Saponins conjugated to epitope-binding proteins

Abstract

The present invention relates to therapeutic combinations, including a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein the first proteinaceous molecule is provided with at least one saponin covalently attached to an amino acid residue of said first proteinaceous molecule, and a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell surface molecule different from the first cell surface molecule and comprising an effector moiety, the second epitope being different from the first epitope. One aspect of the present invention is a composition comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule. The present invention also relates to an antibody-drug conjugate comprising a first proteinaceous molecule of the present invention and an effector moiety. One aspect of the present invention relates to a pharmaceutical composition comprising a composition or antibody-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient. The present invention also relates to a therapeutic combination, or composition, or antibody-drug conjugate, or pharmaceutical composition of the present invention for use as a medicament. The present invention also relates to a therapeutic combination of the present invention for use in the treatment or prevention of cancer.

Description

[Technical Field]

[0001] The present invention relates to a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, wherein the first proteinaceous molecule is provided with at least one saponin covalently attached to an amino acid residue of said first proteinaceous molecule via at least one linker and / or via an oligomeric or polymeric backbone or directly to an amino acid residue of said first proteinaceous molecule. The present invention also relates to a therapeutic combination comprising a first pharmaceutical composition comprising a first proteinaceous molecule of the present invention and a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell surface molecule different from the first cell surface molecule and comprising an effector moiety, wherein the second epitope is different from the first epitope. Furthermore, the present invention relates to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition of the present invention, comprising a first proteinaceous molecule according to the present invention and comprising a first binding site for binding to a first epitope on a first cell surface molecule; and (b) a third pharmaceutical composition comprising a third proteinaceous molecule, wherein the third proteinaceous molecule comprises a first binding site for binding to the first epitope on the cell surface molecule of (a) and an effector moiety. The first binding site of the first proteinaceous molecule and the first binding site of the third proteinaceous molecule are the same. Furthermore, the first epitope on the first cell surface molecule and the first cell surface molecule to which the first proteinaceous molecule can bind, and the first epitope on the first cell surface molecule and the first cell surface molecule to which the third proteinaceous molecule can bind, are the same. One embodiment of the present invention is a composition comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule of the present invention. One aspect of the present invention relates to a composition comprising a first proteinaceous molecule of the present invention and a third proteinaceous molecule of the present invention.The present invention also relates to a composition comprising one or more oligonucleotides, nucleic acids, and xenonucleic acids, preferably selected from at least one of a vector, a gene, a cell suicide-inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), plasmid, glycol nucleic acid (GNA), and threose nucleic acid (TNA), or derivatives thereof, and a first proteinaceous molecule of the present invention. The present invention also relates to an antibody-drug conjugate or a ligand-drug conjugate comprising a first proteinaceous molecule of the present invention and an effector moiety. An aspect of the present invention relates to a pharmaceutical composition comprising a composition of the present invention or an antibody-drug conjugate of the present invention or a ligand-drug conjugate of the present invention, and optionally further comprising a pharmaceutically acceptable excipient. The present invention also relates to a therapeutic combination of the present invention or a composition of the present invention or an antibody-drug conjugate or ligand-drug conjugate of the present invention or a pharmaceutical composition of the present invention for use as a medicament. [Background technology]

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

[0003] Chemotherapy is one of the most important therapeutic options for cancer treatment. However, because it lacks specificity for cancer cells compared to dividing cells in healthy tissues, it is often associated with a narrow therapeutic window. The development of monoclonal antibodies has offered the possibility of exploiting their specific binding properties as a mechanism for targeted delivery of cytotoxic drugs to cancer cells while sparing normal cells. This can be achieved by chemically conjugating a cytotoxic effector (also known as a payload or warhead) to the antibody to create an antibody-drug conjugate (ADC). Typically, highly potent payloads such as emtansine (DM1), which has a limited therapeutic index (the ratio of the toxic dose to the effective dose) in its unconjugated form, are used. Conjugation of DM1 to trastuzumab (ado-trastuzumab-emtansine), also known as Kadcycla, improves the tolerable dose of DM1 by at least two-fold in monkeys. Over the past few decades, enormous effort and investment has been devoted to developing therapeutic ADCs. Despite promising preclinical data, however, bringing ADCs to the clinic remains challenging. The first ADC approved for clinical use was gemtuzumab ozogamicin (CD33-targeted Mylotarg, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML) in 2000. However, Mylotarg was withdrawn from the market at the request of the Federal Drug Administration (FDA) due to several concerns regarding its safety profile. Patients treated with Mylotarg were found to die more often than patients treated with conventional chemotherapy. Mylotarg was reapproved in 2017 with a lower recommended dose, a different schedule in combination with chemotherapy or on its own, and a new patient population. To date, only five ADCs have been approved for clinical use, while approximately 55 ADCs have been discontinued in clinical development. However, interest remains high, and approximately 80 ADCs are currently in clinical development with nearly 600 trials.

[0004] Despite the potential for using toxic payloads that are usually not tolerated by patients, a low therapeutic index (the ratio of toxic dose to efficacious dose) is a major issue that explains the discontinuation of many ADCs in clinical development. This can be caused by several mechanisms, such as off-target toxicity against normal cells, the development of resistance to cytotoxic drugs, and premature release of the drug in the circulation. A systematic review of ADCs by the FDA found that the toxicity profiles of most ADCs can be categorized according to the payload used rather than the antibody used, suggesting that toxicity is largely determined by premature release of the payload. It is estimated that of the approximately 55 ADCs that have been discontinued, at least 23 were due to a poor therapeutic index. For example, development of a trastuzumab-tesirin conjugate (HER-2-targeting ADCT-502, an ADC therapeutic) was recently halted due to a narrow therapeutic index, likely due to on-target normal tissue effects in lung tissue, which expresses significant levels of HER2. In addition, several ADCs in Phase 3 trials have been discontinued due to missing primary endpoints. For example, phase 3 trials of a depatuxizumab mafodotin conjugate (EGFR-targeting ABT-414, AbbVie) tested in patients with newly diagnosed glioblastoma and a mirvetuximab soravtansine conjugate (folate receptor alpha (FRα)-targeting IMGN853, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently halted and failed to demonstrate survival benefit. It is important to note that the clinically used dose of some ADCs may not be sufficient for their full anticancer activity. For example, ado-trastuzumab emtansine has an MTD of 3.6 mg / kg in humans. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels of 3 mg / kg or higher, but more potent efficacy was observed at 15 mg / kg. This suggests that at clinically administered doses, ado-trastuzumab emtansine may not exert its maximum potential antitumor effect.

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

[0006] Examples of ADCs with clinical potential include those being evaluated as treatment options for lymphoid malignancies and multiple myeloma, such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin. Polatuzumab vedotin, which binds to CD79b on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, are being tested in clinical trials. Each ADC has been combined with rituximab, a monoclonal antibody that binds to CD20, and no payload was provided (Non-Patent Document 1). Monoclonal antibody combinations, such as these examples, represent yet another approach, attempting to arrive at a "magic bullet" that combines many or even all of the previously mentioned desirable features of ADCs.

[0007] Meanwhile, nucleic acid-based therapeutics have been under development for the past few decades. 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 approaches such as gene therapy, RNA interference (RNAi). Many of them share the same fundamental basis of action: inhibiting either DNA or RNA expression, thereby preventing the expression of abnormal proteins associated with disease. The largest number of clinical trials are in the field of gene therapy, with nearly 2,600 ongoing or completed trials worldwide, but only about 4% have entered phase 3. This is followed by ASO-based clinical trials. Similar to ADCs, despite the numerous technologies being explored, therapeutic nucleic acids share two major issues during clinical development: intracellular delivery and off-target effects. ASOs, such as peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), locked nucleic acids (LNAs), and bridged nucleic acids (BNAs), are being explored as attractive strategies for specifically inhibiting target genes, particularly those that are difficult to target with small molecule inhibitors or neutralizing antibodies. The efficacy of different ASOs is currently being investigated in many neurodegenerative diseases, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in several cancer stages. The application of ASOs as potential therapeutic agents requires safe and effective methods for their delivery to the cytoplasm and / or nucleus of target cells and tissues. Although the clinical relevance of ASOs has been demonstrated, inefficient cellular uptake both in vitro and in vivo has limited their efficacy and been a barrier to their therapeutic development. Cellular uptake can be <2% of the dose, resulting in ASO concentrations at the active site that are too low for effective and sustained results. This consequently requires increased administration doses, which induce off-target effects. The most common side effects are activation of the complement cascade, inhibition of the coagulation cascade, and toll-like receptor-mediated stimulation of the immune system.

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

[0009] This conjugate principle has attracted considerable attention and has been under investigation for decades. The majority of small molecule drug conjugates in preclinical or clinical development are for oncology indications. However, to date, only one non-cancer drug (Movantik, AstraZeneca, a PEG oligomer conjugate of the opioid antagonist naloxone) has been approved in 2014 for the non-oncology indication of opioid-induced constipation in chronic pain patients. The translational application of drug-scaffold conjugates to the treatment of human subjects has met with little clinical success. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has shown significant anticancer activity in both solid tumors and leukemia in mouse models and is under clinical investigation for oncology indications. Although it demonstrated significantly reduced non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer efficacy proved to be modest in patients, and as a result, further development of PK1 was discontinued.

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

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

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

[0013] [Non-Patent Document 1] B.Yu And D.Liu,Antibody-drug conjugates in clinical trials for lymphoid malignancies And multiple myeloma;Journal of Hematology & Oncology(2019)12:94 Summary of the Invention

[0014] In some embodiments of the present invention, it is a primary goal to provide improved bioactive compounds or compositions containing such improved bioactive compounds.

[0015] It is one of several objects of embodiments of the present invention to provide a solution to the problem of non-specificity faced when administering small molecule therapeutically active compounds to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of drugs having suboptimal specificity for disease-driving biological factors or biological processes. It is one of several objects of embodiments of the present invention to provide a solution to the problem of inadequate safety characteristics of current drugs when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of current drugs being less effective than desired when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of current drugs not being adequately targeted to diseased cells with little or no off-target activity against non-diseased cells when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in the human patient within a certain time frame and should remain at the target site for a certain time frame).It is one of several objects of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently long-lasting therapeutic activity in the patient's body.

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

[0017] While the present invention will be described with reference to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

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

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

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

[0021] One embodiment is a first proteinaceous molecule of the present invention, wherein the first epitope of the first cell surface molecule to which the first binding site of the first proteinaceous molecule binds is a tumor cell-specific first epitope of a tumor cell-specific receptor, preferably CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, or CD27L receptor. The antigen-specific antigen is selected from the group consisting of CD3, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, and VEGFR2, and more preferably selected from the group consisting of CD71, EGFR, and HER2.

[0022] In one embodiment, the first proteinaceous molecule of the present invention, wherein the tumor cell-specific first epitope, first tumor cell surface molecule, or first tumor cell-specific receptor is a first epitope, first molecule, or first receptor that is internalized by a tumor cell after binding of the first proteinaceous molecule of the present invention to the first epitope, first molecule, or first receptor. Preferably, the first proteinaceous molecule, upon binding to the cell surface molecule, tumor cell surface molecule, or tumor cell-specific receptor comprising the first epitope, is subject to tumor cell receptor-mediated internalization, e.g., via endocytosis, or tumor cell surface molecule-mediated internalization, e.g., via endocytosis.

[0023] Certain aspects of the present invention relate to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition comprising a first proteinaceous molecule of the present invention and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, wherein the second proteinaceous molecule comprises a second binding site for binding to a second epitope of the second cell surface molecule different from the first cell surface molecule and comprises an effector moiety, wherein the second pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient, and wherein the second epitope is different from the first epitope.

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

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

[0026] In one embodiment is a first proteinaceous molecule of the invention and / or a 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, preferably cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 antibody in IgG form. comprising or consisting of any one of the following: CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and an 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 proteinaceous molecule is different from the second binding site of the second proteinaceous molecule.

[0027] In one embodiment is a therapeutic combination of the invention, wherein the effector moiety comprised in the second proteinaceous molecule and / or in the third proteinaceous molecule comprises or consists of one or more of an oligonucleotide, a nucleic acid, or a xenonucleic acid.

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

[0029]

[0033] One embodiment is a therapeutic combination of the invention, wherein the first proteinaceous molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1-100 saponins, or any number of saponins in between, for example 7, 9, 12 saponins, covalently attached directly to an amino acid residue of the first proteinaceous molecule, preferably to a cysteine ​​and / or to a lysine, and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1-8 such backbones or 2-4 such backbones, wherein at least one backbone is optionally dendron-based, and wherein 1-32 saponins, for example 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins in between, for example 7, 9, 12 saponins, are covalently attached to the at least one backbone.

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

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

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

[0033] One embodiment is a composition, comprising a first proteinaceous molecule of the invention and, preferably, a vector, a gene, a transgene that induces cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid, or the like. and one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid selected from at least one of BNA, e.g., BNA for silencing HSP27 protein expression, such as BNA for silencing HSP27 protein expression.

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

[0035] One embodiment is an antibody-drug conjugate or ligand-drug conjugate of the invention, wherein the antibody is selected from the group consisting of CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cri and / or the antibody may bind to any one of pto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR; and / or the antibody may bind to any one of cetuximab, daratumumab, gemtuzumab, cetuximab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor-binding fragment thereof and / or at least one The antibody-drug conjugate is or comprises any one of the tumor cell receptor binding domains of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates of Table A2 and Table A3, or the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0036] One embodiment is an antibody-drug 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.

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

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

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

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

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

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

[0043] One embodiment is structure C of the invention, wherein A is an anti-EGFR antibody, e.g., cetuximab, an anti-HER2 antibody, e.g., trastuzumab, an anti-CD71 antibody, e.g., OKT-9, and / or S is a saponin, triterpenoid saponin, and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group on the carbohydrate substituent at the C-3 beta-OH group of the saponin, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and Quillaja. and / or E is any one or more of the saponins in the water-soluble saponin fraction of S. saponaria, and / or E is any one or more of an oligonucleotide, an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), and / or any one or more of a proteinaceous toxin, a ribosome-inactivating protein, a dianthin, and a saporin.

[0044]

[0033] An embodiment is Structure C of the invention, a conjugate of the invention, or a semi-finished conjugate of the invention, or a first proteinaceous molecule of the invention, wherein the saponin, if present, and / or the effector molecule, if present, are covalently coupled via at least one linker, such as a cleavable linker, and / or via at least one oligomeric or polymeric backbone, for example N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) based linkers, and dendron-based backbones, for example G4-dendrons, or trifunctional linkers, such as the trifunctional linkers of Scheme II. and / or at least one lysine and / or cysteine ​​side chain of the first binding site of the first proteinaceous molecule, preferably a monoclonal antibody or fragment or domain thereof, is involved in a covalent bond with the saponin and / or effector molecule and / or linker and / or cleavable linker and / or scaffold, preferably the saponin and / or effector molecule is covalently linked to the first binding site of the first proteinaceous molecule, preferably an antibody, wherein the covalent linkage comprises or consists of an amide bond, hydrazone bond, disulfide bond.

[0045] An aspect of the present invention relates to the use of any of the previously mentioned conjugates of the invention or semi-finished conjugates of the invention or first proteinaceous molecules of the invention as a medicament.

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

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

[0048] The term "linker" has its usual scientific meaning and herein refers to a linear stretch or chemical moiety of amino acid residues conjugated via a peptide bond, which attaches a molecule or atom to another molecule, such as a ligand or an effector molecule or a scaffold. Typically, a linker comprises a chain of atoms linked by a chemical bond. Any linker molecule or linker technology known in the art can be used in the present disclosure. Where indicated, a linker is a linker for covalently attaching molecules via a chemical group on such molecule suitable for forming a covalent bond or covalent linkage with the linker. The linker can be a non-cleavable linker. For example, the linker is stable under physiological conditions. The linker can be a cleavable linker, for example, a linker that is cleavable in the presence of an enzyme, at a specific pH range or value, or under physiological conditions, such as the intracellular conditions of endosomes, such as lysosomes and late endosomes, of mammalian cells, such as human cells. Exemplary linkers that may be used in the context of the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate, or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0049] The term "trifunctional linker" has its usual scientific meaning and refers herein to a linker that attaches three molecules via their respective chemical groups. Those skilled in the art can design such trifunctional linkers based on this disclosure and common general knowledge. Such trifunctional linkers may exhibit, for example, maleimide groups that can be used for conjugation to targeting ligands that exhibit thiol groups for thiol-ene reactions. Additionally, trifunctional linkers may exhibit dibenzocyclooctyne (DBCO) groups for so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with azide-bearing saponins. Finally, trifunctional linkers may acquire a third functional group, such as a trans-cyclooctyne (TCO) group, to undergo so-called inverse electron-demand Diels-Alder (IEDDA) reactions with tetrazine (Tz)-bearing effector molecules. Those skilled in the art will understand that the three chemical groups of a trifunctional linker can all be the same or different, or the linker can contain two of the same chemical groups for linking molecules to the trifunctional linker. The bond formed between the trifunctional linkers can be covalent or non-covalent, with covalent bonds being preferred. The bond formed between the trifunctional linker and one, two, or three binding molecules via each chemical group can be a cleavable (labile) bond, cleavable under acidic conditions in cells, such as the endosomes and lysosomes of mammalian cells, such as human cells, or a non-cleavable bond. It should be understood that a trifunctional linker can contain one or two chemical groups for forming a covalent bond, and two or one additional chemical group(s) for forming a non-covalent bond, respectively. It will be appreciated that a trifunctional linker may include one or two chemical groups for forming a cleavable bond, and an additional two or one chemical group(s), respectively, for forming a non-cleavable bond.

[0050] The term "cleavable," as used, for example, in the term "cleavable linker" or "cleavable bond," has its usual scientific meaning, and herein refers to cleavage under acidic, reductive, enzymatic, or photoinduced conditions. For example, a cleavable linker may be cleaved under acidic conditions. Preferably, the cleavable linker is cleaved in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of 5.5 or less. As another example, the cleavable linker may be cleaved by an enzyme, for example, a cathepsin. Furthermore, an example of a covalent bond cleavable under reductive conditions is a disulfide bond.

[0051] The terms "oligomer" and "polymer" in the context of oligomeric or polymeric backbones have their customary scientific meanings. Here, a polymer refers to a substance having a molecular structure built up primarily or entirely from a large number of equal or similar units linked together; here, an oligomer refers to a polymer whose molecules consist of a relatively small number of repeating units. For example, a structure containing 5-10 or fewer equal or similar units may be referred to as an oligomeric structure, a structure containing 10-50 or more monomeric units may be referred to as a polymeric structure, and a structure of 10 monomeric units may be referred to as either oligomeric or polymeric.

[0052] The term "binding site" has its usual scientific meaning and refers here to a region or epitope on one molecule, such as a protein, DNA, or RNA, to which another molecule can bind.

[0053] The term "scaffold" has its usual scientific meaning and herein refers to an oligomeric or polymeric template or carrier or base (base molecule or base structure) to which one or more molecules, e.g., ligand molecules, effector molecules, can be covalently attached either directly or via a linker, such as a cleavable linker. The scaffold may have a structurally ordered formation, e.g., a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or may comprise an assembled polymeric structure, e.g., a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The backbone may comprise polymeric or oligomeric structures, such as poly- or oligo(amines), e.g., polyethyleneimine and poly(amidoamine); or polyethylene glycol, poly- or oligo(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers; or poly(dextrins), poly- or oligosaccharides, e.g., cyclodextrin or polydextrose; or natural and / or artificial poly- or oligoamino acids, e.g., polylysine, or peptides or proteins, DNA oligo- or polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not exhibit substantial acute or chronic toxicity in organisms and can either be excreted intact or can be completely degraded by the body's metabolism into excretable and / or physiological compounds.

[0054] The term "ligand" has its usual scientific meaning and refers herein to any molecule or molecules that can selectively bind to a target cell surface molecule or target cell surface receptor expressed on a target cell, 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.

[0055] As used herein, the term "antibody" is used in the broadest sense and can refer to an immunoglobulin (Ig), defined as a protein belonging to the classes IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), or to a functional binding fragment or binding domain of an immunoglobulin. In the context of the present invention, a "binding fragment" or "binding domain" of an immunoglobulin is defined as an antigen-binding fragment or domain of a parent immunoglobulin or other derivative, which essentially retains the antigen-binding activity of such parent immunoglobulin. Functional fragments and functional domains are antibodies in the sense of the present invention, even if their affinity for antigen is lower than that of the parent immunoglobulin. "Functional fragments and domains" according to the present invention include, but are not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single-domain antibodies (sdAbs), monovalent IgG, scFv-Fc, reduced IgG (rIgG), minibodies, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanobodies, variable V domains such as VHH and Vh, and other types of antigen-recognizing immunoglobulin fragments and domains. Fragments and domains can be engineered to minimize or completely eliminate intermolecular disulfide interactions that occur between CH1 and CL domains. Due to their smaller size, functional fragments and domains offer the advantage of greater tumor penetration. In addition, functional fragments or domains may be more uniformly distributed in tumor masses compared to whole immunoglobulins.

[0056] The antibodies (immunoglobulins) of the present invention may be bifunctional or multifunctional. For example, a bifunctional antibody has one arm with specificity for one receptor or antigen, and the other arm recognizes a different receptor or antigen. Alternatively, each arm of a bifunctional antibody may have specificity for a different epitope of the same receptor or antigen on a target cell.

[0057] The antibodies (immunoglobulins) of the present invention can be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, resurfaced antibodies, anti-idiotypic antibodies, mouse antibodies, rat antibodies, rat / mouse hybrid antibodies, llama antibodies, heavy chain-only llama antibodies, heavy chain-only antibodies, and veterinary antibodies. Preferably, the antibodies (immunoglobulins) of the present invention are monoclonal antibodies. Resurfaced, chimeric, humanized, and fully human antibodies are also more preferred because they are less likely to cause immunogenicity in humans. The antibodies of the ADCs 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 essentially unchanged (e.g., by not binding to such normal cells or by binding to such healthy cells with a lesser degree of number and / or affinity).

[0058] Specific antibodies that can be used in the ADCs of the invention include anti-HER2 monoclonal antibodies, such as trastuzumab and pertuzumab, anti-CD20 monoclonal antibodies, such as rituximab, ofatumumab, tositumomab, and ibritumomab, anti-CA125 monoclonal antibodies, such as oregovomab, anti-EpCAM (17-1A) monoclonal antibodies, such as edrecolomab, anti-EGFR monoclonal antibodies, such as cetuximab, panitumumab, and nimotuzumab, anti-CD30 monoclonal antibodies, such as brentuximab, anti-CD33 monoclonal antibodies, such as gemtuzumab and huMy9-6, anti-vascular integrin alpha-v beta-3 monoclonal antibodies, such as etaracizumab, and anti-CD52 monoclonal antibodies, such as alemtuzumab. anti-CD22 monoclonal antibodies, such as epratuzumab, anti-CEA monoclonal antibodies, such as labetuzumab, anti-CD44v6 monoclonal antibodies, such as bivatuzumab, anti-FAP monoclonal antibodies, such as sibrotuzumab, anti-CD19 monoclonal antibodies, such as huB4, anti-CanAg monoclonal antibodies, such as huC242, anti-CD56 monoclonal antibodies, such as huN901, anti-CD38 monoclonal antibodies, such as daratumumab, anti-CA6 monoclonal antibodies, such as DS6, anti-IGF-IR monoclonal antibodies, such as cixutumumab and 3B7, anti-integrin monoclonal antibodies, such as CNTO95, and anti-syndecan-1 monoclonal antibodies, such as B-B4.

