Combination comprising ADC or aoc comprising vhh, and saponin or ligand-saponin conjugate

Abstract

To provide a pharmaceutical combination for use in the treatment or the prophylaxis of a cancer, an autoimmune disease such as rheumatoid arthritis, an enzyme deficiency, a gene defect, a disease related to a gene defect, an amyloidosis, a disease related to an enzyme deficiency, an infection such as a viral infection, hypercholesterolemia, primary hyperoxaluria, haemophilia A, haemophilia B, α-1 antitrypsin related liver disease, acute hepatic porphyria, or transthyretin-mediated amyloidosis.SOLUTION: A kit including a first pharmaceutical composition and a second pharmaceutical composition is provided. The first pharmaceutical composition includes a first conjugate including at least one effector molecule and a single-domain antibody (sdAb) for binding to a first cell-surface molecule. The second pharmaceutical composition includes a second conjugate including an antibody for binding to a second cell-surface molecule and a saponin.SELECTED DRAWING: None

Description

Technical Field

[0001] 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, and a second conjugate comprising a binding molecule for binding to saponin, its derivative, or a second cell surface molecule, and saponin and / or its derivative, wherein the saponin or its derivative is a monodesmoside triterpenoid glycoside or a bidesmoside triterpenoid glycoside. The present invention also relates to a composition comprising the first conjugate and saponin (derivative), or a composition comprising the first conjugate and a second conjugate comprising saponin (derivative). Furthermore, the present invention relates to the use of the pharmaceutical combination or pharmaceutical composition of the present invention for use as a medicament, and for the treatment or prevention of cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiencies, gene deficiencies, diseases associated with gene deficiencies, amyloidosis, diseases associated with enzyme deficiencies, infectious diseases such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, α1-antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis. Furthermore, the present invention relates to a method for transferring the first conjugate of the present invention from extracellular to intracellular, preferably to the cytosol of the cell, in vitro or ex vivo. The present invention also relates to an in vitro or ex vivo method for transferring the first conjugate of the present invention from extracellular to intracellular, and an in vitro or ex vivo method for transferring the effector molecule contained in the first conjugate into the cytosol of the cell if the first conjugate is transferred from extracellular to intracellular, for example, to the endosome, endolysosome or lysosome of the cell.

Background Art

[0002] Molecules with therapeutic biological activity are often, in theory, 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 small molecule biologically active moiety. However, many, if not all, of the potential drug-like molecules and therapeutic agents currently in clinical use have at least one of many deficiencies and drawbacks. When administered to the human body, a therapeutic active molecule can exert off-target effects in addition to the biological activity directed towards the aspects underlying the disease or health problem to be treated. Such off-target effects are undesirable and carry the risk of inducing health-threatening or life-threatening side effects of the administered molecule. The occurrence of such adverse events is the cause of failure of many drug-like compounds and therapeutic moieties in Phase III clinical trials or Phase IV clinical trials (post-marketing follow-up). Therefore, there is a strong demand for drug molecules such as small molecule therapeutic agents that have therapeutic effects that are, for example, (1) highly specific for the biological factors or biological processes causing the disease, (2) sufficiently safe, (3) sufficiently effective, (4) sufficiently directed towards diseased cells and have little or no off-target activity against non-diseased cells, (5) have a mode of action that is sufficiently timely (e.g., the administered drug molecule reaches the target site in a human patient within a certain time frame and remains at the target site for a certain time frame), and / or (6) in particular, provide a drug molecule with a therapeutic activity that lasts sufficiently long in the patient's body. Unfortunately, to date, the "ideal" therapeutic agents having many or all of the beneficial features outlined above have not yet become available to patients, despite having been intensively studied for a long time and having made remarkable progress in some areas of the individual difficulties and drawbacks faced.

[0003] Chemotherapy is one of the most important treatment options for cancer. However, it is often associated with a low therapeutic index because it is not specific to cancer cells over dividing cells in healthy tissues. The invention of monoclonal antibodies provided the possibility of utilizing their specific binding properties as a mechanism for targeted delivery of cytotoxic agents to cancer cells while sparing normal cells. This can be achieved by chemical conjugation of a cytotoxic effector (also known as payload or warhead) to an antibody to create an antibody-drug conjugate (ADC). Generally, very potent payloads such as mertansine (DM1), which have a limited therapeutic index (the ratio of toxic dose to effective dose) in their unbound form, are used. The conjugation of DM1 to trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla, enhances the tolerated dose of DM1 by at least twofold in monkeys. Over the past few decades, significant efforts and investments have been made to develop therapeutic ADCs. However, despite promising preclinical data, it remains difficult to translate 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 re-approved for market entry in 2017 at a lower recommended dose, different schedules in combination with chemotherapy or alone, and in a new patient population. To date, only five ADCs have been approved for clinical use, while approximately 55 ADC clinical developments have been discontinued during that time. However, interest remains high and approximately 80 ADCs are still in clinical development in nearly 600 clinical trials.

[0004] Despite the potential for patients to use highly toxic payloads that are normally intolerable, the low therapeutic index (the ratio of the toxic dose compared to the effective dose) is a major problem that has forced the discontinuation of many ADCs in clinical development, which can be caused by several mechanisms such as off-target toxicity to normal cells, the development of resistance to cytotoxic agents, and the early release of drugs in the circulating blood. The systematic review by the FDA of ADCs found that the toxicity profiles of most ADCs are classified by the payload used and may not be classified by the antibody used, suggesting that most of the toxicity is determined by the early release of the payload. At least 23 of the approximately 55 discontinued ADCs were presumably due to an insufficient therapeutic index. For example, the development of trastuzumab tesirine conjugate (ADCT-502, targeting HER-2, an ADC therapeutic) was recently interrupted due to a narrow therapeutic index, probably due to on-target, off-tissue effects in lung tissue expressing a fairly high level of HER-2. Furthermore, several ADCs in phase 3 trials have been discontinued due to missing primary endpoints. For example, the phase 3 trials of depatuxizumab mafodotin conjugate (ABT-414, targeting EGFR, AbbVie) tested in patients with newly diagnosed glioblastoma, and mirvetuximab soravtansine conjugate (IMGN853, targeting folate receptor alpha (FRα), ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently discontinued and did not show a survival benefit. It is important to note that the clinically used doses of some ADCs may not be sufficient for their full anti-cancer activity. For example, ado-trastuzumab emtansine has an MTD of 3.6 mg / kg in humans. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels of 3 mg / kg or higher, but more potent efficacy was observed at 15 mg / kg. This suggests that at clinically administered doses, ado-trastuzumab emtansine may not exert its maximum potential anti-tumor effect.

