of HSP27BNA and on day 15: 25 mg / kg of cetuximab-(Cys-L-SO1861) 3,8 +10 mg of HSP27 BNA, and this showed that HSP27 mRNA expression in tumors was reduced by 25% ( Figure 6 ). This demonstrates and demonstrates that, according to the present invention, conjugation of SO1861 to targeting antibodies effectively induces SO1861-mediated enhanced cytoplasmic delivery of therapeutic antisense oligonucleotides in solid tumor-bearing mice, inducing tumor-targeted gene silencing in vivo.

[0511] DAR2 and DAR4

[0512] DAR3.7 (Cetuximab-(Cys-L-SO1861) 3.7 ) or DAR2 (cetuximab-(Cys-L-SO1861) 2 ) SO1861-EMCH (labile linker, L) was conjugated to cetuximab via a cysteine ​​residue (Cys). These cetuximab-SO1861 conjugates were tested in combination with 10 pM cetuximab-saporin against cells that highly expressed EGFR (A431, EGFR ++ ) enhanced cell killing activity. This revealed that cetuximab-conjugated SO1861, DAR2, and DAR4 had the same cell killing potency ( Figure 22 A). Trastuzumab-(Cys-L-SO1861) 4 and trastuzumab-(Cys-L-SO1861) 2.1 Combined with 50pM trastuzumab saporin for high expression of HER2 cells (SK-BR-3, HER2 ++ ) and obtained the same results ( Figure 22B), which shows that reducing the amount of conjugated SO1861 according to the present invention reduces the efficacy of efficient endosomal escape and toxin-mediated cell killing.

[0513] Stability and instability

[0514] SO1861-HATU (stable linker, S) was conjugated to the anti-EGFR antibody cetuximab via a cysteine ​​residue (Cys) using DAR3.7 (cetuximab-(Cys-S-SO1861) 3.7 ) conjugated with unstable conjugated (cetuximab-(Cys-L-SO1861) 3.7 In comparison, this cetuximab-SO1861 conjugate was combined with a fixed concentration of 10 pM anti-EGFR antibody-protein toxin conjugate (cetuximab-saporin) and tested against cells that overexpress EGFR (MDA-MB-468, EGFR ++ This indicates that the stable or unstable antibody-conjugated SO1861 according to the present invention exhibits the same cell killing activity ( Figure 23 A). When trastuzumab-(Cys-L-SO1861) 4 with trastuzumab-(Cys-S-SO1861) 4 When compared with 50 pM of trastuzumab-saporin combination, the ++ ) reveals the same data ( Figure 23 B). All of these indicate that the stable or unstable conjugated SO1861 according to the present invention works effectively to induce endosomal escape of the antibody-conjugated toxin.

[0515] 2-target 2-component system (in vivo)

[0516] The 2-target 2-component system (2T2C) is a combination treatment of mAb1-SO1861 and mAb2-protein toxin ( Figure 37 SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR 4, resulting in: Cetuximab-(Cys-L-SO1861) 4 As in Figure 37 As shown in A431 (EGFR ++ / HER2 + / - / CD71 + ) Testing Cetuximab-(Cys-L-SO1861) in a xenograft 'nude' mouse tumor model 4The combination of trastuzumab-saporin or CD71mab-saporin was used for EGFR tumor-targeted cell killing. Dose escalation was performed to determine the efficacy of treatment (Day 9: 0.3 mg / kg of trastuzumab-saporin or 0.1 mg / kg of CD71mab-saporin + 5 mg / kg of cetuximab-(Cys-L-SO1861) 4 Day 14, 18: 0.1 mg / kg of trastuzumab-saporin or 0.05 mg / kg of CD71mab-saporin + 5 mg / kg of cetuximab-(Cys-L-SO1861) 4 Day 21: 0.05 mg / kg of trastuzumab-saporin or 0.05 mg / kg of CD71mab-saporin + 15 mg / kg of cetuximab-(Cys-L-SO1861) 4 Day 28: 0.02 mg / kg of trastuzumab-saporin or 0.02 mg / kg of CD71mab-saporin + 15 mg / kg of cetuximab-(Cys-L-SO18) 4 Trastuzumab-saporin / cetuximab-SO1861. Controls received the same dosing regimen, except for cetuximab (iv) at 25 mg / kg daily. On days 32 (dashed line), 35, and 39, we began combining cetuximab-(Cys-L-SO1861) at 25 mg / kg. 4 (2T2C invention with intraperitoneal injection (ip) + 0.02 mg / kg of trastuzumab-saporin or 0.02 CD71mab-saporin (intravenous administration, (iv))) and vehicle control, 25 mg / kg of cetuximab-(Cys-L-SO1861) 4 This revealed strong tumor regression in both 2T2C combination groups compared with 0.02 mg / kg or 0.02 mg / kg of trastuzumab-saporin / CD71mab-saporin monotherapy ( Figure 24 The 2T2C system even outperformed cetuximab, a clinically used monoclonal antibody against EGFR. We then performed the same experiment, but then we started with 25 mg / kg of cetuximab-(Cys-L-SO1861). 4 (Intraperitoneal injection (ip) + 0.03 mg / kg of trastuzumab-saporin or 0.03 mg / kg of CD71mab-saporin (intravenous administration, (iv)) treatment, administered on days 9 and 14, and then once a week. The 2T2C system according to the present invention showed tumor regression in all mice, and even in one mouse in the two 2T2C groups, the tumor was completely eradicated (tumor volume = 0 mm 3) ( FIG. 25 ). Here again, the control showed a strong increase in tumor volume, while the positive control for this A431 mouse model, cetuximab, only showed tumor growth inhibition, but no regression ( FIG. 25 ). This demonstrates and realizes that the 2T2C system approach of cetuximab-conjugated SO1861 + trastuzumab-conjugated protein toxin or CD71mab-conjugated protein toxin according to the present invention induces efficient targeted delivery of therapeutic protein toxins in the cytoplasm of solid tumors in tumor-bearing mice in vivo, thereby inducing or even completely eradicating tumors in some mice, and even in large-sized tumors (2000 mm) in other mice. 3 ) induced strong tumor regression in .