[0059] Any molecule other than an antibody that binds to a cell receptor or antigen on a target cell can also be used as the cell-binding ligand of the ligand-drug conjugate of the present invention. The ligand is provided in accordance with the present invention as a covalently attached saponin. These ligands include, but are not limited to, proteins, polypeptides, peptides, and small molecules. Examples of these non-antibody ligands include interferons (e.g., IFN-α, IFN-β, and IFN-γ), transferrin, lectins, epidermal growth factor (EGF) and EGF-like domains, gastrin-releasing peptide (GRP), platelet-derived growth factor (TGF), transforming growth factor (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGFs, e.g., IGF-1 and IGF-2), other suitable hormones, such as thyrotropin-releasing hormone (TRH), melanocyte-stimulating hormone (MSH), and steroids. Hormones (e.g., estrogen and androgen), somatostatin, lymphokines (e.g., IL-2, IL-3, IL-4, and IL-6), colony-stimulating factors (CSFs, e.g., G-CSF, M-CSF, and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g., AS-1411, GBI-10, RNA aptamers against HIV glycoproteins), small molecules (e.g., folic acid, anisamidophenylboronic acid), vitamins (e.g., vitamin D), carbohydrates (e.g., hyaluronic acid, galactose).

[0060] An "effector molecule" or "effector moiety" or "payload" has its usual scientific meaning and, in the context of the present invention, is any substance that affects cellular metabolism by interacting with an effector molecule target within the cell. This effector molecule target is any molecule or structure within the cell that excludes the lumen of compartments and vesicles of the endocytic and recycling pathways, but includes the membranes of these compartments and vesicles. Said structures within the cell therefore include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol.

[0061] The effector molecule or moiety is a drug substance, such as a toxin, e.g., a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide. A drug substance in the present invention is an effector molecule or moiety used to achieve a beneficial result in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / human subject. Benefits include diagnosis, prognosis, treatment, cure, and prevention (prophylaxis) of a disease and / or condition and / or health problem. A drug substance may also lead to unwanted and possibly even harmful side effects (e.g., adverse events observed during clinical trials). In this case, the pros and cons must be weighed to determine whether the drug substance is suitable in a particular case. If the effect of a drug substance in a cell is overwhelmingly beneficial to the organism as a whole, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the organism as a whole, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, target and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules and moieties are drugs, toxins, polypeptides (eg, enzymes), polynucleotides (including polypeptides and polynucleotides that contain unnatural amino acids or nucleic acids), and any combination thereof.

[0062] Drug effector molecules or effector moieties may include, but are not limited to, anti-cancer agents, anti-inflammatory agents, and anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral) agents. Preferably, the drug molecules of the present invention are anti-cancer or anti-autoimmune agents. Suitable anti-cancer agents include, but are not limited to, alkylating agents, metabolic inhibitors, spindle poison plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. The definition of "anti-cancer agent" also includes, for example, (i) antihormonal agents that act to control or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands; (iii) antiandrogens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation; (vii) ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines; topoisomerase 1 inhibitors; (ix) angiogenesis inhibitors; and pharmaceutically acceptable salts, acids, solvates, and derivatives of any of the above.

[0063] Toxin effector molecules or moieties can include, but are not limited to, proteinaceous toxins (e.g., bacterial and plant-derived toxins), toxins that target tubulin filaments, toxins that target DNA, and toxins that target RNA. Examples of proteinaceous toxins include saporin, dianthin, ricin, modeccin, abrin, volkensin, viscumin, Shiga toxin, Shiga-like toxins, Pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of tubulin filament-targeting toxins include maytansinoids (e.g., DM1 and DM4), auristatins (e.g., monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF)), toxoids, tubulysins, cryptophycins, and rhizoxins. Examples of DNA-targeted toxins are calicheamicin: N-acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycin, doxorubicin, methotrexate, benzodiazepines, camptothecin analogs, and anthracyclines. Examples of DNA-targeted toxins are amanitin, splicehostatin, and Thailanstatin. The toxins used in the present invention are defined as drug substances that can kill or inactivate cells. Preferably, targeted toxins are toxins that are only, or at least predominantly, toxic to target cells, but not to off-target cells. The net effect of the targeted toxin is preferably beneficial to the organism as a whole.

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

[0065] An effector molecule or effector moiety that is a polynucleotide can be, for example, a polynucleotide containing coding information, such as a gene or open reading frame encoding a protein. It can also contain regulatory information, such as a promoter or regulatory element binding region, or a sequence encoding a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include, for example, peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these is distinguished from naturally occurring DNA or RNA by modifications to the backbone of the molecule. Examples of nucleotides as effector molecules include, but are not limited to, DNA: single-stranded DNA (e.g., adenine phosphoribosyltransferase DNA); linear double-stranded DNA (e.g., coagulation factor IX gene); circular double-stranded DNA (e.g., plasmid); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA; antisense oligonucleotides (ASO, AON, e.g., PNA, PMO, LNA, and BNA).

[0066] For example, the term "proteinaceous," as used in "proteinaceous molecule" and "proteinaceous toxin," refers to molecules and toxins comprising at least one string of amino acid residues that can be obtained as an expression product from a single mRNA. Such molecules or toxins can further comprise any post-translational modifications, carbohydrates, e.g., N- or O-linked carbohydrates, disulfide bonds, phosphorylation, sulfation, etc., resulting from any post-translational modification, and / or can further comprise any other modifications, e.g., resulting from chemical modifications (e.g., resulting from the attachment of an effector moiety, saponin, scaffold, ligand, etc., either directly, e.g., to an amino acid side chain, or via at least one linker that is (covalently) bound to the molecule and chemically bound to the proteinaceous molecule to chemically modify the proteinaceous molecule). The term "proteinaceous" also encompasses and includes aggregates of such molecules, e.g., homodimers, heterotrimers, heterohexamers, or complex assemblies, e.g., ribosomes.

[0067] For example, in the context of "specific binding" and "a receptor or molecular target specifically present or expressed on the surface of tumor cells," and the like, the terms "specific" and "specifically" have their ordinary scientific meaning known in the art. Here, for example, it refers to a binding interaction of a first molecule with a second molecule that occurs with a relatively higher affinity relative to any putative binding of the first molecule to an additional molecule different from the second molecule, or, when considering a number of receptors or molecular targets, the expression or higher degree of expression of a cell surface receptor or molecular target on the surface of a first type of cell, such as a tumor cell, autoimmune cell, diseased cell, or abnormal cell, relative to the degree of expression of the same receptor or molecular target on a second type of cell, such as a healthy cell. Expression in a second type of cell may be completely absent or very low relative to any degree of expression on tumor cells. Furthermore, for example, in "specific binding," the term "specific" has its ordinary scientific meaning known in the art, and here refers to a molecule that can interact with another molecule with a binding affinity higher than background interactions between molecules. Similarly, the term "specificity" refers to an interaction, e.g., between two molecules or between a cell and a molecule, that has a higher binding affinity than background interactions between molecules. Binding molecules, such as immunoglobulins, bind via their binding site, e.g., the immunoglobulin variable region of an immunoglobulin, to a binding site on a molecule, e.g., an epitope, cell surface receptor, etc., with a higher binding affinity than background interactions between molecules. In the context of the present invention, background interactions typically have a K of 10E-4 M or less. D Similarly, a "specific binding domain" is a domain that preferentially binds to a binding site on a molecule, e.g., an epitope, cell surface receptor, etc., with a binding affinity higher than background interactions between molecules. In the context of the present invention, a "background interaction" is typically a K of 10E-4 M or less. D Preferably, the specific binding domain has a K of about 10E-5 M. D It binds with higher affinity than

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

[0069] As used herein, the term "fragment" refers to an amino acid sequence that is part of a protein domain or that makes up an intact protein domain. Binding fragments according to the invention must have binding specificity for their respective targets, e.g., cell surface receptors on the surface of diseased cells, e.g., tumor cells.

[0070] The term "ADC" or "antibody-drug conjugate" has its usual scientific meaning known to those of skill in the art and, herein, refers to a class of biopharmaceutical drugs designed as targeted therapies, for example, to treat cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADCs consist of an antibody linked to a bioactive cytotoxic (anticancer) payload or drug. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs. They are designed with the intent of discriminating between healthy cells and diseased tissue, such as tumor cells in tumors.

[0071] The term "Saponinum album" has its ordinary meaning and herein refers to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila Arostii, including SA1657 and primarily SA1641.

[0072] The term "Quillaja saponin" has its ordinary meaning and refers herein to the saponin fraction of Quillaja saponaria, and therefore the source of all other QS saponins, containing primarily QS-18 and QS-21.

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

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

[0075] Similarly, "QS-21B" has its customary scientific meaning and refers herein to a mixture of QS-21 B-apio (-65%) and QS-21 B-xylo (-35%).

[0076] The term "Quill-A" refers to a commercially available semi-purified extract from Quillaja saponaria, which contains variable quantities of more than 50 distinct saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3 beta-OH group, found in QS-7, QS-17, QS18, and QS-21. The saponins found in Quil-A are listed in Table 2 of van Setten (1995) (Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer, and Gideon F. Kersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 660-666 (1995)). Quil-A and Quillaja saponin are also fractions of saponins from Quillaja saponaria, and both contain a large variety of different saponins with largely overlapping contents. Because the two fractions are obtained by different purification procedures, the two fractions differ in their specific composition.

[0077] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. QS1861 has a molecular mass of 1861 daltons, and QS1862 has a molecular mass of 1862 daltons. QS1862 is listed in line 28 of Table 1 in Fleck et al. (2019) (Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira, and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171). The structure shown is that of QS1862, an API variant of QS-7. The molecular mass is 1862 daltons, because this mass is the nominal mass including the proton in the glucuronic acid. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in the negative ion mode, the measured mass is 1861 daltons.

[0078] The terms first, second, third, and the like in the specification and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. The terms are interchangeable in appropriate circumstances. Embodiments of the invention may operate in sequences other than those described or illustrated herein.

[0079] Furthermore, although various embodiments may be referred to as "preferably" or "eg" or "for example" or "specifically," these should not be construed as limiting the scope of the invention, but rather as exemplary ways in which the invention may be implemented.

[0080] The term "comprising" used in the claims should not be construed as being limited to the subsequently listed elements or steps; it does not exclude other elements or steps. It needs to be construed as specifying the presence of a stated feature, integer, step, or component, 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 phrase "a pharmaceutical composition comprising A and B" should not be limited to a pharmaceutical composition consisting solely of components A and B; rather, for the purposes of the present invention, the recited components of the pharmaceutical composition are only A and B, and the claim should be construed to encompass equivalents of those components. Similarly, the scope of the phrase "a method comprising steps A and B" should not be limited to a method consisting solely of steps A and B; rather, for the purposes of the present invention, the recited steps of the method are only A and B, and the claim should be construed to encompass equivalents of those steps.

[0081] Additionally, unless the context clearly requires that there be only one of the feature, reference to a feature by the indefinite article "a" or "an" does not exclude the possibility that more than one of the feature may be present, e.g., a component, excipient, saponin, etc. Thus, the indefinite article "a" or "an" normally means "at least one." [Brief explanation of the drawings]