[0005] An ADC is mainly composed of an antibody, a cytotoxic moiety such as a payload, and a linker. To overcome existing problems, several novel strategies have been proposed and implemented in the design and development of new ADCs that target each of the components of the ADC. For example, by identifying and validating appropriate antigenic targets for the antibody component, antigens with high expression levels in tumors and little or no expression in normal tissues, antigens present on the cell surface accessible to circulating ADCs, and antigens that enable internalization of the ADC into cells after binding, Also, by alternative mechanisms of activity, designing and optimizing linkers that can overcome resistance induced by proteins that enhance the solubility and drug-to-antibody ratio (DAR) of the ADC and transport chemotherapeutic agents extracellularly, enhancing the DAR ratio by including more payloads, and selecting and optimizing antibodies to improve the homogeneity and developability of the antibody. In addition to the technological development of ADCs, new clinical and translational strategies are also being deployed to maximize the therapeutic index, such as changing the dosing schedule by fractionated administration; conducting biodistribution studies; including biomarkers to optimize patient selection, capturing the response signal early, and monitoring the duration and depth of response; and informing combination trials.

[0006] Examples of clinically possible ADCs include ADCs such as 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 the co-administration of rituximab, a monoclonal antibody that binds to CD20, and no payload is provided [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 reach the "magic bullet" that combines many or all of the aforementioned desired characteristics of ADCs.

[0007] On the one hand, in the past few decades, nucleic acid-based therapeutic agents have been under development. Therapeutic nucleic acids can be based on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), and 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 nearly 2,600 clinical trials underway or completed worldwide, but only about 4% have entered phase III trials. Subsequently, clinical trials using ASO are carried out. Similar to ADC, despite a number of technologies being investigated, therapeutic nucleic acids share two major problems during clinical development: delivery to cells and off-target effects. For example, ASOs such as peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), and bridged nucleic acid (BNA) have been studied as attractive strategies for specifically inhibiting target genes, especially genes that are difficult to target with small molecule inhibitors or neutralizing antibodies. Currently, the efficacy of various ASOs is being studied in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in some cancer stages. To apply ASO as a potential therapeutic agent, a safe and effective method for delivering them to the cytoplasm and / or nucleus of target cells and tissues is required. Although the clinical relevance of ASO has been demonstrated, inefficient cellular uptake both in vitro and in vivo limits the efficacy of ASO and poses a barrier to therapeutic drug development. Cellular uptake is less than 2% of the dose, resulting in an ASO concentration at the active site that is too low to obtain an effective and sustained outcome. Therefore, it is necessary to increase the dose, 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 agents are most commonly small molecules, but their effectiveness is hampered by severe off-target side toxicity, as well as their low solubility, rapid clearance, and limited tumor exposure. Scaffold-small molecule drug conjugates, such as polymer-drug conjugates (PDCs), are macromolecular constructs with pharmacological activity that contain one or more molecules of a small molecule drug attached to a carrier scaffold (e.g., polyethylene glycol (PEG)).

[0009] Such conjugate principles have received much attention and have been studied for decades. Most of the small molecule drug conjugates in preclinical or clinical development are for oncology indications. However, in 2014, the only recent drug approved for a non-oncology indication, opioid-induced constipation in patients with chronic pain, was Movantik, a PEG oligomer conjugate of the opioid antagonist naloxone (AstraZeneca). The translation of drug-scaffold conjugates for therapeutic application in human subjects has thus far provided little clinical benefit. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) showed significant anti-cancer activity in both solid tumors and leukemia in mouse models and was under clinical investigation for oncology indications. Despite showing a significant reduction in non-specific toxicity and improved pharmacokinetics in humans, only a slight improvement in the anti-cancer effect was found in patients, and as a result, further development of PK1 was discontinued.

[0010] The cause of the failure of scaffold-small molecule drug conjugates lies, at least in part, in their poor accumulation at the tumor site. For example, in mouse models, PK1 showed 45- to 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 them with self - organizing, emulsifying, and wetting properties, which can be used in the design of new drugs and new drug delivery systems. Drugs encapsulated in liposomal delivery systems can offer several advantages over direct drug administration, such as improved and controlled pharmacokinetics and pharmacodynamics, tissue - targeting properties, reduced toxicity, and enhanced drug activity. Examples of such success are the liposomal encapsulated forms of the small - molecule chemotherapeutic agent doxorubicin (Doxil: pegylated liposomal encapsulated doxorubicin; Myocet: non - pegylated liposomal doxorubicin), which are approved for clinical use.

[0012] Therefore, there is still a need to find a solution that enables drug therapies, such as anti - tumor therapies, applicable for non - systemic use when desired, where the drug has, for example, an acceptable safety profile, low off - target activity, sufficient efficacy, a sufficiently low clearance rate from the patient's body, a sufficiently broad therapeutic window, etc.

[0013] European Patent No. 1623715B1 describes a composition comprising a pharmacologically active agent bound to a target - cell - specific binding molecule in combination with a saponin, wherein the target - cell - specific binding molecule is 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 bound to each other directly or via a linker; - A second conjugate comprising a binding molecule for binding to saponin and / or its derivative and / or a second cell surface molecule, and saponin and / or its derivative, wherein the binding molecule and the saponin or its derivative are covalently bonded to each other directly or via a linker, the saponin or its derivative is a monodesmoside triterpenoid glycoside or a bidesmoside triterpenoid glycoside, and the second cell surface molecule is the same as or different from the first cell surface molecule, saponin and / or its derivative, and / or the second conjugate; and optionally - A pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

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

[0016] One embodiment is a pharmaceutical combination of the present invention in the form of a single pharmaceutical composition comprising the first conjugate and · Saponin; · A saponin derivative; and · A second conjugate at least one, preferably one, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent. A second aspect of the present invention relates to the pharmaceutical combination of the present invention for use as a medicament.

[0017]

[0018] ​The 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 deficiencies, gene deficiencies, diseases associated with gene deficiencies, amyloidosis, diseases associated with enzyme deficiencies, infectious diseases such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, α1-antitrypsin-related liver disease, acute hepatic porphyria, and transthyretin-mediated amyloidosis.

[0019] The fourth aspect of the present invention is an in vitro or ex vivo method of transferring the first conjugate of the present invention from outside the cell into the cell, preferably into the cytosol of the cell, comprising: a) providing a cell that expresses the first cell surface molecule according to the present invention on its surface and optionally expresses the second cell surface molecule according to the present invention on its surface, wherein the cell is preferably selected from abnormal cells such as hepatocytes, virus-infected cells, autoimmune cells, and tumor cells; b) providing the first conjugate of the present invention to be transferred to the cell provided in step a); c) providing the saponin or saponin derivative of the present invention, or providing the second conjugate of the present invention when the cell provided in step a) expresses the second cell surface molecule according to the present invention on its surface; d) contacting the cell of step a) with the first conjugate of step b) and any one or more of the saponin, saponin derivative, and conjugate of step c) in vitro or ex vivo, thereby establishing transfer of the first conjugate from outside the cell into the cell, preferably into the cytosol of the cell.