[0517] 2-target 2-component system (in vitro)

[0518] result

[0519] The 2-target 2-component system (2T2C) is a combination treatment of mAb1-SO1861 and mAb2-protein toxin ( Figure 37 SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) using DAR 3,7 (Cetuximab-(Cys-L-SO1861) 3,7 ).like Figure 37 As shown in Figure 2, a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab, conjugated to the protein toxin, saporin) was used to detect the effect of cetuximab-(Cys-L-SO1861) on the 3,7 Titration was performed and the targeted protein toxin-mediated inhibition of EGFR / HER2 expressing cells (A431), EGFR ++ / HER2 + / - ;CaSKi, EGFR + / HER2 + / - ) cells. This suggests that at low concentrations of cetuximab-(Cys-L-SO1861) 3,7 The results showed that the inhibitory effect of Cetuximab on the cytotoxicity of A431 was strong (A431: IC50 = 3 nM and CaSKi IC50 = 10 nM; Figure 26 A, B), while the equivalent concentrations of Cetuximab, Cetuximab-(Cys-L-SO1861) 3,7 Or cetuximab + 50pM trastuzumab-saporin could not induce any cell killing activity in cells expressing EGFR / HER2. This suggests that relatively low concentrations of cetuximab-SO1861 conjugate effectively enhance the endosomal escape of trastuzumab-conjugated protein toxin (at non-effective concentrations), thereby inducing effective cell killing of high EGFR / low HER2 expressing cells.

[0520] Next, at a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 Trastuzumab-saporin was titrated on the EGFR / HER2 expressing cells and the targeted protein toxin-mediated killing effect was determined. This showed that 75nM cetuximab-(Cys-L-SO1861) 3,7 Combined with low concentrations of trastuzumab-saporin, effective cell killing was induced in cells expressing EGFR / HER2 (A431: IC50 = 5 pM; and CaSKi: IC50 = 1 pM; Figure 26 C, D), while trastuzumab-saporin alone or trastuzumab-saporin + 75 nM cetuximab did not show significant cell killing activity in both cell lines (IC50> 10.000 pM) (Figure 26 C, D). All of this indicates that in high EGFR / low HER2 expressing cells, relatively low concentrations of trastuzumab-saporin can be effective and induce cell killing in combination with low cetuximab-SO1861 conjugate concentrations.

[0521] Next, if Figure 37 As shown in Figure 2, the expression of cetuximab-(Cys-L-SO1861) at a fixed concentration of 50 pM trastuzumab-saporin was detected. 3,7 titrated and determined the targeted protein toxin-mediated inhibition of HeLa (EGFR + / - / HER2 + / - ) or A2058(EGFR - / HER2 + / - ) cell killing. + / - / HER2 + / - ) and A2058 (EGFR - / HER2 + / - ) cells were treated with low concentrations of cetuximab-(Cys-L-SO1861) 3,7 +50 pM trastuzumab-saporin showed no cell killing (HeLa: IC50 = 400 nM; A2058: IC50 > 400 nM; Figure 27 This indicates that in the absence of sufficient receptor expression, an effective intracellular delivery concentration of SO1861 (threshold) is not reached to induce endosomal escape and cytoplasmic delivery of protein toxins. Next, at a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 Trastuzumab-saporin was titrated on the EGFR-targeted protein toxin and the EGFR-targeted protein toxin-mediated inhibition of HeLa cells was determined. + / - / HER2 + / - ) or A2058(EGFR - / HER2 + / - ) cell killing. + / - / HER2 + / - ) and A2058 (EGFR - / HER2 + / - ) cells showed no cell killing activity (HeLa: IC50>10.000pM; A2058: IC50>10.000pM; Figure 27 C, D). All of these indicate that cells with low or no EGFR expression are resistant to cetuximab-(Cys-L-SO1861) 3,7 The combination of +trastuzumab-saporin was insensitive due to the lack of sufficient EGFR receptors to facilitate antibody-mediated delivery of sufficient SO1861 (threshold) to ensure endosomal escape of the cytoplasmic endotoxin.