[0082] [Figure 1] In vivo study of antibody-protein toxin + unconjugated SO1861. BT474 tumor-bearing mice treated with various concentrations of trastuzumab-saporin (iv) + 1.5 mg / kg unconjugated SO1861 (subQ injection 1 hour before trastuzumab-saporin treatment). [Figure 2-1]Enhanced endosomal escape and target cell killing mediated by unconjugated saponins. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904, with or without 1.5 pM EGF dianthine. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthine and fixed concentrations of SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904. Axes and legends for A) and B) are the same. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741, with or without 1.5 pM EGF dianthine. D) Cell viability analysis of HeLa cells (EGFR+) treated with different QS mixes (saponin mixtures from Quillaia Saponaria) with or without 1.5pMEGF dianthin. The Y-axis in C) and D) is the same. [Figure 2-2] Enhanced endosomal escape and target cell killing mediated by unconjugated saponins. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904, with or without 1.5 pM EGF dianthine. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthine and fixed concentrations of SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904. Axes and legends for A) and B) are the same. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741, with or without 1.5 pM EGF dianthine. D) Cell viability analysis of HeLa cells (EGFR+) treated with different QS mixes (saponin mixtures from Quillaia Saponaria) with or without 1.5pMEGF dianthin. The Y-axis in C) and D) is the same. [Figure 2-3]Enhanced endosomal escape and target cell killing mediated by unconjugated saponins. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904, with or without 1.5 pM EGF dianthine. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthine and fixed concentrations of SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904. Axes and legends for A) and B) are the same. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741, with or without 1.5 pM EGF dianthine. D) Cell viability analysis of HeLa cells (EGFR+) treated with different QS mixes (saponin mixtures from Quillaia Saponaria) with or without 1.5pMEGF dianthin. The Y-axis in C) and D) is the same. [Figure 3-1] Activity of unconjugated SO1861 versus SO1861-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-EMCH with or without 1.5 pM EGGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-N3 with or without 1.5 pM EGGF dianthine. The axes and legends for A), B), C), D), and E) are the same. That is, the legends for Figures 3A-C appear next to Figure 3C, and the legends for Figures 3D and 3E appear next to Figure 3E. [Figure 3-2]Activity of unconjugated SO1861 versus SO1861-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-EMCH with or without 1.5 pM EGGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-N3 with or without 1.5 pM EGGF dianthine. The axes and legends for A), B), C), D), and E) are the same. That is, the legends for Figures 3A-C appear next to Figure 3C, and the legends for Figures 3D and 3E appear next to Figure 3E. [Figure 3-3] Activity of unconjugated SO1861 versus SO1861-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-EMCH with or without 1.5 pM EGGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-N3 with or without 1.5 pM EGGF dianthine. The axes and legends for A), B), C), D), and E) are the same. That is, the legends for Figures 3A-C appear next to Figure 3C, and the legends for Figures 3D and 3E appear next to Figure 3E. [Figure 4] Unconjugated SO1861 vs. SO1861-EMCH (unstable) vs. SO1861-S (stable). Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1861-S (S=HATU, stable linker), and SO1861-EMCH (unstable linker) with or without EGF dianthin. [Figure 5-1]EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing. HSP27 mRNA expression analysis of A431 (EGFR++) and A2058 (EGFR-) cells treated with cetuximab-(Cys-L-SO1861)3.9 or cetuximab-(Cys-L-SO1861)3.9 + 100 nM HSP27 BNA. Axes and legends for A) and B) are the same, and the legend appears next to Figure 5B. Y-axes for C) and D) are the same. [Figure 5-2] EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing. HSP27 mRNA expression analysis of A431 (EGFR++) and A2058 (EGFR-) cells treated with cetuximab-(Cys-L-SO1861)3.9 or cetuximab-(Cys-L-SO1861)3.9 + 100 nM HSP27 BNA. Axes and legends for A) and B) are the same, and the legend appears next to Figure 5B. Y-axes for C) and D) are the same. [Figure 5-3] EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing. HSP27 mRNA expression analysis of A431 (EGFR++) and A2058 (EGFR-) cells treated with cetuximab-(Cys-L-SO1861)3.9 or cetuximab-(Cys-L-SO1861)3.9 + 100 nM HSP27 BNA. Axes and legends for A) and B) are the same, and the legend appears next to Figure 5B. Y-axes for C) and D) are the same. [Figure 5-4] EGFR-targeted antisense BNA oligonucleotide delivery and gene silencing. HSP27 mRNA expression analysis of A431 (EGFR++) and A2058 (EGFR-) cells treated with cetuximab-(Cys-L-SO1861)3.9 or cetuximab-(Cys-L-SO1861)3.9 + 100 nM HSP27 BNA. Axes and legends for A) and B) are the same, and the legend appears next to Figure 5B. Y-axes for C) and D) are the same. [Figure 6]Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. Mice treated with HSP27 BNA plus cetuximab-(Cys-L-SO1861)3,9 in A431 tumor-bearing mice demonstrate efficient tumor-targeted gene silencing compared to controls. [Figure 7] In vivo activity of 1T2C. In A431 tumor-bearing mice, the 1T2C combination of 50 mg / kg cetuximab-(Cys-L-SO1861)4 + 25 mg / kg cetuximab-(-L-HSP27BNA)4 demonstrates robust tumor-targeted gene silencing compared to controls. [Figure 8] In vivo activity of 1T2C. The 1T2C combination of 40 mg / kg trastuzumab-(Cys-L-SO1861)4 + 0.02 / 0.03 mg / kg trastuzumab-saporin in a PDX tumor mouse model (high HER2 expression) shows effective tumor growth inhibition. [Figure 9-1] One target, two components. EGFR-targeted cell killing in A431 cells (EGFR++) (A, C) and CaSKi cells (EGFR+) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861) 3,7 titration + fixed concentration of 10 pM cetuximab-saporin and control on A431 (A) and CaSKi (B) cells. C, D) Cetuximab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 and control on A431 (C) and CaSKi (D) cells. [Figure 9-2]One target, two components. EGFR-targeted cell killing in A431 cells (EGFR++) (A, C) and CaSKi cells (EGFR+) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861) 3,7 titration + fixed concentration of 10 pM cetuximab-saporin and control on A431 (A) and CaSKi (B) cells. C, D) Cetuximab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 and control on A431 (C) and CaSKi (D) cells. [Figure 10-1] One target, two components. EGFR-targeted cell killing in HeLa cells (EGFR+ / -) (A, C) and A2058 cells (EGFR-) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861) 3,7 titration + fixed concentration of 10 pM cetuximab-saporin and control on HeLa (A) and CaSKi (B) cells. C, D) Cetuximab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 and control on HeLa (C) and A2058 (D) cells. [Figure 10-2] One target, two components. EGFR-targeted cell killing in HeLa cells (EGFR+ / -) (A, C) and A2058 cells (EGFR-) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861) 3,7 titration + fixed concentration of 10 pM cetuximab-saporin and control on HeLa (A) and CaSKi (B) cells. C, D) Cetuximab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861) 3,7 and control on HeLa (C) and A2058 (D) cells. [Figure 11]One target, two components. HER2-targeted cell killing in SKBR3 cells (HER2++) (A, B) by therapeutic combinations according to the invention. A) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 50 pM trastuzumab-saporin and control on SKBR3 cells. B) Trastuzumab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on SKBR3 cells. [Figure 12-1] One target, two components. HER2-targeted cell killing in JIMT-1 cells (HER2+ / -) (A, C) and MDA-MB-468 cells (HER2-) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 50 pM trastuzumab-saporin and control for JIMT-1 (A) and MDA-MB-468 (B) cells. C, D) Trastuzumab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for JIMT-1 (C) and MDA-MB-468 (D) cells. [Figure 12-2] One target, two components. HER2-targeted cell killing in JIMT-1 cells (HER2+ / -) (A, C) and MDA-MB-468 cells (HER2-) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 50 pM trastuzumab-saporin and control for JIMT-1 (A) and MDA-MB-468 (B) cells. C, D) Trastuzumab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for JIMT-1 (C) and MDA-MB-468 (D) cells. [Figure 13]Chloroquine inhibits two components, one target. HER2- and EGFR-targeted cell killing in SK-BR-3 (HER2++) and A431 cells (EGFR++) by a therapeutic combination according to the invention plus chloroquine. A) Trastuzumab-saporin titration plus a fixed concentration of 5 nM trastuzumab-(Cys-L-SO1861)4 + 0.5 μM chloroquine and control for SK-BR-3 cells. B) Cetuximab-saporin titration plus a fixed concentration of 5 nM cetuximab-(Cys-L-SO1861)3,8 + 0.5 μM chloroquine and control for A431 cells. [Figure 14-1] One target, two components. EGFR-targeted gene silencing in A431 cells (EGFR++) and A2058 cells (EGFR-) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-S01861)3,8 titration + fixed concentration of 100 nM cetuximab-(Lys-L-HSP27BNA)4 and control for A431 cells (A) and A2058 cells (B). C, D) Cetuximab-(Lys-L-HSP27BNA)4 titration + fixed concentration of 77 nM cetuximab-(Cys-L-S01861)3,8 and control for A431 cells (C) and A2058 cells (D). [Figure 14-2] One target, two components. EGFR-targeted gene silencing in A431 cells (EGFR++) and A2058 cells (EGFR-) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-S01861)3,8 titration + fixed concentration of 100 nM cetuximab-(Lys-L-HSP27BNA)4 and control for A431 cells (A) and A2058 cells (B). C, D) Cetuximab-(Lys-L-HSP27BNA)4 titration + fixed concentration of 77 nM cetuximab-(Cys-L-S01861)3,8 and control for A431 cells (C) and A2058 cells (D). [Figure 15-1]Two targets, two components. A) EGFR- and HER2-targeted cell killing in MDA-MB-468 cells (EGFR++) and HeLa cells (EGFR+ / -), and HER2-targeted cell killing in SK-BR-3 cells (HER2++) and JIMT-1 cells (HER2+ / -) by a therapeutic combination according to the invention. A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM cetuximab-saporin and control in MDA-MB-468 cells (A) and HeLa cells (B). C, D) Trastuzumab-(Cys-(Dendron(-L-SO1861)4)4 titration + fixed concentration 50 pM trastuzumab-saporin and control in SK-BR-3 cells (C) and JIMT-1 cells (D). [Figure 15-2] Two targets, two components. A) EGFR- and HER2-targeted cell killing in MDA-MB-468 cells (EGFR++) and HeLa cells (EGFR+ / -), and HER2-targeted cell killing in SK-BR-3 cells (HER2++) and JIMT-1 cells (HER2+ / -) by a therapeutic combination according to the invention. A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM cetuximab-saporin and control in MDA-MB-468 cells (A) and HeLa cells (B). C, D) Trastuzumab-(Cys-(Dendron(-L-SO1861)4)4 titration + fixed concentration 50 pM trastuzumab-saporin and control in SK-BR-3 cells (C) and JIMT-1 cells (D). [Figure 16] One target, two components. SK-BR-3 cells (HER2+ / -) can be efficiently killed by the therapeutic combination of trastuzumab-saporin + 2.5 nM trastuzumab-(Cys-L-SO1861)4 according to the present invention. However, at such low toxin concentrations, titration with T-DM1 + ​​2.5 nM trastuzumab-(Cys-L-SO1861)4 is ineffective. T-DM1 is trastuzumab emtansine (Kadcyla®) and carries 3.5 emtansine (DM1) toxin molecules per antibody (DAR 3.5). [Figure 17-1] One target, two components. EGFR-targeted cell killing in A431 cells (EGFR++) (A), CaSKi cells (EGFR+) (B), and A2058 cells (EGFR-) by a therapeutic combination according to the invention. A, B, C) Cetuximab-(Cys-L-QS mix) 4,1 titration + fixed concentration of 10 pM cetuximab-saporin or 10 pM cetuximab-giantin and controls in A431 cells (A), CaSKi cells (B), and A2058 cells (C). QS mix is ​​a mixture of saponins from the extract Quillaja Saponaria. [Figure 17-2] One target, two components. EGFR-targeted cell killing in A431 cells (EGFR++) (A), CaSKi cells (EGFR+) (B), and A2058 cells (EGFR-) by a therapeutic combination according to the invention. A, B, C) Cetuximab-(Cys-L-QS mix) 4,1 titration + fixed concentration of 10 pM cetuximab-saporin or 10 pM cetuximab-giantin and controls in A431 cells (A), CaSKi cells (B), and A2058 cells (C). QS mix is ​​a mixture of saponins from the extract Quillaja Saponaria. [Figure 18] One-target, two-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 and enables endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 19]One-target, two-component concept: mAb1-SO1861 + mAb2-BNA oligo. SO1861 and antisense BNA oligonucleotides are each independently conjugated to an antibody (mAb1) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb1-BNA oligo bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active and enables endolysosomal escape, 4) release of the BNA oligo into the cytoplasm, and 5) targeted gene silencing occurs. [Figure 20] One-target, two-component concept: mAb1-(scaffold(-SO1861)n)n + mAb1-protein toxin. The dendron (-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 bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active and enables endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 21-1] A) HER2- and EGFR-targeted cell killing in A431 cells (EGFR++) and SK-BR-3 (HER2++) by cetuximab-(-L-SO1861)4 + 10 pM cetuximab-saporin compared to antibody-(-L-SO1861)4 vs. antibody-(-L-SO1861)2 in A431 cells. B) Trastuzumab-(-L-SO1861)4 + 50 pM trastuzumab-saporin compared to trastuzumab-(-L-SO1861)2 + 50 pM trastuzumab-saporin in SK-BR-3 cells. [Figure 21-2]A) HER2- and EGFR-targeted cell killing in A431 cells (EGFR++) and SK-BR-3 (HER2++) by cetuximab-(-L-SO1861)4 + 10 pM cetuximab-saporin compared to antibody-(-L-SO1861)4 vs. antibody-(-L-SO1861)2 in A431 cells. B) Trastuzumab-(-L-SO1861)4 + 50 pM trastuzumab-saporin compared to trastuzumab-(-L-SO1861)2 + 50 pM trastuzumab-saporin in SK-BR-3 cells. [Figure 22] Antibody-(-L-SO1861)4 versus antibody-(-S-SO1861)4. HER2-targeted cell killing in SK-BR-3 (HER2++) cells by a therapeutic combination according to the invention. B) Trastuzumab-(-L-SO1861)4 + 50 pM trastuzumab-saporin compared to trastuzumab-(-S-SO1861)4 + 50 pM trastuzumab-saporin in SK-BR-3 cells. [Figure 23] The 2T2 component system tested in an A431 tumor-bearing mouse model reveals tumor regression. [Figure 24] The 2T2 component system tested in an A431 tumor-bearing mouse model demonstrates tumor regression and eradication. [Figure 25-1]Two targets, two components. EGFR / HER2-targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control for A431 cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control for CaSKi cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 25A and B appear next to Figure 25B; the legends for Figures 25C and 25D appear next to Figure 25D. [Figure 25-2] Two targets, two components. EGFR / HER2-targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control for A431 cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control for CaSKi cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 25A and B appear next to Figure 25B; the legends for Figures 25C and 25D appear next to Figure 25D. [Figure 26-1]Two targets, two components. EGFR / HER2-targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control for HeLa cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control for A2058 cells. Axes and legends are the same for A) and B). That is, the legends for Figures 26A and B appear next to Figure 26B. [Figure 26-2] Two targets, two components. EGFR / HER2-targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by therapeutic combinations according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control for HeLa cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control for A2058 cells. Axes and legends are the same for A) and B). That is, the legends for Figures 26A and B appear next to Figure 26B. [Figure 27] Two targets, two components. HER2 / EGFR-targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by a therapeutic combination according to the invention. Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 1.5 pM EGF dianthine and control on ASKBR3 cells. B) EGF dianthine titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on SKBR3 cells. [Figure 28-1]Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 1.5 pM EGF dianthine and control for JIMT-1 cells. C, D) EGF dianthine titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for MDA-MB-468 cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 28A and 28B appear next to Figure 28B; the legends for Figures 28C and 28D appear next to Figure 28D. [Figure 28-2] Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 1.5 pM EGF dianthine and control for JIMT-1 cells. C, D) EGF dianthine titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for MDA-MB-468 cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 28A and 28B appear next to Figure 28B; the legends for Figures 28C and 28D appear next to Figure 28D. [Figure 29] Two targets, two components. HER2 / EGFR-targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by a therapeutic combination according to the invention. A) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 10 pM cetuximab-saporin and control on SKBR3 cells. B) Cetuximab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on SKBR3 cells. [Figure 30-1]Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 10 pM cetuximab-saporin and control for JIMT-1 cells. C, D) Cetuximab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for MDA-MB-468 cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 30A and 30B are displayed next to Figure 30B; the legends for Figures 30C and 30D are displayed next to Figure 30D. [Figure 30-2] Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by therapeutic combinations according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 10 pM cetuximab-saporin and control for JIMT-1 cells. C, D) Cetuximab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control for MDA-MB-468 cells. Axes and legends are the same for A) and B) and C) and D). That is, the legends for Figures 30A and 30B are displayed next to Figure 30B; the legends for Figures 30C and 30D are displayed next to Figure 30D. [Figure 31-1]Chloroquine inhibits two targets, two components. GFR / HER2, EGFR / CD71, or HER2 / CD71-targeted cell killing in EA431 cells (EGFR++ / HER2+ / - / CD71+) (A, B), MDA-MB-468 cells (EGFR++ / HER2- / CD71+) (C), or SK-BR-3 (HER2++ / EGFR+ / - / CD71+) (D) by a therapeutic combination according to the invention plus chloroquine. A) Trastuzumab-giantine or trastuzumab-saporin titration plus a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,9 + 800 nM chloroquine on A431 cells, and control. B) CD71mab-saporin titration + fixed concentration of 10.5 nM cetuximab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on A431 cells. C) CD71mab-saporin titration + fixed concentration of 10.5 nM cetuximab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on MDA-MB-468 cells. D) CD71mab-saporin titration + fixed concentration of 5 nM trastuzumab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on SK-BR-3 cells. [Figure 31-2]Chloroquine inhibits two targets, two components. GFR / HER2, EGFR / CD71, or HER2 / CD71-targeted cell killing in EA431 cells (EGFR++ / HER2+ / - / CD71+) (A, B), MDA-MB-468 cells (EGFR++ / HER2- / CD71+) (C), or SK-BR-3 (HER2++ / EGFR+ / - / CD71+) (D) by a therapeutic combination according to the invention plus chloroquine. A) Trastuzumab-giantine or trastuzumab-saporin titration plus a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,9 + 800 nM chloroquine on A431 cells, and control. B) CD71mab-saporin titration + fixed concentration of 10.5 nM cetuximab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on A431 cells. C) CD71mab-saporin titration + fixed concentration of 10.5 nM cetuximab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on MDA-MB-468 cells. D) CD71mab-saporin titration + fixed concentration of 5 nM trastuzumab-(Cys-L-SO1861)3,9 + 500 nM chloroquine and control on SK-BR-3 cells. [Figure 32-1] Two targets, two components. EGFR / HER2-targeted gene silencing in A431 cells (EGFR++ / HER2+ / -) (A) and A2058 cells (EGFR- / HER2+ / -) (B) using a therapeutic combination according to the invention. A) Cetuximab-(Cys-L-SO1861)3,9 titration + fixed concentration of 100 nM trastuzumab-(Lys-L-HSP27BNA)4,4 and control for A431 cells (A) and A2058 cells (B). C, D) Trastuzumab-(Lys-HSP27BNA)4,4 titration + fixed concentration of 77 nM cetuximab-(Cys-L-SO1861)3,9 and control for A431 cells (A) and A2058 cells (B). The axes and legends are the same for A) and B) and C) and D), i.e., the legend for Figures 32A and B appears next to Figure 32B; the legend for Figures 32C and 32D appears next to Figure 32D. [Figure 32-2] Two targets, two components. EGFR / HER2-targeted gene silencing in A431 cells (EGFR++ / HER2+ / -) (A) and A2058 cells (EGFR- / HER2+ / -) (B) using a therapeutic combination according to the invention. A) Cetuximab-(Cys-L-SO1861)3,9 titration + fixed concentration of 100 nM trastuzumab-(Lys-L-HSP27BNA)4,4 and control for A431 cells (A) and A2058 cells (B). C, D) Trastuzumab-(Lys-HSP27BNA)4,4 titration + fixed concentration of 77 nM cetuximab-(Cys-L-SO1861)3,9 and control for A431 cells (A) and A2058 cells (B). The axes and legends are the same for A) and B) and C) and D), i.e., the legend for Figures 32A and B appears next to Figure 32B; the legend for Figures 32C and 32D appears next to Figure 32D. [Figure 33-1] Two targets, two components. A) EGFR / CD71 or HER2 / CD71 targeted cell killing in MDA-MB-468 cells (EGFR++ / CD71+) (A), HeLa cells (EGFR+ / - / CD71+), SK-BR-3 cells (HER2++ / CD71+) (B), and JIMT-1 cells (HER2+ / - / CD71+) by therapeutic combinations according to the invention. A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM CD71mab-saporin and control on MDA-MB-468 cells. B) A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM CD71mab-saporin and control on HeLa cells. C) Trastuzumab-Cys-(dendron(-L-SO1861)4)4 titration + fixed concentration 10 pM CD71mab-saporin and control on SK-BR-3 cells. D) Trastuzumab-Cys-(dendron(-L-SO1861)4)4 titration + fixed concentration 10 pM CD71mab-saporin and control on JIMT-1 cells. [Figure 33-2]Two targets, two components. A) EGFR / CD71 or HER2 / CD71 targeted cell killing in MDA-MB-468 cells (EGFR++ / CD71+) (A), HeLa cells (EGFR+ / - / CD71+), SK-BR-3 cells (HER2++ / CD71+) (B), and JIMT-1 cells (HER2+ / - / CD71+) by therapeutic combinations according to the invention. A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM CD71mab-saporin and control on MDA-MB-468 cells. B) A) Cetuximab-Cys-(Dendron(-L-SO1861)4)3,9 titration + fixed concentration 10 pM CD71mab-saporin and control on HeLa cells. C) Trastuzumab-Cys-(dendron(-L-SO1861)4)4 titration + fixed concentration 10 pM CD71mab-saporin and control on SK-BR-3 cells. D) Trastuzumab-Cys-(dendron(-L-SO1861)4)4 titration + fixed concentration 10 pM CD71mab-saporin and control on JIMT-1 cells. [Figure 34] Two targets, two components vs. T-DM1. A431 cells (EGFR++ / HER2+ / -) can be efficiently killed by the therapeutic combination of trastuzumab-saporin + 75 nM cetuximab-(Cys-L-SO1861)3,9 according to the present invention. However, titration of T-DM1 + ​​75 nM cetuximab-(Cys-L-SO1861)3,9 is not effective at such low toxin concentrations. T-DM1 is trastuzumab-emtansine (Kadcyla®), which carries 3.5 emtansine (DM1) toxin molecules per antibody. [Figure 35-1] Control treatments for all cell lines. AD) Cell viability when trastuzumab (A), cetuximab (B), T-DM1, (C) free toxins: saporin and dianthin (D) or saporin coupled to non-cell-binding IgG (D) are treated with the indicated cell lines: SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. [Figure 35-2] Control treatments for all cell lines. AD) Cell viability when trastuzumab (A), cetuximab (B), T-DM1, (C) free toxins: saporin and dianthin (D) or saporin coupled to non-cell-binding IgG (D) are treated with the indicated cell lines: SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. [Figure 36-1] Two targets, two components. EGFR / CD71 and EGFR / HER2-targeted cell killing in A431 cells (EGFR+++ / HER2+ / -) (A), CaSKi cells (EGFR++ / HER2+ / -) (B), and A2058 cells (EGFR- / HER2+ / -) by a therapeutic combination according to the invention. A, B, C) Cetuximab-(Cys-L-QS mix) 4,1 titration + fixed concentration of 10 pM trastuzumab-saporin or 10 pM CD71mab-saporin, and control, in A431 cells (A), CaSKi cells (B), and A2058 cells (C). CaSKi cells (B) and A2058 cells (C). QS mix is ​​a mixture of saponins from the extract Quillaja Saponaria. The legends for Figures 36A and 36B appear after Figure 36B. [Figure 36-2] Two targets, two components. EGFR / CD71 and EGFR / HER2-targeted cell killing in A431 cells (EGFR+++ / HER2+ / -) (A), CaSKi cells (EGFR++ / HER2+ / -) (B), and A2058 cells (EGFR- / HER2+ / -) by a therapeutic combination according to the invention. A, B, C) Cetuximab-(Cys-L-QS mix) 4,1 titration + fixed concentration of 10 pM trastuzumab-saporin or 10 pM CD71mab-saporin, and control, in A431 cells (A), CaSKi cells (B), and A2058 cells (C). CaSKi cells (B) and A2058 cells (C). QS mix is ​​a mixture of saponins from the extract Quillaja Saponaria. The legends for Figures 36A and 36B appear after Figure 36B. [Figure 37]Two-target, two-component concept: mAb1-SO1861 + mAb2-protein toxin. SO1861 and a toxin (ribosome-inactivating protein) are each separately 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 and enables endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 38] Two-target, two-component concept: mAb1-SO1861 + mAb2-BNA oligo. SO1861 and antisense BNA oligonucleotides are each separately conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb1-SO1861 and mAb2-BNA oligo 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 and enables endolysosomal escape, 4) release of the BNA oligo into the cytoplasm, and 5) targeted gene silencing occurs. [Figure 39] Two-target, two-component concept: mAb1-(scaffold(-SO1861)n)n + mAb2-protein toxin. The dendron (-SO1861)n and protein toxin (ribosome-inactivating protein) are each separately conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb1-(dendron (-SO1861)4)1) 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 and enables endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 40-1]Tumor-targeted protein toxin delivery results in tumor volume reduction and tumor growth inhibition in tumor-bearing mice. A) Dose-escalating administration (intraperitoneal i.p.) of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice reveals tumor volume reduction compared to controls. B, C) Dose-escalating administration (intraperitoneal i.p. (B) or intravenous i.v. (C)) of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice reveals tumor growth reduction compared to controls. [Figure 40-2] Tumor-targeted protein toxin delivery results in tumor volume reduction and tumor growth inhibition in tumor-bearing mice. A) Dose-escalating administration (intraperitoneal i.p.) of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice reveals tumor volume reduction compared to controls. B, C) Dose-escalating administration (intraperitoneal i.p. (B) or intravenous i.v. (C)) of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice reveals tumor growth reduction compared to controls. [Figure 41] Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg cetuximab-(Cys-L-SO1861)3,9(Lys-L-HSP27BNA)1,8 in A431 tumor-bearing mice demonstrated efficient tumor-targeted gene silencing compared to controls. [Figure 42] Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 in A431 tumor-bearing mice demonstrated induced efficient tumor-targeted gene silencing compared with controls. [Figure 43-1]HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 43A and B have the same legend outlined next to Figure 43B. Figures 43C and D have the same legend outlined next to Figure 43D. [Figure 43-2] HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 43A and B have the same legend outlined next to Figure 43B. Figures 43C and D have the same legend outlined next to Figure 43D. [Figure 43-3]HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 43A and B have the same legend outlined next to Figure 43B. Figures 43C and D have the same legend outlined next to Figure 43D. [Figure 43-4] HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 43A and B have the same legend outlined next to Figure 43B. Figures 43C and D have the same legend outlined next to Figure 43D. [Figure 44-1] EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 44A and B have the same legend as Figure 44B, as outlined next. [Figure 44-2]EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)1,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). Figures 44A and B have the same legend as Figure 44B, as outlined next. [Figure 45] HER2-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)3,5 treatment and control in SK-BR-3 cells (HER2++). [Figure 46] EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 treatment and control in A431 cells (EGFR++) and A2058 cells (EGFR-). [Figure 47] (S)n-(L)(E) Concept: mAb-(SO1861)n (protein toxin). For delivery and internalization into target cells, SO1861 is conjugated to a cysteine ​​residue (Cys) and a protein toxin (ribosome-inactivating protein) is conjugated to a lysine residue on the same antibody (mAb). 1) mAb-(Cys-L-SO1861)4 (Lys-protein toxin)2 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of the conjugate occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) release of the toxin into the cytoplasm, and 5) the toxin induces cell death. [Figure 48](S)n-(L)(E) Concept: mAb-(SO1861)n (antisense BNA oligo)n. Both SO1861 at a cysteine ​​residue (Cys) and an antisense BNA oligonucleotide at a lysine residue are conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-SO1861)4 (Lys-BNA oligo)2 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) release of the BNA oligo into the cytoplasm, and 5) induction of target gene silencing. [Figure 49] (S)n-(L)(E) Concept: mAb-(SO1861-scaffold-antisense BNA oligo)n. (SO1861-trifunctional linker-BNA oligo)n is conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb-(SO1861-trifunctional linker-BNA oligo)4 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active and enables endolysosomal escape, 4) release of the BNA oligo into the cytoplasm occurs, and 5) targeted gene silencing is induced. [Figure 50]Antibody-SO1861 Conjugation Procedure. The coupling reaction of four moieties of the plant-derived saponin SO1861 to four cysteines on the light chain of an antibody is shown. First, the disulfide bonds on the IgG are blocked under the influence of exposure to TCEP (tris(2-carboxyethyl)phosphine). Second, the saponin SO1861 containing a chemical linker attached to it is added with trifluoroacetic acid, linking the four saponin moieties to the IgG. To produce a cleavable "ready-to-conjugate" saponin, the aldehyde group of SO1861 was reacted with an EMCH (ε-maleimidocaproic acid hydrazide) linker. The hydrazide group of EMCH forms an acid-cleavable hydrazone bond with the aldehyde of SO1861. At the same time, the EMCH linker presents a maleimide group that is thiol (sulfhydryl) reactive and can therefore be conjugated to a thiol of an IgG, i.e., a ligand moiety, thereby providing an endosomal escape-enhancing conjugate of the invention and / or providing a first binding molecule of the invention. [Figure 51] SO1861-EMCH synthesis. [Figure 52-1] Synthesis of dendron-(-L-SO1861)4. [Figure 52-2] Synthesis of dendron-(-L-SO1861)4. [Figure 52-3] Synthesis of dendron-(-L-SO1861)4. [Figure 52-4] Synthesis of dendron-(-L-SO1861)4. [Figure 52-5] Synthesis of dendron-(-L-SO1861)4. [Figure 52-6] Synthesis of dendron-(-L-SO1861)4. [Figure 53-1] Synthesis of dendron (-L-SO1861)8. [Figure 53-2] Synthesis of dendron (-L-SO1861)8. [Figure 53-3] Synthesis of dendron (-L-SO1861)8. [Figure 53-4] Synthesis of dendron (-L-SO1861)8. [Figure 53-5] Synthesis of dendron (-L-SO1861)8. [Figure 54-1] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 54-2] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 54-3] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 54-4] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 55] Synthesis of HSP27BNA-dendron (-L-SO1861)4. [Figure 56] Synthesis of dendron (NEM)4. [Figure 57] A scaffold precursor with four amino groups for saponin conjugation and an azide group for click chemistry. [Figure 58] Evidence of saponin coupling to the model scaffold. The inset shows the theoretically expected peak and intensity distribution for the bound saponin. Experimental data obtained by LC-MS / ESI-MS showed almost exactly the same peak at m / z 758-760 Da, demonstrating successful saponin coupling. [Figure 59] Cytotoxicity assay using the targeted toxin dianthine-epidermal growth factor (dianthine-EGF). Untreated cells were normalized to 1. The polymeric structure (pentrimer) had no effect on cell viability in the presence or absence of dianthine-EGF and saponin (SA1641), indicating the lack of intrinsic cytotoxicity of the polymeric structure. The clickable targeted toxin (dianthine-EGF-alkyne) had significantly reduced activity. This is the result of toxin modification but not any relationship to the scaffold. The functionalized polymeric structure had the same activity as the unclicked targeted toxin, indicating that scaffold functionalization does not impair effector molecule activity. The effect of saponin was identical in the presence and absence of the polymeric structure, indicating that the polymeric structure does not impair the efficacy of saponin in the two-component system. [Figure 60-1] H-NMR spectra of (A) SO1861 and (B) SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide). (A) The peak at 9.43 ppm (Ha) corresponds to the aldehyde proton of SO1861. (B) The peak at 6.79 ppm (Hc) corresponds to the maleimide proton of SO1861-EMCH, and the peak at 7.68 ppm (Hb) corresponds to the hydrazone proton. The absence of the signal at 9.43 ppm indicates quantitative conversion of the aldehyde group. [Figure 60-2] H-NMR spectra of (A) SO1861 and (B) SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide). (A) The peak at 9.43 ppm (Ha) corresponds to the aldehyde proton of SO1861. (B) The peak at 6.79 ppm (Hc) corresponds to the maleimide proton of SO1861-EMCH, and the peak at 7.68 ppm (Hb) corresponds to the hydrazone proton. The absence of the signal at 9.43 ppm indicates quantitative conversion of the aldehyde group. [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 2109 Da ([M+K]+, SO1861-EMCH), m / z 2094 Da ([M+Na]+, SO1861-EMCH). (B) RP mode: m / z 2193 Da ([M+K]+, saponin-EMCH-mercaptoethanol), m / z 2185 Da ([M+K]+, SO1861-EMCH-mercaptoethanol), m / z 2170 Da ([M+Na]+, SO1861-EMCH-mercaptoethanol). [Figure 62]SO1861 structure with highlighted chemical groups for conjugation of saponin to the polymeric structure to enhance endosomal escape. The highlighted groups are aldehyde (black circle), carboxylic acid (dashed circle), alkene (dashed pentagon), and alcohol (dashed box). The aldehyde group (arrow) is the most suitable group for chemoselective and reversible conjugation reactions. [Figure 63] Strategy to produce (A) stable and (B) cleavable "ready-to-conjugate" endosomal escape-enhancing factor saponins. [Figure 64] Hydrolysis of the hydrazone bond of SO1861-EMCH under acidic conditions. [Figure 65] SO1861-EMCH structure. (A) Standard molecular structure and (B) 3D model. Maleimide groups are marked with circles. [Figure 66-1] (A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 66-2] (A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 66-3] (A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 67-1] MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl solution at pH 3. [Figure 67-2] MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl solution at pH 3. [Figure 68] Reaction scheme for conjugation of SO1861-EMCH to either amine-bearing polymer structure. [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). [Figure 70] Reaction scheme for the conjugation of (A) SO1861-EMCH and (B) SO1861-HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) to cyanine 3 dye-labeled polyamidoamine (PAMAM) G5 dendrimer. [Figure 71-1] MALDI-TOF-MS spectra of (A) Cy3-PAMAM and (B-D) Cy3-PAMAM-SO1861 with increasing SO1861-EMCH loading equivalents from (B) to (D). (B) corresponds to Cy3-PAMAM-SO1861 with 5 SO1861 atoms attached per PAMAM, (C) corresponds to Cy3-PAMAM-SO1861 with 13 SO1861 atoms attached per PAMAM, and (D) corresponds to Cy3-PAMAM-SO1861 with 51 SO1861 atoms attached per PAMAM. [Figure 71-2] MALDI-TOF-MS spectra of (A) Cy3-PAMAM and (B-D) Cy3-PAMAM-SO1861 with increasing SO1861-EMCH loading equivalents from (B) to (D). (B) corresponds to Cy3-PAMAM-SO1861 with 5 SO1861 atoms attached per PAMAM, (C) corresponds to Cy3-PAMAM-SO1861 with 13 SO1861 atoms attached per PAMAM, and (D) corresponds to Cy3-PAMAM-SO1861 with 51 SO1861 atoms attached per PAMAM. [Figure 72] MALDI-TOF-MS spectra of (A) Cy3-PAMAM-SO1861 prepared by SO1861-EMCH at a loading of 5 equivalents and (B) Cy3-PAMAM-SO1861 prepared by SO1861-EMCH at a loading of 30 equivalents. [Figure 73] MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 (NC = stable bond (“non-cleavable”)). [Figure 74-1] (A) Reaction scheme and MALDI-TOF-MS spectra of (B) Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO), (C) Cy3-PAMAM-(SO1861)5-DBCO, and (D) Cy3-PAMAM-(SO1861)27-DBCO. [Figure 74-2] (A) Reaction scheme and MALDI-TOF-MS spectra of (B) Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO), (C) Cy3-PAMAM-(SO1861)5-DBCO, and (D) Cy3-PAMAM-(SO1861)27-DBCO. [Figure 75-1] Reaction schemes of (A) dianthine-EGF-Alexa488 and (B) dianthine-EGF-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine-EGF, (D) dianthine-EGF-Alexa488, and (E) dianthine-EGF-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 75-2] Reaction schemes of (A) dianthine-EGF-Alexa488 and (B) dianthine-EGF-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine-EGF, (D) dianthine-EGF-Alexa488, and (E) dianthine-EGF-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 76-1]Reaction scheme of (A) dianthine-Alexa488 and (B) dianthine-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine, (D) dianthine-Alexa488, and (E) dianthine-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 76-2] Reaction scheme of (A) dianthine-Alexa488 and (B) dianthine-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine, (D) dianthine-Alexa488, and (E) dianthine-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 77] Fluorescent image of SDS-PAGE gel performed by VersaDoc imaging system. M = marker, P = Cy3-PAMAM-(SO1861)27-DBCO, D = dianthine-EGF-Alexa488-SS-PEG-N3, C1 = Cy3-PAMAM-(SO1861)5-dianthine-EGF-Alexa488, C2 = Cy3-PAMAM-NC-SO1861-dianthine-EGF-Alexa488, and C3 = Cy3-PAMAM-(SO1861)27-dianthine-EGF-Alexa488. [Figure 78] (A) Synthesis scheme of Cy3-PAMAM-NC-SO1861 by reductive amination. (B and C) Respective MALDI-TOF-MS spectra. [Figure 79] Reaction scheme for the production of poly(SO1861) using SO1861-EMCH as the monomer, the APS / TMEDA system as the polymerization initiator, and aminopropanethiol as the radical quenching agent. [Figure 80] MALDI-TOF-MS spectra of poly(SO1861) reaction batches: (A) SO1861-EMCH at 60 °C, (B) SO1861-EMCH + 11-3 equiv. APS at 60 °C, (C) SO1861-EMCH + 11-3 equiv. APS / TMEDA at 60 °C. [Figure 81]DNA approach. Use of the principles of DNA origami to generate DNA-based scaffolds capable of conjugating and releasing glycoside molecules. In addition, one of the DNA strands yields a click chemistry moiety that can be used for conjugation to targeted toxins to form a functionalized scaffold. bp: base pair. [Figure 82] The poly(peptide-SO1861) approach uses peptide sequences that can conjugate and release glycoside molecules and react with them to form poly(peptide-SO1861) constructs. The terminus of the poly(peptide) chain can be further modified with click chemistry moieties (e.g., BCN-NHS linkers) that can be used for conjugation to toxins. [Figure 83] MALDI-TOF-MS spectra of (A) the native peptide and (B) the peptide-SO1861 conjugate. [Figure 84] Molecular structure of a G4-dendron with a protected amino group. [Figure 85] Synthetic schemes for the generation of dendron-based and functionalized scaffolds. [Figure 86-1] (A) Reaction scheme for partial dye-labeling and deprotection of G4-dendron. (B) MALDI-TOF-MS spectrum of deprotected and partially dye-labeled G4-dendron. [Figure 86-2] (A) Reaction scheme for partial dye-labeling and deprotection of G4-dendron. (B) MALDI-TOF-MS spectrum of deprotected and partially dye-labeled G4-dendron. [Figure 87] MALDI-TOF-MS spectra of G4-dendron-SO1861 scaffolds with (A) 22 equivalents of SO1861-EMCH, (B) 10 equivalents of SO1861-EMCH, and (C) 3 equivalents of SO1861-EMCH. [Figure 88-1](A) Cell viability curves of HeLa cells treated with EGFR cell surface expression determined by FACS analysis of HeLa cells. (B, see Table 19) Cell viability of HeLa cells treated with SO1861 + dianthine-EGF (Dia-EGF), SO1861 + dianthine-EGF + 500 nM chloroquine, SO1861 + dianthine-EGF + 500 nM PAMAM, SO1861 + dianthine-EGF + 667 nM dendron. (C) Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)16, SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)65, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)108. (D) Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)3, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)8, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)18. [Figure 88-2](A) Cell viability curves of HeLa cells treated with EGFR cell surface expression determined by FACS analysis of HeLa cells. (B, see Table 19) Cell viability of HeLa cells treated with SO1861 + dianthine-EGF (Dia-EGF), SO1861 + dianthine-EGF + 500 nM chloroquine, SO1861 + dianthine-EGF + 500 nM PAMAM, SO1861 + dianthine-EGF + 667 nM dendron. (C) Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)16, SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)65, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(SH)108. (D) Cell viability of HeLa cells treated with SO1861 + Gianthin-EGF, SO1861 + Gianthin-EGF + 500 nM chloroquine, SO1861 + Gianthin-EGF + 500 nM PAMAM, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)3, SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)8, and SO1861 + Gianthin-EGF + 500 nM PAMAM-(mPEG)18. [Figure 89-1] (A) Reaction scheme for thiolation of PAMAM using the thiolation reagent 2-iminothiolane. MALDI-TOF-MS spectra of (B) native PAMAM, (C) thiolated PAMAM-(SH)16, (D) thiolated PAMAM-(SH)65, and (E) thiolated PAMAM-(SH)108. [Figure 89-2] (A) Reaction scheme for thiolation of PAMAM using the thiolation reagent 2-iminothiolane. MALDI-TOF-MS spectra of (B) native PAMAM, (C) thiolated PAMAM-(SH)16, (D) thiolated PAMAM-(SH)65, and (E) thiolated PAMAM-(SH)108. [Figure 90-1](A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 90-2] (A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 90-3] (A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 91] A basic scaffold with click chemistry functional groups for linking any desired effector molecule. The user determines the location of the click chemistry position on the effector molecule, as well as any further properties of the effector molecule, such as the choice and location of any ligands. [Figure 92] A functionalized scaffold with a pre-bound effector molecule and click chemistry functional groups for linking any desired ligand. Optionally, a pH-sensitive linker can be provided for releasing the effector molecule from the scaffold after reaching the endosome. DETAILED DESCRIPTION OF THE INVENTION