[0020] Definitions The term "proteinaceous" has its ordinary scientific meaning and herein refers to a molecule that is protein-like, meaning that the molecule has to some extent the physicochemical properties characteristic of proteins, has the properties of proteins, is related to proteins, contains proteins, is associated with proteins, is similar to proteins, or is a protein. The term "proteinaceous", when used, for example, in "proteinaceous molecule", refers to the presence of at least a part of a molecule that is similar to or is a protein, and "protein" should be understood to include a chain of amino acid residues that is at least two residues in length, and thus includes peptides, polypeptides and proteins, as well as aggregates of proteins or protein domains. In a proteinaceous molecule, at least two amino acid residues are linked, for example, by an amide bond such as 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, a proteinaceous molecule is a molecule that contains at least two amino acid residues, preferably from 2 to about 2,000 amino acid residues. In one embodiment, a proteinaceous molecule is a molecule that contains from 2 to 20 (typical of peptides) amino acids. In one embodiment, a proteinaceous molecule is a molecule that contains from 21 to 1,000 amino acids (typical of polypeptides, proteins, protein domains, such as ligands for receptors such as antibodies, Fab, scFv, single domain antibodies, EGF, etc.). Preferably, the amino acid residues are linked (typically) by peptide bonds. According to the present invention, the amino acid residues are (modified) (non) natural amino acid residues or include them.

[0021] The term "effector molecule" or "effector moiety" has its ordinary scientific meaning, for example when referring to an effector molecule as part of a covalent conjugate. In this specification, for example, it refers to a molecule that selectively binds to any one or more of a target molecule such as a protein, peptide, carbohydrate, saccharide such as glycan, (phospho)lipid, nucleic acid such as DNA, RNA, enzyme, and can regulate the biological activity of such one or more target molecules. Effector molecules are, for example, small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, xeno nucleic acids or siRNA, enzymes, peptides, proteins, or any one or more selected from any combination thereof. Thus, for example, an effector molecule or effector moiety is a molecule or moiety selected from any one or more of small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, xeno nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof, and can selectively bind to any one or more of a target molecule such as a protein, peptide, carbohydrate, saccharide such as glycan, (phospho)lipid, nucleic acid such as DNA, RNA, enzyme, and when bound to the target molecule, regulates the biological activity of such one or more target molecules. Typically, effector molecules can exert a biological effect within a cell, such as within the cytosol of a mammalian cell such as a human cell. Thus, typical effector molecules are drug molecules, plasmid DNA, toxins such as those contained in antibody-drug conjugates (ADCs), siRNA, BNA, oligonucleotides such as nucleic acids contained in antibody-oligonucleotide conjugates (AOCs). For example, an effector molecule 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 or a cell surface molecule-binding molecule such as an antibody such as sdAb when the effector molecule is part of a conjugate.

[0022] The term "modified saponin" has its ordinary scientific meaning and, as used herein, refers to a saponin, i.e., a saponin derivative, having one or more chemical modifications at a position where any of an aldehyde group, a carboxyl group, an acetate group, and / or an acetyl group was present in an un-derivatized saponin prior to being subjected to chemical modification to provide a modified saponin. For example, a modified saponin is provided by chemically modifying any one or more of an aldehyde group, a carboxyl group, an acetate group, and / or an acetyl group in the base saponin of the modified saponin. That is, a saponin is provided and any of an aldehyde group, a carboxyl group, an acetate group, and / or an acetyl group is chemically modified to provide a modified saponin. For example, the saponin to be modified to provide a modified saponin is a naturally occurring saponin. Typically, a modified saponin is a synthetic saponin and typically, a modified saponin is a modification of a natural saponin and thus is derived from a natural saponin, but a modified saponin can also be derived from a synthetic saponin that may or may not have a natural counterpart. Typically, a modified saponin does not have a natural counterpart. That is, a modified saponin is not, for example, naturally produced by a plant or a tree.

[0023] The term "aglycone core structure" has its ordinary scientific meaning and, as used herein, refers to the aglycone core or simply the aglycone of a saponin to which one or two carbohydrate antennas or sugar chains (glycans) are not attached. For example, quillaic acid is the aglycone core structure of SO1861, QS-7, and QS21. Typically, the glycan of a saponin is a monosaccharide or an oligosaccharide such as a linear or branched glycan.

[0024] The term "sugar chain" has its ordinary scientific meaning and, as used herein, refers to any chain containing a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide), or multiple sugar moieties (oligosaccharide, polysaccharide). A sugar chain can consist solely of sugar moieties or can also contain additional moieties such as, for example, 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, as used herein, refers to the chemical modification of a first chemical group or a first chemical moiety such that a second chemical group or a second chemical moiety is provided. Examples include the chemical modification of a carbonyl group to a -(H)C-OH group, the chemical modification of an acetate group to a hydroxyl group, and the provision of a saponin in which an N-ε-maleimidocaproic acid hydrazide (EMCH) moiety is attached to its aldehyde group by a chemical reaction.

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

[0027] The term "chemically modified carboxyl group" has its ordinary scientific meaning and, as used herein, refers to a carboxyl group of a saponin such as a carboxyl group of a glucuronic acid moiety that results in substitution of the original aldehyde group with a new chemical group, and to a chemical reaction product obtained by a chemical reaction involving additional molecules. 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-triazol[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 the name of a sugar chain has its ordinary scientific meaning and, as used herein, refers to a sugar chain that contains an apiose (Api) moiety or a xylose (Xyl) moiety.

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

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

[0031] The term "oligonucleotide" has its ordinary scientific meaning and, as used herein, particularly refers to any natural or synthetic string of nucleic acids presented as single-stranded or double-stranded molecules, such as DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, e.g., BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNAs), antisense DNA, antisense RNA, etc.

[0032] The term "antibody-drug conjugate" or "ADC" has its ordinary scientific meaning and, as used herein, refers to any conjugate of an antibody, such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single-domain antibody, V HH , camelid V H and the like, with any molecule capable of exerting a therapeutic effect when contacted with cells of a subject such as a human patient, e.g., an active pharmaceutical ingredient, a toxin, an oligonucleotide, an enzyme, a small molecule drug compound, etc.

[0033] The term "antibody-oligonucleotide conjugate" or "AOC" has its ordinary scientific meaning and, as used herein, refers to any conjugate of an antibody, such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single-domain antibody, V HH , camelid V HAny conjugate of an antibody, such as those described above, with an oligonucleotide molecule capable of exerting a therapeutic effect when contacted with cells of a subject, such as a human patient, for example, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, such as BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNAs), antisense DNA, antisense RNA, etc., for example, BNA, antisense oligonucleotides (ASO), small or small interfering RNAs (siRNA; silencing RNAs), antisense DNA, antisense RNA, etc., selected from any natural or synthetic string of nucleic acids including such.

[0034] The term "bridged nucleic acid" or abbreviated "BNA" or "locked nucleic acid" or abbreviated "LNA" has its ordinary scientific meaning and refers herein to a modified RNA nucleotide. BNA is also sometimes referred to as a "constrained RNA molecule" or "access-restricted RNA molecule". BNA monomers can contain a 5-, 6- or 7-membered bridged structure with a "fixed" C3'-endo sugar packing. The bridge is synthetically incorporated at the 2',4'-positions of ribose, giving a 2',4'-BNA monomer. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNA is a structurally rigid oligonucleotide with high binding affinity and stability.

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

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

[0037] The terms first, second, third, etc. in this specification and the claims are, for example, for distinguishing similar elements, compositions, components in a composition, or individual steps of a method, and are not necessarily used to describe a sequential or chronological order. The terms are interchangeable under appropriate circumstances, and embodiments of the present invention can be operated in other orders than those described or illustrated herein, unless otherwise specified.

