Combinations comprising an ADC or AOC comprising a VHH and a saponin or a ligand-saponin conjugate

Abstract

The present invention includes a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, and a second conjugate comprising saponin and / or a derivative thereof, and a binding molecule for binding to a second cell surface molecule, wherein the saponin or derivative thereof is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside. The present invention also relates to a composition comprising the first conjugate and a saponin (derivative) or a second conjugate comprising a saponin (derivative). Furthermore, the present invention relates to the pharmaceutical combination or pharmaceutical composition of the present invention for use as a medicament, or for use in the treatment or prevention of cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, gene deficiency, diseases associated with gene deficiency, amyloidosis, diseases associated with enzyme deficiency, infectious diseases such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, α1-antitrypsin-associated liver disease, acute hepatic porphyria, and transthyretin-mediated amyloidosis. Furthermore, the present invention relates to a method for transferring the first conjugate of the present invention from outside a cell into said cell, preferably into the cytosol of said cell, in vitro or ex vivo.

Description

[Technical field]

[0001] The present invention comprises a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, and a second conjugate comprising saponin, a derivative thereof, or a binding molecule for binding to a second cell surface molecule and saponin and / or a derivative thereof, wherein the saponin or derivative thereof is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside. The invention also relates to a composition comprising the first conjugate and saponin (derivative), or to a composition comprising the first conjugate and a second conjugate comprising saponin (derivative). Furthermore, the invention relates to a pharmaceutical combination or pharmaceutical composition of the invention for use as a medicament and for use in the treatment or prevention of cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, gene deficiency, diseases associated with gene deficiency, amyloidosis, diseases associated with enzyme deficiency, infectious diseases such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha 1-antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis. Furthermore, the present invention relates to an in vitro or ex vivo method for transferring a first conjugate of the present invention from outside a cell into said cell, preferably into the cytosol of said cell. The present invention also relates to an in vitro or ex vivo method for transferring a first conjugate of the present invention from outside a cell into said cell, and to an in vitro or ex vivo method for transferring an effector molecule comprised in the first conjugate into the cytosol of said cell, once the first conjugate has been transferred from outside a cell into said cell, e.g. into an endosome, endolysosome or lysosome of said cell. [Background technology]

[0002] Molecules with therapeutic biological activity are often theoretically suitable for application to human patients in need thereof as effective therapeutic agents for the treatment of diseases such as cancer. A typical example is a biologically active moiety of a small molecule. However, many, if not all, of the potential drug-like molecules and therapeutic agents currently used in the clinic suffer from at least one of a number of defects and shortcomings. When administered to the human body, therapeutically active molecules may exert off-target effects in addition to the biological activity directed at the underlying aspects of the disease or health problem to be treated. Such off-target effects carry the risk of inducing undesirable, health- or life-threatening side effects of the administered molecule. It is the occurrence of such adverse events that causes many drug-like compounds and therapeutic moieties to fail Phase III or Phase IV clinical trials (post-marketing follow-up). Thus, there is a strong need to provide drug molecules, such as small molecule therapeutics, whose therapeutic effect is, for example, (1) highly specific against disease-causing biological factors or biological processes, (2) sufficiently safe, (3) sufficiently effective, (4) sufficiently directed to diseased cells with little or no off-target activity against non-diseased cells, (5) having a sufficiently timely mode of action (e.g., the administered drug molecule reaches the target site in a human patient in a certain time frame and remains at the target site for a certain time frame), and / or (6) especially, has a sufficiently long therapeutic activity in the patient's body. Unfortunately, to date, the "ideal" therapeutic drug, which has many or all of the beneficial characteristics outlined above, has not yet been available to patients, despite already being long and intensively researched, and despite significant progress in some areas of the difficulties and shortcomings faced individually.

[0003] Chemotherapy is one of the most important therapeutic options for cancer treatment. However, it is often associated with a low therapeutic window, since it is less specific for cancer cells than for dividing cells in healthy tissues. The invention of monoclonal antibodies offered the possibility to exploit their specific binding properties as a mechanism for targeted delivery of cytotoxic agents to cancer cells while sparing normal cells. This can be achieved by chemical conjugation of cytotoxic effectors (also known as payloads or warheads) to antibodies to create antibody-drug conjugates (ADCs). Typically, highly potent payloads are used, such as emtansine (DM1), which has a limited therapeutic index (ratio comparing the toxic dose to the effective dose) in their unconjugated form. Conjugation of DM1 to trastuzumab (ado-trastuzumab-emtansine), also known as Kadcycla, enhances the tolerable dose of DM1 at least two-fold in monkeys. In the past decades, great efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, it remains difficult to introduce ADCs into the clinic. The first ADC approved for clinical use was gemtuzumab ozogamicin (Mylotarg, targeting CD33, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML) in 2000. However, Mylotarg had many concerns, including its safety profile, and was withdrawn from the market at the request of the US Food and Drug Administration (FDA). Patients treated with Mylotarg were found to have a higher rate of death than those treated with conventional chemotherapy. Mylotarg was approved for market reintroduction in 2017 with a lower recommended dose, a different schedule in combination with chemotherapy or alone, and a new patient population. Only five ADCs have been approved for clinical use to date, and clinical development of approximately 55 ADCs has been discontinued in the meantime. However, interest remains high, and approximately 80 ADCs are still in clinical development, with nearly 600 clinical trials currently underway.

[0004] Despite the possibility of using highly toxic payloads that patients usually cannot tolerate, a poor therapeutic index (the ratio of toxic dose compared to effective dose) is a major issue that has forced the discontinuation of many ADCs in clinical development, which can be caused by several mechanisms, such as off-target toxicity against normal cells, development of resistance to cytotoxic drugs, and premature release of the drug in the circulation. A systematic review by the FDA of ADCs found that the toxicity profile of most ADCs could be classified by the payload used and not by the antibody used, suggesting that most of the toxicity is determined by premature release of the payload. It is estimated that at least 23 of the approximately 55 ADCs that have been discontinued were due to a poor therapeutic index. For example, the development of a trastuzumab tesillin conjugate (ADCT-502, an ADC therapeutic targeting HER-2) was recently halted due to a narrow therapeutic index, likely due to on-target, off-tissue effects in lung tissues that express significant levels of HER-2. 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 (ABT-414, targeting EGFR, AbbVie) tested in patients with newly diagnosed glioblastoma, and a mirvetuximab soravtansine conjugate (IMGN853, targeting folate receptor alpha (FRα), ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently halted and showed no survival benefit. It is important to note that the clinically used doses of some ADCs may not be sufficient for their full anticancer activity. For example, ado-trastuzumab emtansine has an MTD of 3.6 mg / kg in humans. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels of 3 mg / kg and above, 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 are mainly composed of an antibody, a cytotoxic moiety such as a payload, and a linker. To overcome the existing problems, several novel strategies have been proposed and implemented in the design and development of new ADCs that target each of the components of the ADC. For example, by identifying and validating suitable antigenic targets for the antibody components, by selecting antigens with high expression levels in tumors and little or no expression in normal tissues, antigens present on the cell surface that are accessible to circulating ADCs, and antigens that allow internalization of ADCs into cells after binding; Also, by alternative mechanisms of activity, new clinical and translational strategies are being developed to maximize therapeutic index, such as modifying dosing schedules, e.g., by split dosing; performing biodistribution studies; including biomarkers to optimize patient selection; capturing early response signals and monitoring duration and depth of response; and informing combination studies.

[0006] Examples of ADCs with clinical potential include brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, which are being evaluated as treatment options for lymphoid malignancies and multiple myeloma. Polatuzumab vedotin, which binds to CD79B on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, are being tested in clinical trials in which each ADC is combined with co-administration of rituximab, a monoclonal antibody that binds to CD20 and does not provide a payload [B. Yu and D. Liu, Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12: 94]. Combinations of monoclonal antibodies such as these examples are further approaches and attempts to arrive at a "magic bullet" that combines many or all of the aforementioned desired characteristics of ADCs.

[0007] Meanwhile, in the past decades, therapeutic drugs based on nucleic acids have been under development. Therapeutic nucleic acids can be based on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), and small interfering RNA (siRNA), microRNA, as well as DNA and RNA aptamers, for approaches such as gene therapy, RNA interference (RNAi). Many of them share the same basic principle of action by inhibiting DNA expression or RNA expression, thereby preventing the expression of disease-related abnormal proteins. The largest number of clinical trials are being conducted in the field of gene therapy, with almost 2600 clinical trials underway or completed worldwide, but only about 4% enter phase 3 trials. Clinical trials with ASOs are then conducted. Similar to ADCs, despite the large number of technologies being explored, therapeutic nucleic acids share two major problems during clinical development: delivery to cells and off-target effects. For example, ASOs such as peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), locked nucleic acids (LNAs) and bridged nucleic acids (BNAs) have been investigated as an attractive strategy to specifically inhibit target genes, especially those that are difficult to target with small molecule inhibitors or neutralizing antibodies. Currently, the efficacy of various ASOs is being investigated in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in several stages of cancer. To apply ASOs as potential therapeutic agents, a safe and effective method is required to deliver them 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 limits their efficacy and represents a barrier to therapeutic development. Cellular uptake is less than 2% of the dose, which may result in ASO concentrations at the active site that are too low to obtain effective and sustained outcomes. Thus, increased dosage is required, which induces 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, but their effectiveness is hindered by severe off-target side toxicities, as well as their low 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] Such conjugate principles have attracted much attention and been studied for decades. The majority of small molecule drug conjugates in preclinical or clinical development are for oncological indications. However, the only current, non-cancer-related drug was approved in 2014 for a non-oncological indication: opioid-induced constipation in chronic pain patients (Movantik, a PEG oligomer conjugate of the opioid antagonist naloxone, AstraZeneca). Translation of drug-scaffold conjugates to therapeutic applications in human subjects has so far provided little clinical benefit. For example, PK1 (N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) showed great anticancer activity in both solid tumors and leukemia in mouse models and was under clinical investigation for oncological indications. Despite showing a significant reduction in nonspecific toxicity and improved pharmacokinetics in humans, it was found to have only marginal improvements in anticancer efficacy in patients, and as a result, further development of PK1 was discontinued.

[0010] The failure of scaffold-small molecule drug conjugates is at least partly due to their poor accumulation at tumor sites. For example, in mouse models, PK1 showed 45-250-fold higher accumulation in tumors than in healthy tissues (liver, kidney, lung, spleen, and heart), but in clinical trials, accumulation in tumors was only observed in a minority of patients.

[0011] A potential solution to the aforementioned problems is the application of nanoparticle systems for drug delivery, such as liposomes. Liposomes are spherical vesicles composed of one or more phospholipid bilayers, which form spontaneously when phospholipids are dispersed in water. The amphiphilic nature of phospholipids provides it with self-organizing, emulsifying and wettability properties, which can be used in the design of new drugs and new drug delivery systems. Drugs encapsulated in liposomal delivery systems may offer several advantages over direct administration of drugs, such as improved and controlled pharmacokinetics and pharmacodynamics, tissue targeting properties, reduced toxicity, and enhanced drug activity. Such successful examples are the liposomal encapsulated forms of the small molecule chemotherapeutic agent doxorubicin (Doxil: pegylated liposomal encapsulated form of doxorubicin; Myocet: non-pegylated liposomal doxorubicin), which have been approved for clinical use.

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

[0013] EP 1 623 715 B1 describes a composition comprising a pharmacologically active agent bound to a target cell specific binding molecule in combination with saponin, the target cell specific binding molecule being a full-length monoclonal immunoglobulin G type antibody. The pharmacologically active agent is, for example, a toxin. Summary of the Invention [Means for solving the problem]

[0014] A first aspect of the present invention relates to a pharmaceutical combination comprising: - a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, which are covalently linked to each other directly or via a linker; - a second conjugate comprising a saponin and / or a derivative thereof, and / or a binding molecule for binding to a second cell surface molecule, and a saponin and / or a derivative thereof, wherein the binding molecule and the saponin or a derivative thereof are covalently bound to each other directly or via a linker, and the saponin or a derivative thereof is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside, and the second cell surface molecule is the same as or different from the first cell surface molecule; and, optionally, - a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent.

[0015] One embodiment is a pharmaceutical combination according to the invention in the form of at least two, preferably two, pharmaceutical compositions comprising: - a first pharmaceutical composition comprising a first conjugate, and optionally a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent; and - a second pharmaceutical composition comprising a saponin and / or a derivative thereof and / or a second conjugate, and optionally a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent.

[0016] One embodiment comprises a first conjugate, and ·saponin; Saponin derivatives; and Second conjugate At least one, preferably one of and optionally a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent, in the form of a single pharmaceutical composition.

[0017] A second aspect of the present invention relates to a pharmaceutical combination of the present invention for use as a medicament.

[0018] A third aspect of the present invention relates to a pharmaceutical combination of the present invention for use in the treatment or prevention of cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, gene deficiency, diseases associated with gene deficiency, amyloidosis, diseases associated with enzyme deficiency, infectious diseases such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha 1-antitrypsin associated liver disease, acute hepatic porphyria, transthyretin mediated amyloidosis.

[0019] A fourth aspect of the present invention relates to an in vitro or ex vivo method for transferring a first conjugate of the invention from outside a cell into said cell, preferably into the cytosol of said cell, comprising the steps: a) providing a cell expressing on its surface a first cell surface molecule according to the invention and optionally expressing on its surface a second cell surface molecule according to the invention, said cell being preferably selected from diseased cells such as liver cells, virally infected cells, autoimmune cells and tumour cells; b) providing a first conjugate of the invention for transfer to the cell provided in step a); c) providing a saponin or saponin derivative of the invention or, if the cell provided in step a) expresses on its surface a second cell surface molecule according to the invention, providing a second conjugate of the invention; d) contacting the cells of step a) in vitro or ex vivo with the first conjugate of step b) and any one or more of the saponins, saponin derivatives, and conjugates of step c); thereby establishing the transfer of a first conjugate from outside a cell into said cell, preferably into the cytosol of said cell.

[0020] definition The term "proteinaceous" has its ordinary scientific meaning and refers herein to a molecule that is proteinaceous, meaning that the molecule has some physicochemical properties characteristic of a protein, has the properties of a protein, is related to a protein, contains a protein, relates to a protein, resembles a protein, or is a protein. The term "proteinaceous", when used, for example, in "proteinaceous molecule", refers to the presence of at least a portion of a molecule that resembles a protein or is a protein, where "protein" should be understood to include a chain of amino acid residues at least two residues long, and thus includes peptides, polypeptides and proteins, as well as ensembles of proteins or protein domains. In a proteinaceous molecule, at least two amino acid residues are linked by an amide bond, for example a peptide bond. In a proteinaceous molecule, the amino acid residues are natural amino acid residues and / or artificial amino acid residues, such as modified natural amino acid residues. In a preferred embodiment, the proteinaceous molecule is a molecule that comprises at least two amino acid residues, preferably from 2 to about 2,000 amino acid residues. In one embodiment, the proteinaceous molecule is a molecule that comprises from 2 to 20 amino acids (typical of peptides). In one embodiment, the proteinaceous molecule is a molecule comprising between 21 and 1,000 amino acids (typical of polypeptides, proteins, protein domains, e.g. antibodies, Fabs, scFvs, single domain antibodies, ligands for receptors such as EGF). Preferably, the amino acid residues are (typically) linked by peptide bonds. According to the invention, said amino acid residues are or comprise (modified) (non-)natural amino acid residues.

[0021] The term "effector molecule" or "effector moiety", when referring to an effector molecule, for example as part of a covalent conjugate, has its ordinary scientific meaning and refers herein to a molecule that can selectively bind to any one or more of target molecules, for example proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, enzymes, and modulate the biological activity of such one or more target molecules. Effector molecules are, for example, molecules selected from any one or more of small molecules, such as drug molecules, toxins, such as protein toxins, oligonucleotides, such as BNAs, xenonucleic acids, or siRNAs, enzymes, peptides, proteins, or any combination thereof. Thus, for example, an effector molecule or effector moiety is a molecule or moiety selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, xenonucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, which can selectively bind to any one or more of target molecules, such as proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, enzymes, and modulates the biological activity of such one or more target molecules upon binding to the target molecule. Typically, an effector molecule is capable of exerting a biological effect within a cell, such as a mammalian cell, such as a human cell, for example within the cytosol of said cell. Thus, typical effector molecules are drug molecules, plasmid DNA, toxins such as toxins contained in antibody drug conjugates (ADCs), oligonucleotides such as siRNAs, BNAs, nucleic acids, etc. contained in antibody oligonucleotide conjugates (AOCs). For example, an effector molecule is a molecule that can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling. In the context of the present invention, an effector molecule or effector moiety is not a saponin and is not a cell surface molecule binding molecule such as an antibody such as an sdAb when the effector molecule is part of a conjugate.

[0022] The term "modified saponin" has its ordinary scientific meaning and is used herein to refer to a saponin, i.e., a saponin derivative, having one or more chemical modifications at the positions where any of the aldehyde, carboxyl, acetate, and / or acetyl groups were present in the underivatized saponin prior to being subjected to chemical modification to provide the modified saponin. For example, a modified saponin is provided by chemically modifying any one or more of the aldehyde, carboxyl, acetate, and / or acetyl groups in the saponin on which the modified saponin is based. That is, a saponin is provided and any of the aldehyde, carboxyl, acetate, and / or acetyl groups are chemically modified to provide the modified saponin. For example, the saponin that is modified to provide the modified saponin is a naturally occurring saponin. Typically, the modified saponin is a synthetic saponin, typically the modified saponin is a modification of a naturally occurring saponin and is therefore derived from a naturally occurring saponin, however the modified saponin can also be derived from a synthetic saponin which may or may not have a natural counterpart. Typically the modified saponin does not have a natural counterpart, i.e. the modified saponin is not naturally produced by, for example, a plant or tree.

[0023] The term "aglycone core structure" has its usual scientific meaning and is used herein to refer to the aglycone core, or aglycone for short, of a saponin to which one or two carbohydrate antennae or sugar chains (glycans) are not attached. For example, chiral acids are the aglycone core structures of SO1861, QS-7 and QS21. Typically, the glycans of a saponin are monosaccharides or oligosaccharides, such as linear or branched glycans.

[0024] The term "glycan" has its usual scientific meaning and is used herein to refer to either a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide), or a chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycan can consist of only sugar moieties or can also contain further moieties such as any one of 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid, and 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, as present in QS-21.

[0025] The term "chemically modified" has its ordinary scientific meaning and refers herein to the chemical modification of a first chemical group or moiety to provide a second chemical group or moiety. Examples are the chemical modification of a carbonyl group to a -(H)C-OH group, the chemical modification of an acetate group to a hydroxyl group, providing a saponin having an N-ε-maleimidocaproic acid hydrazide (EMCH) moiety attached to its aldehyde group by a chemical reaction, etc.

[0026] The term "chemically modified aldehyde group" has its ordinary scientific meaning and, as used herein, refers to a chemical reaction product resulting from a chemical reaction involving the aldehyde group of a saponin that results in the replacement of the original aldehyde group with a new chemical group, e.g., the formation of a -(H)C-OH group from the initial aldehyde group of the saponin.

[0027] The term "chemically modified carboxyl group" has its usual scientific meaning and refers herein to a chemical reaction product obtained by a chemical reaction involving a carboxyl group of a saponin, such as the carboxyl group of a glucuronic acid moiety, and an additional molecule, resulting in the replacement of the original aldehyde group with a new chemical group. For example, the formation of a conjugate between a saponin and any one of 2-amino-2-methyl-1,3-propanediol (AMPD), N-(2-aminoethyl)maleimide (AEM), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (including the carboxyl group of the glucuronic acid of the saponin).

[0028] The term "Api / Xyl-" or "Api- or Xyl-" in the context of glycan names has its ordinary scientific meaning and refers herein to glycans that contain either an apiose (Api) moiety or a xylose (Xyl) moiety.

[0029] The term "saponin on which a modified saponin is based" has its ordinary scientific meaning and is used herein to refer to a saponin that has been modified to provide the modified saponin. Typically, the base saponin for a modified saponin is a naturally occurring saponin that has been subjected to chemical modification to provide the modified saponin.

[0030] The term "modified saponin based on saponin" has its ordinary scientific meaning and, as used herein, refers to a saponin that has been subjected to a chemical modification process to provide a modified saponin, which is typically a naturally occurring saponin.

[0031] The term "oligonucleotide" has its ordinary scientific meaning and refers herein to any natural or synthetic string of nucleic acid, including, inter alia, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, such as BNAs, antisense oligonucleotides (ASOs), short or small interfering RNAs (siRNAs; silencing RNAs), antisense DNA, antisense RNA, etc., presented as single-stranded or double-stranded molecules.

[0032] The term "antibody drug conjugate" or "ADC" has its ordinary scientific meaning and is used herein to refer to any of a wide variety of antibodies, including IgG, Fab, scFv, immunoglobulins, immunoglobulin fragments, one or more V H Domains, Single Domain Antibodies, V HH , Camelidae V H and any molecule, e.g., active pharmaceutical ingredient, toxin, oligonucleotide, enzyme, small molecule drug compound, etc., that can exert a therapeutic effect when contacted with the cells of a subject, such as a human patient.

[0033] The term "antibody oligonucleotide conjugate" or "AOC" has its ordinary scientific meaning and is used herein to refer to any of a wide variety of antibodies, including IgG, Fab, scFv, immunoglobulins, immunoglobulin fragments, one or more VFvs, and the like. H Domains, Single Domain Antibodies, V HH , Camelidae V Hand any conjugate of an antibody, such as an antibody or antibody fragment thereof, with any oligonucleotide molecule capable of exerting a therapeutic effect when contacted with a cell of a subject, such as a human patient, e.g., any conjugate of an antibody or antibody fragment thereof, such as an antibody or antibody fragment thereof, with any oligonucleotide molecule, such as an antibody or antibody fragment thereof, that is ... that is capable of exerting a therapeutic effect when contacted with a cell of a subject, such as a human patient,

[0034] The term "bridged nucleic acid" or abbreviated "BNA" or "locked nucleic acid" or abbreviated "LNA" has its usual scientific meaning and is used herein to refer to modified RNA nucleotides. BNAs are also referred to as "constrained RNA molecules" or "restricted access RNA molecules". BNA monomers can contain 5-, 6- or 7-membered bridge structures with "locked" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2',4' position of the ribose to give a 2',4'-BNA monomer. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNAs are structurally rigid oligonucleotides with high binding affinity and stability.

[0035] The term "S" as used in such linker-containing antibody-saponin conjugates indicates a "stable linker" that remains intact in endosomes, endolysosomes, and lysosomes of mammalian cells, such as human cells, such as human tumor cells, and thus under slightly acidic conditions (pH<6.6, e.g., pH 4.0-5.5).

[0036] The term "L" as used in linker-containing antibody-saponin conjugates and the like, refers to a "labile linker" that is cleaved under the slightly acidic conditions (pH<6.6, e.g., pH 4.0-5.5) of endosomes, endolysosomes, and lysosomes of mammalian cells, such as human cells, such as human tumor cells.

[0037] Terms such as first, second, third, etc. in the specification and claims are used, for example, to distinguish between similar elements, compositions, ingredients in a composition, or individual steps of a method, and not necessarily to describe a sequential or chronological order. The terms are interchangeable under appropriate circumstances, and embodiments of the invention can operate in other sequences than described or illustrated herein, unless otherwise specified.

[0038] The embodiments of the invention described herein can be combined and operated in concert unless otherwise specified.

[0039] Furthermore, although various embodiments may be referred to as "preferably" or "eg" or "for example" or "particularly," these are not intended to limit the scope of the invention but rather are intended to be exemplary ways in which the invention may be practiced.

[0040] The term "comprises" as used in the claims should not be construed as being limited to, for example, the subsequently recited elements or method steps or composition components, and does not exclude other elements or other method steps or other components in a particular composition. It should be construed as specifying the presence of the mentioned feature, integer value, (method) step or component, but not excluding the presence or addition of one or more other features, integer values, steps or components, or groups thereof. Thus, the scope of the expression "a method comprising steps A and B" should not be limited to a method consisting of only steps A and B, but rather, for the present invention, the recited steps of the method are only A and B, and the claims should be construed to include equivalents of those method steps. Thus, the scope of the expression "a composition comprising components A and B" should not be limited to a composition consisting of only components A and B, but rather, for the present invention, the recited components of the composition are only A and B, and the claims should be construed to include equivalents of those components.

[0041] Furthermore, reference to an element or component by the indefinite article "a" or "an" does not exclude the possibility that more than one element or component is present, unless the context clearly requires that one or only one element or component is present. Thus, the indefinite article "a" or "an" normally means "at least one".

[0042] The term "Saponinum album" has its ordinary meaning and, as used 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.

[0043] The term "Quillajasaponin" has its ordinary meaning and, as used herein, refers to the saponin fraction of Quillaja saponaria, and therefore the source of all other QS saponins, containing primarily QS-18 and QS-21.

[0044] "QS-21" or "QS21" has its ordinary scientific meaning and, as used herein, refers to a mixture of QS-21 A-apio (about 63%), QS-21 A-xylo (about 32%), QS-21 B-apio (about 3.3%), and QS-21 B-xylo (about 1.7%).

[0045] Similarly, "QS-21A" has its ordinary scientific meaning and is used herein to refer to a mixture of QS-21 A-apio (about 65%) and QS-21 A-xylo (about 35%).

[0046] Similarly, "QS-21B" has its ordinary scientific meaning and is used herein to refer to a mixture of QS-21 B-apio (about 65%) and QS-21 B-xylo (about 35%).

[0047] The term "Quil-A" refers to a commercially available semi-purified extract from Quillaja saponaria that contains varying amounts of over 50 different saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcAat at the C-3 β-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 FAKersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja Saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.9, 660-666(1995)]. Quil-A, and also Quillaja saponin, are fractions of saponins from Quillaja saponaria, both of which contain a wide variety of different saponins with largely overlapping contents. The two fractions differ in their specific composition because the two fractions are obtained by different purification procedures.