[0083] For a bioactive molecule to work, it must be able to engage its target, for example, in serum, on the cell surface, or within a cell or organelle. The active moiety of nearly all protein-based targeted toxins must enter the cytosol of the target cell to mediate its target-modulating effect. In many constellations, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound to the outside of the cell, (2) it is recycled to the cell surface after internalization, or (3) it is transported to the endolysosome, where it is degraded. These fundamental issues have been known for decades, and more than 500 targeted toxins have been investigated over the past several decades. However, the problem remains unresolved, and only one antibody-targeted protein toxin, moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP), has been approved by the FDA to date for relapsed or refractory hairy cell leukemia.

[0084] Many strategies to overcome these problems have been described, including approaches to target toxins to endogenous membrane transport complexes of the biosynthetic pathway in the endoplasmic reticulum and techniques to block or weaken the membrane integrity of endosomes, i.e., compartments of the endocytic pathway in cells, thereby facilitating endosomal escape. These include the use of lysosomotropic amines, carboxylic acid ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human, and synthetic origin, other organic molecules, and light-inducible techniques. While the efficacy of targeted toxins has typically been increased by 100- or 1,000-fold in cell culture, and in exceptional cases by more than 1,000,000-fold, the requirement to coadminister endosomal escape-enhancing agents with other substances introduces new challenges, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell-type-dependent variability.

[0085] All strategies involving physicochemical techniques require enhancer molecules that interact more or less directly with membranes and essentially include small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not target cell specific themselves and distribute with kinetics other than targeted toxins. This is one major drawback of current approaches.

[0086] While the present invention will be described with reference to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

[0087] While the present invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading and understanding the specification and upon studying the drawings and graphs. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

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

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

[0090] One embodiment is a first proteinaceous 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 that is specifically present on tumor cells.

[0091] One embodiment is a first proteinaceous molecule of the invention, wherein at least one saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, and / or a saponin isolated from Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, e.g., Quillaja saponaria.

[0092] One embodiment is a first proteinaceous 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 the saponins of Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-21B ..., QS-21B-xyl, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS-21B, QS B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum Album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO183 and / or a saponin having a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group, and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group, and / or a saponin having a 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosyl chiral acid. The preferred saponin is 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside, and more preferably the saponin is SO1861 and / or QS-21.

[0093] One embodiment is a first proteinaceous molecule according to the invention, wherein at least one saponin is a bisdesmosidic saponin, has a molecular mass of at least 1,500 daltons, comprises an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, has a first branched carbohydrate side chain at the C-3 position, which first branched carbohydrate side chain optionally contains glucuronic acid, the saponin contains an ester group with a second branched carbohydrate side chain at the C-28 position, which second branched carbohydrate chain preferably contains at least four carbohydrate units, optionally contains at least one acetyl residue, e.g., two acetyl residues, and / or optionally contains a deoxy carbohydrate; and and / or optionally containing quinovose, and / or optionally containing glucose, and / or optionally containing 4-methoxycinnamic acid, and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally containing 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, linked to the carbohydrate via an ester bond, or at least one saponin is selected from the group consisting of QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21A-xyl, QS-21B, QS-21B, QS-21C, QS-21D, QS-21E, QS-21F, QS-21H ... One or more of B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS-18, QS-1861, protonated QS1861 (QS1862), and Quil-A.

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

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

[0096] One embodiment is a first proteinaceous molecule of the invention, wherein the aldehyde functionality at position C-23 of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled via a thio-ether bond to a sulfhydryl group, such as a sulfhydryl group of a cysteine, on the first proteinaceous molecule.

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

[0098] One embodiment is a first proteinaceous molecule of the present invention, wherein the first epitope of the first cell surface molecule to which the first binding site of the first proteinaceous molecule binds is a tumor cell-specific first epitope of a tumor cell-specific receptor, preferably CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, or CD27L receptor. The antigen-specific antigen is selected from the group consisting of CD3, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, and VEGFR2, and more preferably selected from the group consisting of CD71, EGFR, and HER2.

[0099] In one embodiment, the first proteinaceous molecule of the present invention is a first epitope, first tumor cell surface molecule, or first tumor cell-specific receptor that is tumor cell-specific, and is a first epitope, first molecule, or first receptor that is internalized by a tumor cell after binding of the first proteinaceous molecule according to any one of claims 1 to 11 to the first epitope, first molecule, or first receptor, preferably, the first proteinaceous molecule, upon binding to the cell surface molecule, tumor cell surface molecule, or tumor cell-specific receptor comprising the first epitope, is subject to internalization mediated by the tumor cell receptor, e.g., via endocytosis, or internalization mediated by the tumor cell surface molecule, e.g., via endocytosis.

[0100] An embodiment is a first proteinaceous molecule of the invention, wherein the first binding site of the first proteinaceous molecule is selected from the group consisting of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody in IgG form, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD71 monoclonal antibody, The antibody comprises or consists of any one of the D38 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 and / or at least one tumor cell receptor binding domain thereof, preferably at least one tumor cell-specific receptor binding fragment and / or at least one tumor cell-specific receptor binding domain thereof.

[0101] Certain aspects of the present invention relate to a combination of therapeutic agents, the combination of therapeutic agents comprising: (a) a first pharmaceutical composition comprising a first proteinaceous molecule of the present invention and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, wherein the second proteinaceous molecule comprises a second binding site for binding to a second epitope of the second cell surface molecule different from the first cell surface molecule and comprises an effector moiety, wherein the second pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient, and wherein the second epitope is different from the first epitope.

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

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

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

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

[0106] The present inventors have established that the therapeutic window of the second and third proteinaceous molecules in antibody-drug conjugates, e.g., the second or third pharmaceutical compositions of the present invention, respectively, is increased when administered to tumor-bearing mammals (mice) to which the first pharmaceutical composition is also administered. The first proteinaceous protein has at least one glycoside, such as a saponin, attached thereto, preferably covalently, more preferably via a cleavable linker. The saponin increases the therapeutic efficacy of the effector moieties attached to the second and third proteinaceous molecules, presumably by improving endosomal escape of the effector moiety to the cytosol where the effector moiety's activity is desired. In this manner, a therapeutic effect is established in, near, and / or within target cells under the influence of the presence of the first proteinaceous molecule containing the saponin, at a dose already lower than conventional doses of ADC, i.e., the second or third proteinaceous molecule. The target cells may be, for example, diseased cells, such as tumor cells, autoimmune cells, or B cells associated with a B-cell disorder. According to the present invention, the effector moiety is, for example, a toxin as part of an ADC or an oligonucleotide such as a BNA as part of an AOC.

[0107] One embodiment is a therapeutic combination of the invention, wherein the second binding site of the second proteinaceous molecule and / or the first binding site of the third proteinaceous molecule comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin, and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, and / or comprises or consists of at least one ligand for binding to a cell surface molecule such as EGF or a cytokine.

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

[0109] By targeting (two) different cell surface molecules with the first and second proteinaceous molecules, delivery of saponin and effector molecules in and within the cytosol of the very same target cells that expose both different cell surface molecules on the cell surface is improved and more specific compared to exposure of such cells to only the second proteinaceous molecule, such as an ADC or AOC, without the presence of the cell-targeted saponin (first proteinaceous molecule). The diseased cells selected for separate targeting by the binding site of the first proteinaceous molecule and by the binding site of the second proteinaceous molecule (the binding sites are different, the epitopes bound by the first and second proteinaceous molecules are different, and are located on different types and types of cell surface molecules, such as two different receptors) ideally have a high degree of the first epitope and the second epitope on the first cell surface molecule and the second cell surface molecule, respectively (i.e., relatively higher expression of two separate and distinct cell surface molecules on target cells, e.g., tumor cells or autoimmune cells, than on non-target cells, e.g., healthy cells), and / or specifically expose the first and second cell surface molecules when considered in relation to (adjacent) healthy cells of the patient. Preferably, both cell surface molecules targeted by the first and second binding sites are relatively highly and / or specifically expressed in target (disease, tumor) cells compared to healthy cells. In one embodiment, a pharmaceutical combination is provided in which at least one of the first and second binding sites, and therefore at least one of the first and second cell surface molecules, such as the first and second tumor cell receptors, is specifically or relatively highly expressed when compared to the expression of the first and / or second cell surface molecules on the surface of healthy (neighboring) cells. Therefore, the first epitope or the second epitope on the target cell surface molecule, preferably the first and second epitopes, are ideally unique to the target diseased cells and are specifically present and exposed at least on the surface of the target cells. Binding of the first and second proteinaceous molecules to their respective first and second epitopes on the target cells is followed by endocytosis of the complexes of the first proteinaceous molecule with the first target cell surface molecule and the second proteinaceous molecule with the second target cell surface molecule.Because the first and second proteinaceous molecules must enter the same target cell via binding interactions with two different cell surface molecules that are both expressed to a sufficient extent or uniquely on the target cell when compared to healthy cells that should not be targeted, accumulation of therapeutically active amounts of the first and second proteinaceous molecules within the target cell is only possible and will occur if the expression levels of the two distinct target cell surface molecules are both above a certain minimum expression threshold. At the same time, the fact that the effector moiety attached to the second proteinaceous molecule can only exert its intracellular (e.g. cytotoxic or gene silencing) activity in the presence of the first proteinaceous molecule bearing a covalently attached saponin, when both the first and second proteinaceous molecules are able to enter the target cell in sufficient quantities by binding to sufficiently exposed and expressed first and second cell surface molecules, also provides a safeguard against negative and unwanted side effects of the effector moiety on, for example, healthy cells and healthy tissues not meant to be targeted and affected by the effector moiety, when expression of at least one of the first and second cell surface molecules is sufficiently low in healthy cells, preferably when expression of both the first and second target cell surface molecules is sufficiently low in healthy cells. That is, for the first and second cell surface molecules, at least either the first or second cell surface molecule, bound by the first and second binding sites of the first and second proteinaceous molecules, respectively, sufficiently low expression or even absence of the exposed first and second cell surface molecules ideally does not allow entry of both the first and second proteinaceous molecules into (non-target) healthy cells up to amounts that, in concert, result in endosomal escape of the effector moieties under the influence of the saponin bound to the first proteinaceous molecule. Because the ADC or AOC can be used at a lower dose compared to when the first proteinaceous molecule is not added to the treatment regimen, entry of the ADC or AOC into healthy cells to a lower extent already carries a lower risk of causing unwanted side effects when targeting and killing target diseased cells, such as tumor cells and autoimmune cells, is contemplated.

[0110] One embodiment is a therapeutic combination of the invention comprising a first proteinaceous molecule of the invention or a second pharmaceutical composition, wherein said first and second proteinaceous 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, wherein the receptors are different and present on the same tumor cell, the first and second binding sites are different, and the first and second tumor cell-specific epitopes are different.

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

[0112] In one embodiment is a therapeutic combination of the invention comprising a first proteinaceous molecule of the invention or a second pharmaceutical composition, wherein the first receptor and / or 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 alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin, and the like. The antigen is selected from lin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, and preferably selected from CD71, EGFR, and HER2.

[0113] An embodiment is a therapeutic combination of the invention comprising a first proteinaceous molecule of the invention, or a second pharmaceutical composition, which is internalized by the tumor cell after binding of the first and second tumor cell specific receptors to the first proteinaceous molecule of the invention and / or the second proteinaceous molecule of the invention when the therapeutic combination comprises a second pharmaceutical composition, preferably binding of the first proteinaceous molecule and / or the second proteinaceous molecule to the first and second tumor cell specific receptors, respectively, results in tumor cell receptor-mediated internalization of the complex of the first proteinaceous molecule and the first tumor cell specific receptor and of the complex of the second proteinaceous molecule and the second tumor cell specific receptor, e.g. via endocytosis.

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

[0115] Synchronization is the missing link between a successful delivery strategy in mice and its application in humans. Indeed, the inventors have established in a series of in vivo mouse tumor models that separately administering one dose of free saponin and one dose of ADC (a second or third proteinaceous molecule according to the present invention) to mice does not result in any desired antitumor activity, such as delayed tumor growth, tumor regression, diminished, or slower tumor growth, compared to control animals not treated with ADC and free saponin. Free saponin was administered using various routes of administration and at various times (before, during, and after ADC administration) compared to the moment of ADC administration. The ADCs tested in the in vivo tumor models were cetuximab-giantin (with free SO1861) or trastuzumab-saporin (with free SO1861). Varying the dose of free saponin did not result in effective antitumor activity. The referenced ADC was administered at a dose that did not by itself produce any beneficial antitumor effects in tumor-bearing animals. Surprisingly, the inventors have now established that beneficial antitumor activity in various in vitro mammalian cell-based bioassays and / or various in vivo animal tumor models can be achieved by treating animals with a conjugate according to the present invention, i.e., a combination of the first and second or first and third proteinaceous molecules of the present invention, optionally comprising a scaffold according to the present invention. The scaffold is, for example, a trifunctional linker, and has a saponin (e.g., SO1861, QS-21) covalently bound via a cleavable or non-cleavable linkage, and / or an effector moiety (e.g., dianthin, silencing BNA (HSP27)) covalently bound via a non-cleavable or cleavable bond, and / or a covalently bound monoclonal antibody, such as cetuximab, trastuzumab, OKT-9.Alternatively, the scaffold is a dendron, e.g., a dendron to which four moieties, such as four saponin molecules, can be attached, or a dendron for attaching, e.g., two saponins and two effector molecules, the 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 various of these scaffolds according to the invention, which exhibit in vivo and / or in vitro antitumor cell activity, e.g., when considering cytotoxicity exerted by proteinaceous toxins or when considering gene silencing in tumor cells.

[0116] Without wishing to be bound by any theory, in light of the failures observed when treating tumor-bearing animals with ADCs together with free saponins, it is preferable to synchronize the presence of both at least one saponin and an effector moiety, preferably a toxin or oligonucleotide, in compartments or vesicles of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. With ADCs and free saponins, synchronizing the presence of the molecules in late endosomes to achieve a synergistic effect in vivo has not been beneficially achieved according to the inventors' attempts. In one aspect, the present invention preferably solves at least the following problem for the combination of an effector moiety contained in a second proteinaceous molecule and a saponin contained in a first proteinaceous molecule: without wishing to be bound by any theory, for example, only one rational chemical group on the saponin that can be used for (covalent), particularly single and cleavable, retainable coupling is required for endosomal escape activity. Most likely, known limitations are the reason why, although the significant endosomal escape-enhancing effect of saponins, e.g., listed in Table A1 and Scheme I, has been known for more than a decade, saponins have not been used in combination with active pharmaceutical ingredients in clinical studies other than the application of saponins in vaccination regimens involving the use of immune-enhancing adjuvant substances. Providing the first proteinaceous molecule of the present invention with, for example, a covalently conjugated scaffold, at least partially overcomes these difficulties. Surprisingly, saponins previously applied for their immune-enhancing activity in the context of vaccinations involving saponins as adjuvant components are now also suitable for (covalent) coupling to the first proteinaceous molecule of the present invention due to their in vitro and in vivo antitumor activity.

[0117] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effect. Some effectors, such as Pseudomonas exotoxin, are rerouted to other organelles before the "late endosomal stage" and therefore would not normally benefit from coupling to a second proteinaceous molecule according to the present invention. However, such toxins can be adapted for use in the present invention, for example, by deleting the rerouting carried out by the signal peptide. Specifically, toxins that are highly toxic and would require only one molecule to escape the endosome and kill a cell can be modified to be less potent. It is preferred to use toxins that kill cells when at least 2, more preferably at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and most preferably at least 100 toxin molecules escape the endosome. Furthermore, it is preferred that the second proteinaceous molecule of the present invention comprises a covalently conjugated functionalized scaffold, i.e., a scaffold comprising covalently bound effector moiety(s) for targeting the scaffold comprising the bound effector moiety(s) to target cells, such as tumor cells or autoimmune cells. Furthermore, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules compared to cell membrane-permeable toxins.

[0118] The term "ligand" as used herein has its usual meaning, preferably a molecule or structure capable of binding to another molecule or structure on the cell surface of a target cell. The molecule or structure on the cell surface can be endocytosed, preferably absent or less pronounced on off-target cells. Preferably, the molecule or structure on the cell surface is constitutively endocytosed. More preferably, the ligand of the present invention induces endocytosis of the molecule or structure on the cell surface of a target cell after binding to the molecule or structure. This applies, for example, to epidermal growth factor receptor (EGFR), which is present on the surface of various cancer cells. Examples of molecules or structures on the cell surface of a target cell that are constitutively endocytosed include claudin-1 or major histocompatibility complex class II glycoprotein. The ligand can be, for example, an antibody, growth factor, or cytokine. Combining a toxin with a ligand on a carrier molecule is one possibility for creating a targeted toxin. A toxin that is toxic only in target cells because it interferes with processes occurring only in the target cells can also be considered a targeted toxin (it cannot exert its toxic effect in off-target cells, such as apoptin). Preferably, a targeted toxin is a toxin combined with a ligand or, for example, a monoclonal antibody, so that it is active in target cells and not in off-target cells (because it is bound and endocytosed only by target cells). In a functionalized scaffold comprising a ligand and a carrier molecule (i.e., a second or third proteinaceous molecule) comprising an effector moiety, the ligand or monoclonal antibody guides the effector moiety and the scaffold into the target cell. After internalization, at least one glycoside, preferably a saponin contained in the conjugate of the first proteinaceous molecule and the saponin, mediates endosomal escape of the effector moiety. The saponin is typically a saponin listed in Table A1 and Scheme I, preferably the saponin is SO1861 and / or QS-21 and / or SA1641 and / or GE1741.

[0119] Preferably, an effector moiety attached to a second or third proteinaceous molecule, the effect of which is enhanced by a saponin attached to a first proteinaceous molecule, is detached from the second or third proteinaceous molecule, e.g. an antibody, when it is endocytosed. This can be achieved, for example, by a cleavable bond that breaks under acidic, reductive, enzymatic or light-induced conditions.

[0120] An embodiment is a therapeutic combination of the invention comprising a first proteinaceous molecule of the invention and / or 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, preferably cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, IgG type O comprising or consisting of any one of the KT-9 anti-CD71 monoclonal antibody, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, and an 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 proteinaceous molecule is different from the second binding site of the second proteinaceous molecule.