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

[0039] Furthermore, various embodiments are referred to as "preferred" or "e.g." or "for example" or "in particular", etc., but do not limit the scope of the present invention and should be construed as exemplary ways in which the present invention can be implemented.

[0040] The term "comprising" as used in the claims is not to be construed as being limited to the elements, method steps, or components listed thereafter, and does not exclude other elements, other method steps, or other components in a particular composition. It should be construed as identifying the presence of the recited features, integers, (method) steps, or components, but not precluding the presence or addition of one or more other features, integers, 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 only of steps A and B, but rather, with respect to the present invention, the recited steps of the method are only A and B, and further, the claims should be construed to 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 only of components A and B, but rather, with respect to the present invention, the recited components of the composition are only A and B, and further, the claims should be construed to include equivalents of those components.

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

[0042] The term "Saponinum album" has its ordinary meaning and herein refers to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins derived from Gypsophila paniculata and Gypsophila arostii, and containing SA1657 and mainly SA1641.

[0043] The term "Quillaja saponin" has its ordinary meaning and, as used herein, refers to the saponin fraction of Quillaja saponaria and thus to all other sources of QS saponins, and contains mainly 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, as used herein, refers 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, as used herein, refers 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 derived from Quillaja saponaria, containing various amounts of over 50 different saponins, many of which incorporate the triterpene-trisaccharide moiety Gal-(1→2)-[Xyl-(1→3)]-GlcAat at the C-3β-OH group found in QS-7, QS-17, QS18, and QS-21. The saponins found in Quil-A are listed in Table 2 of van Setten (1995) [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon F.A. Kersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja Saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL.9, 660-666 (1995)]. Quil-A, and quillaja saponins also, are fractions of saponins derived from Quillaja saponaria, both containing a wide variety of different saponins with mostly overlapping content. These two fractions have different specific compositions since they 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 described structure is the api variant QS1862 of QS-7. Since the molecular weight is the formal mass including a proton in glucuronic acid, the molecular weight is 1862 daltons. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in the negative ion mode, the measured mass is 1861 daltons.

Brief Description of the Drawings

[0049]

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Mode for Carrying Out the Invention

[0050] A first object of the present invention is to provide an improved ADC and AOC with an increased therapeutic range, and to provide an improved therapeutic combination or an improved pharmaceutical composition for delivering an effective amount or an effective dosage 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 to the cytosol of said target cell is considered. A second object of the present invention is to provide an improved method of treating a (human) patient suffering from a disease to be treated, for example, with respect to target tumor cells, by 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 on target abnormal cells such as tumor cells), that is, to improve the therapeutic range of an ADC or AOC comprising an effector molecule delivered to the cytosol of target tumor cells.

[0051] An object of the present invention is to provide a combination of an effector molecule activity enhancing molecule and an improved ADC or AOC for use in treatments such as anti-cancer therapy. An object of the present invention is also to provide a therapeutic composition comprising such an improved ADC or AOC and comprising an effector molecule activity enhancing molecule, or a therapeutic combination of, for example, two therapeutic compositions. Such an effector molecule activity enhancing molecule can improve the biological effect of the effector molecule when the effector molecule is delivered to, for example, preferably endosomes, endolysosomes and / or lysosomes in a target cell and, for example, subsequently, preferably delivered to the cytosol of said cell. This target cell comprises the molecular target of the effector molecule. When the provided effector molecule activity enhancing molecule is administered to a (human) patient in need thereof and the patient has target cells, the patient experiences a higher therapeutic effect, or at least a sufficient effect, with a preferably lower dosage of the effector molecule than the dosage currently required to reach an effective dosage of the effector molecule. Thus, the therapeutic range of an effector molecule, for example, an effector molecule that is part of a conjugate such as an ADC or AOC, is effectively broadened.

[0052] For a bioactive molecule (e.g., an effector molecule) to act, the molecule must be able to engage its target, for example, in serum, outside the cell surface, or inside a cell or organelle. The active moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell, for example, to mediate its target modulating effect. In many cases, the toxin remains ineffective because (1) the internalization of the targeting moiety is insufficient and it remains bound outside the cell, (2) it is returned to the cell surface after internalization, or (3) it is transported to the endosome where it is degraded. These fundamental problems have been known for decades, and over 500 targeted toxins have been investigated over the past few decades, but the problems remain unsolved, and despite the severe toxicity warning labels, only two antibody-targeted protein toxins have been accepted on the market. Moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP) has been approved by the FDA to date for relapsed or refractory hairy cell leukemia. Other ADCs approved in this way are Elzonris and Ontak.

[0053] To overcome these problems, many strategies have been described, including approaches to redirect the toxin to the endogenous cell membrane transport complexes of the biosynthetic pathway within the endoplasmic reticulum, and techniques to disrupt or weaken the membrane integrity of the endosome, i.e., the compartment of the intracellular endocytosis pathway, and thus promote endosomal escape. This includes the use of lysosomotropic amines, carboxylic acid ionophores, calcium channel antagonists, viruses, bacteria, plants, animals, various cell-penetrating peptides of human and synthetic origin, other organic molecules, and photoinduced techniques. The potency of the targeted toxin typically increases by 100-fold or 1000-fold in cell culture, and in exceptional cases by more than a million-fold, but the need to co-administer an endosomal escape enhancer poses new problems, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell type-dependent variability.

[0054] All strategies involving physical chemistry techniques interact, directly or indirectly, with the membrane to a greater or lesser extent and require enhancer molecules that are essentially small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not themselves target cell-specific and are distributed in a non-targeted manner, except for targeted toxins. This is a 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, and, for example, when delivery from the outside to the inside of target cells is considered, or more specifically when delivery of effector molecules in the cytosol of said target cells is considered, to provide an improved therapeutic combination or an improved pharmaceutical composition for the delivery of an effective amount of effector molecules. A further object of the present invention is to provide an improved method of treating a (human) patient suffering from a disease to be treated, for example against target tumor cells, with a conjugate comprising an effector molecule and a ligand, i.e., an sdAb, i.e., to improve the therapeutic range of an ADC or AOC comprising an sdAb and containing an effector molecule delivered to the cytosol of target tumor cells.

[0056] The object of the present invention is to provide a therapeutic composition or, for example, a therapeutic combination of two therapeutic compositions, comprising an effector molecule enhancing molecule for use in treatments such as anti-cancer therapy, a molecule that can improve the biological effect of an effector molecule when delivered inside a target cell containing the molecular target of the effector molecule, wherein the molecule provided experiences an improved therapeutic effect or a sufficient effect with a lower dose of the effector molecule than the dose currently required to reach the effective dose of the effector molecule when administered to a (human) patient in need thereof and having target cells. Thus, the therapeutic range of effector molecules, for example, effector molecules that are part of a conjugate such as an ADC comprising an sdAb or an AOC comprising an sdAb, is effectively widened.