[0522] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) via a cysteine ​​residue (Cys) using DAR 4 (Trastuzumab-(Cys-L-SO1861) 4 Trastuzumab-(Cys-L-SO1861)4 was titrated against a fixed concentration of 1.5 pM EGF-oleanol (EGFR-targeted ligand-toxin fusion protein), and the targeted protein-toxin-mediated inhibition of HER2 / EGFR-expressing cells (SK-BR-3:HER2 ++ / EGFR + / - This revealed that at low concentrations of trastuzumab-(Cys-L-SO1861) 4 Strong cell killing under the presence of 1.5 pM EGF oleanolic acid (SK-BR-3: IC50 = 1 nM; Figure 28A), while equivalent concentrations of trastuzumab, trastuzumab-(Cys-L-SO1861) 4 Or trastuzumab + 1.5pM EGF oleanolic acid cannot be ++ / EGFR + / - This suggests that trastuzumab-conjugated SO1861 effectively enhances the endosomal escape of the EGF fusion protein toxin (at non-effective concentrations), thereby inducing cell killing in high HER2 / low EGFR expressing cells.

[0523] Next, at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 EGF oleanolic acid was titrated on the SK-BR-3 expressing cells (HER2 ++ / EGFR + / -This indicates that 2.5nM trastuzumab-(Cys-L-SO1861) 4 Combining with low concentrations of EGF-oleanolic acid has induced effective cell killing in cells expressing HER2 / EGFR (SK-BR-3: IC50=1 pM) (Figure 28B), while EGF-oleanolic acid alone or EGF-oleanolic acid + 2.5 nM trastuzumab did not show cell killing activity (IC50>10.000 pM) (Figure 28B). All these indicate that relatively low concentrations of EGF-oleanolic acid can be effective and only in combination with low trastuzumab-(Cys-L-SO1861) 4 The concentration combination induced cell killing in high HER2 / low EGFR expressing cells.

[0524] Next, trastuzumab-(Cys-L-SO1861) was tested on a fixed concentration of 1.5 pM EGF-oleanol. 4 Titration was performed and the targeted protein toxin-mediated inhibition of JIMT-1 (HER2 + / - / EGFR + / - ) or MDA-MB-468 cells (HER2 - / EGFR ++ ) cell killing. 4 Any combination of EGF + 1.5 pM oleanolic acid was insensitive (JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM; Figure 29 This suggests that in the absence of sufficient HER2 receptor expression, an effective intracellular delivery concentration (threshold) of SO1861 is not reached to induce endosomal escape and cytoplasmic delivery of the protein toxin.

[0525] Next, at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 EGF oleanolic acid was titrated and the targeted protein toxin-mediated inhibition of JIMT-1 (HER2 + / - / EGFR + / - ) or MDA-MB-468 cells (HER2 - / EGFR ++ ) cell killing. Whether or not 2,5nM trastuzumab-(Cys-L-SO1861) was present 4 Both cell lines showed cell killing effects at high concentrations of EGF oleanolic acid (JIMT-1: IC50 = 10.000 pM; MDA-MB-468: IC50 = 200 pM). Figure 29 C, D).

[0526] All of these indicate that cells with low or no HER2 receptor expression are resistant to trastuzumab-(Cys-L-SO1861) 3,7 The combination of EGF + 1.5 pM oleanolic acid was insensitive due to the lack of sufficient HER2 receptors to facilitate antibody-mediated delivery of sufficient SO1861 (threshold value) to ensure endosomal escape of the toxin from the cytoplasm of the cells.

[0527] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) via a cysteine ​​residue (Cys) using DAR 4 (Trastuzumab-(Cys-L-SO1861) 4 ).like Figure 37 As shown in Figure 2, the expression of trastuzumab-(Cys-L-SO1861) at a fixed concentration of 5 pM cetuximab-saporin (EGFR-targeted antibody-protein toxin conjugate) was detected by flow cytometry. 4 Titration was performed and the targeted protein toxin-mediated inhibition of HER2 / EGFR-expressing cells (SK-BR-3: HER2 ++ / EGFR + / - This suggests that at low concentrations of trastuzumab-(Cys-L-SO1861) 4 +5pM cetuximab-saporin showed strong cell killing (SK-BR-3: IC50=1nM; Figure 30A), while equivalent concentrations of trastuzumab, trastuzumab-(Cys-L-SO1861) 4 Or trastuzumab + 5pM cetuximab-saporin can not express HER2 ++ / EGFR + / - This suggests that trastuzumab-conjugated SO1861 effectively enhances the endosomal escape of cetuximab-conjugated protein toxin (at non-effective concentrations), thereby inducing cell cytotoxicity against HER2-expressing cells. ++ / EGFR + / - Cell killing of cells.