[0121] An embodiment is a therapeutic combination comprising a third pharmaceutical composition of the invention, or a first pharmaceutical composition according to the invention when comprised in a therapeutic combination comprising a third pharmaceutical composition, wherein the first proteinaceous molecule and the first binding site of the third proteinaceous molecule comprise a monoclonal antibody, or at least one cell surface molecule binding domain and / or fragment thereof, preferably selected from the group consisting of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, and the like. the first proteinaceous molecule is a first binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a first binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a second binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a second binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a third binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a second binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a third binding site of the first proteinaceous molecule, or alternatively, the third proteinaceous molecule is a

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

[0123] The inventors have established that such immunoglobulins, domains thereof, ligands, etc. are particularly suitable for application as first binding sites of a first proteinaceous molecule comprising a first binding site (and the same binding site of a third proteinaceous molecule). Similarly, the inventors have established that such immunoglobulins, domains thereof, ligands, etc. are particularly suitable for application as second binding sites of a second proteinaceous molecule comprising a second binding site. For example, antibodies and antibody binding domains are suitable for targeting epitopes on exposed surfaces of selected cell surface molecules, resulting in targeting of the first and third (and separately, second) proteinaceous molecules to cells expressing the cell surface molecules targeted by the first and third proteinaceous molecules, and / or cells expressing the second cell surface molecule targeted by the second proteinaceous molecule. These cells also express and have the first and third cell surface molecules (which are the same cell surface molecule) on their cell surfaces. Similarly, a ligand such as EGF that targets EGFR on a target cell is suitable for use as a binding site on a first and third proteinaceous molecule or a second binding site on a second proteinaceous molecule, provided that the second binding site is different from both the first and third binding sites, and the first and third binding sites are the same. Binding sites for first and third epitopes or second epitopes that are specific for binding of the first and third proteinaceous molecules to a first cell surface molecule and / or for binding of the second proteinaceous molecule to a second cell surface molecule are preferred. The first and second cell surface molecules are exposed on the very same target cell. For example, binding sites based on antibodies or domains or binding fragments thereof provide such desired specificity for selected first, second, and third epitopes on selected first or second cell surface molecules of selected cells for targeting, such as diseased cells, tumor cells, autoimmune cells, etc. Thus, first, second and third binding sites based on antibodies or binding molecules (fragments, domains) are preferred for the first, second and third proteinaceous molecules.

[0124] Targeting the same cell surface molecule by the first and third proteinaceous molecules improves and makes the delivery of the saponin and effector moiety in and within the cytosol of the very same target cells more specific. The diseased cells selected for targeting by the binding sites of the first and third proteinaceous molecules ideally have a higher degree and / or specificity of the cell surface molecule than the patient's (adjacent) healthy cells are considered. Therefore, the epitope on the target cell surface molecule is ideally unique to the target diseased cells and is at least specifically present and exposed on the surface of the target cells. Binding of the first and third proteinaceous molecules is followed by endocytosis of the complex between the first proteinaceous molecule and the target cell surface molecule and the third proteinaceous molecule and the target cell surface molecule. Because the first and third proteinaceous molecules must enter the same target cell by binding interaction with the very same cell surface molecule, accumulation of therapeutically active amounts of the first and third proteinaceous molecules within the target cell is only possible and will occur if the expression level of the target cell surface molecule is above a certain minimum expression threshold. At the same time, the fact that the effector moiety attached to the third proteinaceous molecule can only exert its intracellular (e.g., cytotoxic or gene silencing) activity in the presence of the first proteinaceous molecule bearing a covalently attached saponin, when both the first and third proteinaceous molecules are able to enter the target cell in sufficient quantities by binding to a sufficiently exposed and expressed cell surface molecule, also provides a safeguard against negative and unwanted side effects of the effector moiety on, for example, healthy cells and tissues not meant to be targeted and affected by the effector moiety, when expression of the target cell surface molecule is sufficiently low in healthy cells. That is, low expression of the cell surface molecules bound by the binding sites of the first and third proteinaceous molecules does not allow the entry of both the first and third proteinaceous molecules to amounts that would cooperatively result in endosomal escape of the effector moieties under the influence of the saponin attached to the first proteinaceous molecule.Since the ADC or AOC can be used at a lower dose compared to when the first proteinaceous molecule is not added to the treatment regimen, the entry of the ADC or AOC in healthy cells to a lower extent already carries a lower risk of causing unwanted side effects when targeting and killing target diseased cells, such as tumor cells and autoimmune cells, is considered.

[0125] In this specification and claims (throughout the application) when first and third epitopes, first and third binding sites, first and third cell surface molecules are considered, the terms "first" and "third" have the same meaning, i.e., for the first and third proteinaceous molecules, the target epitope is the same, the binding site is the same, and the target cell surface molecule, e.g., tumor cell (specific) receptor, is the same.

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

[0127] In one embodiment, the therapeutic combination comprises the second or third pharmaceutical composition of the present invention, wherein the effector moiety comprised in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of one or more of an oligonucleotide, a nucleic acid, or a xenonucleic acid, preferably a vector, a gene, a transgene that induces cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, or a minicircle. The nucleic acid may be selected from any one or more of DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), plasmid, glycol nucleic acid (GNA), and threose nucleic acid (TNA), or derivatives thereof, more preferably BNA, for example, BNA for silencing HSP27 protein expression.

[0128] In one embodiment is a therapeutic combination comprising the second or third pharmaceutical composition of the invention, wherein the effector moiety comprised in the second proteinaceous molecule and / or in the third proteinaceous molecule comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of peptides, proteins, enzymes such as urease and Cre recombinase, ribosome-inactivating proteins, proteinaceous toxins, more preferably proteinaceous toxins selected from Table A5, and / or viral toxins such as apoptin, bacterial toxins such as Shiga toxin, Shiga-like toxins, Pseudomonas Aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins such as alphasarcin; plant toxins including ribosome-inactivating proteins and the A chains of type 2 ribosome-inactivating proteins, such as giantin, e.g., giantin-30 or giantin-32, saporin, e.g., saporin-S3 or saporin-S6, bouganin or a deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or animal or human toxins such as frog RNase, or granzyme B or angiogenin from humans, or any fragment or derivative thereof; preferably, the protein toxin is giantin and / or saporin.

[0129] In one embodiment, the therapeutic combination comprises the second or third pharmaceutical composition of the present invention, wherein the effector molecule comprised in the second proteinaceous molecule and / or the third proteinaceous molecule comprises or consists of at least one payload, preferably any one or more of a 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, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, and / or a derivative thereof. The compound is selected from one or more of CC-1065 analogs, duocarmycins, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepines, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracyclines, camptothecin analogs, SN-38, DX-8951f, exatecan mesylate, a truncated form of Pseudomonas Aeruginosa exotoxin (PE38), duocarmycin derivatives, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine.

[0130] A drug 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 or autoimmune patient. Benefits include diagnosis, prognosis, treatment, cure, and / or prevention of a disease and / or condition. A drug substance may also lead to unwanted and harmful side effects. In this case, the pros and cons must be weighed to determine whether the drug substance is suitable in a particular case. If the effect of the drug substance in a cell is overwhelmingly beneficial to the whole organism, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the whole organism, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, target and off-target cells depend on the purpose and are defined by the user.

[0131] A polypeptide effector moiety can be, for example, a polypeptide that restores a lost function, such as an enzyme replacement, a gene regulatory function, or a toxin.

[0132] One embodiment is a first proteinaceous molecule of the invention, wherein the first proteinaceous molecule comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1-100 saponins, or any number in between, e.g., 7, 9, 12 saponins, covalently linked directly to an amino acid residue of the first proteinaceous molecule, preferably to a cysteine ​​and / or to a lysine, and / or via at least one linker and / or are covalently attached via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1 to 8 such backbones or 2 to 4 such backbones, at least one backbone optionally based on a dendron, and 1 to 32 saponins, e.g., 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins in between, e.g., 7, 9, 12 saponins, are covalently attached to the at least one backbone.

[0133] Table A1 and Scheme I and the embodiments above summarize a series of saponins that have been identified for their endosomal escape-enhancing activity when contacted in free form with mammalian cells, particularly human tumor cells, together with a second molecule (e.g., an effector moiety or molecule, e.g., a toxin, an oligonucleotide). Indeed, in cell-based bioassays using human tumor cells, it has been established for the saponins tabulated in Table A1 and those in Scheme I and various embodiments of the invention described herein that, under the influence of these saponins when conjugated to a first proteinaceous molecule, a second molecule (effector moiety), such as a nucleic acid and / or a toxin, e.g., a protein toxin (e.g., one or more of the protein toxins listed in Table A5), conjugated to a second or third proteinaceous molecule, is delivered to the cytosol with increased efficiency and / or effectiveness, presumably by intracellular release from (late) endosomes and lysosomes. That is, endosomal and / or lysosomal escape of such second molecules (effector moieties attached to the second or third proteinaceous molecules of the invention), e.g. nucleic acids and / or toxins, is less efficient in the absence of saponin.

[0134] Surprisingly, the inventors now demonstrate that water-soluble saponin fractions from Quillaja saponaria, including QS-21 and its family members QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18, and Quil-A, also exhibit the ability to enhance the in vitro biological effect of, for example, a nucleic acid conjugated to a monoclonal antibody or a protein toxin conjugated to a monoclonal antibody (examples of second and / or third proteinaceous molecules of the invention comprising a payload such as a covalently attached oligonucleotide or (protein) toxin). In this case, tumor cells of a mammalian species (human) are administered a monoclonal antibody (first proteinaceous molecule of the invention) in the form of a covalent conjugate comprising a second and / or third proteinaceous molecule (the second and / or third proteinaceous molecules mentioned above) comprising an effector moiety, together with at least one glycoside contained in the first proteinaceous molecule as a covalent conjugate, such as QS-21 and its family member saponin (e.g., the water-soluble fraction of Quillaja saponaria) encapsulated by such QS-21 preparations. The effector molecule and glycoside, such as the saponin fraction of Quillaja saponaria, QS-21, SO1861, SA1641, GE1741, are for example covalently bound to the proteinaceous molecule either directly or via a linker, or via a polymeric or oligomeric structure, either directly or via at least one linker. Without wishing to be bound by any theory, the observed stimulation or enhancement of, for example, antisense BNA-mediated reduction of tumor cell HSP27 expression (HSP27 gene silencing) in the presence of saponin from Quillaja saponaria in vitro may (also) be related to activation of inflammasomes in tumor cells by saponin, leading to, for example, tumor cell pyroptosis.The present inventors have established that second and third proteinaceous molecules, for example conjugated to antisense BNAs or dianthin or saporin, when contacted with cells in a bio-based cell assay in the presence of a first proteinaceous molecule of the invention targeted to the same (tumor) cells as the cell surface molecule targeted by the second and / or third proteinaceous molecules and comprising saponin, exert any or improved anti-tumor cell activity in vitro, whereas in the absence of the first proteinaceous molecule, and therefore in the absence of saponin, no such activity against tumor cells is observed.

[0135] QS-21 and water-soluble saponin fractions containing QS-21 from Quillaja saponaria have also been known for a long time and have been actively applied, for example, due to their immune-enhancing ability as adjuvants for subunit vaccines. For example, QS-21 has been applied in two phase III clinical trials in human patients. They were vaccinated with a subunit vaccine mixed with an adjuvant containing QS-21 (Glaxo-Smith-Kline, MAGRIT trial, DERMA study). The subunit was the MAGE-A3 protein, which is specifically expressed and presented by tumor cells. QS-21-enhanced antitumor vaccination aimed to extend disease-free survival in cancer patients (melanoma; non-small cell lung cancer). Additionally, QS-21 has been tested as an adjuvant in clinical trials for the development of anti-cancer vaccine treatments, vaccines for HIV-1 infection, vaccines for hepatitis B, and anti-malaria vaccines using QS-21 with Glaxo-Smith-Kline's adjuvants AS01 and AS02. Previous studies have demonstrated that under the influence of QS-21 saponin containing adjuvants, immune responses are elicited against MAGE-A3 peptides displayed on the surface of cancer cells (AS15; GSK). Surprisingly, the inventors found that the saponin fraction of Quillaja saponaria, and therefore likely QS-21 (as part of the water-soluble saponin fraction of Quillaja saponaria), enhances the anti-tumor cell activity of a payload, such as a protein toxin (giantin), conjugated to a second proteinaceous molecule (e.g., the ligand EGF).

[0136] The present inventors have demonstrated that a tumor cell-targeting monoclonal antibody provided with a covalently coupled antisense BNA, e.g., BNA(HSP27), and contacted with tumor cells together with a first proteinaceous 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 compared to an AOC (third proteinaceous molecule) without the presence of the first proteinaceous molecule having a coupled saponin. Both the BNA and saponin are coupled to the respective antibodies (e.g., cetuximab) of the first and third proteinaceous molecules via cleavable bonds. Thus, coadministration of an ADC or antibody-oligonucleotide conjugate (AOC), e.g., an antibody-BNA conjugate, with a first proteinaceous molecule having a saponin confers anti-tumor cell activity to the ADC or AOC not seen with the same dose of ADC alone or AOC alone. Remarkably, when administered separately to tumor-bearing mice in separate groups, AOC (second or third proteinaceous molecule) and a monoclonal antibody (first proteinaceous molecule) having a covalently coupled saponin increase HSP27 expression in tumor cells compared to a control group (administered vehicle only). Only co-administration of an AOC (second or third proteinaceous molecule) comprising an effector moiety of the present invention and a first proteinaceous molecule having a covalently coupled saponin results in reduced HSP27 expression when compared to the control.The antisense BNA (HSP27) was a BNA with the oligonucleotide sequence 5'-GGCacagccagtgGCG-3' according to Zhang et al. (2011) (Y Zhang, Z Qu, S Kim, V Shi, B Liao, P Kraft, R Bandaru, Y Wu, LM Greenberger, and ID Horak, "Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection," Gene Therapy (2011) 18, 326-333). Of note, to the best of our knowledge, BNAs have been designed for application as free nucleic acids. Here, we demonstrate for the first time 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 providing BNA-based AOCs opens new avenues for administering targeted BNAs to human (cancer) patients in need thereof.

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

[0138] One embodiment is a first proteinaceous molecule of the invention, comprising a saponin comprising one or some or all of the indicated structural features of a saponin of Structure A of Scheme I (a saponin of Structure A is said to have an "ideal" structure when endosomal escape-enhancing activity for an effector moiety is present in the endosomes of a cell contacted with the first proteinaceous molecule), and / or a saponin selected from any one or more of the additional saponins of Scheme I:

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] [ka]

[0143] [ka]

[0144] According to the present invention, a glycoside such as a saponin according to the present invention attached to a first proteinaceous molecule of the present invention, which has an "ideal" structure for the purpose of enhancing endosomal escape of an effector molecule attached to a second or third proteinaceous molecule of the present invention, is a bisdesmosidic saponin according to Structure A of Scheme I, which has a molecular mass of at least 1,500 daltons, comprises an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, has a first branched carbohydrate side chain at the C-3 position, which first branched carbohydrate side chain optionally contains glucuronic acid, and the saponin contains an ester group with a second branched carbohydrate side chain at the C-28 position, which second branched carbohydrate chain preferably Preferably, it comprises at least four carbohydrate units, optionally containing at least one acetyl residue, for example two acetyl residues, and / or optionally containing a deoxy carbohydrate, and / or optionally containing quinovose, and / or optionally containing glucose, and / or optionally containing 4-methoxycinnamic acid, and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally containing 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, linked to the carbohydrate via an ester bond.

[0145] SO1861 differs from the "ideal structure" represented by Structure A in Scheme I only by having only one acetyl residue on the quinovose and by having an additional xylose. The "ideal structure" of a saponin for enhancing endosomal escape of an effector molecule or effector moiety is preferably a saponin having Structure A in Scheme I, and saponins that exhibit endosomal escape-enhancing activity have one or more structural features represented by Structure A in Scheme I. Without wishing to be bound by any theory, the inventors believe that Structure A in Scheme I represents an "ideal saponin" (and not a minimum requirement saponin) for endosomal escape-enhancing activity. This means that not all of the structures (chemical groups) can or must be present on every saponin that has at least sufficient endosomal escape-enhancing activity to promote accumulation of the effector moiety in the cytosol. This also means that some saponins may have other structural elements, such as acyl chains, and / or that for still other saponins that exhibit endosomal escape-enhancing activity, the sugars may differ from those represented by Scheme I. For example, when considering the idealized structure A in Scheme I, QS-21 saponin and some of the saponins in the water-soluble fraction of Quillaja saponaria (Quillaja saponin; Quil-A) differ in carbohydrate modifications at C-28, such as the presence of an acyl chain in QS-21. Saponins in the water-soluble fraction of Quillaja saponaria, such as QS-7 and QS1862, are similar to the idealized structure A and similar to QS1861.

[0146] One embodiment is a first proteinaceous molecule of the invention, wherein at least one linker is a non-cleavable linker or a cleavable linker, wherein the cleavable linker undergoes cleavage when bound to saponin, for example under acidic, reductive, enzymatic or photoinduced conditions, preferably the cleavable linker comprises a hydrazone or hydrazide bond that undergoes cleavage under acidic conditions when bound to saponin, and / or comprises a bond that is susceptible to proteolysis, for example proteolysis by cathepsin B, and / or is a bond that is susceptible to cleavage under reductive conditions, for example a disulfide bond.

[0147] One embodiment is a first proteinaceous molecule of the invention, wherein when the cleavable linker is attached to a saponin, the cleavable linker undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦5.5.

[0148] An embodiment is a first proteinaceous molecule of the invention, wherein the oligomeric or polymeric backbone comprises a polymeric or oligomeric structure and comprises chemical groups, chemical groups for covalent coupling of the backbone to amino acid residues of said first proteinaceous molecule.

[0149] According to the present invention, the saponin is typically a saponin listed in Table A1, Scheme I. When considering intracellular entry and cytosolic accumulation of an effector moiety covalently coupled to a second or third proteinaceous molecule, the activity of the saponin, e.g., intracellular endosomal escape-enhancing activity, has proven beneficial when the saponin is covalently coupled to a first proteinaceous molecule and involves a hydrazone bond and / or hydrazide bond and / or disulfide bond. Such bond types are readily cleaved under acidic and / or reductive conditions within the (late) endosomes and lysosomes of mammalian cells, e.g., human cells. Alternatively, the inventors have also demonstrated that covalent coupling of a saponin to a first proteinaceous molecule via a bond that is not readily cleavable under physiological conditions within a cell, e.g., in (late) endosomes, lysosomes, or the cytosol, is also beneficial for enhancing the activity of the saponin on the biological effects of effector moieties, such as nucleic acids (e.g., HSP27-silencing BNAs) and proteinaceous toxins such as saporin. In the present application, including the claims, when referring to the conjugates of the present invention, e.g., a first proteinaceous molecule optionally comprising a backbone having a saponin coupled to the first proteinaceous molecule via a linker and / or backbone via a hydrazone bond or disulfide bond, the terms "cleavable linker," "cleavable bond," and the like, are also referred to as "labile linker" ("L") and "labile bond," e.g., in the context of cleavage of such bond or linker in (late) endosomes and / or lysosomes. For example, Figures 6 and 7 show in vivo HSP27 gene silencing in human tumors in mice. Tumor-bearing mice were treated with a first proteinaceous molecule consisting of a monoclonal antibody having a saponin attached to it via a labile linker (hydrazone bond) according to the present invention, while a third proteinaceous molecule contained a conjugated antisense BNA for silencing the HSP27 gene of tumor cells, covalently coupled to a monoclonal antibody (of the same type as the first monoclonal antibody) via a disulfide bond.That is, without wishing to be bound by any theory, once the therapeutic combination of the present invention is internalized, for example by endocytosis, the hydrazone bond and disulfide bond are cleaved in (late) endosomes and / or lysosomes of target tumor cells that express epitopes on the target cell surface molecule, here EGFR, on their cell surface. While bond cleavage likely contributes to the endosomal escape-enhancing activity of saponins when BNAs enter the cytosol from endosomes and / or lysosomes, such cleavage is not essential to observing the gene silencing effects of the cetuximab-SO1861 conjugate and cetuximab-BNA conjugate combinations of the present invention.

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

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

[0152] [ka]

[0153] wherein the saponin is covalently attached to the trifunctional linker backbone via a labile cleavable hydrazone linker (acid sensitive) and / or via a maleimide-containing bond, while attachment of the backbone to a binding site, such as an antibody, is established via a maleimide-containing bond to cysteines of the binding site, such as one, two, three, or four cysteines, and / or via a labile cleavable hydrazone linker (acid sensitive), thereby forming structure B:

[0154] [ka]

[0155] As a result, one to four scaffolds are covalently attached to a single, eg, antibody, eg, a monoclonal antibody.

[0156] One embodiment is a first proteinaceous molecule of the present invention, wherein the glycoside molecule is a saponin, and the linkage between the saponin and the first proteinaceous molecule is preferably an acid-labile bond that is stable at pH 7.4 and preferably releases the saponin below pH 6.5, more preferably between pH 6.5 and 5.0. This is achieved, for example, by an imine formed by an aldehyde group of the saponin and an amino group of the linker connecting the saponin and the first proteinaceous molecule. Other chemical bonds that satisfy the pH conditions can also be used for aldehyde coupling, such as specific hydrazones or acetals, which require hydrazide and hydroxyl groups as functional groups on the linker, respectively. When the bond is a cleavable bond, the saponin is preferably attached to the backbone polymer or oligomeric structure via one of the carboxyl groups or an aldehyde functional group in the saponin, more preferably via an aldehyde functional group, preferably an aldehyde functional group at position 23. Alternatively, the saponin is preferably attached to the first proteinaceous molecule via the backbone polymeric or oligomeric structure, via a linker connecting the backbone polymeric or oligomeric structure, either via an aldehyde functional group or via a carboxylic acid functional group of the glycoside molecule, i.e., the saponin.

[0157] In one embodiment, the first proteinaceous molecule of the present invention comprises at least one saponin attached to the first proteinaceous molecule via a stable bond. In a more preferred embodiment, the stable bond between the saponin and the first proteinaceous molecule preferably occurs via amide coupling or amine formation. This is achieved, for example, by carbodiimide-mediated amide bond formation between an activated glucuronic acid group on the saponin and an amino group on the polymeric or oligomeric backbone structure linking the saponin and the first proteinaceous molecule together. Chemical bonds meeting the definition of a stable bond can also be used for aldehyde coupling. For example, specific amines are derivatized after reductive amination, requiring a primary amino group as a functional group on the polymeric or oligomeric structure of the linker or backbone. When the bond is a stable bond, the saponin is preferably attached to the linker or backbone via one of the carboxyl groups of the saponin, and the linker or backbone is further linked to the first proteinaceous molecule.

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

[0159] One embodiment is a first proteinaceous molecule of the present invention, in which a saponin is coupled to a binding site via a scaffold according to the present invention, and the click chemistry group is a tetrazine, azide, alkene, or alkyne, or a cyclic derivative of any of these groups, preferably azide. The click chemistry group is a chemical functional group suitable for click chemistry, which is defined as a reaction that is modular, broad in scope, gives very high yields, produces only benign by-products, offers high selectivity and high tolerance for different functional groups, and is stereospecific. Required process features include simple reaction conditions, readily available starting materials and reagents, no solvent or the use of a mild (e.g., water) or easily removed solvent, and simple product isolation. The click chemistry group for coupling the saponin, optionally via a scaffold or linker, to a binding site on the first proteinaceous molecule is preferably a tetrazine, azide, alkene, or alkyne, or a reactive derivative thereof, such as a methyl-tetrazine or a maleimide (alkene), more preferably an alkyne, or a cyclic derivative of these groups, such as a cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]non-4-yne, dibenzocyclooctyne).

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

[0161] The scaffold is essentially independent of the type of saponin covalently attached to it, and the scaffold is subsequently (sequentially) covalently coupled to a first proteinaceous molecule. Therefore, the first proteinaceous molecule comprising the scaffold is the foundational product of a new platform technology. Because at least one covalently attached saponin mediates the intracellular delivery of an effector moiety attached to a second proteinaceous molecule, the scaffold technology according to the present invention is the first known system for mediating controlled intracellular effector moiety delivery by saponins. The scaffold provides an optimized and functionally active unit that can be linked to a saponin(s) and to a binding site contained in a first proteinaceous molecule, such as a ligand, antibody, etc., at a single, defined position.