[0057] The present invention is described with respect to specific embodiments, but the present invention is not limited thereto and is limited only by the claims. Although the present invention has been described with respect to several embodiments, those skilled in the art will recognize alternatives, modifications, variations, and equivalents by reading this specification and examining 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 found that the therapeutic scope of the first conjugate of the present invention, such as an antibody-drug conjugate or an antibody-oligonucleotide conjugate, is such that such an ADC or AOC preferably contains a cell surface molecule-binding sdAb for targeting the ADC or AOC to a cell surface molecule, and that the therapeutic scope increases when a second conjugate containing at least one covalently bound saponin is administered to the same subject, such as a human cancer patient or a tumor-bearing mammal in need thereof. (V HH For conjugates containing, for example, an antibody such as an mAb or sdAb, see the Examples section for a series of in vitro and in vivo tumor cell model examples of the effect of saponin covalently bound to an antibody such as IgG or V HH such as, for example, the examples in Tables A5 - A7 and FIGS. 4 - 7, and for conjugates containing a monoclonal antibody (mAb), see FIGS. 2, 3, 8 - 13, 16, 16, 18 - 23, 26, and 27. Saponin is conjugated to an antibody such as IgG or V HH such as, and an effector molecule such as a protein toxin or an oligonucleotide such as BNA is conjugated to a second antibody such as IgG or sdAb. The inventors have found that a ligand for binding the saponin of the present invention to a cell surface molecule, such as an antibody such as a full-length intact IgG, or V HHBy conjugating with antibodies such as sdAbs, saponin is specifically delivered to the cell surface of target cells that expose cell surface molecules on the cell surface, and then to cells, for example, internally, for example, to cell endosomes, endolysosomes, lysosomes, and ultimately to the cell cytosol (released) saponin. A conjugate (preferably a conjugate containing saponin enters cell endosomes, endolysosomes, lysosomes and the bond is cleaved (for example, when the conjugate binds to a cell surface molecule, i.e., a receptor, it enters by receptor-mediated endocytosis), and saponin is covalently bound by a cleavable bond in the endosomes, endolysosomes, lysosomes of mammalian cells such as human tumor cells so that free saponin is present. A saponin-containing conjugate) was first established and determined. Examples of such cell-targeted saponin conjugates are, for example, saponin-V as outlined in the sections of the following examples HH Conjugates and saponin-Mab conjugates are provided in FIGS. 2-27. Saponin is conjugated to ligands such as EGF, V targeting Her2 HH Or IgG, IgG targeting EGFR, etc.

[0059] At least one of the above objects is achieved by providing a combination of an improved ADC or an improved AOC, including an sdAb that binds to a cell surface molecule, such as a second conjugate containing a covalently bound bidesmoside triterpenoid glycoside (triterpenoid saponin), and an effector molecule enhancing molecule of the present 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 bound to each other directly or via a linker; A second conjugate comprising a binding molecule for binding to a saponin and / or its derivative and / or a second cell surface molecule, and the saponin and / or its derivative, wherein the binding molecule and the saponin or its derivative are covalently bonded to each other directly or via a linker, the saponin or its derivative is a monodesmoside triterpene glycoside or a bidesmoside triterpene glycoside, and the second cell surface molecule is the same as the first cell surface molecule or different from the first cell surface molecule, the saponin and / or its derivative, and / or the second conjugate; and optionally o pharmaceutically acceptable excipients and / or pharmaceutically acceptable diluents.

[0061] 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 sdAb, is efficiently delivered into the cell under the influence of saponin such as the saponin contained in the second conjugate of the present invention. More specifically, 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 sdAb, is efficiently delivered into the cytosol of the cell under the influence of the saponin of the present invention such as the saponin contained in the second conjugate of the present invention. That is, for example, in the absence of saponin as part of the second conjugate, contact with a sub-optimal dose of the first conjugate of tumor cells does not result in the activation of the effector molecule in the cell (the target cells are not efficiently killed by the biological activity of the effector molecule). However, when the target tumor cells are contacted with both the first conjugate containing the effector molecule and the free saponin and / or the second conjugate containing saponin, efficient tumor cell death is established.

[0062] To describe the present invention in more detail, the cell's substance uptake process and the terms used in the present invention will be described first. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. The vesicle budding can be characterized by (1) the uptake of receptor-dependent ligands mediated by the cytosolic protein clathrin, (2) the uptake of lipid rafts mediated by the cholesterol-binding protein caveolin, (3) the uptake of non-specific fluids (pinocytosis), or (4) the uptake of non-specific particles (phagocytosis). All types of endocytosis lead to the following cellular processes of vesicle transport and material sorting, called the endocytic pathway. The endocytic pathway is complex and not fully understood. Initially, it was thought that organelles were newly formed and matured into the next organelle along the endocytic pathway. Today, a hypothesis has been proposed that the endocytic pathway includes stable compartments connected by vesicle transport. The compartments are complex and multifunctional membrane organelles specialized for a specific set of functions essential to the cell. Vesicles are considered to be transient organelles with a simple composition and are defined as membrane-enclosed containers newly formed by budding from existing compartments. In contrast to compartments, vesicles can undergo maturation, which is a series of physiologically irreversible biochemical changes. Early endosomes and late endosomes 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 from the plasma membrane, most prominently from clathrin-coated pits, and first fuse with early endosomes, which are the major sorting compartments at approximately pH 6.5. Most of the internalized cargo and membrane are recycled to the plasma membrane via recycling vesicles (the recycling pathway). Components to be degraded are transported by multivesicular bodies to acidic late endosomes (pH less than 6). Lysosomes are vesicles that store mature lysosomal enzymes and can deliver them to the late endosomal compartment as needed.The resulting organelles are called hybrid organelles or endolysosomes. Lysosomes bud from the hybrid cell organelles in a process called lysosome reformation. Late endosomes, lysosomes, and hybrid organelles are highly dynamic organelles, and it is often difficult to distinguish between them. The degradation of endocytosed molecules occurs inside the endolysosomes. Endosomal escape is the active or passive release of substances from the lumen of any type of compartment or vesicle in the endocytosis pathway, preferably clathrin-mediated endocytosis, or 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 substances may move 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, particularly by promoting endosomal escape. The glycoside molecules interact with the membranes of compartments and vesicles in the endocytosis pathway and the recycling pathway to make the effector molecules more leaky, resulting in enhanced endosomal escape.

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

[0064] One of the main drawbacks of target toxin enhancement by free glycosides, such as saponins of the present invention, which are not part of the cell surface molecule target conjugate up to the present invention, is that the target toxin is internalized by receptor-mediated endocytosis (after the target toxin binds to the receptor, the target toxin conjugate / receptor complex is internalized), while the glycoside diffuses passively through the plasma membrane and probably reaches the endosomal membrane by interaction with cholesterol. In principle, glycosides such as saponins in the second conjugate of the present invention, when in the free unconjugated form, can enter any cell and can also enter non-target cells (off-target cells), resulting in low utilization efficiency of the enhancer in target cells for effectively releasing the target toxin and the possibility of side effects in non-target cells. One of the main problems is that the entry of the targeted toxin and the glycoside proceeds in different kinetics, and these kinetics vary between cell (lines) and tissues, and as a result, the correct time difference for applying the two substances (e.g., an ADC or AOC containing an sdAb for binding to a cell surface molecule with free saponin) can vary greatly between tumors (cell (lines)). Furthermore, in organisms, the release, absorption, distribution, metabolism and excretion of these substances also differ. Additionally, non-specific uptake of the glycoside by non-target cells can induce undesirable effects in these cells. This can be, for example, cytosolic delivery of a compound that was supposed to be delivered to the lysosome, disruption of antigen presentation, etc. Non-target administration of the glycoside and the target drug can also be a problem in drug development and can prevent or at least delay manufacturing approval by relevant authorities (e.g., FDA or EMA). A target toxin or a target drug in the context of the present invention means a toxin or a drug that specifically targets a membrane-bound molecule (cell surface molecule) of a target cell, for example, a toxin or a drug conjugated to an antibody such as an sdAb that specifically recognizes a structure (binding site, epitope) on the cell membrane of the target cell.