[0048] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. QS1861 has a molecular weight of 1861 Daltons and QS1862 has a molecular weight of 1862 Daltons. QS1862 is described in line 28 of Table 1 of 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 described is the api variant of QS-7, QS1862. The molecular weight is the formal mass including the proton on the glucuronic acid, so the molecular weight is 1862 daltons. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in negative ion mode, the measured mass is 1861 daltons. [Brief description of the drawings]

[0049] [Figure 1A] Targeted two-component approach (one target): SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) mAb-toxin and VHH-SO1861 bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 is activated allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, inducing cell death. [Figure 1B]Targeted two-component approach (two targets): SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) mAb-toxin and VHH-SO1861 bind to the corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 is activated allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs and induces cell death. [Figure 1C] Targeted two-component approach (two targets): SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH for delivery and internalization into target cells. 1) VHH-toxin and VHH-SO1861 bind to the corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 is activated allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs and induces cell death. [Figure 1D] Targeted two-component approach (two targets): SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH or mAb for delivery and internalization into target cells. 1) VHH-toxin and mAb-SO1861 bind to the corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 is activated allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs and induces cell death. [Figure 1E]Targeted two-component approach (one target): SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) VHH-toxin and mAb-SO1861 bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 is activated allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs and induces cell death. [Figure 2A] Cell killing (MTS) assays of combination treatments according to the invention of VHH(HER2)-SO1861(DAR1)+50pM trastuzumab-saporin (DAR4) or 10pM CD71mab-saporin (DAR4) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 2B] Cell killing (MTS) assays of combination treatments according to the invention of VHH(HER2)-SO1861(DAR1)+50pM trastuzumab-saporin (DAR4) or 10pM CD71mab-saporin (DAR4) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 3A] Cell killing (MTS) assays of combination treatments according to the invention of trastuzumab-saporin (DAR4) or CD71-saporin (DAR4) + 900 nM HER2VHH-SO1861 (DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 3B]Cell killing (MTS) assays of combination treatments according to the invention of trastuzumab-saporin (DAR4) or CD71-saporin (DAR4) + 900 nM HER2VHH-SO1861 (DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 4A] Cell killing (MTS) assays of combination treatment according to the invention of VHH(HER2)-SO1861(DAR1)+50pM CD71VHH-Gianthin(DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 4B] Cell killing (MTS) assays of combination treatment according to the invention of VHH(HER2)-SO1861(DAR1)+50pM CD71VHH-Gianthin(DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 5A] Cell killing (MTS) assay of combination treatment according to the invention of CD71VHH-Gianthin (DAR1) + 900 nM HER2VHH-SO1861 (DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 5B] Cell killing (MTS) assay of combination treatment according to the invention of CD71VHH-Gianthin (DAR1) + 900 nM HER2VHH-SO1861 (DAR1) against SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 6A]Cell killing (MTS) assays of combination treatments according to the invention of CD71 VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) or HER2 VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) against A431 cells (EGFR+= / HER+ / - / CD71+) (A) and A2058 cells (EGFR- / HER+ / - / CD71+) (B). [Figure 6B] Cell killing (MTS) assays of combination treatments according to the invention of CD71 VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) or HER2 VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + Cetuximab-SO1861 (DAR4) against A431 cells (EGFR+= / HER+ / - / CD71+) (A) and A2058 cells (EGFR- / HER+ / - / CD71+) (B). [Figure 7A] Cell killing (MTS) assays of combination treatments according to the invention of CD71 VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) or HER2 VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) against A431 cells (EGFR+= / HER+ / - / CD71+) (A) and MDA-MB-468 cells (HER2- / EGFR++ / CD71+) (B). [Figure 7B] Cell killing (MTS) assays of combination treatments according to the invention of CD71 VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) or HER2 VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + 77 nM cetuximab-SO1861 (DAR4) against A431 cells (EGFR+= / HER+ / - / CD71+) (A) and MDA-MB-468 cells (HER2- / EGFR++ / CD71+) (B). [Figure 8A] 1 target 2 components (EGFR high expression). EGFR targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of 10 pM cetuximab-saporin shows that conjugated SO1861 requires 100-400 fold lower concentration to induce cell killing by cetuximab-saporin compared to unconjugated SO1861. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 kills cells in contrast to unconjugated SO1861 + cetuximab-saporin at 300 nM. Since both cetuximab conjugates compete for the same EGFR receptor, 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 8B also relates to the graph in Figure 8A. Note: The legend shown in Figure 8D also relates to the graph in Figure 8C. [Figure 8B]1 target 2 components (EGFR high expression). EGFR targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of 10 pM cetuximab-saporin shows that conjugated SO1861 requires 100-400 fold lower concentration to induce cell killing by cetuximab-saporin compared to unconjugated SO1861. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 kills cells in contrast to unconjugated SO1861 + cetuximab-saporin at 300 nM. Since both cetuximab conjugates compete for the same EGFR receptor, 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 8B also relates to the graph in Figure 8A. Note: The legend shown in Figure 8D also relates to the graph in Figure 8C. [Figure 8C]1 target 2 components (EGFR high expression). EGFR targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of 10 pM cetuximab-saporin shows that conjugated SO1861 requires 100-400 fold lower concentration to induce cell killing by cetuximab-saporin compared to unconjugated SO1861. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 kills cells in contrast to unconjugated SO1861 + cetuximab-saporin at 300 nM. Since both cetuximab conjugates compete for the same EGFR receptor, 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 8B also relates to the graph in Figure 8A. Note: The legend shown in Figure 8D also relates to the graph in Figure 8C. [Figure 8D]1 target 2 components (EGFR high expression). EGFR targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of 10 pM cetuximab-saporin shows that conjugated SO1861 requires 100-400 fold lower concentration to induce cell killing by cetuximab-saporin compared to unconjugated SO1861. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 kills cells in contrast to unconjugated SO1861 + cetuximab-saporin at 300 nM. Since both cetuximab conjugates compete for the same EGFR receptor, 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 8B also relates to the graph in Figure 8A. Note: The legend shown in Figure 8D also relates to the graph in Figure 8C. [Figure 9A] 1 target 2 components (EGFR non-expression / low expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of cetuximab-saporin at 10 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 fails to induce cell killing. Low EGFR receptor expression prevents antibody-mediated delivery from delivering sufficient SO1861, while 1500 nM of unconjugated SO1861 induces cell killing. Note: The legend shown in Figure 9B also relates to the graph in Figure 9A. Note: The legend shown in Figure 9D also relates to the graph in Figure 9C. [Figure 9B]1 target 2 components (EGFR non-expression / low expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of cetuximab-saporin at 10 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 fails to induce cell killing. Low EGFR receptor expression prevents antibody-mediated delivery from delivering sufficient SO1861, while 1500 nM of unconjugated SO1861 induces cell killing. Note: The legend shown in Figure 9B also relates to the graph in Figure 9A. Note: The legend shown in Figure 9D also relates to the graph in Figure 9C. [Figure 9C] 1 target 2 components (EGFR non-expression / low expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of cetuximab-saporin at 10 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 fails to induce cell killing. Low EGFR receptor expression prevents antibody-mediated delivery from delivering sufficient SO1861, while 1500 nM of unconjugated SO1861 induces cell killing. Note: The legend shown in Figure 9B also relates to the graph in Figure 9A. Note: The legend shown in Figure 9D also relates to the graph in Figure 9C. [Figure 9D]1 target 2 components (EGFR non-expression / low expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with a fixed concentration of cetuximab-saporin at 10 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 fails to induce cell killing. Low EGFR receptor expression prevents antibody-mediated delivery from delivering sufficient SO1861, while 1500 nM of unconjugated SO1861 induces cell killing. Note: The legend shown in Figure 9B also relates to the graph in Figure 9A. Note: The legend shown in Figure 9D also relates to the graph in Figure 9C. [Figure 10A] 1 target 2 components (HER2 high expression). HER2 targeted cell killing in SKBR3 (HER2+++) cells by therapeutic combination. A) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 673 pM trastuzumab-saporin shows that conjugated SO1861 requires 1000-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with 9,4 nM trastuzumab-SO1861 kills cells in contrast to 10 nM unconjugated SO1861 + trastuzumab-saporin. Since both trastuzumab conjugates compete for the same HER2 receptor, 1075nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both trastuzumab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. SPT001 is SO1861. [Figure 10B]1 target 2 components (HER2 high expression). HER2 targeted cell killing in SKBR3 (HER2+++) cells by therapeutic combination. A) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 673 pM trastuzumab-saporin shows that conjugated SO1861 requires 1000-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with 9,4 nM trastuzumab-SO1861 kills cells in contrast to 10 nM unconjugated SO1861 + trastuzumab-saporin. Since both trastuzumab conjugates compete for the same HER2 receptor, 1075nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination. Only the simultaneous targeted delivery of both trastuzumab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. SPT001 is SO1861. [Figure 11A] 1 target 2 components (HER2 non-expression / low expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression and low EGFR receptor expression prevent sufficient SO1861 delivery by antibody-mediated delivery, while 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 11B also relates to the graph in Figure 11A. Note: The legend shown in Figure 11D also refers to the graph in Figure 11C. SPT001 is SO1861. [Figure 11B]1 target 2 components (HER2 non-expression / low expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression and low EGFR receptor expression prevent sufficient SO1861 delivery by antibody-mediated delivery, while 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 11B also relates to the graph in Figure 11A. Note: The legend shown in Figure 11D also refers to the graph in Figure 11C. SPT001 is SO1861. [Figure 11C] 1 target 2 components (HER2 non-expression / low expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression and low EGFR receptor expression prevent sufficient SO1861 delivery by antibody-mediated delivery, while 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 11B also relates to the graph in Figure 11A. Note: The legend shown in Figure 11D also refers to the graph in Figure 11C. SPT001 is SO1861. [Figure 11D]1 target 2 components (HER2 non-expression / low expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression and low EGFR receptor expression prevent sufficient SO1861 delivery by antibody-mediated delivery, while 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 11B also relates to the graph in Figure 11A. Note: The legend shown in Figure 11D also refers to the graph in Figure 11C. SPT001 is SO1861. [Figure 12A]2 targets 2 components (EGFR high expression and HER2 low expression). EGFR / HER2 targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with trastuzumab-saporin at a fixed concentration of 50 pM shows that conjugated SO1861 requires 100-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with cetuximab-SO1861 at 278 nM kills cells in contrast to unconjugated SO1861 + trastuzumab-saporin at 300 nM. Since both conjugates do not compete for the same receptor, unconjugated SO1861 + trastuzumab-saporin at 1500 nM has comparable cell killing efficiency compared to the therapeutic combination, cetuximab-SO1861 + trastuzumab-saporin at 278 nM. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. Note: The legend shown in Figure 12B also relates to the graph in Figure 12A. Note: The legend shown in Figure 12D also relates to the graph in Figure 12C. SPT001 is SO1861. [Figure 12B]2 targets 2 components (EGFR high expression and HER2 low expression). EGFR / HER2 targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with trastuzumab-saporin at a fixed concentration of 50 pM shows that conjugated SO1861 requires 100-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with cetuximab-SO1861 at 278 nM kills cells in contrast to unconjugated SO1861 + trastuzumab-saporin at 300 nM. Since both conjugates do not compete for the same receptor, unconjugated SO1861 + trastuzumab-saporin at 1500 nM has comparable cell killing efficiency compared to the therapeutic combination, cetuximab-SO1861 + trastuzumab-saporin at 278 nM. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. Note: The legend shown in Figure 12B also relates to the graph in Figure 12A. Note: The legend shown in Figure 12D also relates to the graph in Figure 12C. SPT001 is SO1861. [Figure 12C]2 targets 2 components (EGFR high expression and HER2 low expression). EGFR / HER2 targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with trastuzumab-saporin at a fixed concentration of 50 pM shows that conjugated SO1861 requires 100-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with cetuximab-SO1861 at 278 nM kills cells in contrast to unconjugated SO1861 + trastuzumab-saporin at 300 nM. Since both conjugates do not compete for the same receptor, unconjugated SO1861 + trastuzumab-saporin at 1500 nM has comparable cell killing efficiency compared to the therapeutic combination, cetuximab-SO1861 + trastuzumab-saporin at 278 nM. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. Note: The legend shown in Figure 12B also relates to the graph in Figure 12A. Note: The legend shown in Figure 12D also relates to the graph in Figure 12C. SPT001 is SO1861. [Figure 12D]2 targets 2 components (EGFR high expression and HER2 low expression). EGFR / HER2 targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-SO1861 in combination with trastuzumab-saporin at a fixed concentration of 50 pM shows that conjugated SO1861 requires 100-fold lower concentration to induce cell killing by trastuzumab-saporin compared to unconjugated SO1861. C, D) Titration of trastuzumab-saporin in combination with cetuximab-SO1861 at 278 nM kills cells in contrast to unconjugated SO1861 + trastuzumab-saporin at 300 nM. Since both conjugates do not compete for the same receptor, unconjugated SO1861 + trastuzumab-saporin at 1500 nM has comparable cell killing efficiency compared to the therapeutic combination, cetuximab-SO1861 + trastuzumab-saporin at 278 nM. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. Note: The legend shown in Figure 12B also relates to the graph in Figure 12A. Note: The legend shown in Figure 12D also relates to the graph in Figure 12C. SPT001 is SO1861. [Figure 13A]2 targets 2 components (EGFR low expression and HER2 non-expression / low expression). EGFR / HER2 targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-SO1861 does not enhance cell killing, whereas 1500 nM unconjugated SO1861 induces efficient cell killing. Low EGFR receptor expression prevents sufficient SO1861 from being delivered by antibody-mediated delivery. Note: The legend shown in Figure 13B also relates to the graph in Figure 13A. Note: The legend shown in Figure 13D also relates to the graph in Figure 13C. SPT001 is SO1861. [Figure 13B] 2 targets 2 components (EGFR low expression and HER2 non-expression / low expression). EGFR / HER2 targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-SO1861 does not enhance cell killing, whereas 1500 nM unconjugated SO1861 induces efficient cell killing. Low EGFR receptor expression prevents sufficient SO1861 from being delivered by antibody-mediated delivery. Note: The legend shown in Figure 13B also relates to the graph in Figure 13A. Note: The legend shown in Figure 13D also relates to the graph in Figure 13C. SPT001 is SO1861. [Figure 13C]2 targets 2 components (EGFR low expression and HER2 non-expression / low expression). EGFR / HER2 targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-SO1861 does not enhance cell killing, whereas 1500 nM unconjugated SO1861 induces efficient cell killing. Low EGFR receptor expression prevents sufficient SO1861 from being delivered by antibody-mediated delivery. Note: The legend shown in Figure 13B also relates to the graph in Figure 13A. Note: The legend shown in Figure 13D also relates to the graph in Figure 13C. SPT001 is SO1861. [Figure 13D] 2 targets 2 components (EGFR low expression and HER2 non-expression / low expression). EGFR / HER2 targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-SO1861 does not enhance cell killing, whereas 1500 nM unconjugated SO1861 induces efficient cell killing. Low EGFR receptor expression prevents sufficient SO1861 from being delivered by antibody-mediated delivery. Note: The legend shown in Figure 13B also relates to the graph in Figure 13A. Note: The legend shown in Figure 13D also relates to the graph in Figure 13C. SPT001 is SO1861. [Figure 14A]2 targets 2 components (HER2 high expression and EGFR low expression). HER2 targeted cell killing in SKBR3 (HER2+++ / EGFR+ / -) cells by therapeutic combination. A) Titration of trastuzumab-SO1861 in combination with EGF Giantin at a fixed concentration of 1.5 pM shows that the conjugated SO1861 requires 400 times lower concentration to induce cell killing by EGF Giantin compared to unconjugated SO1861. B) Titration of EGF Giantin in combination with trastuzumab-SO1861 at 9.4 nM can kill cells in contrast to unconjugated SO1861 + EG Giantin at 10 nM. Since both conjugates do not compete for the same receptor, 1075nM of unconjugated SO1861+EGF Giantin has equivalent cell killing efficiency compared to the therapeutic combination, 9.4nM of trastuzumab-SO1861+EGF Giantin. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. SPT001 is SO1861. [Figure 14B]2 targets 2 components (HER2 high expression and EGFR low expression). HER2 targeted cell killing in SKBR3 (HER2+++ / EGFR+ / -) cells by therapeutic combination. A) Titration of trastuzumab-SO1861 in combination with EGF Giantin at a fixed concentration of 1.5 pM shows that the conjugated SO1861 requires 400 times lower concentration to induce cell killing by EGF Giantin compared to unconjugated SO1861. B) Titration of EGF Giantin in combination with trastuzumab-SO1861 at 9.4 nM can kill cells in contrast to unconjugated SO1861 + EG Giantin at 10 nM. Since both conjugates do not compete for the same receptor, 1075nM of unconjugated SO1861+EGF Giantin has equivalent cell killing efficiency compared to the therapeutic combination, 9.4nM of trastuzumab-SO1861+EGF Giantin. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, in contrast to monotherapy using either conjugate alone. SPT001 is SO1861. [Figure 15A]2 targets 2 components (HER2 low expression and EGFR low or high expression). HER2 targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-SO1861 in combination with cetuximab-saporin at a fixed concentration of 5 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 cannot induce cell killing. Low HER2 receptor expression prevents antibody-mediated delivery of sufficient SO1861, while 1500 nM of unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels for the delivery of cetuximab-saporin (D) did not change its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck in cell killing activity lies in too low expression levels of HER2, leading to insufficient SO1861 inside the target cells to switch on endosomal escape. Note: The legend shown in Figure 15B also relates to the graph in Figure 15A. Note: The legend shown in Figure 15D also relates to the graph in Figure 15C. SPT001 is SO1861. [Figure 15B]2 targets 2 components (HER2 low expression and EGFR low or high expression). HER2 targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-SO1861 in combination with cetuximab-saporin at a fixed concentration of 5 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 cannot induce cell killing. Low HER2 receptor expression prevents antibody-mediated delivery of sufficient SO1861, while 1500 nM of unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels for the delivery of cetuximab-saporin (D) did not change its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck in cell killing activity lies in too low expression levels of HER2, leading to insufficient SO1861 inside the target cells to switch on endosomal escape. Note: The legend shown in Figure 15B also relates to the graph in Figure 15A. Note: The legend shown in Figure 15D also relates to the graph in Figure 15C. SPT001 is SO1861. [Figure 15C]2 targets 2 components (HER2 low expression and EGFR low or high expression). HER2 targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-SO1861 in combination with cetuximab-saporin at a fixed concentration of 5 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 cannot induce cell killing. Low HER2 receptor expression prevents antibody-mediated delivery of sufficient SO1861, while 1500 nM of unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels for the delivery of cetuximab-saporin (D) did not change its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck in cell killing activity lies in too low expression levels of HER2, leading to insufficient SO1861 inside the target cells to switch on endosomal escape. Note: The legend shown in Figure 15B also relates to the graph in Figure 15A. Note: The legend shown in Figure 15D also relates to the graph in Figure 15C. SPT001 is SO1861. [Figure 15D]2 targets 2 components (HER2 low expression and EGFR low or high expression). HER2 targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-SO1861 in combination with cetuximab-saporin at a fixed concentration of 5 pM does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 cannot induce cell killing. Low HER2 receptor expression prevents antibody-mediated delivery of sufficient SO1861, while 1500 nM of unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels for the delivery of cetuximab-saporin (D) did not change its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck in cell killing activity lies in too low expression levels of HER2, leading to insufficient SO1861 inside the target cells to switch on endosomal escape. Note: The legend shown in Figure 15B also relates to the graph in Figure 15A. Note: The legend shown in Figure 15D also relates to the graph in Figure 15C. SPT001 is SO1861. [Figure 16] Two-target dual component versus T-DM1. Cells with high EGFR expression and low HER2 expression (A431) are efficiently killed with the therapeutic combination, but T-DM1 is not effective at such low toxin concentrations. T-DM1 is trastuzumab-emtansine (Kadcyla®) with approximately 3.5 DM1 toxin molecules per antibody. [Figure 17A]Figure 17 shows relative cell viability when trastuzumab (Figure 17A), cetuximab (Figure 17B) or T-DM1 (Figure 17C), the free toxins saporin (Figure 17D) and dianthin (Figure 17D), saporin conjugated to non-cell-bound IgG (Figure 17D), and saporin conjugated to non-cell-bound IgG combined with free saponin SO1861 (Figure 17E) were contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Note: The legend shown in Figure 17C also refers to the curves in Figures 17A and 17B. [Figure 17B] Figure 17 shows relative cell viability when trastuzumab (Figure 17A), cetuximab (Figure 17B) or T-DM1 (Figure 17C), the free toxins saporin (Figure 17D) and dianthin (Figure 17D), saporin conjugated to non-cell-bound IgG (Figure 17D), and saporin conjugated to non-cell-bound IgG combined with free saponin SO1861 (Figure 17E) were contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Note: The legend shown in Figure 17C also refers to the curves in Figures 17A and 17B. [Figure 17C] Figure 17 shows relative cell viability when trastuzumab (Figure 17A), cetuximab (Figure 17B) or T-DM1 (Figure 17C), the free toxins saporin (Figure 17D) and dianthin (Figure 17D), saporin conjugated to non-cell-bound IgG (Figure 17D), and saporin conjugated to non-cell-bound IgG combined with free saponin SO1861 (Figure 17E) were contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Note: The legend shown in Figure 17C also refers to the curves in Figures 17A and 17B. [Figure 17D]Figure 17 shows relative cell viability when trastuzumab (Figure 17A), cetuximab (Figure 17B) or T-DM1 (Figure 17C), the free toxins saporin (Figure 17D) and dianthin (Figure 17D), saporin conjugated to non-cell-bound IgG (Figure 17D), and saporin conjugated to non-cell-bound IgG combined with free saponin SO1861 (Figure 17E) were contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Note: The legend shown in Figure 17C also refers to the curves in Figures 17A and 17B. [Figure 17E] Figure 17 shows relative cell viability when trastuzumab (Figure 17A), cetuximab (Figure 17B) or T-DM1 (Figure 17C), the free toxins saporin (Figure 17D) and dianthin (Figure 17D), saporin conjugated to non-cell-bound IgG (Figure 17D), and saporin conjugated to non-cell-bound IgG combined with free saponin SO1861 (Figure 17E) were contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, BT-474. Note: The legend shown in Figure 17C also refers to the curves in Figures 17A and 17B. [Figure 18] 1T2C in vivo activity. The 1T2C combination of 50 mg / kg cetuximab-(Cys-L-SO1861)4 + 25 mg / kg cetuximab-(-L-HSP27BNA)4 in A431 tumor-bearing mice reveals potent tumor-targeted gene silencing compared to controls. [Figure 19] 1T2C in vivo activity. The 1T2C combination of 40 mg / kg trastuzumab-(Cys-L-SO1861)4 + 0.02 / 0.03 mg / kg trastuzumab-saporin in PDX tumor mouse models (high HER2 expression) shows effective tumor growth inhibition. [Figure 20] The 2T2 component system tested in the A431 tumor-bearing mouse model demonstrates tumor regression. [Figure 21] The 2T2 component system tested in the A431 tumor-bearing mouse model demonstrates tumor regression and tumor eradication. [Figure 22A] 2 targets 2 components. EGFR / HER2 targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on A431 cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on Caski cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for A and B, legend is the same for figures C and D. [Figure 22B] 2 targets 2 components. EGFR / HER2 targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on A431 cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on Caski cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for A and B, legend is the same for figures C and D. [Figure 22C] 2 targets 2 components. EGFR / HER2 targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on A431 cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on Caski cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for A and B, legend is the same for figures C and D. [Figure 22D]2 targets 2 components. EGFR / HER2 targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on A431 cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on Caski cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for A and B, legend is the same for figures C and D. [Figure 23A] 2 targets, 2 components. EGFR / HER2 targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on HeLa cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on A2058 cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for C and D. [Figure 23B] 2 targets, 2 components. EGFR / HER2 targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on HeLa cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on A2058 cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for C and D. [Figure 23C]2 targets, 2 components. EGFR / HER2 targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on HeLa cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on A2058 cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for C and D. [Figure 23D] 2 targets, 2 components. EGFR / HER2 targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by the therapeutic combination of the invention. A, B) Titration of Cetuximab-(Cys-L-SO1861)3.7 on HeLa cells + Trastuzumab-Saporin at a fixed concentration of 50 pM, and control. C, D) Titration of Trastuzumab-Saporin on A2058 cells + Cetuximab-(Cys-L-SO1861)3.7 at a fixed concentration of 75 nM, and control. Legend and / or axes are the same for C and D. [Figure 24A] 2 targets, 2 components. HER2 / EGFR targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by the therapeutic combination of the present invention. A) Titration of trastuzumab-(Cys-L-SO1861)4 on SKBR3 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. B) Titration of EGF dianthine on SKBR3 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. [Figure 24B]2 targets, 2 components. HER2 / EGFR targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by the therapeutic combination of the present invention. A) Titration of trastuzumab-(Cys-L-SO1861)4 on SKBR3 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. B) Titration of EGF dianthine on SKBR3 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. [Figure 25A] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of Trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. C, D) Titration of EGF dianthine on MDA-MB-468 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same in C and D. [Figure 25B] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of Trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. C, D) Titration of EGF dianthine on MDA-MB-468 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same in C and D. [Figure 25C]2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of Trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. C, D) Titration of EGF dianthine on MDA-MB-468 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same in C and D. [Figure 25D] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of Trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + EGF dianthine at a fixed concentration of 1.5 pM, and control. C, D) Titration of EGF dianthine on MDA-MB-468 cells + Trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same in C and D. [Figure 26A] 2 targets, 2 components. HER2 / EGFR targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by the therapeutic combination of the present invention. A) Titration of trastuzumab-(Cys-L-SO1861)4 on SKBR3 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. B) Titration of cetuximab-saporin on SKBR3 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. [Figure 26B]2 targets, 2 components. HER2 / EGFR targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by the therapeutic combination of the present invention. A) Titration of trastuzumab-(Cys-L-SO1861)4 on SKBR3 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. B) Titration of cetuximab-saporin on SKBR3 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. [Figure 27A] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. C, D) Titration of cetuximab-saporin on MDA-MB-468 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same for A and B, and legends are the same for C and D. [Figure 27B] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. C, D) Titration of cetuximab-saporin on MDA-MB-468 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same for A and B, and legends are the same for C and D. [Figure 27C]2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. C, D) Titration of cetuximab-saporin on MDA-MB-468 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same for A and B, and legends are the same for C and D. [Figure 27D] 2 targets, 2 components. HER2 / EGFR targeted cell killing in JIMT-1 cells (HER2+ / -EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by the therapeutic combination of the present invention. A, B) Titration of trastuzumab-(Cys-L-SO1861)4 on JIMT-1 cells + cetuximab-saporin at a fixed concentration of 10 pM, and control. C, D) Titration of cetuximab-saporin on MDA-MB-468 cells + trastuzumab-(Cys-L-SO1861)4 at a fixed concentration of 2.5 nM, and control. Legends and / or axes are the same for A and B, and legends are the same for C and D. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] A first object of the present invention is to provide improved ADCs and AOCs with an increased therapeutic window, as well as to provide improved therapeutic combinations or improved pharmaceutical compositions for the delivery of an effective amount or effective dose of an effector molecule, for example when delivery from the outside to the inside of a target cell is considered, or more particularly when delivery of the effector molecule in the cytosol of said target cell is considered. A second object of the present invention is to provide an improved method of treatment of a (human) patient suffering from a disease to be treated, with (an effective amount of) a conjugate comprising an effector molecule and a ligand (a cell surface molecule binding molecule for targeting the effector molecule in and within a target abnormal cell, such as a tumor cell), for example to a target tumor cell, i.e. to improve the therapeutic window of an ADC or AOC comprising an effector molecule delivered to the cytosol of a target tumor cell.

[0051] It is an object of the present invention to provide a combination of an effector molecule activity enhancer molecule and an improved ADC or AOC for use in a treatment, such as an anti-cancer therapy. It is also an object of the present invention to provide a therapeutic composition, or a therapeutic combination of, for example, two therapeutic compositions, comprising such an improved ADC or AOC and comprising an effector molecule activity enhancer molecule. Such an effector molecule activity enhancer molecule can improve the biological effect of the effector molecule when the effector molecule is delivered, for example, preferably to an endosome, endolysosome and / or lysosome in a target cell, for example, and then preferably to the cytosol of the cell. The target cell comprises the molecular target of the effector molecule. When the provided effector molecule activity enhancer molecule is administered to a (human) patient in need thereof, and the patient has a target cell, the patient experiences a high therapeutic effect, or at least a sufficient effect, at a preferably lower dose of the effector molecule than is currently required to reach an effective dose of the effector molecule. In this way, the therapeutic window of the effector molecule, for example, an effector molecule that is part of a conjugate such as an ADC or AOC, is effectively widened.

[0052] For a bioactive molecule (e.g., an effector molecule) to act, the molecule must be able to engage its target, e.g., in serum, outside the cell surface, or inside a cell or organelle. The active moiety of almost all protein-based targeted toxins, for example, must enter the cytosol of the target cell to mediate its target-modulating effect. In many cases, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound to the outside of the cell, (2) it is internalized back to the cell surface, or (3) it is transported to the endolysosome, where it is degraded. These fundamental problems have been known for decades, and over 500 targeted toxins have been investigated in the past few decades, but the problem has not yet been solved, and only two antibody-targeted protein toxins have been accepted into the market, albeit with warning labels of severe toxicity. Moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP) has been approved by the FDA to date for relapsed or refractory hairy cell leukemia. Other such approved ADCs are Elzonris and Ontak.

[0053] To overcome these problems, many strategies have been described, including approaches to redirect toxins to endogenous cell membrane transport complexes of biosynthetic pathways in the endoplasmic reticulum, and techniques to disrupt or weaken the membrane integrity of endosomes, i.e., compartments of the endocytic pathway in cells, and thus promote endosomal escape. This includes the use of lysosomotropic amines, carboxylic acid ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light induction techniques. The potency of targeted toxins has typically increased 100- or 1000-fold in cell cultures, and in exceptional cases more than 1 million-fold, but the need to co-administer endosomal escape enhancers with other substances introduces new problems, including additional side effects, loss of target specificity, difficulties in determining therapeutic windows, and cell type-dependent variability.

[0054] All strategies involving physicochemical techniques require enhancer molecules that interact more or less directly with the membrane and essentially include small chemical molecules, secondary metabolites, peptides and proteins. A common feature of all these substances is that they are not themselves target cell specific and distribute with kinetics other than the targeted toxins. This is one major drawback of current approaches.

[0055] A further object of the present invention is to provide improved enhancers for sdAb-based ADCs and sdAb-based AOCs, as well as improved therapeutic combinations or improved pharmaceutical compositions for the delivery of an effective amount of an effector molecule, for example when delivery from the outside to the inside of a target cell is considered, or more particularly when delivery of the effector molecule in the cytosol of said target cell is considered. A further object of the present invention is to provide an improved method of treatment of a (human) patient suffering from a disease to be treated, for example with a conjugate comprising an effector molecule and a ligand, i.e. an sdAb, against a target tumor cell, i.e. to improve the therapeutic window of an ADC or AOC comprising an sdAb, comprising an effector molecule delivered to the cytosol of a target tumor cell.

[0056] It is an object of the present invention to provide a therapeutic composition or therapeutic combination of e.g. two therapeutic compositions comprising an effector molecule-enhancing molecule for use in a treatment, such as an anti-cancer therapy, a molecule capable of improving the biological effect of an effector molecule when delivered inside a target cell comprising the molecular target of the effector molecule, which provided molecule, when administered to a (human) patient in need thereof and having the target cell, experiences an improved therapeutic effect or a sufficient effect at a lower dose of the effector molecule than is currently required to reach an effective dose of the effector molecule. In this way, the therapeutic window of the effector molecule, e.g. an effector molecule that is part of a conjugate such as an ADC comprising an sdAb or an AOC comprising an sdAb, is effectively widened.

[0057] Although the present invention will be described with respect to certain embodiments, the present invention is not limited thereto, but only by the scope of the claims. Although the present invention has been described with respect to several embodiments, alternatives, modifications, variations and equivalents will become apparent to those skilled in the art upon reading the specification and studying the drawings and graphs. The present invention is in no way limited to the illustrated embodiments. Modifications can be made without departing from the scope defined by the appended claims.