[0528] Next, at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 and 75 nM trastuzumab-(Cys-L-SO1861) 4 Cetuximab-saporin was titrated on the HER2 / EGFR expressing SK-BR-3 and the targeted protein toxin-mediated ++ / EGFR + / -) cells. This indicates that 2.5 nM trastuzumab-(Cys-L-SO1861) 4 Combining with low concentrations of cetuximab-saporin already induced effective cell killing in SK-BR-3 cells (SK-BR-3: IC50 = 1 pM; Figure 30B), while cetuximab-saporin alone or cetuximab-saporin + 2.5 nM trastuzumab only showed cell killing at high concentrations of trastuzumab-saporin (SK-BR-3: IC50 > 4000 pM; Figure 30B). All of this suggests that relatively low concentrations of cetuximab-saporin can be effective and only in combination with low concentrations of trastuzumab-(Cys-L-SO1861) 4 Combination in HER2 expression ++ / EGFR + / - induce cell killing in cells.

[0529] Next, trastuzumab-(Cys-L-SO1861) was tested on a fixed concentration of 5 pM cetuximab saporin. 4 Titration was performed and the targeted protein toxin-mediated inhibition of JIMT-1 (HER2 + / - / EGFR + / - ) and MDA-MB-468 (HER2 - / EGFR ++ ) cells. Both cell lines were resistant to trastuzumab-(Cys-L-SO1861) 4 The combination of +5 pM cetuximab-saporin was insensitive (JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM; Figure 31 This suggests that in the absence of sufficient HER2 receptor expression, an effective intracellular delivery concentration (threshold) of SO1861 is not reached to induce endosomal escape and cytoplasmic delivery of the protein toxin.

[0530] Next, at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861) 4 Cetuximab-saporin was titrated and the targeted protein toxin-mediated inhibition of JIMT-1 (HER2 + / - / EGFR + / - ) and MDA-MB-468 (HER2 - / EGFR ++ ) cells. Whether or not 2.5 nM trastuzumab-(Cys-L-SO1861) was present 4, both cell lines showed cell killing at similar cetuximab-saporin concentrations (JIMT-1: IC50 = 80 pM; MDA-MB-468: IC50 = 100 pM; Figure 31 C, D).

[0531] All of this suggests that cells with low or no HER2 receptor expression are resistant to trastuzumab-(Cys-L-SO1861) due to the lack of sufficient HER2 receptors to promote antibody-mediated uptake of sufficient SO1861 (threshold) to ensure endosomal escape of the toxin from the cell's cytoplasm. 4 + Cetuximab-saporin insensitive.

[0532] To demonstrate that the activity of conjugated SO1861 is driven by acidification of the endosomal compartment, the 2T2 component system according to the present invention was tested in combination with the endosomal acidification inhibitor chloroquine. 3,9 or trastuzumab-oleanol + 77nM cetuximab-(Cys-L-SO1861) 3,9 In A431 (EGFR ++ / HER2 + / - ) cells, and according to the present invention, when 800 nM chloroquine was co-administered with the two combinations, this 2T2C activity was inhibited ( Figure 32 A). In A431 (EGFR ++ / CD71 + ) and MDA-MB-468 (EGFR ++ / CD71 + ) cells tested CD71mab-saporin + 10.5nM cetuximab-(Cys-L-SO1861) 3,9 +500nM chloroquine ( Figure 32 B, C of Figure 9), or when CD71mab-saporin + 5nM trastuzumab-(Cys-L-SO1861) 4 +500nM chloroquine in SK-BR-3 (HER2 ++ / CD71 + ) cells when tested ( Figure 32 The same results were observed in D). This indicates that the intracellular activity of conjugated SO1861 in the 2T2C system can be inhibited when endosome acidification is blocked.

[0533] The 2-target 2-component system (2T2C) is also a combination treatment of mAb1-SO1861 and mAb2-antisense BNA oligonucleotide ( Figure 38Therefore, the 2T2C system was also tested in combination with antisense BNA oligonucleotides targeting the mRNA of the cancer-specific target gene heat shock protein 27 (HSP27). After release into the cytoplasm, the antisense BNA recognizes and binds to the mRNA encoding HSP27, targeting the mRNA for destruction, thereby depleting HSP27 expression in cancer cells. Figure 38 As shown in , HSP27BNA was conjugated to trastuzuma...

Claims

1. A first protein molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein the first protein molecule has at least one saponin, the at least one saponin being covalently bound to an amino acid residue of the first protein molecule via at least one linker and / or via an oligomeric or polymeric scaffold or being covalently bound directly to an amino acid residue of the first protein molecule.

2. The first protein molecule according to claim 1, 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, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, a scFv, Fv, dAb, F(ab)2, an Fcab fragment, and / or comprises or consists of at least one ligand that binds to a cell surface molecule, such as EGF or a cytokine.

3. The first protein molecule according to claim 1, wherein the first epitope of the first cell surface molecule is a tumor cell-specific first epitope of a first tumor cell surface molecule, or a tumor cell-specific first epitope of a first tumor cell surface receptor specifically present on tumor cells.