[0065] Therefore, in order for the enhancer to be available at an effective concentration within the acidic compartment of the endocytosis pathway of the target cell and to exhibit a synergistic effect with the toxin, it is highly useful to direct the glycoside (the saponin of the present invention) to the target cell by the same pathway as the effector molecule, for example, by a target ligand. Accordingly, the present invention provides a novel method for redirecting both an effector molecule and an endosome escape enhancer (i.e., the saponin of the present invention) to the acidic compartment of the endocytosis pathway of a target cell by a targeting ligand (binding molecule). The targeting ligand containing saponin in the second conjugate of the present invention may be the same as or different from the targeting ligand contained in the first conjugate containing the effector molecule of the present invention, i.e., the sdAb. 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 a tumor cell. For example, the second conjugate of the present invention containing a saponin moiety can bind to EGFR or CD71 or HER2, and for example, the first conjugate containing an effector moiety and a cell surface molecule-binding sdAb can bind to EGFR or CD71 or HER2, and the first and second conjugates of the present invention bind to the same cell receptor or different cell receptors.

[0066] Considering the effect of the effector molecule inside 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 efficiently delivered into the cell under the influence of the saponin contained in the second conjugate of the present invention. Surprisingly, despite the relatively small size of sdAb such as V HH the binding of the first conjugate of the present invention containing an effector molecule and such an sdAb to the target cell surface receptor still occurs when the effector molecule and sdAb are included in the first conjugate, and the sdAb is preferably V HH is. Surprisingly, V HHDespite the relatively small size of sdAbs such as, the binding of the second conjugate of the invention, which contains saponin and sdAb, to the target cell surface receptor still occurs when saponin and sdAb are included in the second conjugate, and the sdAb is V HH is preferred. The binding of effector molecules to sdAbs such as V HH forming the first conjugate of the invention does not cause, for example, steric hindrance considering the ability of V HH to bind to cell surface molecules. The binding of saponin to sdAbs such as V HH forming the second conjugate of the invention does not cause, for example, steric hindrance considering the ability of V HH to bind to cell surface molecules. That is, in the absence of the second conjugate of the invention containing saponin covalently bound to a cell surface molecule binding ligand such as an antibody such as sdAb, contact of an ADC containing sdAb or an AOC containing sdAb (i.e., the first conjugate of the invention) with, for example, tumor cells at a sub-optimal dose of the sdAb 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 target tumor cells are contacted with the first conjugate of the invention containing an effector molecule and sdAb, such as an ADC or AOC, and free saponin and / or the second conjugate of the invention containing saponin, efficient tumor cell killing is achieved.

[0067] By targeting a single cell surface molecule with the second conjugate and ADC or AOC of the present invention, i.e., the first conjugate of the present invention, or by targeting a second cell surface molecule with the second conjugate of the present invention and targeting the ADC or AOC, i.e., the first conjugate of the present invention, to the first cell surface molecule (wherein the first cell surface molecule and the second cell surface molecule are different and present on the same target cell), the delivery of saponin conjugated to an antibody targeting a cell surface molecule such as sdAb in the second conjugate of the present invention into the cytosol of target cells that expose the cell surface molecule on the cell surface, and the delivery of an ADC or AOC containing an effector molecule and an sdAb that binds to the first cell surface molecule are, for example, by contacting the cells only with the ADC and not contacting the cells simultaneously with the second conjugate containing the saponin of the present invention. Thus, it is improved and the specificity is increased compared to the case where the saponin (the second conjugate of the present invention) targeting the cells is not present. The abnormal cells selected for targeting by the sdAb targeting the cell surface molecule of the first conjugate and for targeting by the cell surface molecule-binding molecule such as an antibody such as sdAb in the second conjugate preferably have a high level (i.e., for example, the expression of the target cell surface molecule in target cells such as tumor cells or autoreactive cells is relatively higher than the expression in non-target cells such as healthy cells) of the epitope to which the molecule targeting the cell surface molecule can bind on the cell surface molecule and / or expose the epitope on the target cell surface molecule for binding of the sdAb targeting the cell surface molecule of the first conjugate and for binding of the antibody targeting the cell surface molecule such as sdAb of the second conjugate, especially when the (adjacent) healthy cells of the patient are considered. Preferably, the cell surface molecule targeted by the sdAb targeting the cell surface molecule of the first conjugate of the present invention and the cell surface molecule targeted by the molecule targeting the cell surface molecule such as an antibody such as sdAb of the second conjugate of the present invention are expressed relatively highly and / or specifically in the target (disease, tumor) cells compared to healthy cells.One embodiment is that the target cell surface molecule for an sdAb that targets the cell surface molecule of a first conjugate such as a tumor cell receptor is expressed specifically or to a relatively high degree compared to the expression of the cell surface molecule on healthy (adjacent) cell surfaces, and is the first conjugate of the present invention. One embodiment is that the target cell surface molecule for a molecule that targets a cell surface molecule such as an antibody such as an sdAb of a second conjugate such as a tumor cell receptor is expressed specifically or to a relatively high degree compared to the expression of the cell surface molecule on healthy (adjacent) cell surfaces, and is the second conjugate of the present invention. Therefore, the epitope of the target cell surface molecule is ideally unique to the target diseased cells and is present and exposed specifically at least 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 translocates internally). After the second conjugate of the present invention binds to the epitope of 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 translocates internally). Since the first and second conjugates can enter the target cells only by binding interactions with cell surface molecules that are expressed specifically enough or uniquely in the target cells compared to healthy cells that should not be targeted, an ADC or AOC (the first conjugate of the present invention) that targets the same cell surface molecule as the second conjugate of the present invention or targets a different cell surface molecule that exists in the same target cells as the cell surface molecule targeted by the second conjugate of the present invention, the accumulation of a therapeutically active amount of the effector moiety contained in the first conjugate of the present invention and the saponin contained in the second conjugate of the present invention is possible only inside the target cells and occurs when the expression level of the target cell surface molecule exceeds a specific minimum expression threshold.At the same time, the fact that the effector portion of the first conjugate, which binds to the sdAb targeting the cell surface molecule, can exert its intracellular (e.g., cytotoxic or gene silencing) activity only in the presence of the second conjugate of the present invention having the covalently bound saponin also provides a safeguard against the negative and undesirable side effects of the effector portion on healthy cells and healthy tissues that are not meant to be targeted and affected by the effector portion, compared to the case where cells are exposed to the ADC without the presence of the second conjugate of the present invention containing the covalently bound saponin. That is, when the expression of the exposed cell surface molecule to which the first conjugate of the present invention, e.g., an ADC or AOC containing an sdAb, and the second conjugate containing saponin can bind is sufficiently low or absent, ideally, the first conjugate of the present invention containing the effector molecule and the second conjugate containing saponin cannot be introduced into (non-target) healthy cells to an amount that results in endosomal escape of the effector portion of the ADC or AOC under the influence of the saponin contained in the second conjugate of the present invention. The ADC or AOC can be used at a lower dose in the presence of the saponin contained in the second conjugate of the present invention compared to the case where the ADC or AOC is applied in a treatment regimen in the absence of the second conjugate of the present invention and thus in the absence of saponin, so the degree of entry of the ADC or AOC into healthy cells is low, and for example, considering the targeting and killing of target disease cells such as tumor cells and autoimmune cells, the risk of occurrence of undesirable side effects is already lower.