[0058] The inventors have invented and established that the therapeutic window of a first conjugate of the invention, such as an antibody drug conjugate or an antibody oligonucleotide conjugate, preferably comprising a cell surface molecule binding sdAb for targeting the ADC or AOC to a cell surface molecule, is increased when a second conjugate comprising at least one covalently attached saponin is administered to the same subject, such as a human cancer patient or tumor-bearing mammal in need thereof (V HH For conjugates containing IgG or V, when combined with an ADC or AOC containing an antibody such as a mAb or sdAb, HH See the Examples section, e.g., Tables A5-A7 and e.g., Figures 4-7 for examples of a series of in vitro and in vivo tumor cell models of the effect of saponin covalently conjugated to antibodies such as V and VL, and see Figures 2, 3, 8-13, 16, 16, 18-23, 26 and 27 for conjugates comprising monoclonal antibodies (mAbs). HH The present inventors have conjugated the saponins of the present invention to ligands for binding to cell surface molecules, for example antibodies such as full length intact IgG, or VNAs, and effector molecules such as protein toxins or oligonucleotides such as BNAs are conjugated to a second antibody such as an IgG or sdAb. HHFor the first time, it has been established and determined that conjugation to an antibody, such as an sdAb, such as , provides a conjugate that cell-specifically delivers saponin to the cell surface of a target cell that exposes a cell surface molecule on the cell surface, and then delivers the (free) saponin to the cell, e.g., interior, e.g., cellular endosomes, endolysosomes, lysosomes, and ultimately the cellular cytosol (preferably a saponin-containing conjugate in which the saponin is covalently attached by a bond that is cleavable in the endosomes, endolysosomes, lysosomes of a mammalian cell, such as a human tumor cell, such that free saponin is present when the saponin-containing conjugate enters the cellular endosomes, endolysosomes, lysosomes and the bond is cleaved (e.g., by receptor-mediated endocytosis when the conjugate binds to a cell surface molecule, i.e., a receptor). An example of such a cell-targeted saponin conjugate is, for example, saponin-V, as outlined in the Examples section below. HH Conjugates and saponin-Mab conjugates are provided in Figures 2-27. Saponin targets EGF, Her2, HH or conjugated to a ligand such as IgG, an IgG that targets EGFR.

[0059] At least one of the above objectives is achieved by providing an improved ADC, or improved AOC combination, comprising an sdAb that binds to a cell surface molecule, such as a second conjugate comprising a covalently attached bidesmoside triterpene glycoside (triterpene saponin), and an effector molecule enhancing molecule of the invention.

[0060] A first aspect of the present invention relates to a pharmaceutical combination comprising: o a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, which are covalently linked to each other directly or via a linker; o a second conjugate comprising a saponin and / or a derivative thereof, and / or a binding molecule for binding to a second cell surface molecule, and a saponin and / or a derivative thereof, wherein the binding molecule and the saponin or a derivative thereof are covalently bound to each other directly or via a linker, and the saponin or a derivative thereof is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside, and the second cell surface molecule is the same as or different from the first cell surface molecule; and, optionally, o A pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent.

[0061] The inventors have demonstrated that, considering the effect of the effector molecule inside the cell, the effector molecule that is part of the first conjugate comprising an sdAb is delivered into the cell with high efficiency under the influence of a saponin, such as the saponin contained in the second conjugate of the present invention. More specifically, the inventors have demonstrated that, considering the effect of the effector molecule inside the cell, the effector molecule that is part of the first conjugate comprising an sdAb is delivered into the cytosol of the cell with high efficiency under the influence of a saponin of the present invention, such as the saponin contained in the second conjugate of the present invention. That is, in the absence of a saponin, for example as part of the second conjugate, contact of the tumor cell with a suboptimal dose of the first conjugate does not result in the activity of the effector molecule inside the cell (the target cell is not efficiently killed by the biological activity of the effector molecule). However, contacting the target tumor cells with both the first conjugate comprising the effector molecule and the second conjugate comprising free saponin and / or saponin ensures efficient tumor cell killing.

[0062] In order to explain the present invention in more detail, the cellular material uptake process and the terms used in the present invention will be described first. The uptake of extracellular material into cells by vesicle budding is called endocytosis. Said 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) non-specific fluid uptake (pinocytosis), or (4) non-specific particle uptake (phagocytosis). All types of endocytosis lead to the following cellular processes of vesicle trafficking and material sorting, called endocytic pathways. The endocytic pathways are complex and not fully understood. In the early days, it was believed that organelles were newly formed and matured into the next organelle along the endocytic pathway. Today, it is hypothesized that the endocytic pathways include stable compartments connected by vesicle trafficking. Compartments are complex, multifunctional membrane organelles that are specialized for a specific set of functions essential for the cell. Vesicles are considered to be transient organelles with simple composition and are defined as membrane-enclosed containers that are newly formed 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 are stable compartments in the endocytic pathway, while primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and even lysosomes are vesicles. Endocytic vesicles originate at the plasma membrane, most notably from clathrin-coated pits, but first fuse with early endosomes, the main sorting compartment at approximately pH 6.5. The majority of internalized cargo and membranes are recycled to the plasma membrane via recycling vesicles (recycling pathway). Components to be degraded are transported by multivesicular bodies to the acidic late endosomes (pH less than 6). Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to the late endosomal compartment when needed.The resulting organelle is called a hybrid organelle or an endolysosome. Lysosomes bud off from the hybrid cell organelle in a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are highly dynamic organelles, and their distinction is often difficult. Degradation of endocytosed molecules occurs inside the endolysosome. Endosomal escape is the active or passive release of a substance from the lumen of any kind of compartment or vesicle of the endocytic pathway, preferably of clathrin-mediated endocytosis, or of the recycling pathway, into the cytosol. Thus, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediate organelles and hybrid organelles. After entering the cytosol, the substance may be transferred to other cellular units, such as the nucleus. Glycoside molecules (saponins) in the context of the present invention are compounds that can enhance the effect of effector molecules, in particular by promoting endosomal escape. Glycoside molecules interact with the membranes of compartments and vesicles of the endocytic and recycling pathways, rendering the effector molecules leaky and thus enhancing endosomal escape.

[0063] The term "improving the effect of an effector molecule" means that the saponin increases the functional efficacy of the effector molecule (e.g. therapeutic index of a toxin or drug; metabolic efficiency of a modifier in a biotechnological process; transfection efficiency of a gene in a cell culture research experiment), preferably by allowing or improving engagement of its target. Acceleration, prolongation, or enhancement of an antigen-specific immune response is preferably not included. Therapeutic efficacy includes, but is not limited to, increased therapeutic efficacy due to lower dosage and / or reduced side effects. "Improving the effect of an effector molecule" may also mean that an effector molecule that could not be used due to lack of efficacy (e.g., was not known to be an effector molecule) becomes effective when used as part of a first conjugate of the invention comprising an sdAb targeting a cell surface molecule and in combination with free saponin (derivative) and / or in combination with a second conjugate of the invention comprising a covalently bound saponin (derivative). Any other effect that may be beneficial or desirable and that may result from the combination of, for example, an AOC comprising a cell-targeting sdAb according to a first conjugate of the invention, or an effector molecule as part of an AOC comprising a cell-targeting sdAb according to a first conjugate of the invention, with a saponin comprised in a second conjugate of the invention, as provided by the present invention, is considered to be an "improved effect". In the context of the present invention, a saponin, such as a saponin comprised in a second conjugate of the invention, is an "enhancer" of the functional efficacy of the effector molecule comprised in a first separate conjugate, such as an AOC comprising a cell surface molecule-binding sdAb according to the present invention, or an ADC comprising a cell surface molecule-binding sdA.

[0064] Up until the present invention, one major drawback of targeted toxin enhancement with free glycosides that are not part of a cell surface molecule targeted conjugate, such as the saponins of the present invention, is that the targeted toxin is internalized by receptor-mediated endocytosis (the targeted toxin binds to the receptor and then the targeted toxin conjugate / receptor complex is internalized), while the glycoside passively diffuses through the plasma membrane and reaches the endosomal membrane, possibly by interaction with cholesterol. In principle, the glycosides, such as the saponins in the second conjugates of the present invention, when in a free, unconjugated form, can enter any cell, even non-target cells (off-target cells), resulting in less efficient utilization of the enhancer in the target cells to effectively release the targeted toxin, and the possibility of side effects in non-target cells. One major problem is that the entry of targeted toxins and glycosides proceeds with different kinetics, and these kinetics vary between cells (lines) and between tissues, so that the correct time difference for applying the two substances (e.g., ADCs or AOCs with free saponins and containing sdAbs for binding to cell surface molecules) can vary greatly between tumors (cell lines). Furthermore, the release, absorption, distribution, metabolism and excretion of these substances are also different in the organism. Furthermore, non-specific uptake of glycosides by non-targeted cells can induce undesirable effects in these cells. This can be, for example, cytosolic delivery of compounds that should have been delivered to lysosomes, impaired antigen presentation, etc. Non-targeted administration of glycosides and targeted drugs can also be problematic in drug development and can prevent or at least delay manufacturing approval by the relevant authorities (e.g., FDA or EMA). A targeted toxin or targeted drug in the context of the present invention means that the toxin or drug is specifically targeted to a membrane-bound molecule (cell surface molecule) of a target cell, e.g., a toxin or drug conjugated to an antibody, such as an sdAb, that specifically recognizes a structure (binding site, epitope) on the cell membrane of a target cell.

[0065] Therefore, it is very useful to direct the glycoside (saponin of the present invention) to the target cell by the same route as the effector molecule, for example by a targeting ligand, so that the enhancer is available in effective concentrations in the acidic compartment of the endocytic pathway of the target cell and shows synergistic effects with the toxin. Thus, the present invention provides a novel method for redirecting both the effector molecule and the endosomal escape enhancer (i.e., saponin of the present invention) to the acidic compartment of the endocytic pathway of the target cell by a targeting ligand (binding molecule). The targeting ligand comprising the saponin in the second conjugate of the present invention may be the same as or different from the targeting ligand, i.e., sdAb, comprised in the first conjugate comprising the effector molecule of the present invention. When the targeting ligands are different, it is understood that both targeting ligands bind to first and second cell surface molecules that are different but present on the same target cell, such as tumor cells. For example, a second conjugate of the invention comprising a saponin moiety can bind to EGFR or CD71 or HER2 and a first conjugate comprising, for example, an effector moiety and a cell surface molecule binding sdAb can bind to EGFR or CD71 or HER2, and the first conjugate and the second conjugate of the invention bind to the same cell receptor or different cell receptors.

[0066] Considering the effect of the effector molecule in the cell, the inventors have demonstrated that the effector molecule, which is part of the first conjugate containing a targeting ligand such as sdAb, is delivered into the cell with high efficiency under the influence of the saponin contained in the second conjugate of the present invention. Surprisingly, V HH Despite the relatively small size of an sdAb, such as V, binding of a first conjugate of the invention comprising such an sdAb to a target cell surface receptor still occurs when the effector molecule and the sdAb are included in the first conjugate, and the sdAb is HH Surprisingly, V HHDespite the relatively small size of the sdAb, such as V, binding of the second conjugate of the invention comprising saponin and comprising an sdAb to a target cell surface receptor still occurs when the saponin and the sdAb are included in the second conjugate, and the sdAb is HH It is preferred that V which forms the first conjugate of the present invention is HH Binding of effector molecules to sdAbs such as V HH Considering the ability of V to form the second conjugate of the present invention, for example, does not result in steric hindrance. HH Binding of saponin to sdAbs such as V, which bind to cell surface molecules, HH Considering the ability of, for example, no steric hindrance is caused. That is, in the absence of the second conjugate of the present invention comprising saponin covalently bound to a cell surface molecule binding ligand, such as an antibody, such as an sdAb, according to the second conjugate of the present invention, contacting, for example, tumor cells with a suboptimal dose of an ADC comprising an sdAb or an AOC comprising an sdAb (i.e., the first conjugate of the present invention) does not result in intracellular effector molecule activity (the target cells are not efficiently killed by the biological activity of the effector molecule). However, when the target tumor cells are contacted with the first conjugate of the present invention comprising an effector molecule and an sdAb, such as an ADC or AOC, together with free saponin and / or the second conjugate of the present invention comprising saponin, efficient tumor cell killing is achieved.

[0067] By targeting a single cell surface molecule with the second conjugate of the invention and an ADC or AOC, i.e. the first conjugate of the invention, or by targeting a second cell surface molecule with the second conjugate of the invention and an ADC or AOC, i.e. the first conjugate of the invention, to a first cell surface molecule, where the first and second cell surface molecules are different and are present on the same target cell, delivery of a saponin bound to an antibody targeting a cell surface molecule such as an sdAb in the second conjugate of the invention, and delivery of an ADC or AOC comprising an effector molecule and an sdAb for binding to the first cell surface molecule, to the interior cytosol of a target cell exposing the cell surface molecule on the cell surface is improved and more specific than, for example, contacting the cell with only the ADC and not simultaneously contacting the cell with the second conjugate comprising the saponin of the invention and therefore in the absence of the cell-targeted saponin (the second conjugate of the invention). The diseased cells selected for targeting by the cell surface molecule targeting sdAb of the first conjugate and for targeting by the cell surface molecule binding molecule comprised in the second conjugate, such as an antibody such as an sdAb, ideally bear a high degree of epitopes on the cell surface molecule to which the cell surface molecule targeting molecule can bind (i.e. expression of the target cell surface molecule in target cells, such as tumor cells or autoimmune cells, is relatively higher than expression in non-target cells, such as healthy cells), and / or expose epitopes on the target cell surface molecule for binding of the cell surface molecule targeting sdAb of the first conjugate and for binding of the cell surface molecule targeting antibody such as an sdAb of the second conjugate, particularly when (adjacent) healthy cells of the patient are considered. Preferably, the cell surface molecule targeted by the cell surface molecule targeting sdAb of the first conjugate of the invention, and the cell surface molecule targeted by a molecule targeting a cell surface molecule such as an antibody such as an sdAb of the second conjugate of the invention, are relatively highly and / or specifically expressed on target (disease, tumour) cells compared to healthy cells.One embodiment is a first conjugate of the present invention, in which the target cell surface molecule for the sdAb targeting the cell surface molecule of the first conjugate, such as a tumor cell receptor, is specifically or relatively highly expressed compared to the expression of the cell surface molecule on the surface of healthy (adjacent) cells. One embodiment is a second conjugate of the present invention, in which the target cell surface molecule for the molecule targeting the cell surface molecule, such as an antibody, such as an sdAb, of the second conjugate, such as a tumor cell receptor, is specifically or relatively highly expressed compared to the expression of the cell surface molecule on the surface of healthy (adjacent) cells. Thus, the epitope of 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. After the first conjugate of the present invention binds to the epitope of the cell surface molecule of the target cell, endocytosis of the complex of the conjugate and the target cell surface molecule occurs (after the conjugate binds to the receptor (target cell surface molecule), the conjugate / receptor complex is internalized). After the second conjugate of the present invention binds to an epitope on the cell surface molecule of the target cell, endocytosis of the complex of the second conjugate and the target cell surface molecule occurs (after the conjugate binds to the target cell surface molecule (cell surface receptor), the conjugate / receptor complex is internalized). Since the first and second conjugates can only enter the target cell through a binding interaction with a cell surface molecule that is specifically or uniquely expressed to a sufficient degree on the target cell compared to healthy cells that should not be targeted, accumulation of a therapeutically active amount of the effector moiety contained in the ADC or AOC (first conjugate of the present invention) that targets the same cell surface molecule as the second conjugate of the present invention or targets a cell surface molecule present on the same target cell but different from the cell surface molecule targeted by the second conjugate of the present invention, and the saponin contained in the second conjugate of the present invention is only possible inside the target cell and occurs when 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 of the first conjugate, linked to an sdAb targeting a cell surface molecule, can only exert its intracellular (e.g. cytotoxic or gene silencing) activity in the presence of a second conjugate with a covalently bound saponin of the invention, also provides a safeguard against negative undesirable side effects of the effector moiety on, for example, healthy cells and healthy tissues that are not meant to be targeted and affected by the effector moiety, compared to exposure of the cells to an ADC without the presence of a second conjugate of the invention comprising a covalently bound saponin. That is, when the expression of the exposed cell surface molecule to which the first conjugate of the invention, e.g. an ADC or AOC comprising an sdAb, and a second conjugate comprising a saponin, can bind is sufficiently low or absent, ideally the first conjugate of the invention comprising the effector molecule and the second conjugate comprising a saponin cannot enter (non-targeted) healthy cells to an amount that would result in endosomal escape of the effector moiety of the ADC or AOC under the influence of the saponin contained in the second conjugate of the invention. Since the ADC or AOC can be used in lower doses in the presence of a saponin comprising a second conjugate of the invention compared to when the ADC or AOC is applied in a treatment regimen in the absence of the second conjugate of the invention and thus in the absence of a saponin, the degree of penetration of the ADC or of the AOC in healthy cells is lower and the risk of occurrence of undesirable side effects is already lower, taking into account the targeting and killing of target disease cells such as, for example, tumor cells and autoimmune cells.

[0068] The inclusion of an sdAb in the first conjugate has many advantages compared to the inclusion of an antibody such as an IgG (full-length immunoglobulin), or a binding fragment or binding domain thereof. Importantly, since the sdAb does not contain the Fc tail present in IgG, the risk of off-target side effects due to binding of the conjugate to Fc receptors of cells, such as endothelial cells, of the host to which the conjugate is administered is not present when the first conjugate of the present invention is administered to a mammal, such as a human subject. Thus, the risk profile of the first conjugate of the present invention is improved compared to IgG-based ADCs and AOCs, or compared to ADCs or AOCs that include an Fc tail. In addition, since the first conjugate of the present invention cannot be bound by Fc receptors, the first conjugate is already effective at lower doses than those required to reach the same effector molecule activity as full-length antibody-based ADCs and AOCs, due to less or no undesired capture of the first conjugate by cell surface receptors that are different from the first cell surface molecule of the intended target. Moreover, due to the relatively small size of sdAbs, as compared to e.g. Fab, scFv, IgG, the tissue penetration of the first conjugate of the invention is good, which is beneficial for reaching the target cells when the first conjugate is administered to a patient in need of a treatment based on the biological activity of the effector molecule inside the (tumor) cell. All these advantages of applying sdAbs in the first conjugate of the invention improve the therapeutic window of the effector molecule when included in the first conjugate of the invention, as compared to the application of a larger antibody or fragment thereof, such as IgG with an Fc tail, in a similar ADC or AOC. When the effector molecule is e.g. a toxin and the ADC is based on IgG and at the same dose with the same effector molecule, the improvement of the target cell killing in the case of target tumor cells is, for example, suboptimal.This aspect of the invention allows for the treatment of patients with lower doses of effector molecules as part of a conjugate comprising an sdAb, i.e., a first conjugate of the invention, to reach the same or improved effector molecule-mediated effects in target cells, compared to the higher doses required when an antibody-based ADC or AOC comprising the same effector molecule is used. The administration of such a first conjugate of the invention at a lower dose reduces the risk of the patient experiencing side effects, for example, due to non-specific entry into non-target healthy cells. This is important, for example, when the first cell surface molecule targeted by the sdAb contained in the first conjugate is highly expressed on target (tumor) cells, but is not uniquely expressed on such target cells. A low dose of the first conjugate reduces the risk of the first conjugate binding to low expressers, such as non-tumor healthy cells.

[0069] The inventors have also found that the therapeutic window of the first conjugate of the present invention is expanded when the first conjugate is contacted with a target cell in the presence of saponin, or more specifically in the presence of a second conjugate of the present invention that comprises saponin. When a target cell that has a first cell surface molecule, i.e. the target of the sdAb contained in the first conjugate, and a second cell surface molecule (which is essentially true when both the first and second cell surface molecules are the same cell surface molecule according to an embodiment of the present invention), i.e. the target of the binding molecule contained in the second conjugate, is contacted with both the first conjugate and the second conjugate, the effective dose of the effector molecule is lower than when the target cell is contacted with the first conjugate in the absence of the second conjugate (or in the absence of free saponin). The presence of the second conjugate that comprises saponin or the presence of free saponin enhances the activity of the effector molecule in the target cell, such that the therapeutic window of the first conjugate and the therapeutic window of the effector molecule are expanded. Full efficiency of the effector molecule is achieved at a lower dose when the target cell is contacted with both the first and second conjugates of the present invention. When the first conjugate is contacted with the target cell in the presence of saponin or its functional derivative, a similar effect is found by the inventors when the effector molecule enhancing activity of saponin is taken into account, but the concentration of free saponin (derivative) is 100-1000 times higher compared to the effective dose established when the second conjugate containing covalently bound saponin is combined with the first conjugate. Thus, providing a binding molecule to saponin or its derivative (i.e., the second conjugate of the present invention), a better effect of enhancing effector molecule activity is obtained when both the first and second conjugates are contacted with a target cell expressing both cell surface molecules on its surface.Targeted saponins are already effective at lower doses than free saponin in delivering effector molecules inside a target cell, and from the endosomes, endolysosomes or lysosomes of said cells to the cytosol, where the effector molecule binds to its target binding partner and exerts its biological activity (e.g. cell killing if the target cell is a tumour cell and the effector molecule is, for example, a toxin).

[0070] At the same time, the inventors provide a combination of a first pharmaceutical composition comprising a first conjugate and a second pharmaceutical composition comprising a free saponin (derivative) or a second conjugate comprising a free saponin (derivative), and The present invention provides pharmaceutical compositions comprising a first conjugate and a free saponin (derivative) or a second conjugate, which improve the therapeutic window, reduce the risk of inducing side effects when an effective dose of the effector molecule contained in the first conjugate is administered to a patient in need of effector molecule-based therapy, and improve the delivery of the first conjugate to the inside of a target cell, more specifically the delivery of the effector molecule to the inside of the cytosol of such a target cell, when compared to current ADCs or AOCs based on Fc containing full-length antibodies or constructs thereof. Although it is part of the present invention to administer such a first conjugate of the present invention together with a free saponin (derivative) or a second conjugate to a patient in need of effector molecule-based therapy, it is preferred to apply the second conjugate in combination with the first conjugate.

[0071] One embodiment is a pharmaceutical combination according to the invention in the form of at least two, preferably two, pharmaceutical compositions comprising: - a first pharmaceutical composition comprising a first conjugate, and optionally a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent; and - a second pharmaceutical composition comprising a saponin and / or a derivative thereof and / or a second conjugate, and optionally a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent.

[0072] One embodiment comprises a first conjugate, and ·saponin; Saponin derivatives; and Second conjugate At least one, preferably one of and optionally a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent, in the form of a single pharmaceutical composition.

[0073] One aspect of the invention relates to a pharmaceutical composition comprising: o a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, which are covalently linked to each other directly or via a linker; o a second conjugate comprising a saponin and / or a derivative thereof, and / or a binding molecule for binding to a second cell surface molecule, and a saponin and / or a derivative thereof, wherein the binding molecule and the saponin or a derivative thereof are covalently bound to each other directly or via a linker, and the saponin or a derivative thereof is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside, and the second cell surface molecule is the same as or different from the first cell surface molecule; and, optionally, o A pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent.

[0074] In one embodiment, the first conjugate comprises a V nucleotide derived from the heavy chain of an antibody, preferably of immunoglobulin G origin, preferably of human origin. H Domain; V derived from the light chain of an antibody, preferably of immunoglobulin G origin, preferably of human origin L Domains: Various heavy chain neoantigen receptors (V NAR ) domain, or heavy chain antibodies (HCAbs) derived from Camelidae origin, such as Ig-NAR origin, HHdomain (preferably the HCAb is from Camelidae origin); preferably the sdAb is from Camelidae origin (Camelidae V H ) derived from HCAb, for example V derived from HCAb of camel, llama, alpaca, dromedary, vicuna, guanaco and Bactrian camel HH The pharmaceutical combination or pharmaceutical composition of the present invention is

[0075] In particular, V HH The V domain is suitable for application in the first conjugate of the invention, and indeed is also suitable for application in the second conjugate of the invention. HH The domains are generally known for their high stability, i.e., resistance to unfolding, their ability to bind to a binding partner without requiring the presence of a second V domain, such as that present in IgG and required for IgG to bind to a binding partner via two V domains, their ease of production by techniques known in the art (camelid immunization techniques, phage display techniques, etc.), and their ability to penetrate tissues to a greater extent than is seen with full-length IgG, which is beneficial when target (tumor) cells are located within or as part of such (organ) tissues. Furthermore, such V domains are well known for their high stability, i.e., resistance to unfolding, their ability to bind to a binding partner without requiring the presence of a second V domain, such as that present in IgG, which requires IgG to bind to a binding partner via two V domains, their ease of production by techniques known in the art (camelid immunization techniques, phage display techniques, etc.), and their ability to penetrate tissues to a greater extent than is seen with full-length IgG, which is beneficial when target (tumor) cells are located within or as part of such (organ) tissues. HH The V domain lacks the Fc tail common to immunoglobulin G antibodies, and as a result HH Administering a conjugate containing an sdAb such as a domain is unlikely to result in Fc receptor mediated side effects (such side effects may occur in subjects administered a therapeutic full length immunoglobulin containing an Fc tail).

[0076] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention wherein the first conjugate comprises at least two sdAbs having a single first sdAb covalently bound to at least one effector molecule, or having two or more sdAbs bound to at least one effector molecule, or having all of the at least two sdAbs bound to at least one effector molecule, e.g. each of the at least two sdAbs is bound to one or more of the at least one effector molecule.

[0077] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention wherein the sdAb comprises at least two sdAbs, preferably 2 to 8 sdAbs, more preferably 2 to 4 sdAbs, which are the same sdAb.

[0078] One embodiment is a pharmaceutical combination or composition of the invention comprising 1 to 8 sdAbs capable of binding to the same binding site on a first cell surface molecule, and wherein at least one effector molecule is bound to a single first sdAb of the 1 to 8 sdAbs, or at least one effector molecule is bound to two or more (if present) of the sdAbs, and / or each of the at least one effector molecule is separately bound to a single sdAb such that one or more of the sdAbs are each bound to a single effector molecule.

[0079] By providing a first conjugate of the invention (and / or a second conjugate of the invention) that comprises a (linear) string of sdAbs covalently linked to each other, it is possible to provide benefit from the ability of the conjugate to bind with high affinity to target cells, thereby improving uptake (endocytosis) of the conjugate by the target cells.

[0080] When considering the application of the endosomal escape enhancing effect of saponin on effector molecules, synchronization is the missing link between the successful delivery strategy in mice and its application in humans. Indeed, the inventors have demonstrated in a series of in vivo mouse tumor models that administration of free saponin and, for example, ADC separately to mice, without the presence of a second conjugate comprising saponin according to the invention, does not result in the desired antitumor activity, such as tumor growth delay, tumor regression, reduced and slowed tumor growth, compared to control animals treated with ADC in the absence of free saponin. See also Figures 18-21 for in vivo tumor models and the examples described in the Examples section below herein. Free saponin was administered via different routes of administration and at different times relative to the administration of ADC (free saponin administered before, during and after 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 provide any effective antitumor activity. The ADCs mentioned were administered at doses that did not themselves confer any beneficial antitumor effect on tumor-bearing animals. Surprisingly, the inventors have demonstrated that beneficial antitumor activity can be achieved by treating cells or animals with the second conjugate according to the invention in combination with the ADC or AOC, i.e. the first conjugate of the invention, in various in vitro mammalian cell-based bioassays using human tumor cells and / or in various in vivo animal tumor models.

[0081] A second conjugate of the invention, optionally comprising a scaffold according to the invention (see below; a scaffold such as a covalent saponin conjugate comprising an oligomeric or polymeric structure to which one or more saponin moieties are covalently attached), e.g. a scaffold which is a trifunctional linker with a saponin (e.g. SO1861, QS-21) covalently attached by a cleavable or non-cleavable bond, a monoclonal antibody such as cetuximab, trastuzumab, OKT-9, but preferably V HHor a scaffold which is a dendron to which four moieties, such as for example four saponin molecules, can be attached, such as for example a G4-dendron or a G5-dendron, or a dendron for attaching for example eight saponins (the dendron comprising a chemical group for (covalent) attachment to an antibody targeting a cell surface molecule, such as an sdAb of the second conjugate). With reference to the further embodiments and examples section, some of these scaffolds according to the invention are illustrated and show in vivo and / or in vitro antitumor cell activity, for example when the cytotoxicity exerted by the proteinaceous toxin comprised in the first conjugate of the invention is considered or when gene silencing in tumor cells is considered.