4. The first protein molecule according to claim 1, wherein the at least one saponin is a triterpenoid saponin and / or a disaccharide-chain triterpenoid saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and optionally comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, and / or a saponin isolated from species of the genus Gypsophila and / or species of the genus Saponaria and / or species of the genus Agrostemma and / or species of the genus Quillaja, such as Quillaja saponaria.

5. The first protein molecule of claim 1, wherein the at least one saponin is a single specific saponin or a mixture of two or more different saponins, or one or more of the following: a saponin in Table A1 or Scheme 1, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B- xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, saponin set, QS-18, Quil-A, Gyp1, Gyp1 saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any one of their stereoisomers and / or any combination thereof, or the saponin is SO1 861 and / or GE1741 and / or SA1641 and / or QS-21 and / or having a sapogenin glycoside core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3β-OH group and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group. substituted saponins, and / or 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-pyranoglucopyranosyl saponin 28-O-β-D-pyranoglucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4OAc-β-D-quinoline-(1→4)]-β-D-fucopyranoside.

6. The first protein molecule of claim 1, wherein the at least one saponin is an oleanolic triterpene having a molecular weight of at least 1,500 Daltons and comprising an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, a disaccharide chain saponin having a first branched carbohydrate side chain at the C-3 position, the first branched carbohydrate side chain optionally comprising glucuronic acid, wherein the saponin contains an ester group and a second branched carbohydrate side chain at the C-28 position, the second branched carbohydrate chain preferably comprising at least four carbohydrate units, which optionally contain at least one acetyl residue, such as two acetyl residues, and / or optionally contain deoxy carbohydrates and / or optionally contain isorhamnose and / or optionally contain glucose and / or optionally contain 4-methoxycinnamic acid and / or optionally contain 5-O-[5- O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid and / or optionally comprising 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyloctanoic acid bound to a carbohydrate via an ester bond, or wherein at least one saponin is QS-21 or any one or more of the following: QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

7. The first protein molecule according to claim 1, wherein the at least one saponin is a disaccharide-chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde functional group in position C-23, wherein the at least one saponin is covalently coupled to an amino acid residue of the first protein molecule via the aldehyde functional group in the saponin, or via the aldehyde functional group in position C-23, or via at least one linker, or via at least one cleavable linker, or wherein the amino acid residue is selected from cysteine ​​and lysine.

8. The first protein molecule according to claim 1, wherein the at least one saponin is a disaccharide-chain triterpene saponin of the 12,13-dehydrooleanane type having an aldehyde function in position C-23 and comprising a glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, wherein the at least one saponin is covalently coupled to an amino acid residue of the first protein molecule via the glucuronic acid function in the carbohydrate substituent at the C-3β-OH group of the saponin, or via at least one linker, or wherein the amino acid residue is selected from cysteine ​​and lysine.

9. The first protein molecule according to claim 4, wherein the aldehyde functional group in position C-23 of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, and the linker is covalently coupled to a sulfhydryl group, or a sulfhydryl group of cysteine ​​in the first protein molecule through a thioether bond.

10. The first protein molecule according to claim 4, wherein the glucuronic acid functional group in the carbohydrate substituent at the C-3β-OH group of the at least one saponin is covalently coupled to a linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidohexafluorophosphate, and the linker is covalently coupled to an amine group or an amine group of lysine in the first protein molecule, or to the N-terminus of the first protein molecule via an amide bond.

11. The first protein molecule according to claim 1, wherein the first epitope of the first cell surface molecule bound by the first binding site of the first protein molecule is a tumor cell-specific first epitope of the tumor cell-specific receptor, or the tumor cell-specific receptor is selected from the group consisting of: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD 138, CD27L receptor, PSMA, CanAg, vascular integrin α-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.

12. The first protein molecule according to claim 3, wherein the tumor cell-specific first epitope, the first tumor cell surface molecule or the first tumor cell-specific receptor is the first epitope or the first molecule or the first receptor internalized by the tumor cell after the first protein molecule according to any one of claims 1 to 11 binds to the first epitope or the first molecule or the first receptor, and / or wherein, When the first protein molecule binds to a cell surface molecule, a tumor cell surface molecule or a tumor cell-specific receptor comprising the first epitope, it undergoes tumor cell receptor-mediated internalization, such as by endocytosis, or tumor cell surface molecule-mediated internalization, such as by endocytosis.

13. The first protein molecule according to claim 11 or 12, wherein the first binding site of the first protein molecule comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mositumomab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, or cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof, or at least one tumor cell-specific receptor binding fragment thereof and / or at least one tumor cell-specific receptor binding domain thereof.

14. A therapeutic combination, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition comprising a first protein molecule according to any one of claims 1 to 13 and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second protein molecule different from the first protein molecule, the second protein 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, wherein the second epitope is different from the first epitope.