[0068] Including sdAb in the first conjugate has many advantages compared to including an antibody such as IgG (full-length immunoglobulin), or its binding fragment or binding domain. Importantly, since sdAb does not contain the Fc tail present in IgG, the risk of off-target side effects due to the binding of the conjugate to the Fc receptor of cells such as endothelial cells of the host to which the conjugate is administered does not exist when the first conjugate of the present invention is administered to a mammal such as a human subject. Therefore, 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 containing an Fc tail. In addition, since the first conjugate of the present invention cannot be bound by the Fc receptor, the first conjugate has little or no unwanted capture by cell surface receptors different from the first cell surface molecule of the target, and thus is already effective at a lower dose than the dose required to achieve the same effector molecule activity as ADCs and AOCs based on full-length antibodies. Furthermore, for example, compared to Fab, scFv, IgG, the size of sdAb is relatively small, so the tissue permeability of the first conjugate of the present invention is good, which is beneficial for reaching the target cells when the first conjugate is administered to a patient in need of treatment based on the biological activity of effector molecules inside (tumor) cells. All of these advantages of applying sdAb in the first conjugate of the present invention improve the therapeutic window of the effector molecule when included in the first conjugate of the present invention compared to the case of applying a larger antibody such as IgG containing an Fc tail or its fragment in a similar ADC or AOC. When the effector molecule is, for example, a toxin, the ADC is based on IgG, and the dose is the same and contains the same effector molecule, the improvement in target cell killing in the case of target tumor cells is, for example, sub-optimal.According to an aspect of the present invention, a conjugate comprising an sdAb, i.e., as part of the first conjugate of the present invention, enables treating a patient with a lower dose of an effector molecule and reaches the same or improved effector molecule-mediated effect in target cells compared to the higher dose required when an antibody-based ADC or AOC containing the same effector molecule is used. Administration of such a first conjugate of the present invention at a lower dose reduces the risk of side effects in the patient, for example, by 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 in target (tumor) cells but its expression in such target cells is not unique. The low dose of the first conjugate reduces the risk of binding of the first conjugate to low expressors such as such non-tumor healthy cells.

[0069] The inventors have also found that when the first conjugate contacts the target cells in the presence of saponin, or more specifically in the presence of the second conjugate of the present invention containing saponin, the therapeutic range of the first conjugate of the present invention is broadened. When a target cell has a first cell surface molecule, i.e., a target of the sdAb contained in the first conjugate, and a second cell surface molecule (which is essentially true when, according to an embodiment of the present invention, both the first and second cell surface molecules are the same cell surface molecule), i.e., a target of the binding molecule contained in the second conjugate, contacts both the first conjugate and the second conjugate, the effective dose of the effector molecule is lower than when the target cell contacts the first conjugate in the absence of the second conjugate (or in the absence of free saponin). The presence of the second conjugate containing saponin or the presence of free saponin enhances the activity of the effector molecule in the target cell such that the therapeutic range of the first conjugate and the therapeutic range of the effector molecule are broadened. Sufficient 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 a functional derivative thereof, a similar effect is found by the inventors, but the concentration of free saponin (derivative) is 100 to 1000 times higher compared to the effective dose established when the second conjugate containing covalently bound saponin is combined with the first conjugate. Therefore, if a binding molecule is provided to saponin or its derivative (i.e., the second conjugate of the present invention), a better effector molecule activity enhancing effect is brought about when both the first and second conjugates are contacted with a target cell expressing both cell surface molecules on its surface.In the delivery of effector molecules into target cells and also in the delivery from endosomes, endolysosomes or lysosomes of said cells into the cytosol, the targeted saponin is already effective at a lower dose than free saponin, and the effector molecule binds to its target binding partner and exerts its biological activity (for example, cell death when the target cell is a tumor cell and the effector molecule is a toxin, for example).

[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 free saponin (derivative) or a second conjugate comprising free saponin (derivative), and also provide a pharmaceutical composition comprising the first conjugate and free saponin (derivative) or the second conjugate, which improves the therapeutic window compared to current Fc-based ADCs or AOCs comprising full-length antibodies or constructs thereof, reduces the risk of inducing side effects when the effective dose of the effector molecule comprised in the first conjugate is administered to patients in need of an effector molecule-based treatment, and improves the delivery of the first conjugate into target cells, more specifically the delivery of the effector molecule into the cytosol of such target cells. Administration of such a first conjugate of the invention together with free saponin (derivative) or the second conjugate to patients in need of an effector molecule-based treatment is part of the invention, but it is preferred to apply the second conjugate in combination with the first conjugate.

[0071] One embodiment is a pharmaceutical combination of 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 pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent; and - a second pharmaceutical composition comprising saponin and / or a derivative thereof and / or a second conjugate and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0072] One embodiment includes a first conjugate, and · saponin; · saponin derivative; and · a second conjugate including at least one, preferably one, and optionally including a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent, the pharmaceutical combination of the present invention in the form of a single pharmaceutical composition.

[0073] One aspect of the present 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 bonded to each other directly or via a linker; o a second conjugate comprising saponin and / or its derivative and / or a binding molecule for binding to a second cell surface molecule and saponin and / or its derivative, wherein the binding molecule and saponin or its derivative are covalently bonded to each other directly or via a linker, the saponin or its derivative is a monodesmoside triterpenoid glycoside or a bidesmoside triterpenoid glycoside, and the second cell surface molecule is the same as or different from the first cell surface molecule, saponin and / or its derivative, and / or a second conjugate; and optionally o a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0074] One embodiment is that the first conjugate is a V derived from the heavy chain of an antibody, preferably an antibody of immunoglobulin G origin, preferably a human origin H domain; a V derived from the light chain of an antibody, preferably an antibody of immunoglobulin G origin, preferably a human origin L domain; various heavy chain new antigen receptors (V NAR ) domains, such as V domains derived from heavy chain antibodies (HCAb) derived from Camelidae origin or Ig-NAR origin, etc. HHcomprising one or more sdAbs of any one of the domains (preferably, HCAb is derived from Camelidae origin); preferably, the sdAb is derived from a HCAb of Camelidae origin (Camelidae animal V H ), for example, derived from the HCAb of camel, llama, alpaca, dromedary, vicuña, guanaco and guanaco V HH is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0075] In particular, V HH the domain is suitable for application in the first conjugate of the present invention, and in fact, is also suitable for application in the second conjugate of the present invention. Such V HH domains are generally known for their high stability, i.e., resistance to unfolding, for example, present in IgG, the ability to bind to binding partners without the need for the presence of a second V domain such as required for IgG to bind to binding partners by two V domains, the ease of their generation by techniques known in the art (such as immunization techniques of camelids, phage display techniques, etc.), and a higher ability to penetrate tissues than that seen in full-length IgG (this ability is beneficial when the target (tumor) cells are located inside or as part of such (organ) tissues). Furthermore, such V HH domains lack the Fc tail common to immunoglobulin G-type antibodies, and as a result, Fc receptor-mediated side effects cannot occur by administering conjugates containing sdAbs such as V HH domains (such side effects can occur in subjects administered with therapeutic full-length immunoglobulins containing the Fc tail).