[0082] Without wishing to be bound by any theory, taking into account the failures observed when treatment of tumor-bearing animals with ADC and free saponin is considered, it is preferred to synchronize the presence of both at least one saponin and an effector moiety, preferably a toxin or oligonucleotide, in a compartment or vesicle of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. Synchronizing the presence of molecules in late endosomes to obtain a synergistic effect in vivo with ADC and free saponin has not been beneficially achieved by the inventors' attempts. In one aspect, the present invention solves at least the following problems with respect to combining a first conjugate (e.g., ADC or AOC) that preferably comprises an effector moiety and an sdAb that binds to a (tumor) cell surface molecule with a second conjugate of the present invention that comprises a saponin: without wishing to be bound by any theory, for example, only rational chemical groups in saponin that can be used for (covalent), particularly single, cleavable, retainable bonds are necessary for endosomal escape activity. The reason why saponins have not been used in combination with pharma- ceutically active substances in clinical trials other than the application of saponins in vaccination regimens, where the use of immune-enhancing adjuvant substances is implied, is most likely due to known limitations, e.g., the remarkable endosomal escape enhancer effect of the saponins of the present invention and exemplified herein has been known for more than a decade. For example, providing a second conjugate of the present invention with a covalently bound saponin, e.g., in association with a scaffold carrying several saponins, at least partially solves these difficulties. For example, providing a second conjugate of the present invention with a covalently bound saponin by a cleavable bond that is cleaved under the influence of a slightly acidic pH (pH<6,5), as evident in the endosomes, endolysosomes and lysosomes of the target cells, at least partially solves these problems, since once the second conjugate is endocytosed and delivered to the interior of the cell, the free saponin cleaved from the second conjugate is present inside said endosomes, endolysosomes or lysosomes.Surprisingly, saponins previously applied for their immune enhancing activity in the context of vaccination comprising saponins as adjuvant component are now also suitable for (covalent) binding to antibodies targeting cell surface molecules such as sdAbs comprised in a second conjugate of the invention for in vitro and in vivo antitumor activity when used in combination with an ADC or AOC comprising an sdAb, i.e. a first conjugate of the invention.

[0083] In one embodiment the sdAb is a single sdAb or at least two, preferably two sdAbs, wherein the sdAb is capable of binding to a first cell surface molecule such as HIV gp41 or the first cell surface molecule is a tumor cell surface receptor such as a tumor cell specific receptor, more preferably CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrins, syndecan-1, vascular integrin alpha V beta 3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate specific membrane antigen (PSMA), CanAg, integrin alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30 , CD239, CD70, CD123, CD352, DLL3, CD25, EphrinA4, MUC-1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5, CD16, CXCR7 (ACKR3), glucocorticoid-inducible TNFR-related protein (GITR), most preferably a receptor selected from: HER2, c-Met, VEGFR2, CXCR7, CD71 and EGFR1.

[0084] It is part of the present invention that the sdAb contained in the first conjugate of the present invention has binding specificity for a cell surface molecule that is specifically expressed on a target cell. By "specifically expressed" it is meant herein that the unique expression of the cell surface molecule only in the target cell (wherein, for example, healthy cells that do not bind to the first conjugate are not targets) or, as used herein, that the upregulated or relatively high expression of the target cell surface molecule in the target cell, as compared to the low expression of the cell surface molecule in, for example, healthy cells that do not bind to the conjugate or at least bind to a very low degree. These listed cell receptors are sufficiently specific cell surface molecules for the cells that are the target of the first conjugate, and are therefore preferred candidates for binding by the first conjugate. When comparing the expression of the cell surface molecule in the target cell with the expression in other cells that are not meant to be targeted by the first conjugate of the present invention, it is understood that the higher the specificity of a particular cell surface molecule, the better the therapeutic window, when considering the activity of the effector molecule inside the cell. For example, suitable targets for the first conjugate to target are HER2, EGFR, eg, EGFR1, and CD71, among other tumor cell specific receptors.

[0085] In one embodiment the sdAb in the first conjugate is a single sdAb or at least two, preferably two sdAbs, the sdAbs being selected from the group consisting of anti-CD71 sdAb, anti-HER2 sdAb, anti-CD20 sdAb, anti-CA125 sdAb, anti-EpCAM(17-1A) sdAb, anti-EGFR sdAb, anti-CD30 sdAb, anti-CD33 sdAb, anti-vascular integrin αvβ3 sdAb, anti-CD52 sdAb, anti-CD22 sdAb, anti-CEA sdAb, anti-CD44v6 sdAb, anti-FAP sdAb, anti-CD19 sdAb, anti-CanAg sdAb, anti-CD56 sdAb, anti-CD38 sdAb, anti-CA6 sdAb, anti-IGF-1R sdAb, anti-integrin sdAb, anti-syndecan-1 sdAb, anti-CD79b, anti-c-Met sdAb, anti-EGFR1 sdAb, anti-VEGFR2 sdAb, anti-CXCR7 sdAb, and anti-HIV gp41, the sdAb being preferably HH and more preferably a camelid V H The pharmaceutical combination or pharmaceutical composition of the present invention is

[0086] In one embodiment, the first conjugate comprises an sdAb capable of binding to HER2, CD71, HIV gp41 and / or EGFR, the sdAb preferably being HH and more preferably camelid V H The pharmaceutical combination or pharmaceutical composition of the present invention is

[0087] In one embodiment, the first conjugate is an sdAb for binding to HER2 selected from the sdAb produced by clone 11A4, clone 18C3, clone 22G12, clone Q17 or clone Q17-C tag; or an sdAb for binding to EGFR produced by clone anti-EGFR Q86-C tag; or an sdAb for binding to CD71 produced by clone anti-CD71 Q52-C tag; or an sdAb for binding to CD71 produced by clone anti-HIV gp41 or an sdAb encoded by the cDNA of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; or an sdAb having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36-72, optionally the first conjugate further comprises a covalently attached sdAb for binding to albumin, for example any one or more of the sdAbs having the amino acid sequence of SEQ ID NOs: 33, 34 and 35, preferably the sdAb is V HH and more preferably camelid V H A pharmaceutical combination or composition of the present invention, which is a compound suitable for incorporation into the first conjugate of the present invention, HH can be found, for example, in the Single Domain Antibody Database (Wilton, EE et al. (2018)), patent applications US 2016 / 0251440 (anti-CD123, anti-CEACAM), US 9683045 (anti-c-Met), US 2009 / 0252681 (anti-EGFR, anti-IGF-1R), US 9969805 (anti-HER2), US 20190023796A1 (anti-HER3), and Kijanka et al. (2013) (for anti-HER2) and Mercier et al. (2019) (for anti-HER2). A series of V-binding domains suitable for anti-HER2, anti-HER3, anti-CD123, anti-CEACAM, anti-c-Met, anti-EGFR, anti-IGF-1R, anti-PD-L1, anti-CTLA-4, anti-CD19, anti-HER1 and anti-VGFR2HH Taking into account their ability to bind to tumor cell-specific receptors, the amino acid sequences and / or cDNA sequences of V are also provided herein below as SEQ ID NOs: 1 to 32 and 36 to 72. In particular, V can bind to the binding site of any of the tumor cell-specific receptors HER2, VEGFR and CD71. HH are suitable for incorporation into the first conjugate of the invention. The inventors have HH have demonstrated that ADCs comprising:

[0088] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the effector molecule comprises or consists of at least one of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, xenonucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof.

[0089] In one embodiment, the effector molecule is selected from the group consisting of vectors, genes, cell suicide inducing transgenes, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), anti-microRNA (anti-miRNA), DNA aptamers, RNA aptamers, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), and the like. com / literaturevault / superior-silencing-by-2-4-bnanc-based-short-antisense-oligonucleotides.aspx, 3'-fluorohexitol nucleic acid (FHNA), plasmid, glycol nucleic acid (GNA) and threose nucleic acid (TNA), or derivatives thereof, more preferably a BNA, for example a BNA for silencing HSP27 protein expression, or a BNA for silencing apolipoprotein B expression.

[0090] In one embodiment, the effector molecule is selected from the group consisting of short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single stranded RNA, aptamer RNA, double stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), mRNA, DNA, antisense DNA, locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-O,4'-aminoethylene bridged nucleic acid (BNA), and the like. NC ), BNA-based siRNA, and BNA-based antisense oligonucleotides (BNA-AONs).

[0091] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the effector molecule is an oligonucleotide selected from any one of anti-miRNA, BNA-AON, or siRNA, e.g., BNA-based siRNA, preferably selected from chemically modified siRNA, metabolically stable siRNA, and chemically modified and metabolically stable siRNA.

[0092] In one embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the effector molecule is an oligonucleotide capable of silencing a gene (when present in a cell containing such a gene), the gene being any one of the following genes: apolipoprotein B (apoB), transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of the hepatitis B virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT) and lactate dehydrogenase (LDH), and / or capable of targeting an aberrant miRNA (when present in a cell containing such miRNA).

[0093] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the effector molecule is an oligonucleotide capable of targeting an mRNA (when present in a cell containing such an mRNA) which is involved in the expression of any one of the following proteins: apoB, PCSK9, TTAS1, AT3, GO, CC5, the expression product of the X gene of HBV, the expression product of the S gene of HBV, AAT and LDH, or capable of antagonizing or restoring miRNA function, such as inhibiting oncogenic miRNAs (onco-miRs) or suppressing the expression of onco-miRs (when present in a cell containing such miRNAs).

[0094] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the effector molecule comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of peptides, proteins, enzymes and protein toxins. The inventors have found that highly effective tumor cell killing is achieved when an sdAb that binds to either HER2, VEGFR, CD71 is selected, which sdAb is combined in the first conjugate with a toxin, such as a protein toxin, such as dianthin or saporin. Examples showing the high efficacy of such first conjugates of the invention are provided in the Examples section and shown in Figures 4-7.

[0095] In one embodiment, the effector molecule comprises or consists of at least one of urease and Cre-recombinase, proteinaceous toxins, ribosome-inactivating proteins, protein toxins, bacterial toxins, plant toxins, more preferably viral toxins such as apoptin; bacterial toxins such as shiga toxins, 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 alpha-sarcin; plant toxins, e.g. ribosome-inactivating proteins and the A chain of type 2 ribosome-inactivating proteins, e.g. 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 A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or an animal or human toxin such as frog RNase, or granzyme B, or human angiogenin, or any toxic fragment or toxic derivative thereof; preferably, the protein toxin is dianthin and / or saporin.

[0096] One embodiment is a pharmaceutical combination or a pharmaceutical composition according to the invention, wherein the effector molecule comprises or consists of at least one payload.

[0097] In one embodiment, the effector molecule is at least one of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, passudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethylauristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, Benzodiazepines, CC-1065 analogs, duocarmycin, 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, Pseudomonas aeruginosa The pharmaceutical combination or pharmaceutical composition of the present invention comprises or consists of any one or more of a truncated form of A. aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or derivatives thereof.

[0098] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the first conjugate comprises or consists of an antibody drug conjugate (ADC), such as an ADC comprising at least one sdAb and a toxin derived from any one of the following ADCs: gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin. When the sdAb is derived from such a human antibody, the V of such a human antibody is H The domains may be selected from domain stability methods known in the art (e.g., human V H It will be appreciated that some refinement with respect to "camelization" of the domain may be required.

[0099] Effector moieties useful as part of an ADC or AOC (i.e., the first conjugate of the invention) in the present invention preferably rely on late endosomal escape to exert their effect. For example, some effector molecules, such as Pseudomonas exotoxin, are redirected to other organelles before the "late endosome" and therefore do not usually benefit from incorporation into an ADC for combination with the second conjugate according to the present invention. However, such toxins can be adapted for use in the present invention, for example, by deleting the signal peptide involved in the redirection. In particular, toxins that are highly toxic and require only one molecule to escape the endosome to kill the cell can be modified to reduce their potency. 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 (enter the cytosol). It is further preferred that the first conjugate of the present invention, which is included in the pharmaceutical composition of the present invention or in the pharmaceutical combination, comprises a covalently conjugated functionalized scaffold, i.e., a scaffold such as an oligomeric or polymeric molecule or a trifunctional linker, which comprises one or more covalently bound effector moieties for targeting the scaffold comprising the bound effector moieties in target cells, such as tumor cells or autoimmune cells. Furthermore, in order to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0100] Preferably, the effector moiety contained in an ADC or AOC (i.e., first conjugate of the invention) for combination with a second conjugate of the invention, whose effector moiety effect is enhanced by a saponin contained in the second conjugate of the invention, is detached from the first conjugate (i.e., ADC, AOC) upon endocytosis (e.g., from an antibody such as an sdAb present in the first conjugate as a cell surface molecule targeting moiety of the first conjugate). This can be achieved, for example, by a cleavable bond that cleaves under acidic, reducing, enzymatic or light-induced conditions.

[0101] The inventors show that when tumor cells are contacted with a tumor cell targeting monoclonal antibody or sdAb (first conjugate of the invention) in combination with a tumor cell targeting monoclonal antibody or sdAb (i.e. second conjugate of the invention) with a covalently attached antisense BNA such as BNA (HSP27) and with the same or different, covalently attached saponin, both the BNA attached (by a cleavable bond) to a cell targeting ligand (i.e. first conjugate) and a saponin attached (by a cleavable bond) to an antibody (e.g. sdAb of the second conjugate of the invention) are able to silence HSP27 in tumors in vivo compared to controls and AOC with the BNA in the absence of the second conjugate of the invention comprising saponin (SO1861). Thus, an ADC (a first conjugate of the invention) may be administered together with a second conjugate comprising a saponin of the invention, or an antibody-oligonucleotide conjugate, such as an antibody-BNA conjugate of the invention (wherein the antibody is a VV HH(HSP27) together with a second conjugate comprising a saponin of the present invention confers antitumor cell activity to the ADC or AOC not seen with the ADC or AOC alone at the same dose in the absence of the second conjugate of the present invention. The antisense BNA (HSP27) was a BNA with an oligonucleic acid sequence by Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333]. Of note, to the best of the inventors' knowledge, BNAs have been designed for application as free nucleic acids. The inventors have demonstrated that antisense BNAs can be used in combination with ligands or antibodies, such as mAbs or sdAbs, such as V HH We have demonstrated for the first time that BNAs can be covalently linked to BNAs via a (non)cleavable linker in a manner that retains gene silencing activity in vitro and, more importantly, in vivo in tumor cells of tumor-bearing animals. This method of providing a BNA-based AOC, i.e., the first conjugate of the present invention, opens a new method for administering targeted BNAs to human (cancer) patients in need thereof.

[0102] In one embodiment, the saponin is 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Gypsogenin; Quilacic acid; Protoesigenin 21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-valingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; Digitogenin; 3,16,28-trihydroxyoleanan-12-ene; and Gypsogenic acid or the saponin derivative comprises an aglycone core structure selected from Preferably, the saponin comprises or the saponin derivative is derived from an aglycone core structure selected from quillic acid and gypsogenin, more preferably the saponin comprises or the saponin derivative is derived from the aglycone core structure quillic acid.

[0103] Without wishing to be bound by any theory, the presence of an aldehyde group (or a derivative thereof) in the aglycone core structure (also referred to herein as "aglycone") of the saponin is beneficial for the ability of the saponin to stimulate and / or enhance the endosomal escape of the effector molecule contained in the first conjugate, such as an ADC or AOC according to the present invention, when such saponins are co-localized in a cell, in the endosomes of said cells, as part of a second conjugate of the present invention, or when they are in free form inside the endosome (e.g., separated from the second conjugate once the second conjugate is delivered inside the endosome or lysosome of the target cell), and when exposed to the cell in the presence of a first conjugate having an effector molecule and an sdAb that targets the cell. Thus, the second conjugate of the present invention comprising a saponin having an aglycone with an aldehyde group is preferred. In quillic acid and gypsogenin, the aldehyde group is located at the C of the aglycone. 23 It's in the atom.

[0104] In one embodiment, the saponin or saponin derivative is preferably attached to the C3 atom or C4 atom of the aglycone core structure of the saponin or saponin derivative. 28 and / or the saponin or saponin derivative is a saponin derivative having a first glycan bonded to the C atom of the aglycone core structure of the saponin or saponin derivative. 28 The pharmaceutical combination or pharmaceutical composition of the present invention optionally comprises a second sugar chain attached to the atom, preferably at least one saponin comprises a first and a second sugar chain. Thus, when the saponin contained in the second conjugate of the present invention has two glycans (sugar chains), the first sugar chain is attached to the C3 position of the aglycone core structure and the second sugar chain is attached to the C4 position of the aglycone core structure. 28 It binds to the position.

[0105] In one embodiment, Saponin or a saponin derivative, GlcA-, Glc-, Gal-, Rha-(1→2)-Ara-, Gal-(1→2)-[Xyl-(1→3)]-GlcA-, Glc-(1→2)-[Glc-(1→4)]-GlcA-, Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-, Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and The first sugar chain is selected from these derivatives, and / or Saponin or a saponin derivative, Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc- (wherein R1 is 4E-methoxycinnamic acid), Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc- (wherein R2 is 4Z-methoxycinnamic acid), Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc- (wherein R3 is 4E-methoxycinnamic acid), Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc- (wherein R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc- (wherein R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- (wherein R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- (wherein R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- (wherein R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- (wherein R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- (wherein R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- (wherein R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- (wherein R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Glc-(1→3)-[Glc-(1→6)]-Gal-, and The pharmaceutical combination or pharmaceutical composition of the present invention comprises a second glycan selected from these derivatives.

[0106] Thus, when the saponin contained in the second conjugate of the present invention has two glycans (sugar chains), the first sugar chain is bound to the C3 position of the aglycone core structure, and the second sugar chain is bound to the C4 position of the aglycone core structure. 28 It binds to the position.

[0107] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the saponin or saponin derivative in the second conjugate comprises a first glycan and comprises a second glycan according to claim 24 or 25, wherein the first glycan comprises two or more saccharide moieties, the second glycan comprises two or more saccharide moieties, and the aglycone core structure is quillic acid or gypsogenin or a derivative thereof, and wherein one, two or three, preferably one or two of the following are satisfied: i. the aldehyde group in the aglycone core structure is derivatized; ii. the carboxyl group of the glucuronic acid moiety in the first glycan is derivatized; and iii. At least one acetoxy (Me(CO)O-) group in the second glycan is derivatized.

[0108] One embodiment is a pharmaceutical combination or composition of the invention, wherein the saponin or saponin derivative in the second conjugate comprises: i. -Reduction to alcohol; - conversion to a hydrazone bond by reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond by reaction with N-[β-maleimidopropionic acid] hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by formation of a thioether bond with mercaptoethanol; or - Conversion to a hydrazone bond by reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is optionally derivatized by formation of a thioether bond with mercaptoethanol. Aglycon core structure containing an aldehyde group derivatized with: ii. a first glycan comprising a carboxyl group, preferably a carboxyl group of a glucuronic acid moiety, that has been derivatized by conversion to an amide bond by reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); iii. a second glycan containing an acetoxy group (Me(CO)O-) that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Any combination of two or three derivatizations i., ii. and / or iii., preferably any combination of two derivatizations i., ii. and / or iii.

[0109] In one embodiment, the saponin or saponin derivative is selected from the group consisting of quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, escinate, AS6.2, NP-005236, AMA-1, AMR, alpha-hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-0 17705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, S O1674, SO1832, SO1904, SO1862, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21B-apio, QS-21B-xylo, β-escin, escin Ia, tea seed saponin I, tea seed saponin J, The pharmaceutical combination or pharmaceutical composition of the present invention is selected from Assamsaponin F, Digitonin, Primular acid 1 and AS64R, or derivatives thereof, or stereoisomers thereof, and / or combinations thereof, preferably the saponin or saponin derivative is any one or more of QS-21 or a QS-21 derivative, SO1861 or a SO1861 derivative, SA1641 or a SA1641 derivative, and GE1741 or a GE1741 derivative, more preferably a QS-21 derivative or a SO1861 derivative, most preferably a SO1861 derivative, wherein preferably such saponin derivative is a saponin derivative according to the present invention.

[0110] In one embodiment, the saponin or saponin derivative is selected from the group consisting of SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypsoside A, AG1, AG2, SO1542, SO158 4, SO1658, SO1674, SO1832, SO1862, SO1904, or derivatives thereof, or stereoisomers thereof, and / or any combination thereof, preferably the saponin derivative is a SO1861 derivative, and / or a GE1741 derivative, and / or a SA1641 derivative, and / or a QS-21 derivative, more preferably the saponin derivative is a SO1861 derivative or a QS-21 derivative, and most preferably the saponin derivative is a SO1861 derivative according to the present invention.

[0111] Such triterpene glycoside type saponins are capable of enhancing endosomal escape of effector molecules present in the endosomes (or lysosomes) of a cell when the saponins are co-localized with such effector molecules within the cell, either as part of a second conjugate or in free form. The inventors have demonstrated that the endosomal escape enhancing activity of these saponins is approximately 100-1000 times more potent when the saponins are contacted with cells as part of a second conjugate of the invention. Free saponins can stimulate the delivery of effector molecules in the cytosol of a cell at concentrations 100-1000 times higher when such cells are contacted with the effector molecule and the saponin as part of a first conjugate, compared to the concentration of the same saponin contained in the second conjugate of the invention required to achieve the same degree of delivery of the effector molecule from outside the target cell to the inside of the endosome and ultimately into the cytosol of said cell. Saponins that exhibit such endosomal escape enhancing activity, as well as saponins with high structural similarity to saponins that have demonstrated the ability to enhance cytosolic delivery of effector molecules included in the first conjugate, are listed in Table A1. When the saponin is part of a second conjugate of the invention, the targeted delivery of the saponin upon binding of the binding molecule of the second conjugate, e.g., an antibody or sdAb, to a second cell surface binding site on the target cell and after endocytosis into the endosomes of said cell is therefore about 100-1000 times more effective than when the same cell is contacted with a free non-targeted saponin (derivative) that is not provided with a binding molecule, such as an antibody or sdAb, for binding to the second cell surface molecule of the target cell. In a particular embodiment, the binding molecule included in the second conjugate is an sdAb. The small size of the sdAb of the second conjugate of the present invention, e.g. compared to an IgG type antibody or fragment thereof, e.g. Fab, scFv, contributes to efficient uptake, e.g. by endocytosis, by the target cell exposing a second binding site for binding of the sdAb comprised in the second conjugate.Typically the sdAb in the second conjugate of the invention is capable of binding to a cell surface receptor on a target cell that is the same or different from the cell surface receptor targeted by the sdAb comprised in the first conjugate of the invention, for example a tumor cell specific cell surface receptor. Thus, the first and second conjugates of the invention are particularly suitable for endocytosis into, for example, tumor cells expressing the first and second cell surface receptors, which may be the same or different.

[0112] In one embodiment, the second conjugate comprises a binding molecule for binding to a second cell surface molecule, the binding molecule being any one or more of a proteinaceous ligand for binding to a second cell surface molecule, such as EGF or a cytokine, or a ligand such as an immunoglobulin or at least one binding fragment or binding domain of said immunoglobulin capable of binding to a second cell surface molecule, wherein the immunoglobulin is preferably a monoclonal antibody, preferably a human antibody, IgG, sdAb, at least one V HH Domain or at least one V H A molecule comprising or consisting of a domain, the variable heavy chain novel antigen receptor (V NAR ) domain, Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment or any other antibody.

[0113] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the second conjugate comprises a binding molecule for binding to a second cell surface molecule, the second cell surface molecule being a cell surface receptor present on a diseased cell such as a tumor cell, an autoimmune cell, an infected cell such as a virally infected cell.

[0114] In one embodiment, the second conjugate comprises a binding molecule for binding to a second cell surface molecule, such as an antibody, or a binding fragment or binding domain thereof, e.g., an sdAb, or a receptor ligand, the second cell surface receptor being preferably a tumor cell surface receptor, such as HIV gp41, or a tumor cell specific receptor, more preferably CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrins, syndecan-1, vascular integrin αVβ3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate specific membrane antigen (PSMA), CanAg, integrin αV, CA6, CD33, mesothelin, Cripto, CD3, CD3 0, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC-1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5, CD16, CXCR7 (ACKR3), glucocorticoid-inducible TNFR-related protein (GITR), and most preferably, a cell surface receptor selected from HER2, c-Met, VEGFR2, CXCR7, CD71, EGFR and EGFR1.

[0115] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the second conjugate comprises a binding molecule for binding to a second cell surface molecule, said binding molecule being an immunoglobulin, preferably an sdAb, selected from: anti-CD71 antibodies such as IgG OKT-9, anti-HER2 antibodies such as trastuzumab (Herceptin), pertuzumab, anti-CD20 antibodies such as rituximab, ofatumumab, tositumomab, obinutuzumab, ibritumomab, anti-CA125 antibodies such as oregovomab, anti-EpCAM (17-1A) antibodies such as edrecolomab, cetuximab, matuzumab, panitumumab, nimotuzumab, anti-EGFR antibodies such as huMy9-6, anti-CD30 antibodies such as brentuximab, anti-CD33 antibodies such as gemtuzumab and huMy9-6, anti-vascular integrin αvβ3 antibodies such as etaracizumab, anti-CD52 antibodies such as alemtuzumab, anti-CD22 antibodies such as epratuzumab and pinatuzumab, binding fragments (Fv) of the anti-CD22 antibody moxetumomab, humanized monoclonal antibodies inotuzumab, anti-CEA antibodies such as labetuzumab, anti-CD44v6 antibodies such as bivatuzumab, anti-FAP antibodies such as sibrotuzumab, anti-CD19 antibodies such as huB4, anti-CanAg antibodies such as huC242, anti-CD56 antibodies such as huN901, anti-CD38 antibodies such as daratumumab, OKT-10 anti-CD38 monoclonal antibody, anti-CA6 antibody such as DS6, anti-IGF-1R antibody such as cixutumumab, anti-integrin antibody such as 3B7, CNTO 95, anti-syndecan-1 antibody such as B-B4, anti-CD79b such as polatuzumab, anti-HIV gp41 antibody, preferably any one of anti-HIV gp41 antibody, anti-CD71 antibody, anti-HER2 antibody and anti-EGFR antibody, more preferably any one of trastuzumab, pertuzumab, cetuximab, matuzumab, anti-CD71 antibody and OKT-9, most preferably trastuzumab, cetuximab and anti-CD71 antibody OKT-9.

[0116] In one embodiment, the second conjugate comprises an sdAb capable of binding to HER2, CD71, HIV gp41 and / or EGFR, the sdAb preferably being HH and more preferably camelid VH The pharmaceutical combination or pharmaceutical composition of the present invention is

[0117] In one embodiment, the second conjugate is an sdAb for binding to HER2 selected from sdAbs produced by clone 11A4, clone 18C3, clone 22G12, clone Q17 or clone Q17-C tag; or an sdAb for binding to EGFR produced by clone anti-EGFR Q86-C tag; or an sdAb for binding to CD71 produced by clone anti-CD71 Q52-C tag; or an sdAb for binding to CD71 produced by clone anti-HIV gp41 or an sdAb encoded by the cDNA of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; or an sdAb having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36-72, optionally the first conjugate further comprises a covalently attached sdAb for binding to albumin, for example any one or more of the sdAbs having the amino acid sequence of SEQ ID NOs: 33, 34 and 35, preferably the sdAb is V HH and more preferably camelid V H A pharmaceutical combination or composition of the invention, which is a compound suitable for incorporation into a second conjugate of the invention, HHcan be found, for example, in the Single Domain Antibody Database (Wilton, EE et al. (2018)), patent applications US 2016 / 0251440 (anti-CD123, anti-CEACAM), US 9683045 (anti-c-Met), US 2009 / 0252681 (anti-EGFR, anti-IGF-1R), US 9969805 (anti-HER2), US 20190023796A1 (anti-HER3), and Kijanka et al. (2013) (for anti-HER2) and Mercier et al. (2019) (for anti-HER2). A series of V-binding domains suitable for anti-HER2, anti-HER3, anti-CD123, anti-CEACAM, anti-c-Met, anti-EGFR, anti-IGF-1R, anti-PD-L1, anti-CTLA-4, anti-CD19, anti-HER1 and anti-VGFR2 HH Taking into account their ability to bind to tumor cell-specific receptors, the amino acid sequences and / or cDNA sequences of V are also provided herein below as SEQ ID NOs: 1 to 32 and 36 to 72. In particular, V can bind to the binding site of any of the tumor cell-specific receptors HER2, VEGFR and CD71. HH are suitable for incorporation into the second conjugate of the invention. The inventors have combined saponin with a V HH It has been shown that conjugates with binding molecules including are effective for delivery of saponin bound to sdAb. See, for example, the Examples section and Figures 2-4.