15. The therapeutic combination according to claim 14, wherein the therapeutic combination comprises: (a) the first pharmaceutical composition of claim 14, comprising the first protein molecule of any one of claims 1-13, wherein the first epitope on the first cell surface molecule is a tumor cell-specific first epitope on a first tumor cell-specific surface molecule, or a tumor cell-specific first epitope specifically present on a first cell surface receptor of a tumor cell; and (b) The second pharmaceutical composition of claim 14, wherein the second cell surface molecule is a second tumor cell-specific surface molecule different from the first tumor cell-specific surface molecule, or a second cell surface receptor specifically present in tumor cells is different from the first cell surface receptor specifically present in the tumor cells, and wherein the second epitope is a tumor cell-specific second epitope.

16. The therapeutic combination according to claim 14, wherein the first binding site for binding to the first epitope on the first cell surface molecule is a binding site for a tumor cell-specific first epitope on a first cell surface receptor specifically present on tumor cells.

17. The therapeutic combination according to claim 15, wherein the first binding site for binding to the first epitope on the first cell surface molecule is a binding site for a tumor cell-specific first epitope on a first cell surface receptor specifically present on tumor cells.

18. A therapeutic combination, wherein the therapeutic combination comprises: (a) a first pharmaceutical composition according to claim 14 or 15, comprising a first protein molecule according to any one of claims 1 to 13 and comprising the first binding site for binding to the first epitope on the first cell surface molecule, the first pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient; and (b) a third pharmaceutical composition comprising a third protein molecule, said third protein molecule comprising said first binding site for binding to said first epitope on the cell surface molecule of (a) and an effector portion, said third pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, wherein the first binding site of the first protein molecule is the same as the first binding site of the third protein molecule, and wherein the first cell surface molecule to which the first protein molecule is capable of binding and the first epitope on the first cell surface molecule are the same as the first cell surface molecule to which the third protein molecule is capable of binding and the first epitope on the first cell surface molecule.

19. A therapeutic combination, wherein the therapeutic combination comprises: (a) the first pharmaceutical composition of claim 18; and (b) the third pharmaceutical composition according to claim 18, The first cell surface molecule is expressed on the surface of a tumor cell, and / or the first cell surface molecule is a tumor cell-specific surface molecule, and / or the first epitope is a first tumor cell-specific epitope.

20. The first protein molecule according to claim 1, wherein the first binding site for binding to the first epitope on the first cell surface molecule is a binding site for a tumor cell-specific first epitope on a first cell surface receptor specifically present on tumor cells.

21. The therapeutic combination of claim 14, wherein the second binding site of the second protein molecule and / or the first binding site of the third protein molecule comprises or consists of: An immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, such as an antibody, IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, a scFv, Fv, dAb, F(ab)2, an Fcab fragment, and / or comprises or consists of at least one ligand that binds to a cell surface molecule such as EGF or a cytokine.

22. The therapeutic combination of claim 14, wherein the second binding site of the second protein molecule for binding to the second epitope is a second binding site for a tumor cell-specific second epitope specifically present on a second cell surface receptor of the tumor cell, wherein the second binding site is different from the first binding site.

23. The first protein molecule of claim 1, wherein the first and second protein molecules comprise the first and second binding sites for binding to first and second tumor cell-specific epitopes on first and second tumor cell-specific receptors, respectively, the receptors being different and present on the same tumor cell, wherein the first and second binding sites are different and the first and second tumor cell-specific epitopes are different.

24. The therapeutic combination of claim 14, wherein the first and second protein molecules comprise the first and second binding sites for binding to first and second tumor cell-specific epitopes on first and second tumor cell-specific receptors, respectively, the receptors being different and present on the same tumor cell, wherein the first and second binding sites are different and the first and second tumor cell-specific epitopes are different.

25. The first protein molecule of claim 1, wherein the first and third protein molecules comprise the same first binding site for binding to a first tumor cell-specific epitope on a first tumor cell-specific receptor.

26. The therapeutic combination of claim 18, wherein the first and third protein molecules comprise the same first binding site for binding to a first tumor cell-specific epitope on a first tumor cell-specific receptor.

27. The first protein molecule according to claim 23, wherein the first receptor and / or the second receptor are selected from: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, vascular integrin αV, CA6, CD33, Mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, Ephrin A4, MUC1, Trop2, CEACAM5, CEAC AM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2.

28. The therapeutic combination of claim 23, wherein the first receptor and / or the second receptor is selected from the group consisting of: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, vascular integrin α V, CA6, CD33, Mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, Ephrin A4, MUC1, Trop2, CEACAM5, CEAC AM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2.

29. The first protein molecule according to claim 23, wherein the first and second tumor cell-specific receptors are internalized by the tumor cell after binding to the first protein molecule of any one of claims 1-23 or 27 and / or the second protein molecule of claim 14, and / or wherein the binding of the first protein molecule and / or the second protein molecule to the first and second tumor cell-specific receptors, respectively, results in tumor cell receptor-mediated internalization of the complex of the first protein molecule and the first tumor cell-specific receptor and the complex of the second protein molecule and the second tumor cell-specific receptor, and / or by endocytosis.