[0162] One embodiment is a first proteinaceous molecule comprising a scaffold according to the present 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 structures such as poly(amines), e.g., polyethyleneimines and poly(amidoamines), or polyethylene glycols; structures such as poly(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins); or peptides or proteins; or structures such as natural and / or artificial polyamino acids, e.g., polylysine, DNA polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. They may appear as either pure or mixed linear, branched, or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates of these structures. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not exhibit substantial acute or chronic toxicity in living organisms and can either be excreted intact or completely degraded by the body's metabolism into excretable and / or physiological compounds. The aggregates can be assembled by covalent crosslinking or non-covalent bonds and / or attractive forces. Thus, they can also form nanogels, microgels, or hydrogels. Alternatively, they can be attached to carriers, such as inorganic nanoparticles, colloids, liposomes, micelles, or particle-like structures containing cholesterol and / or phospholipids. The polymeric or oligomeric structures preferably have a precisely defined number or range of coupling moieties for 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 a precisely defined number or range of coupling moieties on a polymeric or oligomeric structure are occupied by glycoside molecules in the backbone according to the present invention.

[0163] Preferably, a dendron is a branched, well-defined dendritic polymer with a single chemically addressable group at the origin of the tree, called the focal point. A dendrimer is the connection of two or more dendrons at their focal points. A dendronized polymer is the connection of the focal points of one or more dendrons to a polymer. In 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, dendronized polymer, dendronized oligomer, or aggregates of these structures, either pure or mixed. The aggregates can be assembled by covalent crosslinking or non-covalent attractive forces to form nanogels, microgels, or hydrogels. Preferably, the polymer is a derivative of poly(amine), such as polyethyleneimine and poly(amidoamine), and polyethylene glycol, a poly(ester) structure, such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), and a structure such as natural and / or artificial polyamino acids, such as polylysine, or a peptide or protein or DNA polymer, stabilized RNA polymer, or PNA (peptide nucleic acid) polymer. Preferably, the polymer or oligomeric structure is biocompatible.

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

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

[0166] An embodiment is a first proteinaceous molecule of the invention, wherein the chemical group of the oligomeric or polymeric backbone for covalent coupling of the oligomeric or polymeric backbone to an amino acid residue of said first proteinaceous molecule is a click chemistry group, preferably selected from tetrazine, azide, alkene, or alkyne, or cyclic derivatives of these groups, more preferably said chemical group is azide.

[0167] One embodiment is a first proteinaceous molecule of the invention, wherein the polymer or oligomeric structure of the oligomeric or polymeric backbone comprises a linear, branched, and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures, which assembly is preferably assembled by covalent crosslinking.

[0168] The inventors have now established that covalent coupling of a saponin, preferably via a cleavable bond or linker, to a first proteinaceous molecule according to any of the above embodiments provides efficient and cell-targeted enhancement of the activity of effector moieties attached to second and third proteinaceous molecules, wherein the first and third proteinaceous molecules comprise the same first binding site, and the first and second proteinaceous molecules comprise different first and second binding sites. When effector moieties are delivered to the same target cell by using second and / or third proteinaceous molecules that comprise the same first binding site as the first proteinaceous molecule when a third proteinaceous molecule is considered, or different first and second binding sites when the first and second proteinaceous molecules are considered, coupling a saponin directly or via a linker to a cysteine ​​or lysine side chain of a first proteinaceous molecule, such as a monoclonal antibody, has proven to be a valuable way of specific and efficient delivery of effector moiety-enhancing activity in target cells.

[0169] To explain the present invention in more detail, the process of cellular uptake of substances (although the inventors do not wish to be bound by any theory) and the terminology used herein will be described. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. Such vesicle budding can be 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) nonspecific fluid uptake (pinocytosis), or (4) nonspecific particle uptake (phagocytosis). All types of endocytosis enter into subsequent cellular processes of vesicle trafficking and material sorting, referred to as endocytic pathways. Endocytic pathways are complex and incompletely understood. Without wishing to be bound by any theory, organelles can form de novo and mature into subsequent organelles along the endocytic pathway. However, it is hypothesized here that endocytic pathways involve stable compartments connected by vesicle trafficking. Compartments are complex, multifunctional membrane organelles specialized for specific sets of essential cellular functions. Vesicles are considered transient organelles, are simpler in composition, and are defined as membrane-enclosed containers formed de novo by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early and late endosomes represent stable compartments in the endocytic pathway, while primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and even lysosomes represent vesicles. Endocytic vesicles, most notably those originating from clathrin-coated pits at the plasma membrane, first fuse with early endosomes, the major sorting compartment at approximately pH 6.5. The majority of internalized cargo and membranes are recycled to the plasma membrane via recycling vesicles (the recycling pathway). Components to be degraded are transported via multivesicular bodies to acidic late endosomes (pH below 6). Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to late endosomal compartments when needed.The resulting organelles are called hybrid organelles or endolysosomes. Lysosomes bud from hybrid organelles through a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are extremely dynamic organelles, and it is often difficult to distinguish between them. Degradation of endocytosed molecules occurs within endolysosomes or lysosomes. Endosomal escape is the active or passive release of materials from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably from clathrin-mediated endocytosis or recycling pathways to the cytosol. Therefore, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediates and hybrid organelles.

[0170] Unless specifically indicated otherwise, and particularly when relating to the endosomal escape mechanisms of glycoside molecules such as the saponins of the present invention, whenever the words "endosome" or "endosomal escape" are used herein, it also encompasses escape from endolysosomes and lysosomes and from endolysosomes and lysosomes, respectively. After entering the cytosol, the material may translocate to other cellular units, such as the nucleus.

[0171] In formal terms, a glycoside is any molecule in which a sugar group is linked to another group via its anomeric carbon by a glycosidic bond. Without wishing to be bound by any theory, glycoside molecules such as saponins in the context of the present invention are such molecules that can further improve the efficacy of an effector moiety, in particular by facilitating the 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 membranes of compartments and vesicles of the endocytic and recycling pathways, making them leaky for the effector molecule, resulting in increased endosomal escape. By the term "scaffold capable of increasing endosomal escape of the effector moiety," it is meant that at least one saponin (glycoside molecule) coupled to the polymeric or oligomeric structure of the scaffold can improve endosomal escape of the effector moiety. At this time, both molecules are in an endosome, e.g., a late endosome, and optionally and preferably after at least one glycoside, such as a saponin, has been released from the first proteinaceous molecule, e.g., from a linker or polymeric or oligomeric structure comprised in said first proteinaceous molecule, e.g., by cleavage of a cleavable bond between the at least one glycoside (saponin) and the first proteinaceous molecule (e.g., via the backbone polymeric or oligomeric structure and / or via the linker). The bond between the at least one glycoside, such as a saponin according to the present invention, and the first proteinaceous molecule, optionally via the linker or backbone, may be a "stable bond," but this does not mean that such a bond cannot be cleaved, e.g., by an enzyme, in the endosome. For example, the glycoside or saponin, optionally together with a linker or a portion of the backbone oligomeric or polymeric structure, may be cleaved from the remaining linker fragment or oligomeric or polymeric structure. For example, it is possible that a protease cleaves a (proteinaceous) linker or proteinaceous polymer structure, such as albumin, thereby releasing at least one glycoside, saponin.However, it is preferred that the glycoside molecule (preferably saponin) is released in an active form, preferably in the original form it had before it was (prepared to be) coupled to the first proteinaceous molecule, optionally via a linker and / or oligomeric or polymeric backbone; therefore, the glycoside (saponin) will have its native structure after such cleavage, or the glycoside (saponin) will have (part of) a chemical group or linker attached to it after such cleavage. On the other hand, the glycoside biological activity (saponin biological activity), e.g., endosomal / lysosomal escape-enhancing activity towards an effector moiety present in the same endosome or lysosome, is maintained or restored by said cleavage of the bond between the carrier molecule, i.e., the first proteinaceous molecule, and the glycoside (saponin), optionally comprising a linker and / or backbone of the invention. In the present invention, the term "stable", for example, between a saponin and an amino acid residue of a first proteinaceous molecule, a linker, a polymeric or oligomeric structure (backbone), a ligand, a (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or an effector (effector moiety, effector molecule), means that the bond is not easily broken, or at least is not designed to be easily broken, for example, by pH differences, salt concentrations, or UV light, or reducing conditions. In the present invention, the term "cleavable", for example, between a saponin and a first proteinaceous molecule, a linker, an amino acid residue, a backbone polymeric or oligomeric structure, a ligand, an antibody, and / or an effector, means that the bond is designed to be easily cleaved, for example, by pH differences, salt concentrations, under reducing conditions, etc. Those skilled in the art are familiar with such cleavable bonds and how to prepare them.

[0172] Prior to the present invention, one of the major hurdles to bringing ADCs and AOCs to market was their small therapeutic window: therapeutically effective doses of ADCs or AOCs were accompanied by (unacceptable) side effects, hindering their development and value in treating patients with ADCs. The application of the first proteinaceous molecule of the present invention now makes it possible to guide one or more glycoside molecules (saponins) together with a payload-bearing 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 proteinaceous molecule of the present invention) to a (target) cell. In particular, it was not previously possible to simultaneously and specifically guide the effector moiety of a second or third proteinaceous molecule and a (predetermined and controllable) specific number or range of glycoside molecules (saponins) per effector moiety to the cytosol of a cell, for example, via the endocytic pathway of the cell.

[0173] The solution provided by the present invention involves the covalent attachment of at least one saponin to a first proteinaceous molecule. A further solution provided by the present invention involves (first) polymerizing a glycoside molecule (saponin) with an oligomeric or polymeric backbone and providing a cluster of covalently attached saponins to the first proteinaceous molecule, allowing for re-monomerization of one or more saponins at the intracellular site where the saponin's mechanism of action is desired, for example after endocytosis. In this context, "polymerizing" refers to the reversible and / or irreversible multiple conjugation of saponin molecules to the first proteinaceous molecule, either via a linker, directly, or via a polymeric or oligomeric structure to form the backbone, or the reversible and / or irreversible multiple conjugation of a (modified) saponin thereby forming a polymeric or oligomeric structure to form the backbone. In this context, "remonomerization" refers to the cleavage of a saponin from the first proteinaceous molecule, from the linker linking the saponin(s) to the first proteinaceous molecule, or from the backbone, e.g., after endocytosis, and regaining the (native) chemical state of the unbound saponin. These unbound saponins may or may not contain additional chemical groups, e.g., chemical groups for linking the saponin to the linker, amino acid residue, or backbone of the first proteinaceous molecule, and / or (chemical) linkers attached to chemical groups of the saponin, such as aldehyde or carboxylic acid groups. Due to the complex chemistry of saponins, for example, "polymerization" of saponins on the backbone or other linking linker and their "remonomerization" at a desired location, e.g., within a cell, e.g., after endocytosis, has been a difficult task. In particular, the chemical reaction (glycoside polymerization) used to provide a linker and scaffold comprising a covalently linked glycoside, e.g., a triterpenoid saponin, for covalent attachment to a first proteinaceous molecule typically occurs in an organic solvent that is free of water, while the saponin and, e.g., the biocompatible polymer applied as a scaffold to carry the attached saponin, are water-soluble molecules.The chemical properties of unmodified saponin further prevented its polymerization by itself. Another solution for (directly) conjugating multiple saponins to effector molecules was viewed as less promising because the effector molecules (drugs, toxins, polypeptides, or polynucleotides) typically did not provide sufficient conjugation sites, and because the coupling products were highly heterogeneous, and / or coupling a biologically active molecule such as a saponin to, for example, a peptide, toxin, or nucleic acid carried the risk of affecting and interfering with the activity of one or even both molecules conjugated together in a conjugate comprising such a saponin. Furthermore, there was a significant risk that the effector moiety contained in a second or third proteinaceous molecule would lose its function after coupling of the saponin to, for example, an ADC or antibody-oligonucleotide conjugate (AOC). Embodiments of the present invention address at least one of these drawbacks.

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

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

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

[0177] One aspect of the present invention relates to a composition, comprising a first proteinaceous molecule of the present invention and, preferably, a vector, a gene, a transgene inducing cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2' and one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid selected from at least one of deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acid, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA), and threose nucleic acid (TNA), or a derivative thereof, more preferably a BNA, such as a BNA for silencing HSP27 protein expression (antisense BNA (HSP27)).

[0178] In the context of the present invention, an effector molecule or effector moiety is any substance that affects cellular metabolism by interacting with an effector molecule target within the cell, which may be any molecule or structure within the cell, excluding the compartments of the endocytic and recycling pathways and the lumen of vesicles, but including the membranes of these compartments and vesicles. Therefore, such structures within the cell include the nucleus, mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol. Cytosolic delivery of an effector moiety in the context of the present invention preferably means that the effector moiety is capable of escaping endosomes (and / or lysosomes) (which also includes escaping endolysosomes and lysosomes, as defined above) and preferably is capable of reaching the effector moiety target described herein. The present invention also encompasses a new type of molecule called a scaffold. This serves to simultaneously bring both the effector moiety and at least one glycoside molecule, such as a saponin of the invention, into the endosome in a predetermined ratio when the effector moiety is comprised in a second or third proteinaceous molecule of the invention and the saponin is comprised in a first proteinaceous molecule. In the context of the present invention, the backbone polymeric or oligomeric structure is a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or it is an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The terms "polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer" have their usual meaning. Specifically, a polymer is a substance having a molecular structure made up primarily or entirely of many equal or similar units linked together, while an oligomer is a polymer whose molecules consist of a relatively small number of repeating units. There is no consensus on a single specific cutoff for the terms "many" and "few" used in the above definitions of polymer and oligomer, respectively.However, because the backbone can comprise a polymeric or oligomeric structure, or both, the full range of numbers of similar units linked together applies to such structures, i.e., from 2 monomeric units to 100 monomeric units, 1000 monomeric units, and more. For example, a structure containing 5 or fewer monomeric units may be referred to as an oligomeric structure, and a structure containing 50 monomeric units may be referred to as a polymeric structure. A structure of 10 monomeric units may be referred to as either an oligomer or a polymer. The backbone as defined herein further comprises at least one glycoside molecule, such as a saponin of the present invention. The backbone preferably includes polymeric or oligomeric structures, such as poly- or oligo(amines), e.g., polyethyleneimine and poly(amidoamines), and biocompatible structures, e.g., polyethylene glycol, poly- or oligo(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, and poly(dextrins), poly- or oligosaccharides, e.g., cyclodextrins or polydextrose, and poly- or oligoamino acids, e.g., polylysine or peptides or proteins, or DNA oligo- or polymers. The assembled polymeric structure defined herein comprises at least one backbone and, optionally, other individual polymeric or oligomeric structures. The other individual polymeric or oligomeric structures of the assembly can be (a) a scaffold (thus comprising at least one glycoside molecule, e.g., a saponin of the invention), (b) a functionalized scaffold (thus comprising at least one glycoside molecule, e.g., a saponin, and a ligand, antibody, etc. as a first proteinaceous molecule), or (c) a polymeric or oligomeric structure without a glycoside molecule, such as a saponin of the invention (see, e.g., Table A1), without a ligand, antibody, etc. as the first proteinaceous molecule. A functionalized aggregated polymeric structure is an assembled polymeric structure containing (a) at least one functionalized scaffold or (b) at least one polymeric structure comprising at least one scaffold and at least one ligand, antibody, etc. as a first proteinaceous molecule.The polymer or oligomeric structure on the assembled polymer structure, which does not contain any of the molecules mentioned above (i.e., no glycosides such as saponins, no first proteinaceous molecules such as ligands, antibodies, etc.), is specifically added as a structural component of the assembled structure, which helps to assemble or stabilize the assembled structure ("glue-like"). Without wishing to be bound by any theory, an acidic environment appears to be a prerequisite for a synergistic action between the glycoside (saponin) and the effector moiety.

[0179] Whether a first proteinaceous molecule comprising a saponin, either with or without one or more (cleavable) linkers and / or optionally a backbone, can disrupt the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be easily determined by assays as described in Example 3 and as known in the art. Inhibition is described as a "-fold increase in the amount of glycoside required to induce 50% cell kill." Preferably, the backbone does not result in an increase that is at least the increase in glycoside molecules (saponin) required to obtain 50% cell kill observed when chloroquine is used as a positive control. Alternatively and preferably, a first proteinaceous molecule comprising a saponin, either with or without one or more (cleavable) linkers and / or optionally a backbone, does not result in at least a four-fold increase, more preferably at least a two-fold increase, of glycoside molecules to induce 50% cell kill. The -fold increase should be measured by assays essentially as described in Example 4. Chloroquine, as a positive control, induces a two-fold increase in the amount of glycoside, preferably the amount of saponin, to observe 50% cell killing, where the saponin is any one or more of the saponins of the present invention (see Table A1, Scheme I, previous embodiments).

[0180]

[0043] By the term "improving or enhancing the effectiveness of an effector moiety", it is meant that the glycoside molecule, preferably a saponin of the present invention, increases the functional effectiveness of the effector moiety (e.g. the therapeutic index of a toxin or drug or oligonucleotide such as BNA; the metabolic effectiveness of a modulator in a biotechnology process; the transfection effectiveness of a gene in a cell culture research experiment), preferably by enabling or improving its target engagement. Acceleration, prolongation or enhancement of an antigen-specific immune response is preferably not encompassed. Therapeutic efficacy includes, but is not limited to, a stronger therapeutic effect, preferably with a lower dosage and / or fewer side effects. "Improving the effectiveness of an effector moiety" may also mean that an effector moiety that could not be used due to lack of efficacy (and for example was not known to be an effector moiety) becomes effective when used in combination with the present invention. Any other effect provided by the present invention that is beneficial or desirable and that can be attributed to the combination of the effector moiety with a second or third proteinaceous molecule is considered to be an "improved effect". In certain embodiments, a scaffold comprising an attached saponin(s) and comprised in a first proteinaceous molecule enhances the effect of an effector moiety comprised in a second proteinaceous molecule, whose effect is intended and / or desired. In the case of a first proteinaceous molecule comprising a saponin attached to a proteinaceous scaffold, the proteinaceous polymer structure of the scaffold itself may have an effect, for example, on colloid osmotic pressure in the bloodstream. If such an effect is not the intended or desired effect of such a functionalized scaffold comprised in the first proteinaceous molecule, then the proteinaceous structure of the scaffold is not an effector moiety as defined herein. Alternatively, for example, in the case of a DNA- or RNA-based scaffold bearing an attached saponin and comprised by the first proteinaceous molecule, the DNA or RNA parts may have an (unintended) function, for example, by interfering with expression. If such interference is not the intended or desired effect of the ultimate functionalized scaffold, then the DNA or RNA polymer structure of the scaffold is not an effector moiety as defined herein.

[0181] A number of preferred characteristics can be formulated for the endosomal escape-enhancing factors, i.e., glycosides or saponins, preferably saponins according to the present invention, contained in the first proteinaceous molecule: (1) they preferably are not toxic and do not provoke an immune response, (2) they preferably do not mediate cytosolic uptake of the effector moiety into off-target cells, (3) their presence at the site of action preferably is synchronized with the presence of the effector moiety, (4) they preferably are biodegradable or excretable, and (5) they preferably do not substantially interfere with biological processes of an organism unrelated to the biological activity of the effector molecule with which the endosomal escape-enhancing factor is combined, e.g., interact with hormones. Examples of glycoside molecules, such as saponins, according to the present invention that meet, at least to some extent, the previously mentioned criteria are bisdesmosidic triterpenes, preferably bisdesmosidic triterpene saponins, such as SO1861, SA1641, QS-21, GE1741, and the saponins of Table A1, Scheme I.

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

[0183] One embodiment is an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate of the invention, wherein the antibody is selected from the group consisting of CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, C A6, 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, HER2, EGFR, and / or cetuximab, daratum mab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of IgG type, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor-binding fragment thereof and / or at least one antibody thereof and / or the antibody-drug conjugate comprises gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates of Table A2 and Table A3; or the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule such as EGF or a cytokine.

[0184] 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.

[0185] Certain aspects of the present invention relate to pharmaceutical compositions, including compositions comprising a first proteinaceous molecule of the present invention and a second proteinaceous molecule of the present invention, or comprising a first proteinaceous molecule of the present invention and a third proteinaceous 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.

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

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

[0188] As previously stated, at least one saponin contained in the first proteinaceous molecule according to the present invention increases the efficacy of at least the current and new effector moieties defined in the present invention. Potential side effects will be reduced without reducing efficacy due to a lower dosage of the effector moiety contained in the second or third proteinaceous molecule. Thus, the present invention provides a first proteinaceous molecule according to the present invention for use in medicine or as a medicament. Therefore, one aspect of the present invention relates to a first proteinaceous molecule according to the present invention for use as a medicament, wherein the first proteinaceous molecule comprises at least a saponin. Also provided is the use of a first proteinaceous molecule according to the present invention for the manufacture of a medicament. In particular, cancer medicine, and in particular classical chemotherapy medicines, are notorious for their side effects. Because the first and third proteinaceous molecules have the same binding site for the same epitope on the same cell surface molecule, or because the first and second proteinaceous molecules have different binding sites for different first and second epitopes on the first and second cell surface molecules, respectively, and because of the temporal and spatial synchronization and targeting of both the drug substance contained in the second or third proteinaceous molecule and the saponin contained in the first proteinaceous molecule, the therapeutic combination according to the present invention is particularly advantageous for use as a pharmaceutical, and in particular for use in a method for treating cancer. Therefore, the present invention provides a therapeutic combination according to the present invention or a first proteinaceous molecule of the present invention for use in a method for treating cancer. The present invention also provides a therapeutic combination according to the present invention or a first proteinaceous molecule of the present invention for use in a method for treating acquired or inherited disorders, in particular monogenic deficiencies. Therefore, the therapeutic combination comprises the first and second proteinaceous molecules and / or comprises the first and third proteinaceous molecules. Therefore, one aspect of the present invention relates to a therapeutic combination according to the present invention for use in a method for the treatment of cancer or an autoimmune disease, wherein the second or third proteinaceous molecule comprises a covalently attached effector moiety.

[0189] A further application of the first, second, and third proteinaceous molecules of the present invention in medicine is the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting disease can be hereditary or acquired. In most cases, only symptomatic treatment is possible, and in several rare diseases, insufficient treatment options lead to a shortened lifespan for patients. An example of such a disease is phenylketonuria, an inborn error of metabolism resulting in impaired metabolism of the amino acid phenylalanine. The disease is characterized by mutations in the gene encoding the hepatic enzyme phenylalanine hydroxylase. To date, phenylketonuria is incurable. The incidence is approximately 1:10,000, with the highest known incidence being 1:2,600 in Turkey. A second or third proteinaceous molecule, preferably an antibody, carrying an attached phenylalanine hydroxylase or an attached polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells and replace the defective enzyme in the liver cells by using a suitable specific antibody. This is one example of the use of a therapeutic agent combination of the present invention for replacement or gene therapy, comprising a first proteinaceous molecule having a saponin attached thereto and a second or third proteinaceous molecule having an enzyme or oligonucleotide attached thereto in accordance with the present invention. In a preferred embodiment there is provided a therapeutic agent combination according to the present invention for use in a method of gene therapy or replacement therapy.

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

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

[0192] The appropriate dosage form will depend in part on the use or route of entry, for example, transdermal or injection. Such a dosage form should allow the compound to reach the target cells, whether or not the target cells are present in a multicellular host. Other factors are known in the art and include considerations such as dosage forms that delay the compound or composition from exerting its effect and toxicity.

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

[0194] One aspect of the invention relates to a kit comprising a first container containing an endosomal escape-enhancing conjugate in accordance with the invention (i.e., a first proteinaceous molecule) and a second container containing a binding moiety in accordance with the invention (i.e., a second and / or third proteinaceous molecule). The kit further comprises instructions for using the binding molecules (i.e., a therapeutic combination comprising the first and second or first and third pharmaceutical compositions).