[0118] These cell surface molecules are typically present on tumor cells with at least some degree of tumor cell specificity. Tumor cell specificity makes these receptors suitable targets for the first and second conjugates of the present invention, and thus the sdAb in the first conjugate can bind to such cell surface receptors, and thus the binding molecule contained in the second conjugate of the present invention, such as an antibody or sdAb, can bind to such cell surface receptors. Tumor cells are generally targeted by the second conjugate, which comprises a binding molecule that targets a tumor cell receptor, such as an antibody or a binding fragment or domain thereof, and comprises a saponin that is delivered to the interior of the target tumor cell, more specifically, into the cytosol of said tumor cell. Since the saponin contained in the second conjugate of the invention can stimulate the release and delivery of the effector molecule contained in the first conjugate of the invention in the cytosol of a cell, e.g. a (tumor) cell targeted by the binding molecule of the second conjugate, e.g. an sdAb contained in the second conjugate of the invention, it is particularly suitable to select a cell surface receptor known to be suitable to serve as a target for ADCs and AOCs, as a target (tumor) cell surface molecule for a binding molecule such as an sdAb. Accordingly, the first and second conjugates of the invention are suitable for co-delivery of an effector molecule that is part of the first conjugate, which is an ADC or AOC comprising an sdAb, together with the saponin contained in the second conjugate of the invention. Targeting a tumor cell specific receptor with the second conjugate of the invention promotes endocytosis and delivery of the saponin as part of the second conjugate to the target cell endosomes and / or lysosomes. When the tumor cells are also contacted with an ADC or AOC, such as the first conjugate of the invention, the effector molecule contained in such ADC or AOC or the first conjugate of the invention is co-delivered to the endosome or lysosome and, under the influence of saponin, subsequently delivered to the cytosol of the target cell.As mentioned above, the application of a targeted saponin as part of the second conjugate of the invention results in an approximately 100-fold to 1000-fold enhancement of the stimulatory effect of the saponin compared to the application of free saponin lacking a cell-targeting binding molecule such as a receptor ligand, antibody or sdAb, when taking into account the biological activity of the effector molecule comprised in the first conjugate of the invention in camelids V in the second conjugate of the invention. H The application of small sdAbs such as , prevents or delays the clearance of the second conjugate of the present invention from the circulation and body of a human subject to which the second conjugate is administered, compared to the clearance rates typically observed with antibody-based ADCs. This is also evident for the first conjugate comprising an sdAb, as described above. In addition, the relatively small size of the sdAb limits the risk of restricting or interfering with saponin activity inside the target cell due to the presence of linked protein domains, compared to the larger size of, for example, an antibody linked to a saponin. In general, the smaller the size of the molecule linked to the saponin, the greater the potential for the binding of the linked molecule, for example, V. HHThe risk of interference with intracellular saponin activity due to the presence of antibody domains such as sdAbs is reduced. Furthermore, the relatively small size of sdAbs results in their rapid distribution in tissues, such as tumor tissue, improving the target cell reach of the second conjugates of the invention, and thus the (extent of) binding to the target cells, compared to the relatively large size of IgGs that are commonly applied, for example, in ADCs, OACs. One of the many advantages of applying sdAbs in the second conjugates of the invention (which also applies, for example, to applying sdAbs in ADCs and AOCs, i.e., the first conjugates of the invention) is the absence of an Fc tail, which is common to conventional antibodies, for example of the IgG type. The absence of an Fc tail in the sdAbs of the first and second conjugates of the invention prevents the occurrence of Fcγ receptor-mediated off-target effects, e.g. undesirable side effects related to Fcγ receptor activation, when the first and second conjugates are administered to a patient in need thereof. The lack of an Fc tail eliminates the risk of side effects caused by Fc binding to the cells of a patient to whom the antibody-based ADC is administered, for example. The sdAb containing conjugates of the invention do not carry the risk of this Fc-mediated undesirable side effect, but the inventors have demonstrated that when tumor cells are contacted with both an ADC (at a relatively low dose; see also the Examples section) and a second conjugate of the invention (at a relatively low dose; see also the Examples section), such first and second conjugates are highly efficient and effective in enhancing the effector molecule contained in the first conjugate.

[0119] In one embodiment, the second conjugate comprises a saponin or saponin derivative covalently attached to a sulfhydryl group in the binding molecule via a thio-ether bond, and a covalent bond via the linker N-ε-maleimidocaproic acid hydrazide (EMCH) at the C of the aglycone core structure of the saponin or saponin derivative. 23 In another aspect, the pharmaceutical combination or pharmaceutical composition of the present invention is a pharmaceutical composition comprising a carboxyl group and a carboxyl group covalently bonded to an aldehyde group at the position of the carboxyl group and a sulfhydryl group in the linking molecule, such as a sulfhydryl group of cysteine.

[0120] In one embodiment, the second conjugate is C of the aglycone core structure of the saponin or derivative thereof. 23 The pharmaceutical combination or pharmaceutical composition of the present invention comprises a bidesmosidic triterpene saponin or derivative thereof belonging to the 12,13-dehydrooleanane type having an optional aldehyde functional group at position 1 of the saponin aglycone core structure, the saponin or derivative thereof comprising a glucuronic acid unit in a first saccharide chain of the C3β-OH group of the aglycone core structure of the saponin, the saponin or derivative thereof being covalently attached to an amino acid residue of a binding molecule via a carboxyl group of the glucuronic acid unit of the first saccharide chain, preferably via at least one linker, the amino acid residue being preferably selected from cysteine ​​and lysine.

[0121] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the second conjugate comprises at least one saponin or derivative thereof comprising a glucuronic acid unit in a first saccharide chain of the C3β-OH group of the aglycone core structure of the saponin or derivative thereof, said glucuronic acid unit being covalently attached to a linker, preferably covalently attached to an amine group in the binding molecule, e.g., an amine group of a lysine or the N-terminus of the binding molecule via an amide bond, preferably wherein the glucuronic acid unit is covalently attached to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0122] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the second conjugate comprises two or more covalently attached saponins or derivatives thereof, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100 saponins or derivatives thereof, or any number in between, for example 7, 9, 12.

[0123] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, in which two or more covalently linked saponins or saponin derivatives are covalently linked to amino acid residues of the binding molecule, preferably directly to cysteine ​​and / or lysine, and / or via at least one linker and / or at least one cleavable linker. Linking the saponin to a binding molecule, such as an antibody, sdAb or ligand for a second cell surface molecule, via a linker provides flexibility when considering the binding site of the binding molecule for binding the saponin. Furthermore, such a linker can act as a spacer between the binding molecule, such as an sdAb, and the saponin, so that the binding molecule maintains its ability to bind to the binding site of the cell surface molecule, and the saponin maintains its ability to enhance endosomal escape of the effector molecule contained in the first conjugate (when the binding molecule, such as an sdAb, binds to the cell surface molecule and the second conjugate co-localizes with the effector molecule in the endosome or lysosome of the cell targeted by the first conjugate).

[0124] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, in which the two or more covalently bound saponins or saponin derivatives are part of a covalent saponin conjugate comprising at least one oligomeric or polymeric molecule to which the two or more saponins or saponin derivatives are covalently bound, the covalent saponin conjugate being covalently bound to a binding molecule, such a covalent saponin conjugate serving as a carrier for multiple saponin moieties that can be attached via a single bond, preferably via a (cleavable) linker, to a binding molecule, such as an antibody or sdAb, contained in a second conjugate. The application of such covalent saponin conjugates offers flexibility when considering the number of saponin moieties in the second conjugate of the invention, since the covalent saponin conjugates can have any selected number of covalently bound saponin moieties, for example 1 to 200 saponin moieties, in relation to the selected type of oligomeric or polymeric structure that contains the binding sites for covalently binding these saponins. For example, for cytosolic delivery of a selected effector molecule comprised in a first conjugate of the invention, the number of saponins present in the second conjugate of the invention can be adapted by providing a covalent saponin conjugate having a sufficient number of saponin moieties to stimulate cytosolic delivery of the effector molecule (when the covalent saponin conjugate is part of the second conjugate of the invention and when the effector molecule co-localizes with the second conjugate in the endosomes or lysosomes of the target cell in which it is to exert its biological activity).

[0125] One embodiment is a pharmaceutical combination or composition of the invention, wherein 1-8 such covalent saponin conjugates, more preferably 2-4 such covalent saponin conjugates are attached to a binding molecule, at least one covalent saponin conjugate is optionally dendron based, and optionally 1-32 saponins or saponin derivatives, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 or any number in between, such as 7, 9, 12 saponins or saponin derivatives, are covalently attached, directly or via a linker, to the at least one covalent saponin conjugate oligomeric or polymeric molecule. Preferably, one or two of the covalent saponin conjugates are attached to a single binding molecule such as an sdAb in the second conjugate of the invention. For many purposes, the binding of a single saponin or a single covalent saponin conjugate to a single binding molecule, such as an antibody or sdAb, contained in a second conjugate is sufficient to efficiently stimulate the delivery of effector molecules into the target cell and the cytosol of said cells, where the effector molecule is contained in the first conjugate of the present invention. Typically, 4, 8 or 16 saponins are contained in the second conjugate of the present invention, for example, 4 or 8 saponins are contained in a single covalent saponin conjugate bound to a binding molecule, such as an sdAb, in the second conjugate of the present invention. Typically, such a second conjugate of the present invention comprises a single antibody or a single sdAb to which a saponin or a saponin or a covalent saponin conjugate is attached, preferably a single saponin or a single covalent saponin conjugate is part of the second conjugate.

[0126] One embodiment is a pharmaceutical combination or composition of the invention, wherein the saponin or saponin derivative is covalently attached to the binding molecule via a cleavable linker.

[0127] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the cleavable linker is cleaved under acidic, reducing, enzymatic and / or light-induced conditions, preferably the cleavable linker comprises a cleavable bond selected from hydrazone and hydrazide bonds, which are cleaved under acidic conditions, and / or a bond susceptible to proteolysis, e.g. proteolysis by cathepsin B, and / or a bond susceptible to cleavage under reducing conditions, such as a disulfide bond.

[0128] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the cleavable linker is cleaved in vivo, for example under acidic conditions such as those present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0-6.5, more preferably at a pH of ≦5.5. Such a cleavable linker, being cleavable under conditions such as those found in endosomes and lysosomes, facilitates the delivery of the free saponin inside the endosome or lysosome upon cleavage of the saponin from the remainder of the second conjugate of the invention. Thus, the second conjugate of the invention combines the benefits of cell-targeted delivery of the saponin upon specific binding of a binding molecule, such as an antibody or sdAb, to a second cell surface molecule on a target cell, and the presence of the free saponin intracellularly, i.e. in the endosome (or lysosome), which contributes to the ability of the free saponin to stimulate and / or facilitate the delivery of an effector molecule comprised in the first conjugate of the invention from the endosome (or lysosome) to the cytosol of the target cell.

[0129] One embodiment is a pharmaceutical combination or composition of the invention, wherein the oligomeric or polymeric molecule of the covalent saponin conjugate is covalently attached to the binding molecule, preferably to an amino acid residue of the binding molecule.

[0130] In one embodiment, the saponin or saponin derivative is covalently attached to the oligomeric or polymeric molecule of the covalent saponin conjugate via a cleavable linker according to the present invention.

[0131] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the saponin or saponin derivative is covalently attached to the oligomeric or polymeric molecule of the covalent saponin conjugate via any one or more of an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thioether bond, an amide bond, a peptide bond or an ester bond, preferably via a linker.

[0132] In one embodiment, the saponin or saponin derivative is 23The pharmaceutical combination or pharmaceutical composition of the present invention comprises an aglycone core structure comprising an aldehyde functional group at position 1 of the saccharide chain of the C3β-OH group of the aglycone core structure of the saponin, the aldehyde functional group optionally comprising a glucuronic acid functional group in the first saccharide chain of the C3β-OH group of the aglycone core structure of the saponin, the aldehyde functional group being involved in a covalent bond to an oligomeric or polymeric molecule of the covalent saponin conjugate, and / or the glucuronic acid functional group, if present, being involved in a covalent bond to an oligomeric or polymeric molecule of the covalent saponin conjugate, the attachment of the saponin or saponin derivative being via a direct covalent bond or via a linker, the linker being a cleavable linker or a stable linker, where stable refers to the bond between the saponin and the binding molecule, such as an sdAb, or between the saponin and the oligomeric or polymeric structure, which bond remains intact (not cleaved) under acidic conditions within a cell, particularly under acidic conditions within the endosomes or lysosomes of such cells. Moreover, such stable bonds remain intact (i.e., are not cleaved) in the circulation and organs of a human subject to which, for example, a second conjugate of the invention comprising a covalent saponin conjugate is administered. In contrast, a cleavable linker associated with the attachment of a saponin to a binding molecule comprised in a second conjugate, such as an antibody or sdAb, or to an oligomeric or polymeric structure, refers to a bond that is cleaved under acidic conditions such as those found inside endosomes and lysosomes of human cells, e.g., mammalian cells, such as tumor cells, whereas such a cleavable linker remains intact (not cleaved) when a second conjugate comprising such a cleavable bond is present in the circulation or organs, i.e., in external cells of, for example, a human subject to which, the second conjugate of the invention is administered.

[0133] One embodiment is the C of the aglycone core structure of the saponin or saponin derivative. 23The pharmaceutical combination or pharmaceutical composition of the present invention is a pharmaceutical combination or pharmaceutical composition in which the aldehyde functional group at position 1 is covalently linked to a linker EMCH, which is covalently linked to a sulfhydryl group, such as a sulfhydryl group of cysteine, in an oligomeric or polymeric molecule of the covalent saponin conjugate via a thio-ether bond. The binding of the EMCH linker to the aldehyde group of the aglycon of the saponin results in the formation of a hydrazone bond. Such a hydrazone bond is a typical example of a bond that is cleavable under acidic conditions in endosomes and lysosomes. A saponin bound to a binding molecule contained in a second conjugate, such as an antibody or sdAb in the second conjugate of the invention, or to an oligomeric or polymeric structure of a covalent saponin conjugate, where such a covalent saponin conjugate is bound to a binding molecule of the second conjugate, for example an sdAb in the second conjugate of the invention, can be released from the second conjugate of the invention when delivered to an endosome or lysosome of a target cell that exposes a second cell surface molecule to which the binding molecule, such as an antibody or sdAb of the second conjugate, can bind. Thus, the saponin bound to a binding molecule, such as an sdAb in the second conjugate of the invention, is transferred from outside the cell to an endosome (or lysosome) where it is released from the second conjugate upon pH-driven cleavage of the hydrazone bond. In the endosome (or lysosome), the free saponin can exert its stimulatory activity, taking into account the delivery to the cytosol of the effector molecule contained in the first conjugate of the invention, which is co-localized in the endosome (or lysosome). Surprisingly, the inventors have demonstrated that for saponin, the presence of the saponin in free form in the endosome or lysosome is not essential for the endosomal escape promoting activity of the saponin.Also, for example, a saponin contained in a second conjugate of the present invention enhances delivery of the effector molecule contained in the first conjugate from the endosome / lysosome to the cytosol of a target cell when both the effector molecule and the saponin as part of the first and second conjugates, respectively, contact the same target cell expressing the first and second cell surface molecules (which may be the same or different).

[0134] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the glucuronic acid functionality of the first sugar chain at the C3β-OH group of the aglycone core structure of the saponin or saponin derivative is covalently attached to a linker HATU, which is covalently attached via an amide bond to an amine group in an oligomeric or polymeric molecule of the covalent saponin conjugate, such as an amine group of a lysine or the N-terminus of a protein. When the HATU linker is attached to the saponin and to a binding molecule of the second conjugate, such as an sdAb, the saponin is attached, for example, to the N-terminus of the sdAb or to an amine group of a lysine present in the sdAb.

[0135] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein the polymeric or oligomeric molecule of the covalent saponin conjugate is linked to a binding molecule, preferably an amino acid residue of the binding molecule, and the polymeric or oligomeric molecule of the covalent saponin conjugate comprises a click chemistry group, preferably selected from a tetrazine, an azide, an alkene or an alkyne, or a cyclic derivative of these groups, more preferably the click chemistry group is an azide.

[0136] One embodiment is a pharmaceutical combination or pharmaceutical composition of the invention, wherein the polymeric or oligomeric molecules of the covalent saponin conjugate comprise polymeric and / or oligomeric structures selected from linear polymers, branched polymers and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, poly-lysine, poly-ethylene glycols, oligo-ethylene glycols (OEGs), such as OEG3, OEG4 and OEG5, or assemblies of these polymeric and / or oligomeric structures, the assemblies being preferably constructed by covalent crosslinking, preferably the polymeric or oligomeric molecules of the covalent saponin conjugate are dendrons, such as poly-amidoamine (PAMAM) dendrimers. The type and size or length of the oligomeric or polymeric structure is selected driven by the number of selected saponins to be incorporated into the second conjugate of the invention. That is, depending on the number of saponins attached to a binding molecule contained in the second conjugate, such as an sdAb, to form the second conjugate of the invention, a suitable oligomeric or polymeric structure can be selected with a sufficient amount of attachment sites to attach the desired number of saponins, thereby providing a covalent saponin conjugate having a selected number of saponin moieties attached to a binding molecule, such as an antibody, receptor ligand or sdAb, to provide a second conjugate of the invention. For example, the length of the OEG or the size of the dendron or polylysine molecule will determine the maximum number of saponins that can be covalently attached to such an oligomeric or polymeric structure.

[0137] Thus, the second conjugate according to the invention comprises at least one saponin. "At least one" in this context means that the second conjugate 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 second conjugate may comprise a covalently attached scaffold (covalently attached saponin conjugate) comprising a covalently attached saponin, and the scaffold may be designed to comprise a defined number of saponins. Preferably, the second conjugate according to the invention comprises a defined number or range of saponins, rather than a random number. This is particularly advantageous for pharmaceutical development in connection with marketing authorization. In this respect, a defined number means that the second conjugate preferably comprises a previously defined number of saponins. This is achieved, for example, by designing a scaffold comprising a polymer structure with a specific number of possible moieties for saponins to be (covalently) attached. Under ideal circumstances, all of these moieties will bind to the saponin, and the scaffold will contain a previously defined number of saponins. It is envisaged to provide a standard set of scaffolds, e.g., containing 2, 4, 8, 16, 32, 64, etc. saponins, so that the user can easily test the optimal number as needed. One embodiment is a second conjugate of the invention, comprising a scaffold of the invention (a covalent saponin conjugate of the invention), where the saponins are present in a defined range, e.g., under non-ideal circumstances, not all moieties present in the polymer structure are bound to the saponin. Such a range can be, for example, 2-4 saponin molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules per scaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules per scaffold, etc. In such cases, the second conjugate comprising a scaffold according to the invention (a covalent saponin conjugate of the invention) will therefore comprise 2, 3 or 4 saponins when the range is defined as 2 to 4.

[0138] The scaffold is essentially independent of the type of saponin covalently attached to it, which in turn is subsequently covalently attached to a cell surface molecule binding molecule, such as a receptor ligand, an antibody, such as an sdAb, in the second conjugate of the invention. Thus, the second conjugate of the invention comprising a scaffold (a covalently attached saponin conjugate of the invention) is the basis product for platform technology. Since at least one covalently attached saponin in the second conjugate of the invention mediates intracellular delivery of an effector moiety bound to a cell surface molecule-targeting sdAb comprised in a first conjugate of the invention, such as an ADC or AOC comprising an sdAb according to the invention, the scaffold technology according to the invention is a system for mediating controlled intracellular effector moiety delivery by saponin by combining an ADC or AOC (i.e., the first conjugate of the invention) with a second conjugate of the invention, which comprises a saponin and, for example and preferably, an antibody such as an sdAb for binding to a target cell surface molecule, for example and preferably, a receptor and a receptor involved in the endocytosis of the conjugate bound thereto. The scaffold provides an optimized functionally active unit that can be linked to a ligand targeting a cell surface molecule, such as an antibody such as an sdAb comprised in the second conjugate, at a single and defined location in the ligand such as an sdAb.

[0139] One embodiment is a second conjugate of the invention comprising a scaffold according to the invention (a covalent saponin conjugate of the invention), in which the number of monomers of the polymeric or oligomeric structure is a precisely defined number or range. Preferably, the polymeric or oligomeric structure comprises structures such as poly(amines), e.g. polyethyleneimines and poly(amidoamines), or structures such as polyethylene glycols, 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. poly-lysine, DNA polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers, in the form of linear, branched or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates (single or mixtures) of these structures. Preferably, the polymeric or oligomeric structures are biocompatible, where biocompatible means that they do not show substantial acute or chronic toxicity in the organism and are excreted intact or can be excreted by the body's metabolism and / or completely degraded into physiological compounds. The aggregates can be constructed by covalent cross-linking or non-covalent bonds and / or by attractive forces. Thus, they can also form nanogels, microgels or hydrogels or they can be bound 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 binding moieties (chemical groups) for the binding of glycoside molecules (and / or carrier molecules such as ligands, monoclonal antibodies or fragments thereof such as sdAbs (wherein sdAbs are preferred in the present invention).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 (approximately) 100% of a precisely defined number or range of binding moieties (chemical groups) in a polymeric or oligomeric structure are occupied by glycoside molecules (saponin of the invention) of a scaffold according to the invention (covalent saponin conjugate of the invention).

[0140] Preferably, dendrons are branched, well-defined, tree-like polymers with a single chemically addressable group at the origin of the tree, called the focal point. Dendrimers are conjugates in which two or more dendrons are connected at their focal points. Dendronized polymers are conjugates in which the focal points of one or more dendrons are connected 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 an aggregate (single or mixture) of these structures, which aggregate can be assembled by covalent cross-linking or non-covalent attraction and can form a nanogel, microgel or hydrogel, preferably the polymer is a poly(amine), such as a derivative of polyethyleneimine and poly(amidoamine), as well as structures such as polyethylene glycol, poly(ester), such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), as well as natural and / or artificial polyamino acids, such as poly-lysine, or peptides or proteins, or structures such as DNA polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers. Preferably, the polymeric or oligomeric structure is biocompatible.

[0141] It will be understood that when the binding molecules, such as the sdAb in the second conjugate of the invention and the sdAb in the first conjugate of the invention, i.e. the ADC or AOC, target different cell surface molecules, such as different tumor cell specific receptors, the two different targets are both present on the surface of the target cell to which the effector molecule comprised in the first conjugate, i.e. the ADC or AOC, and the saponin comprised in the second conjugate of the invention are to be co-delivered. When the ADC or AOC (first conjugate of the invention) and the second conjugate of the invention target different (tumor) cell receptors, the advantage is that when the doses of the first conjugate of the invention (ADC or AOC) and the second conjugate of the invention are contacted with a mixture of different cell types, including target cells, the risk of off-target effects, e.g. on cells that have one of the two cell surface molecules on their surface, is reduced, as would occur if a combination of the first conjugate of the invention (ADC or AOC) and the second conjugate of the invention were (co-)administered to a patient in need thereof. By administering a dose of the first conjugate (ADC or AOC) of the invention that is too low to deliver a therapeutically effective amount of the effector molecule into the target cell, the entry of cells that may express a cell surface molecule to which the ADC or AOC (first conjugate of the invention) can bind, but that do not express the target of the binding molecule, such as the sdAb in the second conjugate of the invention, when the ADC or AOC binds to such a target cell in the absence of a targeting saponin in the form of a second conjugate of the invention, will therefore not induce cytotoxicity, for example when considering the ADC. Only in target cells that have both a receptor for binding the ADC or AOC (first conjugate of the invention) and a receptor for binding the second conjugate of the invention, if endosomal escape occurs under the influence of a saponin co-localized inside said target cell expressing both receptors, will the dose of the effector molecule be sufficient to exert its biological effect in the cytosol of said cells.

[0142] In some embodiments, the binding molecule, such as the sdAb, contained in the second conjugate of the invention and the sdAb contained in the first conjugate of the invention (i.e., ADC or AOC) target the same cell surface molecule, such as the same tumor cell specific receptor. In this way, if only a single tumor cell specific receptor is present, simultaneous delivery of both the ADC or AOC (first conjugate of the invention) and the second conjugate of the invention to the inside of a target cell having such a single type of target receptor is possible. The advantage of such simultaneous delivery to a target cell using a single cell specific surface molecule, such as a tumor cell specific receptor, e.g., HER2, EGFR, CD71, is the possibility of targeting tumor cells expressing only a single receptor that is sufficiently specific for specific targeting of such target tumor cells by the second conjugate of the invention with the saponin and the ADC or AOC (i.e., first conjugate of the invention). By selecting and targeting such a single specific receptor, it becomes possible to simultaneously deliver the targeted saponin and the targeted effector molecule into the tumor cell, so that such difficult to treat tumor cells can still be effectively provided with an effective dose of the effector molecule.

[0143] One aspect of the invention relates to a pharmaceutical combination according to the invention or a pharmaceutical composition according to the invention for use as a medicament.

[0144] One aspect of the present invention relates to a pharmaceutical combination of the present invention or a pharmaceutical composition according to the present invention for use in the treatment or prevention of cancer, an autoimmune disease such as rheumatoid arthritis, an enzyme deficiency, a gene deficiency, a disease associated with a gene deficiency, amyloidosis, a disease associated with an enzyme deficiency, an infectious disease such as a viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha1-antitrypsin associated liver disease, acute hepatic porphyria, transthyretin mediated amyloidosis.

[0145] Surprisingly, the inventors have found that a dose of the first conjugate of the invention, i.e., ADC, that does not result in any tumor cell killing is sufficient for efficient tumor cell killing when the first conjugate of the invention comprising an effector molecule is contacted with tumor cells in the presence of the second conjugate of the invention. Examples are shown in Figures 4 to 7. For example, the ADC anti-CD71 V HH -Toxin, anti-HER2 V HH -Toxin or anti-EGFR V HH A dose of the first conjugate of the present invention, such as a toxin, does not exert a toxic effect on tumor cells when contacted with the tumor cells. Typical toxins are protein toxins, such as dianthin and saporin. However, when the first conjugate of the present invention is co-administered with the second conjugate of the present invention, efficient tumor cell killing is achieved. The second conjugate of the present invention is, for example, an anti-HER2 V toxin. HH -SO1861, anti-CD71 V HH -SO1861, anti-EGFR V HH The first conjugate of the present invention is, for example, a V-type conjugate capable of binding to HER2, CD71 or EGFR. HHFor example, the ADC contains a protein toxin such as dianthin or saporin. As described above, the dose of saponin required to achieve efficient biological activity of the effector molecule contained in the first conjugate of the present invention, i.e., ADC or AOC, in a target cell by applying the second conjugate of the present invention is about 100 to 1000 times lower when the second conjugate containing saponin is co-administered with the first conjugate of the present invention, i.e., ADC or AOC, to a target cell, compared to the dose of free saponin required when the first conjugate of the present invention is co-administered with free saponin to a target cell. Thereby, the second conjugate of the invention enhances the first conjugate of the invention, i.e., an sdAb comprising an ADC or AOC, at a dose of ADC or AOC that would not be effective against tumor cells when administered to a patient in need thereof in the absence of a targeted saponin (the second conjugate of the invention), and furthermore, the second conjugate of the invention is already sufficiently effective at relatively low doses, i.e., at doses where free saponin not provided with a tumor cell targeting binding molecule such as an sdAb is not sufficiently effective in enhancing the effector molecule, when considering the enhancement of the effector molecule of the first conjugate of the invention, i.e., the ADC or AOC. Together, this provides an improved method for treating a human patient in need of treatment with a therapeutically effective amount of the first conjugate of the invention, i.e., an ADC or AOC comprising an sdAb targeting tumor cells, since the ADC or AOC is already effective at low doses when co-administered to a patient in the presence of the second conjugate of the invention. One of the many advantages of the first and second conjugates of the present invention is the absence of an Fc tail in the sdAb portion of the first conjugate, and preferably also in the second conjugate. The lack of an Fc tail prevents unwanted binding of the conjugate to off-target cells of the patient that have Fc receptors, which may otherwise result in side effects as seen with many conventional ADCs, AOCs, if such an Fc tail is present.Antibody-based ADCs containing Fc tails and AOCs containing Fc tails suffer from reduced efficacy due to undesired binding of such IgG-based ADCs, AOCs to Fc receptors. As a result of Fc receptor binding, the effective dose of such IgG-based ADCs and AOCs is reduced. Accordingly, the lack of an Fc tail provides several advantages in this respect. The first conjugate, and preferably also the second conjugate, cannot have off-target and undesired binding to Fc receptors. This allows the first and second conjugates of the present invention to have a lower effective dose when considering the target receptor-mediated endocytosis and delivery of the first and second conjugates of the present invention into endosomes, since the conjugate is not "lost" due to Fc receptor binding, a drawback seen with IgG-based conjugates. As a result, the therapeutic window of the first conjugate of the present invention is wider than the therapeutic window that would be achieved if the sdAb was replaced by a conventional IgG containing an Fc tail. Similarly, as a result, the therapeutic window of the second conjugate of the invention is broader than would be achieved if the sdAb in the second conjugate were replaced by a binding molecule for binding to a second cell surface molecule on the target cell, e.g. a conventional IgG comprising an Fc tail.

[0146] One embodiment is a pharmaceutical combination for use according to the invention, or a pharmaceutical composition for use according to the invention, wherein the saponin is SO1861 or QS-21, or the saponin derivative is a SO1861 derivative or a QS-21 derivative, preferably a saponin derivative of the invention.

[0147] One embodiment is a pharmaceutical combination for use according to the invention, or a pharmaceutical composition for use according to the invention, comprising - the use is in the treatment or prevention of cancer in a human subject; and / or - said use is in the treatment or prevention of cancer in a patient in need thereof, wherein the first and second cell surface molecules are the same or different tumour cell surface molecules, preferably tumour cell specific surface molecules, and the binding molecule of the sdAb and the second conjugate binds to the tumour cell surface molecule, preferably the tumour cell specific surface molecule; and / or - the pharmaceutical combination, preferably a therapeutically effective amount of the pharmaceutical combination, is administered to a patient, preferably a human patient, in need thereof A pharmaceutical combination or pharmaceutical composition for use according to the present invention.