30. The therapeutic combination of claim 23, wherein the first and second tumor cell-specific receptors are internalized by the tumor cell after binding to the first protein molecule of any one of claims 1-23 or 27 and / or the second protein molecule of claim 14, and / or wherein the binding of the first protein molecule and / or the second protein molecule to the first and second tumor cell-specific receptors, respectively, results in tumor cell receptor-mediated internalization of the complex of the first protein molecule and the first tumor cell-specific receptor and the complex of the second protein molecule and the second tumor cell-specific receptor, and / or by endocytosis.

31. The therapeutic combination according to claim 25, wherein the first tumor cell receptor, or the first tumor cell-specific receptor, is internalized by the tumor cell after binding to the first protein molecule of any one of claims 1-20 and / or after binding to the third protein molecule of claim 18, and / or wherein the binding of the first protein molecule and / or the third protein molecule to the first tumor cell receptor, such as the first tumor cell-specific receptor, is followed by tumor cell receptor-mediated internalization, such as by endocytosis, of the complex of the first protein molecule and the first tumor cell receptor and the complex of the third protein molecule and the first tumor cell receptor.

32. The first pharmaceutical composition according to claim 25, wherein the first tumor cell receptor, or the first tumor cell-specific receptor, is internalized by the tumor cell after binding to the first protein molecule of any one of claims 1-20 and / or after binding to the third protein molecule of claim 18, and / or wherein the binding of the first protein molecule and / or the third protein molecule to the first tumor cell receptor, such as the first tumor cell-specific receptor, is followed by tumor cell receptor-mediated internalization, such as by endocytosis, of the complex of the first protein molecule and the first tumor cell receptor and the complex of the third protein molecule and the first tumor cell receptor.

33. The therapeutic combination of claim 14, 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, and / or comprises any one of the following or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxifloxacin, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody and the antibody of Table A4, or cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is different from the second binding site of the second protein molecule.

34. The first protein molecule according to claim 1, 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, and / or comprises any one of the following or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mositumomab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody and the antibody of Table A4, or cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is different from the second binding site of the second protein molecule.

35. The therapeutic combination of claim 18, wherein the first binding site of the first protein molecule and the third protein molecule comprises at least one of a monoclonal antibody or a cell surface molecule binding domain and / or fragment thereof, and / or comprises or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mositumomab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, or cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is the same as the first binding site of the third protein molecule.

36. The first pharmaceutical composition according to claim 18, wherein the first binding site of the first protein molecule and the third protein molecule comprises at least one of a monoclonal antibody or a cell surface molecule binding domain and / or fragment thereof, and / or comprises any one of the following or consists of any one of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, mositumomab, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, or cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof, with the proviso that the first binding site of the first protein molecule is the same as the first binding site of the third protein molecule.

37. The therapeutic combination of claim 14, wherein the second binding site of the second protein molecule and / or the first binding site of the third protein molecule is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment or domain thereof, and / or comprises or consists of any one of: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxifloxacin pasudotox and potuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3.

38. The therapeutic combination according to claim 14, wherein the effector moiety comprised by the second protein molecule and / or the third protein molecule comprises any one or more of the following, or consists of any one or more of the following: Oligonucleotides, nucleic acids, heterologous nucleic acids, and / or any one or more selected from vectors, genes, cell suicide-inducing transgenes, deoxyribonucleic acids (DNA), ribonucleic acids (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamers, RNA aptamers, mRNA, minicircle DNA, peptide nucleic acids (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acids (LNA), bridged nucleic acids (BNA), 2'-deoxy-2'-fluoroarabinoic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNA), plasmids, glycol nucleic acids (GNA) and threose nucleic acids (TNA) or their derivatives.

39. The therapeutic combination of claim 14, wherein the effector portion comprised by the second protein molecule and / or the third protein molecule comprises or consists of at least one protein molecule and / or is selected from any one or more of the following: a peptide, a protein, an enzyme such as urease and Cre-recombinase, a ribosome inactivating protein, a protein toxin, and / or is selected from any one or more of the following: any one or more of the protein toxins 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 fungal toxin, Such as α-sarcin; plant toxins including ribosome inactivating proteins and the A chain of type 2 ribosome inactivating proteins, such as oleanolic acid, for example oleanolic acid-30 or oleanolic acid-32, saporin, for example saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, American pokeweed antiviral protein, ricin, ricin A chain, modicin, modicin A chain, abrin, abrin A chain, modeccin, modeccin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog ribonuclease, or granzyme B or angiogenin from humans, or any fragment or derivative thereof.

40. The therapeutic combination of claim 14, wherein the effector moiety comprised by the second protein molecule and / or the third protein molecule comprises or consists of at least one payload and / or is selected from any one or more of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, and / or any one or more of emtansine, pasudotox, a maytansine derivative DM1, a maytansine derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analogs, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolebenzodiazepine, AZ13599185, scutellarin, rhizotomycin, methotrexate, anthracycline, camptothecin analogs, SN 38. DX 8951f, exitecan mesylate, a truncated form of Pseudomonas aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, spliceostatin, talanstatin, ozogamicin, tesirine, Amberstatin 269 and soravtansine, or derivatives thereof.