[0195] [Table A1-1]

[0196] [Table A1-2]

[0197] [Table A1-3]

[0198] [Table A1-4]

[0199] [Table A1-5]

[0200] [Table A1-6]

[0201] [Table A2-1]

[0202] [Table A2-2]

[0203] Table A2-3

[0204] Table A2-4

[0205] Table A2-5

[0206] Table A2-6

[0207] Table A2-7

[0208] Table A2-8

[0209] Table A2-9

[0210] Table A2-10

[0211] Table A2-11

[0212] Table A2-12

[0213] Table A3-1

[0214] Table A3-2

[0215] Table A4

[0216] Table A5-1

[0217] Table A5-2

[0218] Table A5-3

[0219] Table A5-4

[0220] Table A5-5

[0221] Table A5-6

[0222] Table A5-7

[0223] Table A5-8

[0224] Table A5-9

[0225]

Table A5-10

[0226]

Table A5-11

[0227] Table A5-12

[0228] It is part of the present invention that the therapeutic combinations, first pharmaceutical compositions, first proteinaceous molecules, second or third pharmaceutical compositions, or second or third proteinaceous molecules of the present invention may be further combined with a covalent conjugate (complex) of a binding molecule or binding moiety with a saponin, or further combined with a pharmaceutical compound, antibody, etc. Thereby, there is provided a composition comprising three or more enhancers, drug substances, etc., of the present invention, e.g., a conjugate (e.g., a first proteinaceous molecule and / or a second or third proteinaceous molecule), which may or may not be linked to a saponin, and which may or may not be coupled to a ligand, e.g., a targeting immunoglobulin, domain or fragment thereof, in combination with a binding moiety complexed with an effector molecule, and further combined with a pharmaceutical. Additionally, an embodiment is a therapeutic combination, first pharmaceutical composition, first proteinaceous molecule, second or third pharmaceutical composition, or second or third proteinaceous molecule of the invention, wherein the second or third proteinaceous molecule is provided with two or more effector moieties, e.g., toxins or immunotoxins, and the two or more effector moieties are the same or different.

[0229] Illustrative Embodiments One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position 23, the saponin is preferably a saponin that can be isolated from a Gypsophila or Saponaria species, and more preferably the saponin is saponin SO1861 or any of its diastereomers.

[0230] One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the binding site is at least a ligand, eg, an immunoglobulin, to which is attached at least an effector moiety.

[0231] In one embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the binding moiety is an immunoglobulin or at least a binding domain thereof for binding to a cell surface molecule, preferably selected from HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, and CD71.

[0232] In one embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the linker is coupled to the glycoside via a cleavable bond and the ligand is an immunoglobulin. Preferably, the cleavable bond undergoes cleavage under acidic, reductive, enzymatic, or photoinduced conditions, preferably the cleavable bond is a covalent bond, preferably an imine bond, a hydrazone bond, an oxime bond, a 1,3-dioxolane bond, or an ester bond, preferably the cleavable bond is a disulfide bond or a peptide bond.

[0233] One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the saponin moiety is a terminal saponin, preferably saponin SO1861, the linker is a chemical linker that covalently links the saponin to a binding site on a first proteinaceous molecule, the same first binding site on the third and first proteinaceous molecules is an immunoglobulin, e.g., trastuzumab or cetuximab, and the linker preferably provides a cleavable bond between the terminal saponin moiety and the first binding site comprised in the first and third proteinaceous molecules.

[0234] One embodiment is a combination of an endosomal escape-enhancing conjugate (i.e., a first proteinaceous molecule) in accordance with the invention with a binding moiety (i.e., a second or third proteinaceous molecule), wherein the binding moiety comprises at least one effector moiety, and wherein the endosomal escape-enhancing conjugate and the binding moiety, independently of each other, are 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 of the complex of the binding moiety and the target cell-specific surface molecule.

[0235] In 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 target cell-specific surface molecule or structure when the binding moiety is a third proteinaceous molecule.

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

[0237] 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.

[0238] One embodiment is a combination according to the invention, wherein the endosomal escape-enhancing conjugate is capable of specifically binding to a first epitope, which is the same as the first epitope to which the binding moiety, when the binding moiety is a third proteinaceous molecule, is capable of specifically binding.

[0239] One embodiment is a combination according to the invention, where the binding moiety is a second proteinaceous molecule, the endosomal escape-enhancing conjugate and the binding moiety are capable of specifically binding to different target cell-specific surface molecules or structures.

[0240] In one embodiment is a combination according to the invention, wherein the target cell-specific surface molecule or structure is selected from HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD71.

[0241] An embodiment is a combination according to the invention, wherein the glycoside molecule is a bisdesmosidic triterpene, preferably a saponin.

[0242] In one embodiment is a combination according to the invention, wherein the glycoside molecule is a bisdesmosidic triterpene saponin.

[0243] An embodiment is a combination according to the invention, wherein the saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at the 23-position.

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

[0245] In one embodiment is a combination according to the invention, wherein the saponin is SO1861 or any of its diastereomers.

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

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

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

[0249] In one embodiment is a combination according to the invention, the ranges specified are 1 to 30 glycoside(s), preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 6, more preferably 2 to 6, more preferably 2 to 5, more preferably 3 to 5, more preferably 3 to 4 glycosides.

[0250] In one embodiment is a combination according to the invention, wherein the effector moiety is a drug substance, eg a toxin, eg a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide.

[0251] In one embodiment is a combination according to the invention, wherein the target cells are diseased or disease-associated cells, preferably tumor cells or tumor-associated cells (e.g. tumor vascular cells), or immune cells (e.g. regulatory T cells), or autoimmune cells.

[0252] An embodiment is a combination according to the invention, wherein at least one effector moiety is linked to a binding moiety (second or third proteinaceous molecule) via a cleavable bond, preferably said cleavable bond undergoes cleavage under acidic, reductive, enzymatic or photoinduced conditions, and / or the cleavable bond is a disulfide bond or a peptide bond.

[0253] One embodiment is a combination according to the invention, wherein the glycoside (saponin) is capable of increasing the endosomal escape of the effector molecule.

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

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

[0256] An embodiment is a pharmaceutical composition according to the invention, further comprising at least one additional drug substance, for example an additional immunoglobulin.

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

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

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

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

[0261] One embodiment is a kit comprising a first container containing an endosomal escape-enhancing conjugate in accordance with the invention and a second container containing a binding moiety in accordance with the invention, the kit further comprising instructions for using the binding molecule.

[0262] The first proteinaceous molecule is suitable for use as a semi-finished product for the production of a functionalized ADC or OAC, the functionalized ADC or OAC comprising at least one covalently coupled saponin of the present invention and at least one effector moiety of the present invention. One embodiment is a first proteinaceous molecule of the present invention further comprising a payload or effector moiety of the present invention, e.g., a toxin or oligonucleotide, covalently attached to the first proteinaceous molecule of the present invention either directly or via a linker of the present invention, preferably a cleavable linker of the present invention, and / or via an oligomeric or polymeric backbone according to the present invention. For example, such a functionalized ADC or OAC may comprise two to four saponins covalently coupled, e.g., directly or via a (cleavable) linker, to cysteine ​​side chains on the first proteinaceous molecule, such as a ligand or antibody (fragment). Alternatively, it comprises e.g. a dendron comprising 1 to 16 covalently coupled saponins attached thereto, the dendron being covalently coupled to e.g. a cysteine ​​and / or lysine side chain of the first proteinaceous molecule according to the invention.

[0263] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. [Example]

[0264] Example A - Treatment of mammalian tumor-bearing animals with a conjugate of the invention in combination with an ADC results in survival and tumor regression Female Balb / c nude mice were subcutaneously injected with a suspension of human A431 tumor cells. Human epidermal carcinoma was developed under the skin of the mice in a xenograft animal tumor model. After tumor cell injection, xenograft tumors grew to approximately 170-180 mm. 3 A431 tumor cells have the following characteristics: high EGFR expression, intermediate CD71 expression, and low HER2 expression.

[0265] Table A presents the results of treatment of control and tumor-bearing mice. Tumor-bearing mice were treated with the indicated antibodies directed against either human Her2 / neu, human EGFR, or human CD71, cell surface receptors on xenograft tumors. Cetuximab was covalently conjugated to the saponin SO1861. SO1861 was first provided with the linker EMCH (N-ε-maleimidocaproic acid hydrazide), a maleimide and hydrazide crosslinker used to covalently conjugate a sulfhydryl (reduced cysteine ​​of an antibody) to a carbonyl (aldehyde or ketone; in this case, the carbonyl of the aldehyde at position C-23 of the saponin). Saponin-EMCH was covalently coupled to the reduced cysteine ​​of cetuximab, forming a covalent thioether bond between EMCH and the cysteine ​​side chain. The ADCs trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9, IgG)-saporin (covalent conjugate) were tested for their tumor-attacking efficacy in mice. Tumor volume was measured over time after the initiation of treatment with the ADC. The dose of the ADC was suboptimal in this tumor model. Previous experiments established that at a suboptimal dose of the ADC, tumor regression or cessation of tumor growth would not be observable.

[0266] [Table A]

[0267] These results demonstrate that combination therapy of tumor-bearing mice with an ADC at a dose that is ineffective when treatment of the ADC alone is considered (tumor growth, mouse death (euthanasia) is not prevented) with a conjugate of the present invention consisting of an antibody targeting a tumor cell-specific receptor covalently linked to a saponin, i.e., SO1861, provides an efficient and effective treatment regimen, expressed as regressing tumors and prolonged survival of treated animals (over the duration of the experiment). The covalent conjugate is administered to cancer-bearing mice at a dose that is ineffective when administered alone (tumor growth, mouse death (euthanasia) is not prevented). Therefore, a suboptimal dose of an ADC combined with a conjugate containing a covalently linked saponin of the present invention that has no antitumor activity when administered alone provides an effective treatment option for cancer patients, and a relatively low dose of the ADC is effective. A lower dose of the ADC holds the promise of a lower risk of adverse events, or even no side effects at all. Additionally, the stimulatory effect of conjugates bearing saponins of the present invention on the efficacy of ADCs indicates that ADCs previously found to lack efficacy when treating tumor patients may gain renewed attention and value because their efficacy is improved in combination therapy settings, as demonstrated in this example. Reference is made to Tables A2 and A3, which summarize ADCs that have previously been investigated in human clinical settings, but some ADCs have since been withdrawn from further clinical investigation. In particular, ADCs whose clinical development has been terminated due to an observed lack of efficacy and / or the occurrence of unacceptable adverse events are ADCs that may gain renewed value for cancer patients when combined with conjugates containing covalently attached saponins of the present invention, such as the tested cetuximab-saponin.

[0268] Example B - Quillaja saponaria saponin mixture containing QS-21 with endosomal / lysosomal escape-enhancing activity Scheme I shows the common molecular structure of a series of QS-21 saponins (excerpted from Conrado Pedebos, Laercio Pol-Fachin, Ramon Pons, Cilaine V. Teixeira, Hugo Verli, Atomic Model and Micelle Dynamics of QS-21 Saponin, Molecules 2014, 19, 3744-3760). Mixtures of water-soluble saponins obtained from Quillaja saponaria (Sigma-Aldrich, Product No. S4521; Roth, Item No. 6857; InvivoGen, Product "Quil-A") can be applied to the endosomal / lysosomal escape-enhancing conjugates, compositions, and combinations of the present invention based on the endosomal / lysosomal escape-enhancing properties of at least one individual saponin present in the mixture, e.g., QS-21, or based on a combination of two or more saponins contained in the mixture, e.g., QS-21 and QS-7.

[0269] The present inventors demonstrated that a 2.5 microgram / ml dose of a mixture of saponins from Quillaja saponaria can enhance the endosomal escape of dianthin when tested in mammalian tumor cells using a cell-based bioassay. The effector moiety exposed to the cells was dianthin covalently coupled to the ligand EGF: EGF-dianthin. The cells tested were the tumor cell lines HeLa for free saponin, and A431, MDA-MB-468, CaSki, and A2058 for testing saponin when covalently coupled to cetuximab.

[0270] Example 1 Various concentrations of trastuzumab-saporin (a HER2-targeting protein-toxin conjugate; intravenous) were administered intravenously to BT474 (HER2 ++ ) Enhanced efficacy in a xenograph mouse model was tested in combination with 1.5 mg / kg SO1861 (1 hour prior to antibody-toxin injection; subcutaneous). 3Dosing began on day 13, when tumors reached a size of 1 mg / kg, and tumor volumes were determined after each treatment. Tumor growth inhibition was observed in mice treated with 1 mg / kg and 0.3 mg / kg trastuzumab-saporin, but no improved tumor growth inhibition was observed in mice treated with the combination of trastuzumab-saporin and SO1861. This indicates that unconjugated SO1861 is unable to improve antibody-protein toxins in the current conditions and mouse model.

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

[0272] Previously, the efficacy of various saponins (SO1861, SO1642) was demonstrated when co-administered to cells as "free" unconjugated molecules in combination with ligand-toxin fusions (e.g., EGF dianthin) or antibody-protein toxin conjugates, resulting in enhanced cell-killing activity of target-expressing cells. Here, three different saponin molecules (SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904) isolated from the root extract of Saponaria officinalis were administered to HeLa (EGFR 1) cells. +() cells were titrated in the presence and absence of a non-effective fixed concentration of 1.5 pM EGF-gianthin. This revealed a strong enhancement of cell-killing activity for all tested saponin variants compared to treatment without EGF-gianthin (IC50 = 300 nM; Figure 2A). EGF-gianthin was then titrated with a fixed concentration of saponin (~1000 nM), revealing a strong enhancement of target cell killing at low pM concentrations of EGF-gianthin (IC50 = 0.4 pM; Figure 2B). This was observed for all saponins used: SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904. EGF-gianthin alone could induce cell killing only at very high concentrations (IC50 = 10,000 pM). This indicates that these particular types of saponins all have the inherent ability to efficiently induce endosomal escape with only very low amounts of the targeted toxin available.

[0273] To extend this study, saponins from other sources were analyzed. A saponin purified from the root extract of Gypsophila elegans (M. Bieb.) (GE1741) was titrated against HeLa cells in the presence and absence of 1.5 pM EGF-dianthine and compared with purified SO1861. GE1741 also potentiated EGF-dianthine-induced killing of HeLa cells, but showed slightly lower efficacy than SO1861 (GE1741 IC50 = 800 nM; Figure 2C) and higher general toxicity (IC50 = 5,000 nM in the absence of EGF-dianthine; Figure 1C). A similar study in which different partially purified mixtures of Quillaja saponaria saponins (QS Mix 1–3) were coadministered with 1.5 pM EGF-gianthin in HeLa cells revealed activity similar to that of SO1861 for two of the three (QS Mix 1 and QS Mix 3) (IC50 QS Mix / QS Mix 3 = 300 nM; Figure 1D). QS Mix (2) was less effective in enhancing cell killing induced by 1.5 pM EGF-gianthin (IC50 = 2000 nM; Figure 2D). However, no general toxicity was observed. This indicates that specific types of saponins are also available from QS extracts that efficiently induce endosomal escape of the targeted toxin ligand, EGF-gianthin.

[0274] Example 3 To conjugate the SO1861 molecule to antibodies, a labile / acid-sensitive linker (-EMCH or -N3) was conjugated to SO1861 via the aldehyde group to produce SO1861-EMCH or SO1861-N3 (Figures 60-66). To verify the activity of SO1861-EMCH, the molecule was titrated in EGFR-expressing (A431, HeLa) and non-expressing (A2058) cells in the presence and absence of a fixed, non-effective (1.5 pM) concentration of EGF dianthine. In all three cell lines, SO1861 alone showed a strong reduction in cell viability, whereas SO1861-EMCH as a single compound showed no toxicity up to 25,000 nM (Figures 3A-C). When SO1861-EMCH was combined with 1.5 pM EGF dianthin, a strong target-specific reduction in cell viability was observed in the EGFR + This was observed in A431 cells and HeLa cells (IC50 = 3,000 nM; Figures 2A and 2B). - A2058 cells were completely unaffected (Fig. 3C). Similar results were obtained with SO1861-N3. Coadministration of SO1861-N3 with 1.5 pM EGF-gianthine also showed efficient cell killing in A431 and HeLa cells (IC50 = 3,000 nM), whereas without EGF-gianthine, general toxicity was observed above 10,000 nM (Fig. 3D, 2E).

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

[0276] The one-target, two-component system (1T2C) is a combination treatment of mAb1-protein toxin and mAb1-SO1861, as illustrated in Figure 19. SO1861-EMCH was conjugated via a cysteine ​​residue (Cys) and HSP27BNA oligo was conjugated via a lysine residue to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR), both via DAR4, resulting in two conjugates: cetuximab-(Cys-L-SO1861). 4 and cetuximab-(Lys-L-HSP27BNA) 4 This resulted in the production of cetuximab (Cys-L-SO1861). 4 (intraperitoneal administration (ip)) and cetuximab-(Lys-L-HSP27BNA) 4 (intravenous administration (iv)) was tested for EGFR tumor-targeted gene silencing activity in the A431 xenograph mouse tumor model. 3 Dosing was initiated on day 12 when ≥ 100 mg / kg cetuximab-(Cys-L-SO1861) was reached, and tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression compared to control gene mRNA expression levels (reference gene). 4 +25mg / kg cetuximab-(Lys-L-HSP27BNA) 4 One of the medications was cetuximab (Cys-L-SO1861) 4 or cetuximab-(Lys-L-HSP27BNA) 4 Compared to a single dose of monotherapy, this resulted in a 50% reduction in HSP27 gene expression in A431 tumors (Figure 7). A 40% reduction in HSP27 gene silencing was observed compared to vehicle control tumors. This demonstrates and enables the combination of cetuximab-conjugated SO1861 and cetuximab-conjugated HSP27 BNA oligos according to the 1T2C invention to induce efficient targeted delivery of therapeutic antisense oligonucleotides in the cytoplasm of solid tumor cells, thereby inducing tumor-targeted gene silencing in vivo.

[0277] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) via the cysteine ​​residue (Cys) in DAR4 to form trastuzumab-(Cys-L-SO1861). 4 This resulted in the production of trastuzumab-(Cys-L-SO1861) 4 The combination of 40 mg / kg trastuzumab-(Cys-L-SO1861) and trastuzumab-saporin (a trastuzumab protein-toxin conjugate) was tested in a mouse tumor model (patient-derived xenograph tumor model PDX) that has high HER2 expression levels and is resistant to trastuzumab monotherapy. 4 The combination according to the 1T2C invention, trastuzumab-saporin (intraperitoneal (ip)) + 0.03 (days 1 and 8) / 0.02 (days 15, 22, 30, 36, and 43) mg / kg (intravenous (iv))), was compared with vehicle control and 40 mg / kg trastuzumab-(Cys-L-SO1861). 4 Compared with 0.03 / 0.02 mg / kg trastuzumab-saporin monotherapy, the combination at a lower dose (40 mg / kg trastuzumab-(Cys-L-SO1861) or 0.03 / 0.02 mg / kg trastuzumab-saporin monotherapy) demonstrated strong tumor growth inhibition (Figure 8). 4 No tumor growth inhibitory activity was observed in tumor-bearing mice treated with trastuzumab-conjugated SO1861 + 0.01 mg / kg trastuzumab-saporin (Figure 8). This demonstrates and enables that the 1T2C combination of trastuzumab-conjugated SO1861 + trastuzumab-conjugated protein toxin induces efficient targeted delivery of the therapeutic protein toxin in the cytoplasm of solid tumor cells, thereby inducing tumor cell death and tumor growth inhibition in vivo.

[0278] The one-target, two-component system (1T2C) is a combination treatment of mAb1-SO1861 and mAb1-protein toxin (FIG. 19). SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) at DAR 3,7 (Cetuximab-(Cys-L-SO1861) 3,7 ). Cetuximab-(Cys-L-SO1861) 3,7 was titrated with a fixed concentration of 10 pM cetuximab-saporin (cetuximab conjugated to the protein toxin saporin) and expressed in EGFR-expressing cells (A431, EGFR ++ ;CaSKi, EGFR + We determined cell killing mediated by the targeted protein toxin in IL-16 at low concentrations of cetuximab-(Cys-L-SO1861). 3,7 Although cetuximab and cetuximab-(Cys-L-SO1861) demonstrated potent cell killing in A431 (IC50 = 0.6 nM and Caski IC50 = 1 nM; Figures 9A and 9B), 3,7 Neither cetuximab nor cetuximab + 10 pM cetuximab-saporin could induce any cell-killing activity in EGFR-expressing cells. This indicates that cetuximab-conjugated SO1861 (at non-effective concentrations) effectively enhances the endosomal escape of cetuximab-conjugated protein toxins, thereby inducing cell-killing of EGFR-expressing cells. The cell-killing activity in A431 is more effective than that in CaSki, and correlates with the EGFR expression level in these cell lines. EGFR receptor binding competition between both conjugates of 1T2C is also observed. Cetuximab-(Cys-L-SO1861) 3,7 As the concentration increases, the cell-killing activity declines due to overcoming receptor binding and internalization of cetuximab-saporin (FIGS. 9A, 9B).

[0279] Cetuximab-saporin was then added to a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861). 3,7 The targeted protein toxin-mediated cell killing in EGFR-expressing cells was determined by titrating 75 nM cetuximab-(Cys-L-SO1861) 3,7Although we found that cetuximab-saporin alone or cetuximab-saporin plus 75 nM cetuximab induced efficient cell killing in EGFR-expressing cells (A431: IC50 = 0.4 pM; and CaSKi: IC50 = 2 pM; Figures 9C and 9D), cetuximab-saporin alone or cetuximab-saporin plus 75 nM cetuximab only exhibited cell killing at higher concentrations of cetuximab-saporin (IC50 = 40 pM and IC50 = 1000 pM, respectively) in both cell lines (Figures 9C and 9D). All of this indicates that relatively low concentrations of cetuximab-saporin may be effective and induce cell killing in highly EGFR-expressing cells only in combination with low cetuximab-SO1861 concentrations. Receptor competition between both conjugates in the 1T2C system is also observed in the cetuximab-toxin titration procedure when comparing the cell killing activity of cetuximab-saporin with and without 75 nM cetuximab (Figures 9C, 9D).

[0280] Next, cetuximab-(Cys-L-SO1861) 3,7 was titrated with a fixed concentration of 10 pM cetuximab-saporin and in cells with low or no EGFR expression (HeLa, EGFR + / - ;A2058, EGFR - We determined cell killing mediated by targeted protein toxins in cells expressing EGFR. Cells with low (HeLa) or no EGFR expression (A2058) were completely insensitive to either the combination of cetuximab-(Cys-L-SO1861)3,7 or 10 pM cetuximab-saporin (HeLa: IC50 > 1000 nM; A2058: IC50 > 1000 nM; Figures 10A and 10B). This indicates that in the absence of sufficient EGFR 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, cetuximab-saporin was administered at a fixed concentration of 75 nM cetuximab-(Cys-L-SO1861). 3,7The targeted protein toxin-mediated cell killing in cells with low EGFR expression (HeLa) or no EGFR expression (A2058) was determined by titrating the antibody with 75 nM cetuximab-(Cys-L-SO1861). 3,7 Although cetuximab-saporin showed cell killing only at high concentrations in combination with EGFR (HeLa: IC50 = 60 pM, Figure 10C), it was not effective in A2058 cells (EGFR - ) was not sensitive at any of the concentrations tested (A2058: IC50>10,000 pM; Figure 10D). This all suggests that cells with low or no EGFR receptor expression are sensitive to cetuximab-(Cys-L-SO1861). 3,7 + indicates insensitivity to the cetuximab-saporin combination due to lack of sufficient EGFR receptors to facilitate antibody-mediated delivery of SO1861 within the endolysosomal compartment and facilitate escape of the protein toxin.

[0281] Next, SO1861-EMCH was conjugated to trastuzumab (a monoclonal antibody that recognizes and binds to human HER2) at DAR4 via a cysteine ​​residue (Cys) (trastuzumab-(Cys-L-SO1861)). 4 ). Trastuzumab-(Cys-L-SO1861) 4 was titrated with a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin saporin) and administered to HER2-expressing cells (SK-BR-3, HER2 ++ We determined cell killing mediated by the targeted protein toxin in trastuzumab-(Cys-L-SO1861) at low concentrations. 4 Although the inhibitory effect of trastuzumab on SK-BR-3 was potent (IC50 = 0.8 nM; Figure 11A), the inhibitory effect of trastuzumab on SK-BR-3 was not potent. 4Neither trastuzumab nor trastuzumab + 50 pM trastuzumab-saporin could induce any cell-killing activity in HER2-expressing cells. This indicates that trastuzumab-conjugated SO1861 (at non-effective concentrations) effectively enhances the endosomal escape of trastuzumab-conjugated protein toxins, thereby inducing cell-killing of HER2-expressing cells. Receptor competition between both conjugates of 1T2C was also observed. Trastuzumab-(Cys-L-SO1861) 4 As the concentration increases, the cell-killing activity declines due to overcoming receptor binding and internalization of trastuzumab-saporin (FIG. 11A).