[0148] A further aspect of the present invention relates to an in vivo or ex vivo method for transferring a first conjugate of the invention from outside a cell into said cell, preferably into the cytosol of said cell, comprising the steps: a) providing a cell expressing on its surface a first cell surface molecule according to the invention and optionally expressing on its surface a second cell surface molecule according to the invention, said cell being preferably selected from diseased cells such as liver cells, virally infected cells, autoimmune cells and tumour cells; b) providing a first conjugate of the invention for transfer to the cell provided in step a); c) providing a saponin or saponin derivative of the invention and / or, if the cell provided in step a) expresses on its surface a second cell surface molecule according to the invention, providing a second conjugate of the invention; d) contacting the cells of step a) in vitro or ex vivo with the first conjugate of step b) and any one or more of the saponin, saponin derivative, and conjugate of step c); thereby establishing the transfer of a first conjugate from outside a cell into said cell, preferably into the cytosol of said cell.

[0149] One embodiment is an in vitro or ex vivo method for transferring a first conjugate of the invention from outside a cell according to the invention into said cell, comprising, once the first conjugate has been transferred into said cell, further transferring an effector molecule comprised in the first conjugate from an endosome, endolysosome or lysosome of the cell into the cytosol of the cell, a) providing a cell expressing on its surface a first cell surface molecule according to the invention and optionally expressing on its surface a second cell surface molecule according to the invention, said cell being preferably selected from diseased cells such as liver cells, virally infected cells, autoimmune cells and tumour cells; b) providing a first conjugate of the invention for transfer to the cell provided in step a); c) providing any one of the saponins or saponin derivatives of the invention or, preferably, if the cell provided in step a) expresses on its surface a second cell surface molecule according to the invention, providing a second conjugate of the invention; d) contacting the cells of step a) in vitro or ex vivo with the first conjugate of step b) and any one or more of the saponin, saponin derivative, and conjugate of step c); thereby establishing the transfer of a first conjugate from outside the cell into said cell, and thereby establishing the delivery of an effector molecule comprised by the first conjugate to the cytosol of said cell, optionally as part of the first conjugate or as a free effector molecule cleaved from the first conjugate in an endosome, endolysosome or lysosome.

[0150] 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 enhance the effect of an effector moiety, in particular by promoting endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (such as the saponins of the present invention, for example those exemplified herein and in the claims) interact with the membranes of compartments and vesicles of the endocytic and recycling pathways, making them leaky and increasing the increased endosomal escape of the effector moiety. The term "the scaffold is capable of increasing endosomal escape of the effector moiety" means that in the second conjugate of the invention, at least one saponin (glycoside molecule), which is attached via a linker or directly to an antibody targeting a cell surface molecule, such as an sdAb, or via the polymeric or oligomeric structure of the scaffold (covalent saponin conjugate of the invention), is capable of increasing endosomal escape of the effector moiety comprised in the first conjugate of the invention, once both molecules are in an endosome, e.g. a late endosome, and optionally and preferably after the at least one saponin has been released from the second conjugate of the invention, e.g. from the linker or the polymeric or oligomeric structure comprised in said second conjugate, e.g. by cleavage of a cleavable bond between the at least one glycoside (saponin) and the second conjugate (e.g. via the polymeric or oligomeric structure of the scaffold, and / or via the linker). The bond, optionally via a linker or scaffold, between at least one saponin according to the invention and a ligand that targets a cell surface molecule, such as an antibody such as an sdAb of the second conjugate of the invention may be a "stable bond", but this does not mean that such a bond cannot be cleaved, for example by an enzyme, within the endosome.For example, the saponin, optionally together with part of the linker or scaffold oligomeric or polymeric structure, may be cleaved from the remaining linker fragment or oligomeric or polymeric structure. For example, a protease may cleave the (proteinaceous) linker or proteinaceous polymeric structure, such as albumin, thereby releasing at least one saponin. It is however preferred that the glycoside molecule (preferably saponin) is released in an active form, preferably in its original form before being conjugated (prepared to be conjugated) to a cell surface molecule targeting molecule such as an sdAb of the second conjugate of the invention, optionally via a linker and / or an oligomeric or polymeric scaffold (covalent saponin conjugate of the invention), such that the glycoside (saponin) has its native structure after such cleavage or the glycoside (saponin) has (part of) a chemical group or linker attached to it after such cleavage, while 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 upon said cleavage of the bond between the glycoside (saponin) and the cell surface molecule targeting antibody such as an sdAb, optionally comprising a linker and / or scaffold of the invention. In the context of the present invention, the term "stable" with reference to, for example, the bond between the saponin and the amino acid residues of an sdAb targeting a cell surface molecule in the second conjugate, the linker, the polymeric or oligomeric structure (of the scaffold, i.e., of the covalent saponin conjugates of the present invention), the ligand, the (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or the effector (effector moiety, effector molecule), means that the bond is not easily disrupted, or is at least designed not to be easily disrupted, e.g. by pH differences, salt concentrations, or UV light, reducing conditions.In the context of the present invention, the term "cleavable" with reference to, for example, the bond between a saponin and an sdAb targeting a cell surface molecule, a linker, an amino acid residue, a polymeric or oligomeric structure of a covalent saponin conjugate, a ligand, an antibody, and / or an effector means that the bond is designed to be easily broken, for example, by pH difference, salt concentration, under reducing conditions, etc. Those skilled in the art are familiar with such cleavable bonds and methods for preparing them.

[0151] Prior to the present invention, one of the main hurdles to introduce ADCs and AOCs on the market was the small therapeutic window: therapeutically effective doses of ADCs or AOCs are accompanied by (unacceptable) side effects, which hinder the progress and value of ADC treatment in patients. The application of the first conjugate of the present invention, including an sdAb targeting an effector molecule and a cell surface molecule and a second conjugate of the present invention, such as an sdAb-saponin conjugate, makes it possible to target one or more glycoside molecules (saponins) to a (target) cell together with an ADC carrying a payload or together with an sdAb conjugated to an oligonucleotide such as a BNA according to the present invention. In particular, it was not previously possible to simultaneously and specifically target an effector moiety of an ADC or AOC, or any other conjugate of a payload and a molecule targeting a (proteinaceous) cell surface molecule, and a (predefined, controllable) specific number or range of glycoside molecules (saponins) per effector moiety, to the cytosol of the cell, such as via the endocytic pathway of the cell. As discussed above, the ADC or AOC of the invention (i.e., the first conjugate of the invention) and the second conjugate can target the same cell surface molecule, or different cell surface molecules, where the two different cell surface molecules are expressed on the surface of the same target cell, such as a tumor cell or an autoimmune cell.

[0152] The solution provided by the present invention includes the covalent attachment of at least one saponin to a cell surface molecule targeting molecule, such as an antibody, preferably an sdAb, of the second conjugate of the present invention. A further solution provided by the present invention includes (firstly) polymerizing glycoside molecules (saponins) using an oligomeric or polymeric scaffold, and providing a cluster of covalently attached saponins to the cell surface molecule targeting molecule comprised in the second conjugate of the present invention, allowing remonomerization of one or more saponins at the intracellular site where the mode of action of the saponin is desired, for example after endocytosis. In this context, "polymerize" means the reversible and / or irreversible multiple conjugation of a saponin molecule to a cell surface molecule binding molecule of a second conjugate, for example an antibody such as an sdAb, for example via a linker, or directly, or via a polymeric or oligomeric structure that forms a scaffold (covalent saponin conjugate of the invention), or the reversible and / or irreversible multiple conjugation of a (modified) saponin that forms a polymeric or oligomeric structure to form a scaffold (covalent saponin conjugate of the invention). "Remonomerization" in this context means cleaving the saponin from the second conjugate, from the linker linking the saponin to a ligand targeting a cell surface molecule such as the sdAb of the second conjugate, or from the scaffold, e.g. from the scaffold after endocytosis, and returning it to the (native) chemical state of unbound saponin, which may or may not contain further chemical groups such as chemical groups for linking the saponin to the linker, amino acid residues of the second conjugate, or the scaffold, and / or (chemical) linkers attached to chemical groups of the saponin such as aldehyde or carboxylic acid groups. Due to the complex chemical nature of saponins, for example, "polymerization" of saponins on the scaffold or other linking linkers, and their "remonomerization" at the desired location, e.g. inside the cell after endocytosis, has been a difficult challenge.In particular, the chemical reactions used to provide covalently bound glycoside-containing linkers and scaffolds, e.g., triterpenoid saponins (polymerization of glycosides), for covalent attachment to cell surface molecule-binding molecules to provide second conjugates, typically occur in organic solvents that do not contain water, whereas the saponins and, e.g., the biocompatible polymers applied as scaffolds to carry the bound saponins, are water-soluble molecules. The chemical properties of unmodified saponin, by itself, prevent polymerization, and one possible alternative solution for (directly) conjugating multiple saponins to cell surface molecule binding molecules such as antibodies, e.g. sdAbs, was presumed to be less promising, since antibodies such as sdAbs typically do not provide sufficient binding sites and the conjugation products would be quite heterogeneous and / or conjugating a biologically active molecule such as a saponin with an antibody targeting a cell surface molecule, such as an sdAb, would risk affecting and inhibiting the activity of the saponin and / or the binding of the sdAb to the cell surface molecule, which are for example conjugated together in such a saponin-containing second conjugate of the invention. Embodiments of the present invention overcome at least one of these drawbacks.

[0153] Whether the second conjugate of the present invention comprising saponin further comprises one or more (cleavable) linkers and / or optional scaffolds (covalent saponin conjugates of the present invention) can interfere with the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be easily determined by the assays described in the Examples section and known in the art. Inhibition is described as "fold increase in glycoside (saponin of the present invention) required to induce 50% cell death". It is preferred that the scaffold or second conjugate of the present invention does not result in an increase that at least increases the glycoside molecule (saponin) required to obtain 50% cell death observed when using chloroquine as a positive control. Alternatively and preferably, the second conjugate comprising saponin, whether or not it further comprises one or more (cleavable) linkers and / or optional scaffolds (covalent saponin conjugates of the invention), does not result in at least a 4-fold increase in glycoside molecules to induce 50% cell death, more preferably at least a 2-fold increase. The fold increase must be measured in an assay where chloroquine as a positive control induces a 2-fold increase in glycoside amount, preferably saponin amount, where the saponin is any one or more of the saponins of the invention to observe 50% cell death (previous embodiment).

[0154] As mentioned above, at least one saponin contained in the second conjugate according to the invention increases the efficacy of at least the current and new effector moieties defined in the present invention. The dosage of the effector moiety contained in the first conjugate according to the invention, which further comprises a cell surface molecule binding sdAb such as ADC or AOC, can be reduced without reducing efficacy, thus reducing potential side effects. Thus, the present invention provides a combination of the second conjugate according to the invention with the first conjugate according to the invention, and the present invention also provides the first conjugate according to the invention for use in medicine or for use as a medicament. One aspect of the present invention relates to a pharmaceutical composition according to the invention for use as a medicament, the pharmaceutical composition comprising the first conjugate according to the invention. One aspect of the present invention relates to a pharmaceutical composition according to the invention for use as a medicament, the pharmaceutical composition comprising the first conjugate according to the invention and comprising the second conjugate according to the invention.

[0155] For the endosomal escape enhancers contained in the second conjugate of the present invention, i.e., the saponins of the present invention, the following several preferred features can be mentioned: (1) they are preferably non-toxic and do not cause an immune response, (2) they preferably do not mediate the uptake of the effector moiety into the cytosol of off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector moiety, (4) they are preferably biodegradable or excretable, and (5) they preferably do not substantially interfere with the biological processes of the organism that are unrelated to the biological activity of the effector molecule with which the endosomal escape enhancer is combined, e.g., do not interact with hormones. Examples of saponins of the present invention that meet the above criteria at least to some extent are bidesmosidic triterpenes, preferably bidesmosidic triterpene saponins, such as SO1861, SA1641, QS-21, GE1741, and further saponins according to the present invention, which are listed throughout this specification.

[0156] Also provided is the use of the first conjugate according to the invention for the manufacture of a medicament. Also provided is the use of the first conjugate according to the invention and the second conjugate according to the invention for the manufacture of a medicament. In particular, cancer therapeutics, especially classical chemotherapy drugs, are notorious for side effects. Due to the time and location targeting and synchronization of both the pharmaceutically active substance contained in the first conjugate, such as ADC or AOC, and the saponin contained in the second conjugate molecule of the invention, the first conjugate according to the invention is particularly useful for use as a medicament, or the combination of the first conjugate according to the invention and the second conjugate according to the invention is particularly useful for use as a medicament, especially for use in a method for treating cancer, where the medicament is a single composition comprising the ADC or AOC of the invention (first conjugate of the invention) and the second conjugate of the invention, or a therapeutic combination of a first pharmaceutical composition comprising the first conjugate of the invention, such as ADC or AOC, and a second pharmaceutical composition comprising the second conjugate of the invention. Thus, the present invention provides the first conjugate according to the invention for use in a method for treating cancer. The present invention also provides a first conjugate according to the present invention for use in a method for treating acquired or inherited disorders, in particular single gene deficiency disorders. Thus, the second conjugate comprises at least one saponin and an sdAb for targeting the second conjugate in abnormal target cells, such as tumor cells or autoimmune cells, and the second conjugate of the present invention is combined with at least one first conjugate of the present invention comprising an effector moiety and an sdAb, such as an ADC or AOC of the present invention. Thus, one aspect of the present invention relates to a second conjugate according to the present invention, which comprises a covalently attached saponin and a cell surface molecule binding antibody, such as an sdAb, for use in a method for treating cancer or an autoimmune disease, the method further comprising administering the first conjugate according to the present invention to a subject, such as a cancer patient, in need thereof.

[0157] A further application of the first and second conjugates of the invention in medicine is their use in a method for replacing intracellular enzymes in target cells that produce insufficient amounts or functionality of the enzyme. The second conjugate of the invention is administered to a patient in need thereof, for example in combination with the first conjugate of the invention comprising an oligonucleotide for the enzyme to be replaced or for gene therapy, for replacement of the target intracellular enzyme. The resulting disease can be hereditary or acquired. In most cases, only symptomatic treatment is possible, and in some rare diseases, insufficient treatment options lead to a shortened life span of the patients involved. An example of such a disease is phenylketonuria, which is an inborn error of metabolism that results in a reduced metabolism of the amino acid phenylalanine. The disease is characterized by a mutation in the gene for the hepatic enzyme phenylalanine hydroxylase. Phenylketonuria is incurable to date. The incidence is about 1:10,000, with 1:2,600 in Turkey, where the highest incidence is known. An antibody, preferably V, targeting the cell surface molecule contained in the second conjugate of the invention together with the bound saponin is administered to the patient in need thereof. HH Such an sdAb, and a first conjugate comprising the same or a different cell surface molecule binding sdAb and phenylalanine hydroxylase, or a first conjugate comprising the same or a different cell surface molecule binding sdAb with a bound polynucleotide encoding phenylalanine hydroxylase, can be used to target hepatocytes and replace a missing enzyme in hepatocytes by using a suitable specific antibody or sdAb in the second conjugate of the invention and using a suitable specific sdAb in the first conjugate according to the invention. This is an example of the use of a second conjugate of the invention comprising a saponin bound thereto, and a first conjugate of the invention comprising an effector molecule, such as an enzyme or oligonucleotide, bound thereto in this example according to the invention, for replacement or gene therapy. In a preferred embodiment, a second conjugate according to the invention for use in a method of gene therapy or replacement therapy is provided in combination with a first conjugate of the invention comprising a cell surface molecule binding sdAb and an enzyme or a nucleic acid encoding an enzyme.

[0158] Using the first and second conjugates of the present invention, it is now possible to design and manufacture two-component non-viral clinically applicable gene delivery technologies. For example, the second conjugate of the present invention allows the development of a non-viral-based combination of the first and second conjugates for gene delivery technologies, which enhances therapeutic efficacy at lower therapeutic doses, thereby improving patient health. The second conjugate of the present invention comprises, in particular, a covalently bound cell surface molecule targeting antibody, such as a monoclonal antibody or sdAb for binding to a (tumor, autoimmune) cell surface specific molecule, an effector moiety, such as an oligonucleotide, such as a BNA, and a V HH When combined with the first conjugate containing sdAb targeting cell surface molecules such as , it overcomes a long-standing major bottleneck in the field of gene delivery, i.e., allows efficient, safe, and cost-effective transfer of gene therapy products through the endosomal membrane into the cytosol / nucleosol. Indeed, gene therapy is one of the most promising therapeutic options for future advanced treatment of a wide range of diseases. Successful gene delivery requires recognition of the target cell and incorporation of the gene into the cytosol and nucleosol. One of the major problems in the field of non-viral gene therapy is the inefficient and insufficient safe delivery of genetic material for therapeutic use in patients.

[0159] Thus, when applying the second conjugate of the invention, such as AOC or sdAb, which comprises a first conjugate comprising a cell targeting molecule, such as a ligand or preferably an antibody (fragment, domain, preferably sdAb), targeting a cell surface molecule, comprising an oligonucleotide such as an antisense BNA, comprising an sdAb targeting a cell surface molecule, the inventors have now made it possible to overcome the problem of safe transfer of gene therapy products into the cytosol / nucleosol via the endosomal membrane, which has been a major bottleneck in the field of gene delivery for many years. The combination of the first and second conjugates of the invention represents a technology designed to allow the targeting of any addressable cell type with all known genetic drugs and the subsequent efficient delivery of genes into the cytosol, thereby ensuring better patient treatment, which is important not only for genetic disorders, but also for cancer therapy and therefore for a large patient population. The technology based on the first and second conjugates of the invention is based on the targeting ligand, or (monoclonal) (tumor cell specific) antibody or fragment thereof, or preferably V HHThe present invention may comprise a polymer or oligomer scaffold (covalent saponin conjugate of the present invention) that serves as a carrier for an endosomal escape enhancer (EEE), such as a saponin as exemplified herein, for a molecule targeting a cell surface molecule, such as an sdAb, and a second conjugate of the present invention comprising a saponin of an embodiment according to the present invention. In this case, the first conjugate of the present invention comprises a first cell surface molecule binding molecule, such as an sdAb, and an effector moiety, here an effector gene, such as an LNA or BNA. The use of the second conjugate of the present invention, for example comprising a cell-targeting antibody (fragment) or sdAb, in combination with the first conjugate of the present invention comprising an oligonucleotide, such as an sdAb and BNA, targeting the same or a different cell surface, has the potential to bring any kind of biological macromolecule into the cytosol and nucleus. The development of new targeting ligands, sdAbs and monoclonal (human, humanized) antibodies is being continuously carried out by many research groups and companies around the world. The same is true for oligonucleotides intended for delivery in the cytosol of diseased cells, such as cancer cells. Thus, the first and second conjugates of the present invention also exist as molecular tools that allow current and future targeting sdAbs and antibodies to be used by click chemistry, enabling customized drug applications and enabling future developments in the field of tissue and cell targeting technologies; thus, the first conjugates according to the present invention also exist as molecular tools that allow current and future therapeutic oligonucleotides (as well as payloads such as protein toxins) to be linked or connected, for example, to sdAbs by click chemistry, enabling customized drug applications and enabling future developments in the field of tissue and cell targeting technologies. The first and second conjugates of the present invention can include antibodies and ligands as molecules targeting cell surface molecules, although sdAbs are preferred. The global market for gene therapy drugs is growing rapidly and targets potential treatments for a wide range of disease areas, such as cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, HIV and many rare (monogenic) diseases.Current viral vector-based gene therapy technology has significant challenges such as safety, manufacturing logistics, and associated high costs. The second conjugate of the present invention, when combined with the first conjugate of the present invention, which contains the gene to be delivered and the cell surface targeting molecule, i.e., sdAb, allows for the use in a technology platform that is an alternative to current viral gene delivery technology. Thus, the first and second conjugates of the present invention are suitable for incorporation into approaches to develop non-viral gene therapy for diseases such as cancer, cardiovascular disease, Parkinson's disease, Alzheimer's disease, HIV infection, and many rare (monogenic) diseases. The first and second conjugates of the present invention are suitable for the development of novel therapies to transform the field of antibody drug conjugates (ADCs) and oligonucleotide-based therapeutics by creating non-viral vector-based gene therapy drugs, such as those based on targeted antisense BNAs. The application of the second conjugate of the invention, especially a covalent conjugate comprising an antibody such as an sdAb and a saponin, in combination with a first conjugate comprising a cell surface molecule binding molecule, i.e. an oligonucleotide such as an sdAb and a BNA, is one of many beneficial approaches made possible by the invention. For example, the use of the first and second conjugates of the invention allows the exploitation of the endocytosis pathway of mammalian cells. Endocytosis is exploited for the delivery of therapeutic agents, and the second conjugate of the invention contributes to improved uptake and endosomal escape of, for example, the siRNA contained in the first conjugate. Suitably, the first and second conjugates of the invention are used together with a small molecule that acts as a delivery enhancer for, for example, the payload, the oligonucleotide. This allows the delivery of a covalently attached cell-targeting moiety such as a ligand, preferably an antibody (domain or fragment, preferably V. HH ) in combination with a second conjugate of the invention, having a covalently attached oligonucleotide such as a BNA, and a covalently attached cell-targeting moiety, i.e. an sdAb, preferably V HHThe first conjugate of the invention having the formula (I) provides a solution to the current problems seen with current endosomal escape enhancers and gene therapy products, with regard to applying them as two components with a free non-targeting saponin, thus complicating therapeutic approval and clinical applicability, since such a second conjugate of the invention, comprising a saponin, is combined with the first conjugate of the invention, comprising a gene product such as a BNA and a (tumor) cell targeting moiety, i.e. an sdAb. Thus, the present invention provides a non-viral gene delivery technology in which an endosomal escape enhancer (e.g., a glycoside according to the embodiments of the present invention and the examples provided) and a second targeting ligand or antibody (e.g., an sdAb according to the embodiments of the present invention and as exemplified in the Examples section herein below) are included in a second conjugate of the present invention, combined with a first conjugate of the present invention comprising a gene therapy product (an oligonucleotide according to the present invention such as a BNA) and a sdAb targeting a first cell surface molecule, such as an sdAb targeting the same or a different cell surface molecule, encompassed in the second conjugate of the present invention, the sdAb targeting the first cell surface molecule binding to the same or a different cell surface molecule as the cell surface molecule targeting molecule of the second conjugate of the present invention. Thus, such first and second conjugates of the present invention provide current and future macromolecular drug therapeutic opportunities for a wide range of diseases and large patient populations. The application of such a second conjugate of the invention comprising at least one saponin and at least one specific cell targeting moiety such as an immunoglobulin or sdAb in combination with a first conjugate of the invention comprising at least one oligonucleotide and at least one specific cell targeting moiety, i.e. an sdAb, addresses an issue that is apparent with current methods of applying a non-cell surface molecule targeted free endosomal escape enhancer (e.g. a saponin that is not conjugated to a cell surface molecule binding antibody) and a gene therapy product separately, which current methods do not ensure that both compounds are simultaneously present at the interaction site.This problem has been solved by using a second conjugate of the present invention in combination with a first conjugate of the present invention comprising a nucleic acid. Here, both the second conjugate of the present invention and the first conjugate of the present invention comprise a molecule that targets a cell surface molecule, for example, an antibody such as an sdAb according to the present invention when considering the second conjugate, and an sdAb when considering the first conjugate. That is, such a combination of the second conjugate of the present invention with the first conjugate comprising an oligonucleotide provides a non-viral gene delivery technique with increased synchronization (in time and place) of the gene products of both compounds, i.e., saponin and BNA.

[0160] Gene therapy can help with inherited, previously incurable diseases such as cystic fibrosis, chorea, Huntington's disease or hemophilia. However, currently, several problems remain to be solved. For example, the therapeutic gene must arrive precisely at a specific target cell in the body. On the other hand, the therapeutic gene must be absorbed by the target cell, but not destroyed. Current gene therapy is a method that uses a virus as a ferry for the gene. However, these procedures involve considerable risks and cannot be repurposed for the introduction of other biomolecules. One embodiment is a second conjugate of the present invention that includes a (plant-derived) glycoside (e.g., any one of the saponins of the present invention), for using platform technology that allows not only the delivery of a gene, when such a gene is included in the first conjugate of the present invention as a carrier molecule, but also the delivery of a different therapeutic biomolecule to be introduced into the target cell. Thus, the second conjugate of the present invention is used to develop a nucleic acid-based therapy for cystic fibrosis, chorea, Huntington's disease or hemophilia. This allows new gene therapy strategies to be utilized to improve the health of patients with genetic diseases, including those with cystic fibrosis, Huntington's disease, and hemophilia, using the second conjugates of the present invention. As part of the present invention, a plant-derived endosomal escape enhancer (glycoside; i.e., saponin of the present invention) and a targeting ligand (e.g., an antibody such as an sdAb) contained in a single second conjugate of the present invention can be combined in a V HHA non-viral gene delivery technique is developed to combine with the first conjugate of the present invention, which comprises a cell surface molecule binding sdAb such as and a gene therapy product. The non-viral gene therapy obtained based on the first and second conjugates of the present invention shows about 40-fold delivery efficiency with the first conjugate containing a lower dose of gene than currently available strategies. This makes the second conjugate of the present invention for use in clinical applications, such as for repairing or replacing defective genes, such as in cystic fibrosis patients, and for targeted delivery of specific genes, for example to destroy cancer cells. In fact, the first and second conjugates of the present invention are suitable for application in treatment regimens for any disease that is caused by genetic defects and cannot currently be cured, such as cystic fibrosis, Huntington's disease, and hemophilia. Gene therapy using the first and second conjugates of the present invention helps to solve the following two current problems: Firstly, the second conjugate of the invention allows the delivery of the first conjugate comprising a therapeutic gene to a specific target cell in the body, and secondly, the therapeutic gene gets inside the target cell but is not destroyed due to the presence of a targeting moiety such as an antibody or sdAb (second conjugate of the invention) to bind to the saponin and the target cell, and the presence of a targeting sdAb (first conjugate of the invention) to bind to the oligonucleotide product and the target cell, for example by using an oligomeric or polymeric scaffold of the invention (covalent effector molecule conjugate of the invention) as part of the first and second conjugates of the invention.

[0161] The invention also provides a method of treating cancer comprising administering a medicament comprising a second conjugate according to the invention in combination with a first conjugate of the invention, i.e. an ADC or AOC comprising an effector molecule and a cell surface molecule binding sdAb, to a patient in need thereof, preferably administering an effective dose of said medicament to a patient in need thereof, preferably a human cancer patient.

[0162] Considerations regarding suitable forms for administration are known in the art and include toxicity, solubility, route of administration, and maintenance of activity. For example, a pharmacological composition injected into the blood stream must be soluble.

[0163] The suitable dosage form depends on the use or route of administration, such as transdermal or injection. Such dosage form must 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 toxicity and dosage forms that slow the compound or composition from exerting its effect.

[0164] [Table 1]

[0165] [Table 2]

[0166] [Table 3]

[0167] [Table 4]

[0168] [Table 5] EXAMPLES

[0169] Examples and illustrative embodiments Example 1.V HH -SO1861+mAb-saporin (1T2C and 2T2C) One-target, two-component systems (1T2C) are HH -SO1861 and mAb-protein toxin combination therapy,HH and mAb recognize and bind to the same cell surface receptor (Figure 1A). HH -SO1861 and mAb-protein toxin combination therapy, HH recognizes and binds to a different cell surface receptor than mAbs (Figure 1B). SO1861-EMCH was coupled to the anti-HER2V antibody via the terminal cysteine ​​residue (Cys). HH conjugated with DAR1 (unstable) (HER2V HH -SO1861). HER2V HH -SO1861 was titrated against a fixed concentration of 10 pM CD71mab-saporin (CD71 monoclonal antibody conjugated to the protein toxin saporin with DAR4) or a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin saporin with DAR4). ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. This demonstrated that the combination of both 10 pM CD71mab-saporin or 50 pM trastuzumab-saporin inhibited low concentrations of HER2V in SK-BR-3. HH - Enhanced cell killing was observed with SO1861 (IC50 = 300 nM; Figure 2A). HH -Comparable concentrations of SO1861 alone induced cell death at high concentrations (IC50=4,000nM), but not HER2V HH , HER2V HH +CD71mab-saporin, or HER2V HH + Equivalent concentrations of trastuzumab-saporin failed to induce cell killing activity (IC50>5000nM; Figure 2A). MDA-MB-468 (HER2 - / CD71 + ) in HER2V HH -SO1861 + 10 pM CD71mab-saporin combination showed cell killing activity at high concentrations (IC50 = 2.000 nM; Figure 2B), whereas HER2VHH -SO1861 + 50 pM trastuzumab-saporin combination showed cell killing activity at much higher concentrations (IC50>5,000 nM; Figure 2B). HH , HER2V HH +CD71mab-saporin or HER2V HH Equivalent concentrations of +trastuzumab-saporin failed to induce cell killing activity in MDA-MB-468 cells (IC50>5.000 nM; Figure 2B).

[0170] All of this was achieved through the use of SO1861-EMCH, a HER2-targeting HH They have shown that conjugation to enhances endosomal escape and cytoplasmic delivery of targeted protein toxins (targeted to the same or different cell surface receptors), resulting in cell killing of HER2-expressing cells.