41. A first protein molecule according to claim 1, wherein the first protein molecule comprises more than one saponin, and / or 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100 saponins, or any number therebetween, such as 7, 9, 12 saponins, which are covalently bound directly to an amino acid residue of the first protein molecule, and / or to a cysteine ​​and / or a lysine, and / or covalently bound via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric scaffold, and / or 1-8 such scaffolds or 2-4 such scaffolds, wherein the at least one scaffold is optionally based on a dendron, wherein 1-32 saponins, such as 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number therebetween, such as 7, 9, 12 saponins, are covalently bound to at least one scaffold.

42. The first protein molecule of claim 7, wherein the at least one linker is a non-cleavable linker or a cleavable linker, wherein the cleavable linker undergoes cleavage, for example, under acidic conditions, reducing conditions, enzymatic conditions or light-induced conditions, and / or the cleavable linker comprises a hydrazone bond or a hydrazide bond that undergoes cleavage under acidic conditions when bound to a saponin, and / or comprises a bond that is susceptible to proteolysis, or proteolysis by cathepsin B, and / or is a bond that is susceptible to cleavage under reducing conditions when bound to a saponin, such as a disulfide bond.

43. The first protein molecule of claim 7, wherein when the cleavable linker is conjugated to a saponin, the cleavable linker undergoes cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, and / or human cells, and / or at pH 4.0-6.5, and / or at pH ≤ 5.

5.

44. The first protein molecule of claim 1, wherein the oligomeric or polymeric scaffold comprises a polymeric or oligomeric structure and comprises a chemical group for covalently linking the scaffold to an amino acid residue of the first protein molecule.

45. The first protein molecule of claim 1, wherein the at least one saponin is covalently bound to the polymeric or oligomeric structure of the oligomeric or multimeric scaffold via at least one cleavable linker of any one of claims 32-34.

46. ​​The first protein molecule according to claim 1, wherein the chemical group of the oligomeric or polymeric scaffold used to covalently couple the oligomeric or polymeric scaffold to the amino acid residues of the first protein molecule is a click chemistry group, and / or is selected from tetrazines, azides, alkenes or alkynes, or cyclic derivatives of these groups.

47. The first protein molecule according to claim 1, wherein the polymeric or oligomeric structure of the oligomeric or multimeric scaffold comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendron polymer, dendrimer, DNA, polypeptide, polylysine, polyethylene glycol or an assembly of these polymeric or oligomeric structures, said assembly being established by covalent cross-linking.

48. A composition comprising the first protein molecule of claim 1 and the second protein molecule of claim 14.

49. A composition comprising the first protein molecule of claim 1 and the third protein molecule of claim 16.

50. The composition of claim 48 or 49, wherein the effector moiety comprised by the second protein molecule or the third protein molecule is any one of the effector moieties of claim 38.

51. A composition comprising the first protein molecule of claim 1 and any one or more of an oligonucleotide, a nucleic acid and a heterologous nucleic acid, and / or any one selected from the following: a vector, a gene, a cell suicide-inducing transgene, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), an aminophosphoryl morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a 2'-deoxy-2'-fluoroarabinoic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA) or derivatives thereof.

52. An antibody-drug conjugate or ligand-drug conjugate comprising the first protein molecule of claim 1 and an effector moiety.

53. The antibody-drug conjugate or ligand-drug conjugate of claim 52, wherein the antibody is capable of binding to any of the following: CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin α-Vβ-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin, α-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, and / or Or comprising any of the following: cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxifloxacin, potuzumab, obinutuzumab, IgG type OKT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, antibodies of Table A2 or Table A3 or Table A4, or cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof, and / or wherein the antibody-drug conjugate comprises any one of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxifloxacin pasudotox and potuzumab vedotin and the antibody-drug conjugates of Table A2 and Table A3, or wherein the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule such as EGF or a cytokine.

54. The antibody-drug conjugate or ligand-drug conjugate of claim 52, wherein the effector moiety is any one or more of the effector moieties of claims 38-40.

55. A pharmaceutical composition comprising the composition of any one of claims 48-51, and optionally further comprising a pharmaceutically acceptable excipient.

56. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 52-54 or the ligand-drug conjugate of any one of claims 52-54, and optionally further comprising a pharmaceutically acceptable excipient.

57. The therapeutic combination of any one of claims 14-47, or the composition of any one of claims 48-51, or the antibody-drug conjugate or ligand-drug conjugate of any one of claims 52-54, or the pharmaceutical composition of claim 55, for use as a medicament.

58. The therapeutic combination of any one of claims 14-47, or the composition of any one of claims 48-51, or the antibody-drug conjugate or ligand-drug conjugate of any one of claims 52-54, or the pharmaceutical composition of claim 55, for use in treating or preventing cancer or an autoimmune disease.

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