[0282] Trastuzumab-saporin was then added to the 2.5 nM trastuzumab-(Cys-L-SO1861) solution according to the present invention. 4 The targeted protein toxin-mediated cell killing in HER2-expressing cells was determined by titrating 2.5 nM trastuzumab-(Cys-L-SO1861) 4 revealed that a combination of trastuzumab-saporin with a low concentration already induced efficient cell killing in HER2-expressing cells (SK-BR-3: IC50 = 2 pM; Figure 11B). However, trastuzumab-saporin alone or trastuzumab-saporin plus 2.5 nM trastuzumab only showed cell killing at a high concentration of trastuzumab-saporin (Figure 11B). All this suggests that a relatively low concentration of trastuzumab-saporin can suppress the activity of low trastuzumab-(Cys-L-SO1861) in highly HER2-expressing cells. 4 These results indicate that only in combination with a high concentration of α-glucan can be effective and induce cell killing.

[0283] Next, trastuzumab-(Cys-L-SO1861) 4 was titrated with a fixed concentration of 50 pM trastuzumab-saporin in accordance with the present invention to induce targeted protein toxin-mediated cell killing in low HER2-expressing cells (A431 HER2 + / -) or cells without HER2 expression (JIMT-1:HER2 + ;MDA-MB-468:HER2 - Cells with low or no HER2 expression were treated with trastuzumab-(Cys-L-SO1861). 4 The cells were completely insensitive to either combination of +50 pM trastuzumab-saporin (JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM; Figures 13A and 13B). This indicates 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, trastuzumab-saporin was administered at a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861). 4 The HER2-low expressing cells (JIMT-1) were titrated with 2.5 nM trastuzumab-(Cys-L-SO1861) to determine cell killing mediated by the targeted protein toxin in cells with low or no HER2 expression. 4 Only high trastuzumab-saporin concentrations in combination with HER2 (JIMT-1: IC50 > 10,000 pM; Figure 13C) showed cell killing in MDA-MB-468 cells (HER2 - ) was not sensitive at any of the concentrations tested (MDA-MB-468: IC50 > 10,000 pM; Figure 13D).

[0284] All of this suggests that cells with low or no HER2 receptor expression can tolerate trastuzumab-(Cys-L-SO1861). 3,7 + Insensitivity to the trastuzumab-saporin combination is due to the lack of sufficient HER2 receptors to facilitate antibody-mediated delivery of SO1861 within the endolysosomal compartment and facilitate escape of the protein toxin.

[0285] To demonstrate that the activity of the 1T2C system is driven by acidification of the endolysosomal compartment, the 1T2C system according to the invention was tested in combination with the endosomal acidification inhibitor chloroquine. Trastuzumab-saporin was tested in combination with 5 nM trastuzumab-(Cys-L-SO1861) in the absence or presence of chloroquine. 4 Trastuzumab-saporin + 5 nM trastuzumab-(Cys-L-SO1861) 4 showed strong cytotoxic activity in highly HER2-expressing cells (SK-BR-3, HER2 ++ ; IC50 = 0.2 pM). However, trastuzumab-saporin + 5 nM trastuzumab-(Cys-L-SO1861) 4 +0.5 μM chloroquine inhibited SK-BR-3 (HER2 ++ ) cells (IC50 = 40 pM), indicating that the activity of antibody-conjugated SO1861 is reduced / blocked when endolysosomal acidification is prevented (Figure 14A). The same results were obtained with cetuximab-saporin + 5 nM cetuximab-(Cys-L-SO1861). 3,8 Cetuximab-saporin + 5 nM cetuximab-(Cys-L-SO1861) compared to + 0.5 μM chloroquine (IC50 = 200 pM) 3,8 The 1T2C combination according to the present invention showed an IC50 of 1 pM in EGFR-expressing cells (A431, EGFR ++ ; Figure 14B).

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

[0287] The one-target, two-component system (1T2C) is illustrated in Figure 21, which uses mAb1-(scaffold (-SO1861) n ) n A combination treatment with mAb1-protein toxin is also possible. 4 was conjugated to cetuximab via cysteine ​​residue (Cys) conjugation by DAR3,9 to form cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 was tested for enhanced cell-killing activity in combination with an anti-EGFR antibody-protein toxin conjugate (cetuximab-saporin) in EGFR-expressing cells (MDA-MB-468). Cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 +10 pM cetuximab-saporin efficiently induced toxin-mediated cell killing in high EGFR-expressing cells (IC50 = 0.4 nM; Figure 16A), which was consistent with cetuximab-Cys-(dendron(-L-SO1861) 4 ) 3,9 This was not induced by cetuximab + 10 pM cetuximab-saporin or cetuximab (Figure 16A). This indicates that, according to the 1T2C invention, cetuximab-conjugated dendron (-L-SO1861) 4We show that cetuximab effectively enhances endosomal escape of cetuximab-conjugated protein toxins (at subeffective concentrations), thereby inducing cell killing in highly HER2-expressing cells. + / - ) performed a similar 1T2C experiment, which revealed no cell-killing activity when using the 1T2C combination according to the invention (IC50>100 pM; Figure 16B), indicating that in the absence of sufficient EGFR receptor expression, the effective intracellular SO1861 concentration is not optimal (threshold) for inducing cytoplasmic delivery of the protein toxin, resulting in toxin-mediated cell killing.

[0288] Next, dendron (-L-SO1861) 4 DAR4, trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 The antibody was conjugated to the anti-HER2 antibody trastuzumab by cysteine ​​conjugation (Cys), and then injected into HER2-expressing cells (SK-BR-3, HER2 ++ ) was tested for enhanced cell-killing activity by combining it with an anti-HER2 antibody-protein toxin conjugate (trastuzumab-saporin). Trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 +50 pM trastuzumab-saporin efficiently induced toxin-mediated cell killing (IC50 = 2 nM, Figure 16C), which was consistent with trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 This was not induced by trastuzumab + 50 nM trastuzumab-saporin or trastuzumab (Figure 16C). 4 We show that trastuzumab effectively enhances the endosomal escape of trastuzumab-conjugated protein toxins (at subeffective concentrations), thereby inducing cell killing in highly HER2-expressing cells. + / -Similar experiments in trastuzumab-Cys-(dendron(-L-SO1861) 4 ) 4 +50 pM trastuzumab-saporin revealed no activity (IC50 > 200 nM; Figure 16D), 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.

[0289] The clinically approved ADC trastuzumab-emtansine (T-DM1) is a conjugate of the anti-Her2 antibody trastuzumab and the small molecule toxin emtansine (DAR 3.5). T-DM1 was designated as trastuzumab-(Cys-L-SO1861). 4 and titrated in combination with the antibody protein toxin conjugate trastuzumab-saporin + trastuzumab-(Cys-L-SO1861) according to the present invention. 4 Trastuzumab-saporin + 2.5 nM trastuzumab-(Cys-L-SO1861) 4 showed improved activity compared to trastuzumab-saporin + 2.5 nM trastuzumab or trastuzumab-saporin alone (IC50 = 2 pM, Figure 17), whereas T-DM1 + ​​25.6 nM trastuzumab-(Cys-L-SO1861) 4 did not exhibit enhanced cell-killing activity (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, since small molecules can already passively cross (endolysosomal) membranes.

[0290] The one target two component system (1T2C) can also be a combination treatment of mAb1-QS mix (a mixture of saponins from Quillaja Saponaria) and mAb1-protein toxin.

[0291] QS mix-EMCH was coupled via a cysteine ​​residue (Cys) to DAR 4.1 (cetuximab-(Cys-L-QS mix) 4,1) conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR). Cetuximab-(Cys-L-QS mix) 4,1 The EGFR β-gamma antibody was titrated with a fixed concentration of 10 pM cetuximab-saporin or 10 pM cetuximab-giantin. ++ ), CaSKi(EGFR + ), and A2058 (EGFR - We determined cell killing mediated by targeted protein toxins in A431 (EGFR) cells. ++ ) and CaSKi (EGFR + ) cells at low concentrations of cetuximab-(Cys-L-QS mix) 4.1 While we demonstrated strong cell killing with +10 pM cetuximab-saporin or +10 pM cetuximab-giantin (A431: IC50 = 3 nM, Figure 18A; CaSKi: IC50 = 1 nM, Figure 18B), all control treatments failed to induce any cell killing in EGFR-expressing cells. - ), HSP27 gene silencing is not observed with the combination according to the present invention (IC50>1000nM; Figure 18C). This indicates that the cetuximab-conjugated QS21 mix (at non-effective concentrations) effectively enhances the endosomal escape of cetuximab-conjugated protein toxins, thereby inducing cell killing only in EGFR-expressing cells.

[0292] Example 4 The unstable SO1861 was conjugated via a cysteine ​​residue (Cys) to the anti-EGFR antibody cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) at a DAR of 3.9 (cetuximab-(Cys-L-SO1861)). 3.9) was tested for its enhanced delivery of antisense BNA oligonucleotides, resulting in enhanced target gene silencing. In this study, we used antisense BNA oligonucleotides against the mRNA of the cancer-specific target gene heat shock protein 27 (HSP27). In the cell cytoplasm, HSP27 BNA binds to the mRNA encoding HSP27 and targets the mRNA for destruction, thereby reducing HSP27 expression in cancer cells. Cetuximab-(Cys-L-SO1861) 3.9 EGFR ++ (A431) cells and EGFR - The combination according to the present invention showed efficient HSP27 silencing in A431 (IC50 = 2 nM; Figure 5A), whereas cetuximab-(Cys-L-SO1861) showed no significant effect on HSP27 silencing in A431 (A2058) cells. 3.9 No silencing was observed when cetuximab (Cys-L-SO1861) was used alone. 3.9 +100nM HSP27BNA inhibits EGFR - The antibody-conjugated SO1861 showed no gene silencing activity in target-expressing cells (A2058) (Figure 5B), indicating that low concentrations of the antibody-conjugated SO1861 can effecti...

Claims

1. a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first cell surface molecule, said first proteinaceous molecule being covalently bound to at least one saponin, said at least one saponin being covalently bound to an amino acid residue of said first proteinaceous molecule via at least one linker and / or via an oligomeric or polymeric backbone or directly to an amino acid residue of said first proteinaceous molecule; the first binding site comprises or consists of an immunoglobulin or comprises or consists of at least one ligand for binding to a cell surface molecule; A first proteinaceous molecule, wherein said at least one saponin is selected from the group consisting of SO1861, SA1641, GE1741, QS-21, QS-21A, QS21-B, Quil-A, SO1832, and SO1862.

2. The first proteinaceous molecule of claim 1 , wherein the first epitope of the first cell surface molecule is a tumor cell-specific first epitope of the first tumor cell surface molecule.

3. The first proteinaceous molecule described in claim 1 or 2, wherein the at least one saponin is SO1861.

4. The at least one saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23, 4. The first proteinaceous molecule according to any one of claims 1 to 3, wherein said at least one saponin is covalently coupled to an amino acid residue of the first proteinaceous molecule via an aldehyde functional group on the saponin.

5. The at least one saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin; 5. The first proteinaceous molecule according to any one of claims 1 to 4, wherein said at least one saponin is covalently coupled to an amino acid residue of the first proteinaceous molecule via a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin.

6. A first proteinaceous molecule described in claim 4 or 5, wherein an aldehyde functional group at position C-23 of the at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled to a sulfhydryl group on the first proteinaceous molecule via a thio-ether bond.

7. The first proteinaceous molecule of claim 5 or 6, wherein a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the at least one saponin is covalently coupled to a linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled via an amide bond to an amine group on the first proteinaceous molecule.

8. The first proteinaceous molecule according to any one of claims 1 to 7, wherein the first epitope of the first cell surface molecule to which the first binding site of the first proteinaceous molecule binds is a tumor cell-specific first epitope of a tumor cell-specific receptor.

9. The first binding site of the first proteinaceous molecule is selected from the group consisting of the anti-EGFR monoclonal antibody cetuximab, the anti-CD38 monoclonal antibody daratumumab, the anti-CD33 monoclonal antibody gemtuzumab, the anti-HER2 monoclonal antibody trastuzumab, the anti-EGFR monoclonal antibody panitumumab, the anti-CD30 monoclonal antibody brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, the IgG type OKT-9 anti-CD71 monoclonal antibody ...HER2 monoclonal antibody brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, the IgG type OKT-9 anti-HER2 monoclonal antibody monoclonal antibody pertuzumab, anti-CD20 monoclonal antibody rituximab, anti-CD20 monoclonal antibody ofatumumab, anti-CD52 monoclonal antibody alemtuzumab, pinatuzumab, OKT-10, anti-CD38 monoclonal antibody, anti-CD20 monoclonal antibody tositumomab, anti-CD20 monoclonal antibody ibritumomab, anti-CA125 monoclonal antibody oregovomab, anti-EpCAM (17-1A) monoclonal antibody edrecolomab, anti-EGFR monoclonal antibody monoclonal antibody nimotuzumab, anti-CD33 monoclonal antibody huMy9-6, anti-vascular integrin alpha-v beta-3 monoclonal antibody etaracizumab, anti-CD22 monoclonal antibody epratuzumab, anti-CEA monoclonal antibody labetuzumab, anti-CD44v6 monoclonal antibody bivatuzumab, anti-FAP monoclonal antibody sibrotuzumab, anti-CD19 monoclonal antibody huB4, anti-CanAg monoclonal antibody huC242, anti-CD56 monoclonal antibody 9. The first proteinaceous molecule of claim 8, comprising or consisting of any one of huN901, anti-CA6 monoclonal antibody DS6, anti-IGF-IR monoclonal antibody cixutumumab, anti-IGF-IR monoclonal antibody 3B7, anti-integrin monoclonal antibody CNTO95, or anti-syndecan-1 monoclonal antibody B-B4, or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof.

10. 10. The first proteinaceous molecule according to any one of claims 1 to 9, wherein the first proteinaceous molecule comprises more than one saponin, said saponins being covalently attached directly to amino acid residues of the first proteinaceous molecule and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone.

11. A first proteinaceous molecule described in any one of claims 4 to 10, wherein at least one linker is a non-cleavable linker or a cleavable linker.

12. The first proteinaceous molecule according to any one of claims 4 to 11, wherein the cleavable linker undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of a mammalian cell.

13. 13. The first proteinaceous molecule according to any one of claims 1 to 12, wherein the oligomeric or polymeric backbone comprises a polymeric or oligomeric structure and comprises chemical groups, chemical groups for covalent coupling of the backbone to amino acid residues of said first proteinaceous molecule.

14. A first proteinaceous molecule described in any one of claims 1 to 13, wherein the at least one saponin is covalently linked to a polymer or oligomeric structure of an oligomeric or polymeric backbone via at least one cleavable linker described in claim 10 or 11.

15. 15. The first proteinaceous molecule according to any one of claims 1 to 14, wherein the chemical group of the oligomeric or polymeric backbone for covalent coupling of said oligomeric or polymeric backbone to an amino acid residue of said first proteinaceous molecule is a click chemistry group.

16. 16. The first proteinaceous molecule according to any one of claims 1 to 15, wherein the polymer or oligomeric structure of the oligomeric or polymer backbone comprises a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymer or oligomeric structures.

17. A therapeutic composition for simultaneous administration, the therapeutic composition comprising (a) and (b) or (c). (a) a first pharmaceutical composition comprising a first proteinaceous molecule according to any one of claims 1 to 16 and optionally a pharma- ceutically acceptable excipient. (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, A second pharmaceutical composition, wherein the second proteinaceous molecule comprises a second binding site for binding to a second epitope of a second cell surface molecule that is different from the first cell surface molecule and comprises an effector moiety, the second pharmaceutical composition optionally further comprising a pharma- ceutically acceptable excipient, and the second epitope is different from the first epitope. (c) a third pharmaceutical composition comprising a third proteinaceous molecule and optionally further comprising a pharma- ceutically acceptable excipient, A third pharmaceutical composition, wherein the third proteinaceous molecule comprises a third binding site for binding to the first epitope on the cell surface molecule of (a) and comprises an effector moiety. Here, the first binding site and the third binding site are the same, the first cell surface molecule and the first epitope on the first cell surface molecule to which the first proteinaceous molecule can bind, and the first cell surface molecule and the first epitope on the first cell surface molecule to which the third proteinaceous molecule can bind are the same.

18. 18. The therapeutic composition of claim 17, which is (a) or (b). (a) the therapeutic composition comprises the first pharmaceutical composition and the second pharmaceutical composition; the first epitope on the first cell surface molecule is a tumor cell-specific first epitope on a first tumor cell-specific surface molecule; and A therapeutic composition wherein the second cell surface molecule is a second tumor cell-specific surface molecule different from the first tumor cell-specific surface molecule and the second epitope is a tumor cell-specific second epitope. (b) said therapeutic composition comprises said first pharmaceutical composition and said third pharmaceutical composition; The therapeutic composition, wherein the first cell surface molecule is expressed on the surface of a tumor cell.

19. 19. The therapeutic composition of claim 17 or 18, wherein the second binding site of the second proteinaceous molecule and / or the first binding site of the third proteinaceous molecule comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin, and / or at least one binding fragment of an immunoglobulin.

20. the first binding site binds to a first tumor cell-specific epitope on a first tumor cell-specific receptor; the second binding site binds to a second tumor cell-specific epitope on a second tumor cell-specific receptor; said first and second tumor cell-specific receptors are different but present on the same tumor cell; the first and second binding sites are different; and The therapeutic composition of any one of claims 17 to 19, wherein the first and second tumor cell-specific epitopes are different.

21. A therapeutic composition according to any one of claims 17 to 20, wherein the first and third binding sites, which are the same, are binding sites for binding to a first tumor cell-specific epitope on a first tumor cell-specific receptor.

22. The first receptor and / or the second receptor is CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin , Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2.

23. A first proteinaceous molecule according to any one of claims 1 to 16, or a therapeutic composition according to any one of claims 17 to 22, 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, with the proviso that the first binding site of the first proteinaceous molecule is different from the second binding site of the second proteinaceous molecule.

24. The therapeutic composition according to any one of claims 17 to 23, which is the following (a) or (b): (a) the first binding site of the first proteinaceous molecule and the third proteinaceous molecule comprise a monoclonal antibody, or at least one cell surface molecule binding domain and / or fragment thereof, with the proviso that the first binding site of the first proteinaceous molecule is the same as the third binding site of the third proteinaceous molecule; (b) said second binding site of said second proteinaceous molecule or said first binding site of said third proteinaceous molecule is or comprises a monoclonal antibody, or at least one cell surface molecule binding fragment or domain thereof.

25. A therapeutic composition described in any one of claims 17 to 24, wherein the effector portion contained in the second proteinaceous molecule or the third proteinaceous molecule consists of the following (i) to (iii): (i) comprises or consists of one or more of an oligonucleotide, a nucleic acid, a xenonucleic acid or a derivative thereof; (ii) comprises or consists of at least one proteinaceous molecule; (iii) comprises or consists of at least one payload;

26. A composition comprising a first proteinaceous molecule according to any one of claims 1 to 16 and either: A second proteinaceous molecule as defined in any one of claims 17 to 25, and optionally a pharma- ceutically acceptable excipient, or A third proteinaceous molecule as defined in any one of claims 17 to 25, and optionally a pharma- ceutically acceptable excipient, 26. The effector moiety as defined in any one of claims 17 to 25, wherein optionally the effector moiety comprised in said second proteinaceous molecule or said third proteinaceous molecule is any one of the effector moieties: oligonucleotide, nucleic acid, xenonucleic acid, or derivatives thereof.

27. A first proteinaceous molecule according to any one of claims 1 to 16 or as defined in any one of claims 17 to 23, A composition comprising one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid, or a derivative thereof.

28. 17. An antibody-drug conjugate or a ligand-drug conjugate comprising a first proteinaceous molecule according to any one of claims 1 to 16 and an effector moiety, which is (i) and / or (ii) below: (i) the antibody is selected from the group consisting of CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, and / or may bind to any one of the following: MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2; and / or may bind to any one of the following: the anti-EGFR monoclonal antibody cetuximab, the anti-CD38 monoclonal antibody daratumumab, the anti-CD33 monoclonal antibody gemtuzumab, the anti-HER2 monoclonal antibody trastuzumab, the anti-EGFR monoclonal antibody panitumumab, the anti-C D30 monoclonal antibody brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type, anti-HER2 monoclonal antibody pertuzumab, anti-CD20 monoclonal antibody rituximab, anti-CD20 monoclonal antibody ofatumumab, anti-CD52 monoclonal antibody alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, anti-CD20 monoclonal antibody tositumomab, anti-CD20 monoclonal antibody ibritumomab, anti-CA125 monoclonal the antibody oregovomab, the anti-EpCAM (17-1A) monoclonal antibody edrecolomab, the anti-EGFR monoclonal antibody nimotuzumab, the anti-CD33 monoclonal antibody huMy9-6, the anti-vascular integrin alpha-v beta-3 monoclonal antibody etaracizumab, the anti-CD22 monoclonal antibody epratuzumab, the anti-CEA monoclonal antibody labetuzumab, the anti-CD44v6 monoclonal antibody bivatuzumab, the anti-FAP monoclonal antibody sibrotuzumab, the anti-CD19 monoclonal antibody huB4, the anti-CanAg monoclonal antibody huC242,and / or the antibody-drug conjugate is or comprises any one of the following: anti-CD56 monoclonal antibody huN901, anti-CA6 monoclonal antibody DS6, anti-IGF-IR monoclonal antibody cixutumumab, anti-IGF-IR monoclonal antibody 3B7, anti-integrin monoclonal antibody CNTO95, or anti-syndecan-1 monoclonal antibody B-B4, or at least one tumor cell receptor binding fragment thereof and / or at least one tumor cell receptor binding domain thereof; and / or the antibody-drug conjugate is or comprises any one of the following: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, polatuzumab vedotin, afiltin, IMGN-779, neuradiab, AGS-67E, ASG-15ME, bundletuzumab Vedotin, CDX-014, AGS-16M18, Borsetuzumab mafodotin, Denintuzumab mafodotin, SGN-CD70A, RG-7636, SC-006, MM-310, PF-06647263, PF-06263507, PF-06650808, XMT-1522, AMG-595, Pinatuzumab vedotin, Cantuzumab ravtansine, AVE-9633, BIWI-1, RG-7882, ASG-5ME, DCDS-0780A, SC-004, RG -7600, Sofituzumab Vedotin, IMGN-289, SAR-428926, SGNCD-19B, SGNCD-123A, SGNCD-352A, RG-7841, IMGN-388, lorvotuzumab mertansine, BAY-794620, RG-7598, oncolycin B, ADCT-502, AMG-172, ImmuRAIT-LL2, indusatumab vedotin, clivatuzumab tetraxetan, depatuximab mafodotin, vadatuximab taliline, MLN-2704, cortuximab ravtansine, rifatuzumab At least one of vedotin, visumab-A, avicidin, SGN-15, ASP-6183, SAR-566658, oncolysin S, glenbatumumab vedotin, MM-302, HTI-1511, ZW-33, HuMax-CD74-ADC, sacituzumab govitecan, ibritumomab tiusetan, and rovalpituzumab tesirin. (ii) the effector moiety is any one or more of the effector moieties according to claim 26;

29. A pharmaceutical composition comprising the antibody-drug conjugate or ligand-drug conjugate of claim 28, and optionally further comprising a pharma- ceutically acceptable excipient.

30. A therapeutic composition according to any one of claims 17 to 25, or a composition according to claim 26 or 27, or an antibody-drug or ligand-drug conjugate according to claim 28, or a pharmaceutical composition according to claim 29, for use as a medicament or for use in the treatment or prevention of cancer or an autoimmune disease.

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