[0171] Next, trastuzumab-saporin or CD71mab-saporin was administered to HER2V HH -Titered against SO1861 and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. This resulted in the targeted protein toxin-mediated cell killing of HER2V at 900 nM in combination with low concentrations of trastuzumab-saporin or CD71mab-saporin. HH -SO1861 already induced efficient cell killing of SK-BR-3 (IC50 = 0.0001 pM; Figure 3A), whereas CD71mab-saporin + 900 nM HER2V HH or trastuzumab-saporin + 900nM HER2V HH It was found that at high concentrations (IC50 = 50 pM; IC50 = 400 pM, respectively; Figure 3), it was possible to induce cell death in MDA-MB-468 cells (HER2 - / CD71 + ) in CD71mab-saporin + 900nM HER2V HH-SO1861 demonstrated cell killing with IC50=0.01pM, whereas trastuzumab-saporin + 900nM HER2V HH -SO1861 showed activity with IC50=2,000pM. Trastuzumab-saporin + HER2V at 900nM HH or CD71mab-saporin + 900nM HER2V HH (IC50>10,000 pM and IC50=20 pM, respectively; Fig. 3B). ++ / CD71 + In cells expressing HER2V, relatively low concentrations of trastuzumab-saporin or CD71mab-saporin were effective, and relatively low concentrations of HER2V HH - shows that it can induce cell death in combination with SO1861(DAR1).

[0172] Note: Throughout the description, including the examples, and in the claims, HH " and "VHH" should be understood to refer to the same type of single domain antibody (sdAb).

[0173] Example 2.V HH -SO1861+V HH -Jianqing (2T2C) Two-target, two-component systems (2T2C) are HH 1-SO1861 and V HH It is a combination treatment of two protein toxins, each V HH recognizes a different cell surface receptor (Figure 1C). HH conjugated to the terminal cysteine ​​residue of HER2V HH -SO1861(DAR1) was generated. HER2V HH -SO1861 was added to CD71V at a fixed concentration of 50 pM HH -Titrated against Giantin, SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 +) was measured. This allowed the detection of targeted protein toxin-mediated cell killing at relatively low concentrations of V HH Enhanced cell killing was observed with HER2-L-SO1861 (SK-BR-3: IC50 = 300 nM; Figure 4A). HER2V HH -Comparable concentrations of SO1861 alone induced cell death at high concentrations (IC50=4,000nM), but not HER2V HH , HER2V HH +50pM CD71V HH Comparable concentrations of -dianthine failed to induce cell death (IC50>5,000 nM; Figure 4A). MDA-MB-468 (HER2 - / CD71 + In the case of HER2V HH -SO1861+50pMCD71V HH The combination of -dianthine showed cell killing activity at high concentrations (IC50 = 600 nM; Figure 4B), but not HER2V HH , HER2V HH -SO1861 or HER2V HH +50pMCD71V HH Comparable concentrations of -dianthine failed to induce cell killing activity (IC50>5,000 nM; Fig. 4B).

[0174] Next, CD71V HH -Dianthin was administered at a fixed concentration of 900 nM to HER2V HH -Titered against SO1861 and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. This resulted in targeted protein toxin-mediated cell killing against low concentrations of CD71V HH -HER2V 900nM in combination with Gianthin HH -SO1861 induced efficient cell killing of SK-BR-3 cells (IC50 = 0.05 pM; Fig. 5A), whereas CD71V HH Giantin or CD71VH HH -Giantin + 900nM HER2V HHIt was found that CD71V was able to induce cell death at high concentrations (IC50>10,000 pM; Fig. 5A). HH -Gianthin was also titrated against a fixed concentration of 77 nM trastuzumab-SO1861 (DAR4), which reduced the expression of SK-BR-3 (HER2 ++ / CD71 + A strong enhancement of cell killing activity in MDA-MB-468 cells (HER2+) was also observed (IC50<0.0001pM). - / CD71 + ) in the CD71V HH -Giantin + 900nM HER2V HH -SO1861 caused cell death at very high concentrations (IC50 = 10 pM, Fig. 5B), whereas CD71V HH - Jianqing, CD71V HH -Giantin + 900nM HER2V HH , or CD71V HH -Gianthin + trastuzumab-SO1861 (DAR4) demonstrated cell killing with an IC50 of 2,000 pM (Figure 5B).

[0175] All of this was observed in high HER2 / CD71 expressing cells at relatively low concentrations of V HH CD71-gianthin is effective and low concentrations of V HH 4 shows that it can induce cell death in combination with the HER2-SO1861 conjugate.

[0176] The combination according to the present invention was used in MDA-MB-468 cells (HER2 - / CD71 + ), did not show any cell killing activity, indicating that without sufficient receptor expression, SO1861 delivered into cells does not reach an effective concentration (threshold) that induces endosomal escape and cytoplasmic delivery of the protein toxin.

[0177] Example 3.V HH -Giantin + mAb-SO1861 (1T2C and 2T2C) The one-target, two-component system (1T2C) is a combination of mAb-SO1861 and V HH - A combination therapy of a protein toxin, a mAb and a V HH The two-target two-component system (2T2C) recognizes and binds to the same cell surface receptor (Figure 1E). HH - A combination therapy of a protein toxin, a mAb and a V HH recognize and bind to the same cell surface receptor (Fig. 1D).

[0178] Gianthin-C (giantin with a terminal cysteine) is a HER2-targeting V HH , targeting CD71 HH , targeting EGFR HH conjugated to the terminal cysteine ​​residue of HER2V HH -Jianting (DAR1), CD71V HH -Dianthin (DAR1) and EGFRV HH -Gianthin (DAR1) was generated.

[0179] CD71V HH -Jianqing, HER2V HH -Dianthin or EGFRV HH - Gianthin was titrated against a fixed concentration of cetuximab-SO1861 (DAR4) and A431 (EGFR ++ / HER2 + / - / CD71 + ) and A2058 (EGFR - / HER2 + / - / CD71 + ) was measured for target-specific protein toxin-mediated cell killing. This resulted in the detection of very low concentrations of CD71V in combination with 77 nM cetuximab-SO1861. HH -Dianthin induced efficient cell killing of A431 cells (IC50<0.0001pM; Fig. 6A), whereas CD71V HH -Dianthin alone was found to be active with an IC50 of 2000 pM. HH -Gianthin + 77nM Cetuximab -SO1861, and HER2V HHThe other two combinations, Gianthin + 77 nM cetuximab-SO1861, showed efficient cell killing with IC50 = 20 pM and IC50 = 50 pM, respectively, but EGFRV HH -Giantin or HER2V HH -Dianthin alone was unable to induce efficient cell killing in A431 cells (IC50>10,000 pM; Figure 6A). A2058 cells (EGFR - / HER2 + / - / CD71 + ) in the CD71V HH -Giantin, and CD71V HH -Gianthin + 77 nM cetuximab -SO1861 showed cell killing activity with IC50 = 3.000 pM and IC50 = 1.000 pM, respectively, while all other treatments or combinations showed a V50 of IC50 = 10,000 pM in A2058 cells. HH -toxin did not cause cell death (Figure 6B).

[0180] This indicates that cetuximab-SO1861 (DAR4) is a potent inhibitor of three different V HH These results show that the endosomal escape of the -dianthine conjugate can be efficiently induced, thereby enhancing the induction of cell death in A431 cells.

[0181] Next, CD71V HH -Jianqing, HER2V HH -Dianthin or EGFRV HH - Gianthin was titrated against a fixed concentration of trastuzumab-SO1861 (DAR4) and SK-BR-3 (HER2 ++ / EGFR = / CD71 + ) and MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + ) was measured. This resulted in the targeted protein toxin-mediated cell killing of CD71V in combination with 77 nM cetuximab-SO1861 at very low concentrations. HH-Dianthin induced efficient cell killing of SK-BR-3 cells (IC50<0.0001pM; Fig. 7A), whereas CD71V HH -Dianthin alone was found to be active with an IC50 of 10,000 pM. HH -Gianthin + 77nM trastuzumab -SO1861, and HER2V HH The other two combinations, Gianthin + 77 nM trastuzumab-SO1861, showed efficient cell killing with IC50 = 400 pM and IC50 = 6 pM, respectively, but EGFRV HH -Giantin or HER2V HH -Dianthin alone was unable to induce efficient cell killing in SK-BR-3 cells (IC50>10,000 pM; Fig. 7A). MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + ) in the CD71V HH -Giantin, and CD71V HH -Dianthin + 77 nM cetuximab -SO1861 showed cell killing activity with IC50 = 3000 pM and IC50 = 2000 pM, respectively, whereas all other treatments or combinations showed a V50 of IC50 = 10,000 pM in MDA-MB-468 cells. HH -giantin did not show any cell death (Figure 7B). This is because trastuzumab-SO1861 (DAR4) was able to inhibit the three different V HH These results show that the endosomal escape of the -dianthine conjugate can be efficiently induced, thereby enhancing the induction of cell death in SK-BR-3 cells.

[0182] Materials and Methods material SO1861 was isolated and purified from a raw plant extract obtained from Saponaria officinalis by Analyticon Discovery GmbH. HH was purchased from QVQ, Utrecht, The Netherlands (HER2VHH :Clone name: Q17c;CD71V HH :Clone name: Q52c;EGFRV HH : Clone name: Q86c). Trastuzumab (Tras, Herceptin®, Roche), cetuximab (Cet, Erbitux®, Merck KGaA) were purchased from a pharmacy (Charite, Berlin). CD71 monoclonal antibody was purchased from BioCell (Okt9, #BE0023). Custom trastuzumab-saporin and anti-CD71mab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA). Dianthine-Cys, a dianthine variant with a single C-terminal cysteine ​​(Dia-Cys), was manufactured by Proteogenix, France.

[0183] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, 99%, Sigma-Aldrich), Zeba™ spin desalting columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standards (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Superdex G25 (GE Healthcare), Superdex G50M (GE Healthcare), Superdex 200P (GE Healthcare), isopropyl alcohol (IPA, 99.6%, VWR), tris(hydroxymethyl)aminomethane (Tris, 99%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane hydrochloride (Tris·HCL, Sigma-Aldrich), L-histidine (99%, Sigma-Aldrich), D-(+)-trehalose dehydrate (99%, Sigma-Aldrich), polyethylene glycol sorbitan monolaurate (TWEEN® 20, Sigma-Aldrich), Dulbecco's phosphate buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetate dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filtered 0.2 μm and 0.45 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP, Thermo-Fisher), HSP27 BNA disulfide oligonucleotides (Biosynthesis), [O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium-hexafluorophosphate] (HATU, 97%, Sigma-Aldrich), dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich), N-(2-aminoethyl)maleimide trifluoroacetate (AEM, 98%, Sigma-Aldrich), L-cysteine ​​(98.5%, Sigma-Aldrich), and deionized water (DI) were used as Ultrapure Lab Water. Freshly taken from the Biosystems (MilliQ, Merck), nickel-nitrilotriacetic acid agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5-dithiobis(2-nitrobenzoic acid) (Ellman's Reagent, DTNB, 98%, Sigma-Aldrich), S-acetylmercaptosuccinic anhydride fluorescein (SAMSA Reagent, Invitrogen), sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), PD MiniTrap desalting columns packed with Sephadex G-25 resin (GE Healthcare), PD10 G25 desalting columns (GE Healthcare), Zeba spin desalting columns 0.1–0.2 mm thick.5, 2, 5 and 10mL (Thermo-Fisher), Vivaspin centrifugal filters T4 10kDa MWCO, T4 100kDa MWCO and T15 (Sartorius), Biosep s3000 aSEC columns (Phenomenex), Vivacell ultrafiltration units 10 and 30kDa MWCO (Sartorius), Nalgene Rapid-Flow filters (Thermo-Fisher).

[0184] method SO1861-EMCH synthesis To SO1861 (121 mg, 0.065 mmol) and EMCH·TFA (110 mg, 0.325 mmol) was added methanol (excess dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 h, the reaction mixture was subjected to preparative MP-LC. 1 Fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS: 96%. LRMS(m / z):2069[M-1] 1- LC-MSr.t.(min):1.08 4

[0185] Cell viability assay After treatment, cells were incubated at 37°C for 72 hours, after which cell viability was determined by MTS assay performed according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Briefly, MTS solution was diluted 20x in phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS. Cells were washed once with 200 μL PBS per well, after which 100 μL of diluted MTS solution was added per well. Plates were incubated at 37°C for approximately 20-30 minutes. OD at 492 nm was subsequently measured in a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" wells was subtracted from all other wells, and then cell viability of treated / untreated cells was calculated by dividing the background-corrected signal of treated wells by the background-corrected signal of untreated wells (×100).

[0186] FACS analysis Cells were seeded at 500,000 c / plate in 10 cm dishes with DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) and incubated for 48 h (5% CO2, 37 °C) until 90% confluency was reached. Cells were then trypsinized (TryplE Express, Gibco Thermo Scientific) to single cells. 0.75 × 10 6 The cells were transferred to a 15 mL Falcon tube and centrifuged (1,400 rpm, 3 min). The supernatant was discarded, leaving the cell pellet submerged. The pellet was dissociated by gently tapping the Falcon tube on a vortex shaker, and the cells were resuspended in 4 mL of cold PBS (with Mg 2+ and Ca 2+ After washing, the cells were washed with 3 mL of cold PBS (without MgO, 2% FBS). 2+and Ca 2+ The cells were resuspended in 200 μL of cold PBS (without Mg, 2% FBS) and aliquoted into three round-bottom FACS tubes (1 mL / tube). The cells were centrifuged again and resuspended in 200 μL of cold PBS (without Mg 2+ and Ca 2+ 195 μL of cold PBS (without MgCl, 2% FBS) or 195 μL of cold PBS (without MgCl 2+ and Ca 2+ The cells were resuspended in 200 μL of antibody solution containing 5 μL of antibody in 2% FBS (without Mg). APC Mouse IgG1, Kappa APC Anti-Human EGFR (#352906, Biolegend) was used to stain the EGFR receptor. PE Anti-Human HER2 APC Anti-Human CD340 (erbB2 / HER-2) (#324408 Biolegend) was used to stain the HER2 receptor, and PE Mouse IgG2a, Kappa Isotype Ctrl FC (#400212, Biolegend) was used as its corresponding isotype control. PE Anti-Human CD71 (#334106, Biolegend) was used to stain the CD71 receptor, and PE Mouse IgG2a, Kappa Isotype Ctrl FC (#400212, Biolegend) was used as its corresponding isotype control. Samples were incubated for 30 min at 4°C on a tube roller mixer. Cells were then transferred to cold PBS (without Mg) and washed with PBS (without Mg). 2+ and Ca 2+ The cells were washed three times with PBS (without FBS, 2% FBS) and fixed with 2% PFA solution in PBS for 20 min at room temperature. The cells were washed twice with cold PBS and resuspended in 250-350 μL of cold PBS for FACS analysis. Samples were analyzed using a BD FACSCanto II flow cytometry system (BD Biosciences) and FlowJo software. The results of the analysis of cell surface expression of EGFR, HER2, and CD71 in the various cells are summarized in Table A2.

[0187] [Table 6]

[0188] V HH -SO1861 conjugation procedure VHH To the aliquot of TCEP was added an aliquot of freshly prepared TCEP solution (10.0 mg / ml), the mixture was vortexed briefly, and then incubated at 20° C. for 30 min with roller mixing. After incubation, the resulting V HH -SH was purified by gel filtration into TBS (pH 7.5) using a Zeba spin desalting column. HH To the -SH was added the freshly prepared SPT-EMCH solution, the mixture was vortexed briefly, and then incubated at 20° C. overnight.

[0189] After incubation, V HH An aliquot of the -SO1861 mixture was removed and characterized by Ellman's assay to confirm incorporation of SO1861. The conjugate was purified on a 1.6 x 35 cm Superdex 200PG column eluted with DPBS (pH 7.5) to give purified V HH An aliquot was filtered to 0.2 μm, concentrated, and standardized to 1.0 mg / ml to give V HH -SO1861 was obtained.

[0190] V HH -Dianthine conjugation procedure Gianthin-Cys was concentrated by ultrafiltration using a vivaspin T15 10KDa MWCO centrifugal filter and buffer exchanged into TBS (pH 7.5). An aliquot of freshly prepared TCEP solution (10.0mg / ml) was added to the concentrated Gianthin-Cys, the mixture was vortexed briefly, and then incubated at 20°C for 60 minutes with roller mixing. After incubation, the resulting Gianthin-SH was purified by gel filtration using a zeba spin desalting column, followed by repeated centrifugation-wash cycles into TBS (pH 7.5) using a vivaspin T15 10KDa MWCO centrifugal filter. The resulting Gianthin-SH was reacted with freshly prepared DTME solution (10mg / ml) in DMSO, the mixture was vortexed briefly, and then incubated at 20°C for 60 minutes. The product was then purified by gel filtration into TBS (pH 7.5) using a Zeba spin desalting column to obtain Gianthin-DTME, which was stored at 20°C until conjugation. HH An aliquot of V was concentrated by ultrafiltration using a vivaspin T15 10KDa MWCO centrifugal filter and buffer exchanged into TBS (pH 7.5). HH An aliquot of freshly prepared TCEP solution (10.0 mg / ml) was added to the mixture, which was vortexed briefly and then incubated for 60 min at 37° C. with roller mixing. After incubation, the resulting V HH was purified by gel filtration using a zeba spin desalting column, followed by repeated centrifugation-wash cycles into TBS (pH 7.5) using a vivaspin T4 5KDa MWCO centrifugal filter. HH An aliquot of -SH was reacted with dianthine-DTME, the mixture was vortexed briefly, and then incubated overnight at 20° C. The reaction mixture was then concentrated using a vivaspin T4 10 KDa MWCO centrifuge tube and purified by gel filtration using a 1.6×35 cm Superdex 200PG column and eluted in DPBS (pH 7.5).

[0191] Antibody-(L-SO1861) 4 Trastuzumab, cetuximab are hereafter referred to as "Ab". Ab was conjugated to saponin SO1861-EMCH by Michael-type thiol-ene conjugation reaction with DAR of 1, 2, 3, 4, 5 and 6. SO1861-EMCH molecule obtains a labile (L) hydrazone bond between its structure and its maleimide functional group, generating a labile bond between saponin and Ab. The procedure is exemplarily described for trastuzumab-(L-SO1861)4.

[0192] To a solution of cetuximab (40 mg, 8.0 ml), 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris·HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) was added to obtain a 50 mM TBS, 2.5 mM EDTA buffer (pH 7.5).

[0193] Cetuximab was divided into four portions (9.73 mg, 4.864 mg / ml, 65 nmol, respectively) and freshly prepared aliquots of TCEP solution (0.5–2.0 mg / ml, 1.15–7.02 molar equivalents, 75–455 nmol) were added, the mixtures were vortexed briefly, and then incubated for 300 min at 20°C with roller mixing. After incubation (before addition of SO1861-EMCH), an aliquot of approximately 1 mg (0.210 ml) of Ab-SH was removed from each mixture and purified by gel filtration into TBS (pH 7.5) using a zeba spin desalting column. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-Ab ratios = 2.0, 4.2, 5.9 and 6.8, respectively). To each of the bulk Ab-SHs, an aliquot of freshly prepared SO1861-EMCH solution (2 mg / ml, 1.3 molar equivalents per "thiol", 0.15-0.61 μmol, 0.16-0.63 ml) was added, the mixture was vortexed briefly and then incubated at 20°C for 120 min. Besides each conjugation reaction, two aliquots (0.25 mg, 1.67 nmol) of desalted Ab-SH were reacted with NEM (1.3 molar equivalents per "thiol", 4.3-17.4 nmol, 2.2-8.7 μl of 0.25 mg / ml solution) or TBS (pH 7.5) buffer (2.2-8.7 μl) as positive and negative controls, respectively, for 120 min at 20 °C. After incubation (before addition of NEM), an aliquot of 0.200 ml Ab-SO1861-EMCH was removed and purified by gel filtration into TBS (pH 7.5) using a zeba spin desalting column. This aliquot was characterized by UV-vis and, in parallel, the positive and negative controls were characterized by Ellman's assay to obtain SO1861-EMCH incorporation. To the bulk Ab-SO1861-EMCH mixture, an aliquot of freshly prepared NEM solution (2.5 mg / ml, 2.5-10 molar equivalents, 0.15-0.58 μmol) was added, and the mixture was purified by zeba spin desalting column eluted with DPBS (pH 7.5) to obtain purified cetuximab-(L-SO1861) conjugate. This product was standardized to 2.5 mg / ml, filtered to 0.2 μm, and then distributed for biological evaluation. The reaction conditions and results of the trastuzumab-L-SO1861 conjugate and the cetuximab-L-SO1861 conjugate are summarized in Table A3 and Table A4.

[0194] [Table 7]

[0195] [Table 8]

[0196] Summary of Multiple Embodiments of Antibodies and Receptor Ligands Covalently Attached to Protein Toxins or Saponins mAb: trastuzumab (HER2) or cetuximab (EGFR) Ligand: EGF Protein toxins: ribosome-inactivating proteins, saporin, dianthin Endosomal escape-enhancing conjugates of saponins and ligands: mAb-SO1861 endosomal escape enhancing conjugate Contains a cleavable hydrazone linker Trastuzumab-SO1861 DAR 4.0 · Cetuximab-SO1861 DAR 3.7; In an in vitro or in vivo test model, the endosomal escape enhancing conjugate of saponin and ligand is combined with: mAb / ligand-protein toxin conjugates Contains a non-cleavable chemical linker or is a recombinant fusion protein Trastuzumab-saporin DAR 3.0 Cetuximab-saporin DAR 2.6 Trastuzumab-Giantin DAR 1.0 EGF-Giantin (fusion protein) DAR 1.0 IgG-Saporin DAR 2.2

[0197] Regarding Examples 4 to 9: material: Trastuzumab and cetuximab were purchased from a pharmacy (Charite, Berlin). SO1861 was isolated and purified from a raw plant extract obtained from Saponaria officinalis L by Analyticon Discovery GmbH. method SO1861-EMCH synthesis SO1861 was obtained from Saponaria officinalis L (Analyticon Discovery GmbH) and conjugated to EMCH by conventional procedures known in the art. Conjugation of SO1861 to antibodies Custom production of trastuzumab-SO1861 and cetuximab-SO1861 was carried out by FleetBioprocessing (UK). SO1861-EMCH was conjugated to cysteines of the antibodies. Conjugation of saporin to trastuzumab and cetuximab Custom trastuzumab-saporin and cetuximab-saporin conjugates were manufactured by and purchased from Advanced Targeting Systems (San Diego, CA). IgG-saporin and saporin were purchased from Advanced Targeting Systems. FACS analysis FACS analysis was performed on a BD FACSCanto II and data analysis was performed with FlowJo V10 software. FACS antibodies were as follows: 1) Isotype: APC mouse IgG1, kappa isotype Ctrl (FC) (400122, Biolegend). EGFR: APC anti-human EGFR (352906, Biolegend) HER2: APC anti-human CD340 (erbB2 / HER-2) (324408, Biolegend). Gianthin formation Gianthin was expressed in bacterial culture and the protein was purified according to conventional cell culture and protein purification processes known in the art. Conjugation of antibodies to dianthine Conjugation of antibodies to dianthine followed standard procedures known in the art. cell culture Cells were cultured at 37° C. and 5% CO in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (FBS) (PAN-Biotech GmbH). Cell viability assay Cells were seeded at 5.000-10.000 cells per well in 100 μL / well in 96-well plates and incubated overnight at 37°C. The next day, 10× concentrated treatment mixture samples were prepared in PBS containing SO1861 conjugated to an antibody (i.e., the “binding molecule” or “endosomal escape enhancing conjugate” of the invention) and a targeted toxin (i.e., the “binding molecule”), both at 10× final concentration. The medium was removed from the cell culture plate and replaced with 180 μL of culture medium, followed by the addition of 20 μL of treatment mixture per well. For controls, 10× treatment mixture samples were prepared containing the corresponding concentrations of antibody-conjugated SO1861 only, antibody only, SO1861 only, targeted toxin only, or PBS without compound as a vehicle control. When endosomal acidification inhibitors (chloroquine (Sigma Aldrich) or bafilomycin A1 (Enzo Life Sciences)) were used, the cell culture medium in treatment step 1 was replaced with 180 μL of medium containing 1 μM chloroquine or 0.2 μM bafilomycin A1. Plates were incubated at 37° C. for 1 hour before adding 10× treatment mixture samples. The remaining incubation and treatment steps were performed according to standard procedures known in the art.

[0198] After treatment, cells were incubated at 37°C for 72 hours, after which cell viability was determined by MTS assay performed according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Briefly, MTS solution was diluted 20x in phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS. Cells were washed once with 200 μL PBS per well, after which 100 μL of diluted MTS solution was added per well. Plates were incubated at 37°C for approximately 20-30 minutes. Subsequently, optical density at 492 nm was measured with a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" wells was subtracted from all other wells, and the percentage of treated / untreated cells was calculated by dividing the background-corrected signal of the untreated wells by the background-corrected signal of the treated wells.

[0199] result Example 4.1 Targeted Two-Component Systems The one-target two-component system was a combination treatment of mAb1-protein toxin and mAb1-SO1861 (see Figures 1A,E), and the two-target two-component system was a combination of mAb1-protein toxin and mAb2-SO1861, or mAb2-protein toxin and mAb1-SO1861 (Figures 1B-D).

[0200] Cetuximab-SO1861 (a monoclonal antibody that recognizes and binds EGFR conjugated to the saponin molecule SO1861; an endosomal escape enhancing conjugate) was titrated against a fixed concentration of 10 pM cetuximab-saporin (a monoclonal antibody that recognizes and binds EGFR conjugated to a protein toxin, saporin) (calculated at the concentration of SO1861) and cell killing against high EGFR expressing cells was measured. High EGFR expressing cells (A431 or CaSKi) showed efficient cell killing when 10 pM cetuximab-saporin was combined with high concentrations of non-targeted unconjugated SO1861 (A431: [SO1861] IC50 = 600 nM and Caski: [SO1861] IC50 = 700 nM; Figures 8A, 8B; Table A6). However, when cetuximab-saporin was combined with cetuximab-SO1861, potent cell killing was already induced at low concentrations of SO1861 (A431: [SO1861] IC50 = 5 nM and Caski [SO1861] IC50 = 8 nM; Figure 8A, 8B; Table A6). This indicates that the targeted conjugate SO1861 is more effective at inducing endosomal escape compared to the non-targeted unconjugated SO1861. Next, cetuximab-saporin was titrated against a fixed concentration of 300 nM cetuximab-SO1861 to measure targeted protein toxin-mediated cell killing against EGFR-expressing cells. High EGFR expressing cells (A431 or CaSKi) showed cell killing only at high concentrations of cetuximab-saporin in combination with non-targeted unconjugated 300 nM SO1861 (A431: [toxin] IC50 = 40 pM; CaSki: [toxin] IC50 = 40 pM; Figures 8C, 8D; Table A7), whereas 300 nM cetuximab-SO1861 in combination with low concentrations of cetuximab-saporin already induced efficient cell killing (A431: [toxin] IC50 = 0.4 pM; CaSKi: [toxin] IC50 = 2 pM; Figures 8C and 8D; Table A7).The highest cell killing efficiency is achieved when high concentration of non-targeted non-conjugated SO1861 (1500 nM) is combined with low concentration of cetuximab-saporin (A431: [toxin] IC50 = 0.03 pM; CaSki: [toxin] IC50 = 0.02 pM; Figure 8C, 8D; Table A7). All this indicates that relatively low concentration of SO1861 conjugated to cetuximab can be combined with relatively low concentration of cetuximab-saporin to efficiently kill high EGFR expressing cells. High concentration (1500 nM) non-targeted non-conjugated SO1861 combined with low concentration of cetuximab-saporin remains the most effective because in a one-target two-component system, both conjugates compete for the same EGFR receptor, so receptor competition does not play a role in SO1861's entry into cells. The receptor competition principle is also clearly demonstrated in the cetuximab-toxin titration procedure both in the absence (A431: [toxin] IC50 = 40 pM; CaSki: [toxin] IC50 = 40 pM) and presence (A431: [toxin] IC50 = 1000 pM; CaSki: IC50 = 1000 pM; Figures 8C, 8D) of 75 nM cetuximab.

[0201] Next, cetuximab-SO1861 was titrated against a fixed concentration of 10 pM cetuximab-saporin (calculated at the concentration of SO1861) and cell killing against low / non-EGFR expressing cells was measured. Low EGFR expressing cells (HeLa) only showed cell killing when 10 pM cetuximab-saporin was combined with high concentrations of non-targeted unconjugated SO1861, whereas A2058 cells, which do not express EGFR (A2058), were completely insensitive (HeLa: [SO1861] IC50 = 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figure 9A, 9B; Table A6). 10 pM cetuximab-saporin and increasing concentrations were used to determine cell killing against low / non-EGFR expressing cells. Combination of cetuximab-SO1861 with cetuximab did not induce significant cell death in both cell lines (HeLa: [SO1861] IC50 = 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figure 9A, 9B; Table A6). This indicates that without sufficient receptor expression, SO1861 in cells does not reach the effective concentration (threshold) to induce endosomal escape of the protein toxin and toxin-mediated cell death. Next, cetuximab-saporin was titrated against a fixed concentration of 300 nM cetuximab-SO1861 to measure targeted protein toxin-mediated cell death against low / non-EGFR expressing cells. Low EGFR expressing cells (HeLa) only showed cell killing at very high concentrations of cetuximab-saporin in combination with 278 nM cetuximab-SO1861 or 300 nM unconjugated SO1861, whereas A2058 cells (EGFR) were not sensitive at the concentrations tested (HeLa: [toxin] IC50 = 60 pM; A2058: [toxin] IC50 > 10,000 pM; Figures 9C, 9D; Table A7).In low EGFR expressing cells (HeLa), high concentrations of unconjugated SO1861 (1500 nM) in combination with low concentrations of cetuximab-saporin show efficient cell killing, whereas in A2058 cells, only very high concentrations of cetuximab-saporin in combination with 1500 nM of untargeted SO1861 induce specific cell killing (Hela: [toxin] IC50 = 0.03 pM; A2058: [toxin] IC50 = 20 pM; Figure 9C, 9D; Table A6). All this indicates that cells with low or no EGFR receptor expression are insensitive to the combination of cetuximab-SO1861 + cetuximab-saporin due to lack of sufficient EGFR receptors to facilitate the entry of sufficient SO1861 and toxin into the cells.

[0202] Trastuzumab-SO1861 (monoclonal antibody that recognizes and binds HER2 conjugated to the saponin molecule SO1861; an endosomal escape enhancing conjugate according to the invention) was titrated against a fixed concentration of 50 pM trastuzumab-saporin (monoclonal antibody that recognizes and binds HER2 conjugated to a protein toxin, saporin) (calculated at the concentration of SO1861) and cell killing against high HER2 expressing cells was measured. High HER2 expressing cells (SKBR3) showed efficient cell killing when 50 pM trastuzumab-saporin was combined with high concentrations of non-targeted unconjugated SO1861 (SKBR3; Figures 10A, 10B; Table A6). However, when trastuzumab-saporin was combined with trastuzumab-SO1861, strong cell killing was already induced at low concentrations of SO1861 (SKBR3; Figure 10A, 10B; Table A6). This indicates that the targeted conjugate SO1861 is more effective at inducing endosomal escape compared to the non-targeted non-conjugated SO1861. Trastuzumab-saporin was then titrated against a fixed concentration of 50 nM trastuzumab-SO1861 to measure targeted protein toxin-mediated cell killing against HER2-expressing cells. High HER2 expressing cells (SKBR3 or BT474) showed cell killing only when high concentrations of trastuzumab-saporin were combined with non-targeted unconjugated 10 nM SO1861 (Table A7), while 10 nM trastuzumab-SO1861 combined with low concentrations of trastuzumab-saporin already induced efficient cell killing (Table A7). The highest cell killing efficiency was achieved when high concentrations of non-targeted unconjugated SO1861 (1500 nM) were combined with low concentrations of trastuzumab-saporin (Table A7). All this indicates that low concentrations of SO1861 conjugated to trastuzumab can be combined with relatively low concentrations of trastuzumab-saporin to efficiently kill high HER2 expressing cells.High concentrations (1500 nM) of untargeted unconjugated SO1861 in combination with low concentrations of trastuzumab-saporin remain the most effective because in a one-target two-component system, both conjugates compete for the same EGFR receptor, so receptor competition plays no role in SO1861 entry into cells. The receptor competition principle is also clearly demonstrated in titrations of trastuzumab toxin with or without 2.5 nM trastuzumab (SKBR3: [toxin] IC50 = 1000 nM).

[0203] Trastuzumab-SO1861 was then titrated against a fixed concentration of 50 pM trastuzumab-saporin (calculated as the concentration of SO1861) to measure cell killing against low / non-EGFR expressing cells. Low EGFR expressing cells (JIMT1; A431) only demonstrated cell killing when 50 pM trastuzumab-saporin was combined with high concentrations of non-targeting, unconjugated SO1861 (JIMT1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figures 11A, 11B; Table A6). Trastuzumab-saporin at 50 pM and increasing concentrations of trastuzumab-SO1861 were titrated against a fixed concentration of 50 pM trastuzumab-saporin (calculated as the concentration of SO1861) to measure cell killing against low / non-EGFR expressing cells. Combination with 61 did not induce significant cell death in both cell lines (JIMT1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figure 11A, 11B; Table A6). This indicates that without sufficient receptor expression, SO1861 in cells does not reach the effective concentration (threshold) to induce endosomal escape of the protein toxin and toxin-mediated cell death. Next, trastuzumab-saporin was titrated against a fixed concentration of 10 nM trastuzumab-SO1861 to measure targeted protein toxin-mediated cell death against low / non-HER2 expressing cells. Low HER2 expressing cells (JIMT1; A431) do not show significant cell killing at high concentrations of trastuzumab-saporin in combination with 10 nM trastuzumab-SO1861 (JIMT-1: [toxin] IC50 > 10,000 pM; A431: [toxin] IC50 > 10,000 pM; Figures 11C, 11D; Table A7). In low HER2 expressing cells, high concentrations of unconjugated SO1861 in combination with low concentrations of trastuzumab-saporin (15 00 nM) show efficient cell killing (JIMT1: [toxin] IC50 = 0.1 pM; A431: [toxin] IC50 = 0.8 pM; Figures 11C, 11D; Table A5). All this indicates that cells with low HER2 receptor expression are insensitive to the combination of trastuzumab-SO1861 + trastuzumab-saporin due to lack of sufficient HER2 receptors to facilitate entry of sufficient SO1861 and toxin into the cells.

[0204] Example 5.2 Targeted Two-Component Systems Cetuximab-SO1861 was titrated against a fixed concentration of 50 pM trastuzumab-saporin (calculated at the concentration of SO1861) to measure cell killing against high EGFR / low HER2 expressing cells. A431 and CaSki cells showed efficient cell killing when 50 pM trastuzumab-saporin was combined with high concentrations of non-targeted unconjugated SO1861 (A431 and Caski: [SO1861] IC50 = 1000 nM; Figures 12A, 12B; Table A6). However, when trastuzumab-saporin was combined with cetuximab-SO1861, potent cell killing was already induced at low concentrations of SO1861 (A431: [SO1861] IC50 = 12 nM and Caski [SO1861] IC50 = 40 nM; Figures 12A, 12B; Table A6). This indicates that the targeted conjugate SO1861 is more effective at inducing endosomal escape compared to the non-targeted unconjugated SO1861. Next, trastuzumab-saporin was titrated against a fixed concentration of 300 nM cetuximab-SO1861 to measure targeted protein toxin-mediated cell killing against high EGFR / low HER2 expressing cells (A431 and CaSki). At high concentrations of trastuzumab-saporin in combination with 300 nM of non-targeted, unconjugated SO1861, no efficient cell killing was observed (A431 and Caski::[toxin] IC50 > 10,000 pM; Figures 12C, 12D; Table A7), whereas 300 nM of cetuximab-SO1861 in combination with low concentrations of trastuzumab-saporin already induced efficient cell killing (A431: [toxin] IC50 = 3 pM; CaSKi: [toxin] IC50 = 1 pM; Figures 12C and 12D; Table A7). In A431 cells, comparable cell killing efficiency is achieved when high concentrations (1500 nM) of untargeted unconjugated SO1861 are combined with low concentrations of cetuximab-saporin (A431: [toxin] IC50 = 1 pM; Figure 12C; Table A7).In Caski cells, the response was slightly stronger than that of the combination of cetuximab-SO1861 and trastuzumab-saporin due to the fact that EGFR expression in these cells is significantly lower than that in A431, and therefore the targeted delivery of SO1861 to Caski cells is less sufficient (Caski: [toxin] IC50 = 0.2 pM; Figure 12D; see Table A7). All this indicates that low concentrations of SO1861, when conjugated to cetuximab, can be combined with relatively low concentrations of trastuzumab-saporin to efficiently kill high EGFR expressing cells. High concentrations (1500 nM) of untargeted unconjugated SO1861 in combination with low concentrations of trastuzumab-saporin have comparable activity since in a two-target two-component system, both conjugates are delivered by different receptors, i.e., SO1861 by EGFR and toxin by HER2 receptor, and thus receptor competition does not play a role in SO1861 entry into cells.

[0205] Cetuximab-SO1861 was then titrated against a fixed concentration of 50 pM trastuzumab-saporin (calculated at the concentration of SO1861) to measure cell killing against low / non-EGFR / HER2 expressing cells. Low EGFR / HER2 expressing cells only showed cell killing when 50 pM trastuzumab-saporin was combined with high concentrations of non-targeting unconjugated SO1861 (HeLa: [SO1861] IC50 > 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figure 13A, 13B; Table A6). Combination of 50 pM trastuzumab-saporin with increasing concentrations of cetuximab-SO1861 showed significant cell killing only at high concentrations of cetuximab-SO1861 in both cell lines (HeLa: [SO1861] IC50 > 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figures 13A, 13B; Table A6). This indicates that without sufficient receptor expression, intracellular SO1861 does not reach an effective concentration (threshold) to induce endosomal escape of the protein toxin and toxin-mediated cell killing. Trastuzumab-saporin was then titrated against a fixed concentration of cetuximab-SO1861 to measure targeted protein toxin-mediated cell killing against low / non-EGFR / HER2 expressing cells. Low / non-EGFR / HER2 expressing cells (HeLa and A2058) do not show significant cell killing at high trastuzumab-saporin concentrations in combination with 278 nM cetuximab-SO1861 (HeLa) (HeLa: [toxin] IC50>10,000 pM; A2058: [toxin] IC50>10,000 pM; Figures 13C, 13D; Table A7). High concentrations of unconjugated SO1861 (1500 nM) in combination with low concentrations of trastuzumab-saporin show efficient cell killing (HeLa: [toxin] IC50=0.4 pM; A2058: [toxin] IC50=0.5 pM; Figures 13C, 13D; Table A5). All this indicates that cells with low / non-EGFR / low HER2 expression are insensitive to the cetuximab-SO1861+trastuzumab-saporin combination due to lack of sufficient EGFR receptors to facilitate sufficient entry of SO1861 to ensure efficient cytoplasmic delivery of the toxin in the cells.

[0206] Next, trastuzumab-SO1861 was titrated against a fixed concentration of 1.5 pM EGF-Giantin (calculated at the concentration of SO1861) to measure cell killing against high HER2 / low EGFR expressing cells. SKBR3 showed efficient cell killing when 1.5 pM EGF-Giantin was combined with high concentrations of non-targeted non-conjugated SO1861 (SKBR3: [SO1861] IC50 = 800 nM; Figure 14A; Table A6). However, when EGF-Giantin was combined with trastuzumab-SO1861, strong cell killing was already induced at low concentrations of conjugated SO1861 (SKBR3: [SO1861] IC50 = 2 nM; Figure 14A; Table A5). This indicates that the targeted conjugate SO1861 is more effective at inducing endosomal escape compared to the non-targeted unconjugated SO1861. EGF-gianthin was then titrated against a fixed concentration of trastuzumab-SO1861 and the targeted protein toxin-mediated cell killing against SKBR3 was measured. At high concentrations of EGF-Giantin in combination with 10 nM of non-targeted non-conjugated SO1861, no efficient cell killing was observed (SKBR3 (shown) and BT474 (not shown): [toxin] IC50>10,000 pM; FIG. 14B; Table A7), whereas 9.4 nM of trastuzumab-SO1861 in combination with low concentrations of EGF-Giantin already induced efficient cell killing (SKBR3: [toxin] IC50=3 pM (shown); BT474: [toxin] IC50=1 pM (not shown); FIG. 14B; Table A7). Comparable cell killing efficiency is achieved when high concentrations (1075 nM) of non-targeted non-conjugated SO1861 are combined with low concentrations of EGF-Giantin; FIG. 14B; Table A7). All this indicates that low concentrations of SO1861, when conjugated to trastuzumab, in combination with relatively low concentrations of EGF-gianthin, can efficiently kill high HER2 expressing cells.High concentrations (1500 nM) of untargeted unconjugated SO1861 in combination with low concentrations of EGF-gianthin have comparable activity since in a two-target two-component system, both conjugates are delivered by different receptors, i.e., SO1861 by HER2 and toxin by EGFR receptor, and thus receptor competition does not play a role in SO1861 entry into cells.

[0207] Trastuzumab-SO1861 was then titrated against a fixed concentration of 5 pM cetuximab-saporin (calculated at the concentration of SO1861) and cell killing was measured against HER2-low, EGFR-low / high expressing cells, demonstrating cell killing when 5 pM cetuximab-saporin was combined with high concentrations of non-targeted unconjugated SO1861 (JIMT-1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figures 15A, 22B; Table A6). The combination of 5 pM cetuximab-saporin with increasing concentrations of trastuzumab-SO1861 showed significant cell killing only at high concentrations of trastuzumab-SO1861 in both cell lines (JIMT-1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figures 15A, 15B; Table A6). This indicates that without sufficient receptor expression, intracellular SO1861 does not reach an effective concentration (threshold) to induce endosomal escape of the protein toxin and toxin-mediated cell killing. Next, cetuximab-saporin was titrated against a fixed concentration of trastuzumab-SO1861 to measure targeted protein toxin-mediated cell killing against JIMT-1 and A431. Only when high concentrations of cetuximab-saporin were combined with 10 nM of trastuzumab-SO1861 was cell death observed (JIMT-1: [toxin] IC50 > 90 pM; A431: [toxin] IC50 > 20 pM; Figures 15C, 15D; Figure A7). High concentrations of unconjugated SO1861 (1500 nM) combined with low concentrations of cetuximab-saporin show efficient cell killing (JIMT-1: [toxin] IC50 = 0.02 pM; A431: [toxin] IC50 = 0.03 pM; Figures 15C, 15D; Table A5). All this indicates that cells with low HER2, low / high EGFR expression are insensitive to the trastuzumab-SO1861+cetuximab-saporin combination due to lack of sufficient HER2 receptors to facilitate sufficient entry of SO1861 to ensure efficient cytoplasmic delivery of the toxin in the cells.In A431 cells, even very high EGFR expression did not reach the threshold for SO1861 due to the lack of HER2 receptors that could facilitate uptake of SO1861 by trastuzumab-SO1861, and therefore did not result in efficient cell killing by cetuximab-saporin.

[0208] Example 6. The two-targeted two-component system results in cell killing of very low target expressing cells. In A431 cells, T-DM1 kills cells at nanomolar concentrations, whereas the targeted two-component system can efficiently kill cells at picomolar concentrations (a approx. 7000-fold reduction in toxin concentration) (Figure 16).

[0209] Example 7. Mechanism of action When endosomal acidification is blocked, the targeted two-component system ...

Claims

1. - a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, which are covalently linked to each other directly or via a linker, said effector molecule comprising or consisting of a natural, synthetic or modified oligonucleotide; a second conjugate comprising a binding molecule for binding to a second cell surface molecule and a saponin, said binding molecule and said saponin being covalently linked to each other either directly or via a linker, said saponin being a bidesmosidic triterpene glycoside belonging to the type of 12,13-dehydrooleanane, said saponin having a branched carbohydrate side chain at position C3 containing a glucuronic acid functional group and said saponin having a branched carbohydrate side chain at position C28 containing a deoxy carbohydrate, said second cell surface molecule being the same or different from said first cell surface molecule, and wherein when said first cell surface molecule and said second cell surface molecule are different, said first cell surface molecule and said second cell surface molecule are present on the same target cell; and, optionally - Pharmaceutically acceptable excipients and / or pharmaceutically acceptable diluents 2. A single pharmaceutical composition comprising:

2. The sdAb V derived from the heavy chain of an antibody Ｈ domain; V derived from the light chain of an antibody Ｌ Domain; and V derived from heavy chain antibodies (HCAb) derived from Camelidae origin or Ig-NAR origin ＨＨ domain 2. The method of claim 1, wherein the single pharmaceutical composition is any one or more of the following:

3. 3. A single pharmaceutical composition according to claim 1 or 2, wherein the first conjugate comprises at least two sdAbs covalently linked to the at least one effector molecule.

4. The sdAbs are directed against HIV gp41, CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrins, syndecan-1, vascular integrin αV beta 3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate specific membrane antigen (PSMA), CanAg, integrin αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, C D25, ephrinA4, MUC-1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5, CD16, CXCR7 (ACKR3), glucocorticoid-induced TNFR-related protein (GITR), or Anti-CD71 sdAb, anti-HER2 sdAb, anti-CD20 sdAb, anti-CA125 sdAb, anti-EpCAM (17-1A) sdAb, anti-EGFR sdAb, anti-CD30 sdAb, anti-CD33 sdAb, anti-vascular integrin αvβ3 sdAb, anti-CD52 sdAb, anti-CD22 sdAb, anti-CEA sdAb, anti-CD44v6 sdAb, anti-FAP sdAb, anti-CD19 sdAb, anti-CanAg sdAb, anti-CD56 sdAb, anti-CD38 sdAb, anti-CA6 sdAb, anti-IGF-1R sdAb, anti-integrin sdAb, anti-syndecan-1 4. A single pharmaceutical composition according to any one of claims 1 to 3, capable of binding to a first cell surface molecule which is a receptor selected from: anti-CD79b, anti-c-Met sdAb, anti-EGFR1 sdAb, anti-VEGFR2 sdAb, anti-CXCR7 sdAb and anti-HIV gp41.

5. the first conjugate comprises an sdAb capable of binding to HER2, CD71, HIV gp41 and / or EGFR; or The first conjugate is an sdAb for binding to HER2 selected from the sdAb produced by clone 11A4, clone 18C3, clone 22G12, clone Q17 or clone Q17-Ctag; or an sdAb for binding to EGFR produced by clone anti-EGFR Q86-Ctag; or an sdAb for binding to CD71 produced by clone anti-CD71 Q52-Ctag; or an sdAb for binding to CD71 produced by clone anti-HIV gp41 5. A single pharmaceutical composition according to any one of claims 1 to 4 comprising an sdAb for binding to HIV gp41 produced by a Q8-C tag; or an sdAb encoded by the cDNA of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; or an sdAb having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36-72.

6. 6. A single pharmaceutical composition according to any one of claims 1 to 5, wherein the effector molecule comprises or consists of a BNA, a xenonucleic acid or a siRNA and / or is selected from any one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), anti-microRNA (anti-miRNA), DNA aptamers, RNA aptamers, peptide nucleic acid (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 3'-fluorohexitol nucleic acid (FHNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA), or derivatives thereof, such as a BNA for silencing HSP27 protein expression or a BNA for silencing apolipoprotein B expression.

7. The oligonucleotide in the effector molecule may be selected from the group consisting of short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), mRNA, DNA, antisense DNA, locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-O,4'-aminoethylene bridged nucleic acid (BNA), and the like. ＮＣ ), BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-AON), and / or and / or selected from any one of anti-miRNA, BNA-AON, or siRNA, e.g., BNA-based siRNA; It may silence a gene (if present in a cell containing such a gene), said gene being any one of the following genes: apolipoprotein B (apoB), transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), the X gene of the hepatitis B virus (HBV), the S gene of HBV, alpha-1 antitrypsin (AAT) and lactate dehydrogenase (LDH), and / or it may target an aberrant miRNA (if present in a cell containing such a miRNA), and / or A single pharmaceutical composition according to any one of claims 1 to 6, capable of targeting (when present in a cell containing such an mRNA) an mRNA involved in the expression of any one of the following proteins: ApoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the X gene of HBV, the expression product of the S gene of HBV, AAT and LDH, or capable of antagonizing or restoring miRNA function (when present in a cell containing such an miRNA).

8. The saponin is 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; Gypsogenin; Quila acid; Protoesigenin 21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-barringtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-barringtogenol C-21 (2-methylbut-2-enoate)-16,22-diacetate; Digitogenin; 3,16,28-trihydroxyoleanan-12-ene; and Gypsogenic acid 8. The single pharmaceutical composition of any one of claims 1 to 7, comprising an aglycone core structure selected from:

9. A single pharmaceutical composition according to any one of claims 1 to 8, wherein the saponin comprises an aglycone core structure selected from quillaric acid and gypsogenin.

10. The saponin is a C-substituted saponin aglycone core structure. ３ Atom or C ２８ and / or the saponin comprises a first glycan bonded to an atom at C of the aglycone core structure of the saponin. ２８ The single pharmaceutical composition of any one of claims 1 to 9, comprising a second sugar chain attached to the atom.

11. The saponin is GlcA-, Glc-, Gal-, Rha-(1→2)-Ara-, Gal-(1→2)-[Xyl-(1→3)]-GlcA-, Glc-(1→2)-[Glc-(1→4)]-GlcA-, Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-, Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, comprises a first sugar chain selected from and / or said saponin is Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc-, where R1 is 4E-methoxycinnamic acid; Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc-, where R2 is 4Z-methoxycinnamic acid; Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc-, where R3 is 4E-methoxycinnamic acid; Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-) (1→3)-(Ara- or Xyl-) (1→4)-(Rha- or Fuc-) (1→2)-[4-OAc-(Rha- or Fuc-) (1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc-, where R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc- (wherein R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc-, where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc-, where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc-, where R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid; Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- (wherein R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- (wherein R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- (wherein R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)); Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc-, where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and Glc-(1→3)-[Glc-(1→6)]-Gal-, The single pharmaceutical composition of claim 10, further comprising a second glycan selected from the group consisting of:

12. The saponins include Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, escinate, AS6.2, NP-005236, AMA-1, AMR, α-hederin, NP-012672, NP-017777, NP-017778, NP-01 7774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, NP- 017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-0 12. The single pharmaceutical composition according to any one of claims 1 to 11, wherein the active ingredient is selected from the group consisting of 17773, NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, SO1862, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-api, QS-21A-xyl, QS-21B-api, QS-21B-xyl, β-escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primulic acid 1 and AS64R, or stereoisomers thereof, and / or combinations thereof.

13. the second conjugate comprises a binding molecule for binding to the second cell surface molecule, the binding molecule being any one or more of a proteinaceous ligand for binding to the second cell surface molecule, for example an EGF or a cytokine, or a ligand such as an immunoglobulin or at least one binding fragment or binding domain of an immunoglobulin capable of binding to the second cell surface molecule, or The single pharmaceutical composition of any one of claims 1 to 12, wherein the second cell surface molecule is a cell surface receptor present on an abnormal cell.

14. 14. The single pharmaceutical composition according to any one of claims 1 to 13, wherein the second conjugate comprises a binding molecule for binding to the second cell surface molecule, such as an antibody, or a binding fragment or domain thereof, or a receptor ligand, wherein the second cell surface receptor is HIV gp41, or a tumor cell specific receptor.

15. The second conjugate may be selected from the group consisting of CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin αV beta3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate specific membrane antigen (PSMA), CanAg, integrin αV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD 25, ephrinA4, MUC-1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5, CD16, CXCR7 (ACKR3), glucocorticoid-induced TNFR-related protein (GITR).

16. The second conjugate comprises a binding molecule for binding to the second cell surface molecule, the binding molecule being selected from the group consisting of anti-CD71 antibody OKT-9, trastuzumab (Herceptin), pertuzumab, rituximab, ofatumumab, tositumomab, obinutuzumab, ibritumomab, oregovomab, edrecolomab, cetuximab, matuzumab, panitumumab, nimotuzumab, brentuximab, gemtuzumab, huMy9-6, , etaracizumab, alemtuzumab, epratuzumab, pinatuzumab, moxetumomab, inotuzumab, labetuzumab, bivatuzumab, sibrotuzumab, anti-CD19 antibody huB4, anti-CanAg antibody huC242, anti-CD56 antibody huN901, anti-CD38 antibody OKT-10, daratumumab, anti-CD38 monoclonal antibody, anti-CA6 antibody DS6, anti-IGF-1R antibody 3B7, cixutumumab, anti-integrin antibody CNTO 95, anti-syndecan-1 antibody B-B4, polatuzumab, and anti-HIV gp41 antibody.

17. The second conjugate comprises the saponin covalently attached to a sulfhydryl group in the binding molecule via a thio-ether bond, and a covalent bond via a linker N-ε-maleimidocaproic acid hydrazide (EMCH) at the C of the aglycone core structure of the saponin. ２３ covalently bonded to an aldehyde group at one position of the carboxyl group ... The saponin is a C-substituted saponin aglycone core structure. ２３ It is a bidesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at the C position of the aglycone core structure of said saponin. ３ The saponin or its derivatives contain a glucuronic acid unit in a first sugar chain of a β-OH group, and the saponin or its derivatives are linked to the saponin or its derivatives via a carboxyl group of the glucuronic acid unit of the first sugar chain, or The saponin may comprise a C 2 -substituted hydroxyl group of the aglycone core structure of the at least one saponin. ３ 17. A single pharmaceutical composition according to any one of claims 1 to 16, comprising a saponin comprising a glucuronic acid unit in the first sugar chain of the β-OH group, said glucuronic acid unit being covalently attached to a linker.

18. A single pharmaceutical composition according to any one of claims 1 to 17, wherein the second conjugate comprises two or more covalently attached saponins.

19. 19. The single pharmaceutical composition of claim 18, wherein the two or more covalently linked saponins are covalently linked via at least one cleavable linker.

20. 20. The single pharmaceutical composition of claim 19, wherein the two or more covalently linked saponins comprise at least one oligomeric or polymeric molecule, are part of a covalent saponin conjugate to which the two or more saponins are covalently linked, and the covalent saponin conjugate is covalently linked to the linking molecule.

21. 21. The single pharmaceutical composition of claim 20, wherein the polymeric or oligomeric molecules of the covalent saponin conjugate comprise polymeric and / or oligomeric structures selected from linear polymers, branched and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, poly-lysine, poly-ethylene glycol, oligo-ethylene glycol (OEG), or collections of these polymeric and / or oligomeric structures.

22. 22. The single pharmaceutical composition of claim 21, wherein the polymeric or oligomeric molecule of the covalent saponin conjugate is a dendron that is a poly-amidoamine (PAMAM) dendrimer.

23. 23. A single pharmaceutical composition according to any one of claims 20 to 22, wherein 1 to 8 of the covalent saponin conjugates are attached to the binding molecule, and wherein at least one of the covalent saponin conjugates is based on a dendron.

24. A single pharmaceutical composition according to any one of claims 1 to 23, wherein the saponin is covalently attached to the binding molecule via a cleavable linker.

25. 25. The single pharmaceutical composition of claim 24, wherein the cleavable linker is cleaved under acidic, reducing, enzymatic and / or photoinduced conditions.

26. 26. The single pharmaceutical composition of claim 25, wherein the cleavable linker comprises a cleavable bond selected from a hydrazone bond and a hydrazide bond that are cleaved under acidic conditions, and / or a bond that is susceptible to proteolysis, and / or a bond that is susceptible to cleavage under reducing conditions.

27. The single pharmaceutical composition according to any one of claims 24 to 26, wherein the cleavable linker is cleaved in vivo under acidic conditions.

28. A single pharmaceutical composition according to any one of claims 24 to 27, wherein the cleavable linker is cleaved at a pH of ≦5.

5.

29. A medicine comprising a single pharmaceutical composition according to any one of claims 1 to 28 as an active ingredient.

30. A single pharmaceutical composition according to any one of claims 1 to 28 for the treatment of a disease associated with a gene defect.

31. 31. The single pharmaceutical composition of claim 30, wherein the saponin is SO1861 or QS-21.

32. An in vitro method for transferring a first conjugate according to any one of claims 1 to 28 from outside a cell into said cell, comprising: a) providing a cell expressing on its surface a first cell surface molecule according to any one of claims 1 to 28 and, optionally, expressing on its surface a second cell surface molecule according to any one of claims 1 to 28; b) providing a first conjugate according to any one of claims 1 to 28 for transfer to the cells provided in step a); c) providing a second conjugate according to any one of claims 1 to 28; d) contacting the cells of step a) in vitro with the first conjugate of step b) and with the conjugate of step c); thereby establishing transfer of the first conjugate from outside the cell into the cell.

33. - a first pharmaceutical composition comprising a first conjugate comprising at least one effector molecule and a single domain antibody (sdAb) for binding to a first cell surface molecule, which are covalently linked to each other directly or via a linker, said effector molecule comprising or consisting of a natural, synthetic or modified oligonucleotide, optionally comprising a pharma- ceutically acceptable excipient and / or a pharma- ceutically acceptable diluent; and a second pharmaceutical composition comprising a second conjugate comprising a binding molecule for binding to a second cell surface molecule and a saponin, said binding molecule and said saponin being covalently linked to each other either directly or via a linker, said saponin being a bidesmosidic triterpene glycoside belonging to the type of 12,13-dehydrooleanane, said saponin having a branched carbohydrate side chain at position C3 comprising a glucuronic acid functional group and said saponin having a branched carbohydrate side chain at position C28 comprising a deoxy carbohydrate, said second cell surface molecule being the same or different from said first cell surface molecule, and where said first cell surface molecule and said second cell surface molecule are different, said first cell surface molecule and said second cell surface molecule are present on the same target cell, and optionally comprising a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent; A kit comprising two or more pharmaceutical compositions comprising:

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