[0076] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, which has a single first sdAb in which a first conjugate is covalently bound to at least one effector molecule, or has two or more sdAbs bound to at least one effector molecule, or includes at least two sdAbs having all of at least two sdAbs bound to at least one effector molecule. For example, 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 present invention, in which the sdAbs include at least two sdAbs that are the same sdAb, preferably 2 to 8 sdAbs, more preferably 2 to 4 sdAbs.

[0078] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, which includes 1 to 8 sdAbs that can bind to the same binding site of a first cell surface molecule. At least one effector molecule is bound to a single first sdAb among 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 molecules 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 (and / or a second conjugate of the present invention) comprising a (linear) string of a plurality of sdAbs covalently bonded to each other, the benefit of the ability of the conjugate to bind to target cells with high affinity can be provided, thereby improving the 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 delivery strategy that has been successful in mice and its application in humans. In fact, the inventors have shown that in the absence of the second conjugate according to the invention comprising saponin, even when free saponin and, for example, an ADC are administered separately to mice, no desired anti-tumor activity such as delay of tumor growth, tumor regression, decrease and slowdown of tumor growth is obtained compared to control animals treated with the ADC without the presence of free saponin. This has been demonstrated in a series of in vivo mouse tumor models. See also FIGS. 18-21 for the in vivo tumor models and the examples described in the Examples section below of this specification. Free saponin was administered at various time points relative to the administration of the ADC from various routes of administration (administering free saponin before, during, and after the administration of the ADC). The ADCs tested in the in vivo tumor models were cetuximab-diantin (together with free SO1861), or trastuzumab-saporin (together with free SO1861). Varying the dose of free saponin did not provide effective anti-tumor activity. The ADCs mentioned were administered at a dose that did not confer any beneficial anti-tumor effect on the animals bearing tumors. Surprisingly, the inventors have demonstrated that beneficial anti-tumor activity can be achieved by treating cells or animals with an ADC or an AOC, i.e., the first conjugate according to the invention in combination with the second conjugate according to the invention, in various in vitro mammalian cell-based bioassays using human tumor cells and / or in various in vivo animal tumor models.

[0081] The 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 bound). For example, a scaffold that is a trifunctional linker having saponin (e.g., SO1861, QS-21) covalently bound by a cleavable or non-cleavable bond, monoclonal antibodies such as cetuximab, trastuzumab, OKT-9, but preferably V HHA scaffold covalently linked to a molecule targeting a cell surface molecule of a second conjugate such as an sdAb, or a dendron to which four moieties such as four saponin molecules can bind, such as a G4-dendron or a G5-dendron, or a scaffold that is a dendron for binding eight saponins (the dendron contains a chemical group for (covalently) binding to an antibody targeting a cell surface molecule such as the sdAb of the second conjugate). Referring to the sections on further embodiments and examples, some of these scaffolds according to the invention are exemplified, for example, when considering the cytotoxicity exerted by the proteinaceous toxin contained in the first conjugate of the invention, or when considering gene silencing in tumor cells, they exhibit anti-tumor cell activity in vivo and / or in vitro.

[0082] While not wishing to be bound by any theory, considering the failures observed when treating animals having tumors with ADCs and free saponins, it is preferred to synchronize the presence of at least one saponin and an effector moiety, preferably both a toxin or an oligonucleotide, in compartments or vesicles of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. Synchronizing the presence of molecules in late endosomes in vivo to obtain a synergistic effect using ADCs and free saponins was not beneficially obtained according to the attempts of the inventors. In one aspect, the invention relates to combining a first conjugate (e.g., an ADC or AOC) preferably comprising an effector moiety and an sdAb that binds to a (tumor) cell surface molecule, with a second conjugate of the invention comprising a saponin, to solve at least the following problems: While not wishing to be bound by any theory, for example, the only reasonable chemical group within a saponin that can be used for a (covalent), in particular singly cleavable, retainable bond is required for endosomal escape activity. In clinical trials other than the application of saponins in vaccination regimens where the use of immunostimulatory adjuvant substances is implied, the reason saponins have not been used in combination with pharmaceutically active substances is most likely due to known limitations, for example, the significant endosomal escape enhancer effect of the saponins of the invention and the saponins exemplified herein has been known for over a decade. For example, providing a second conjugate of the invention having a covalently bound saponin, for example, in relation to a scaffold carrying several saponins, solves these difficulties at least in part. For example, as is apparent in the endosomes, endolysosomes and lysosomes of target cells, providing a second conjugate of the invention having a covalently bound saponin by a cleavable bond that is cleaved under the influence of a slightly acidic pH (pH < 6.5) solves these problems at least in part because, when the second conjugate is endocytosed and delivered inside the cell, the free saponin cleaved from the second conjugate is present inside said endosome, endolysosome or lysosome.Surprisingly, the saponins previously applied for their immune-enhancing activities in the context of vaccination containing saponin as an adjuvant component are now also suitable for (covalent) conjugation to antibodies targeting cell surface molecules such as sdAbs contained in the ADC or AOC of the present invention, i.e., the second conjugate of the present invention, when used in combination with the first conjugate of the present invention, for anti-tumor activity in vitro and in vivo.

[0083] In one embodiment, the sdAb is a single sdAb or at least two, preferably two sdAbs, and the sdAb can bind to a first cell surface molecule such as HIVgp41, 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, 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, CD25, ephrin A4, 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), most preferably: a receptor selected from HER2, c-Met, VEGFR2, CXCR7, CD71 and EGFR1, which is a pharmaceutical combination or pharmaceutical composition of the present invention.

[0084] It is part of the present invention that the sdAb contained in the first conjugate of the present invention has binding specificity for cell surface molecules that are specifically expressed on target cells. As used herein, "specifically expressed" should be understood to mean the exclusive expression of cell surface molecules only on target cells (where, for example, healthy cells that do not bind to the first conjugate are not targets), or, as used herein, no binding to the conjugate or at least a very low level of binding, i.e., compared to the low expression of cell surface molecules in healthy cells, upregulated or relatively high expression of target cell surface molecules in target cells should be understood. These listed cell receptors are cell surface molecules that are sufficiently specific for the cells targeted by the first conjugate and are thus preferred candidates for binding by the first conjugate. When comparing the expression of cell surface molecules in target cells with their expression in other cells that are not meant to be targeted by the first conjugate of the present invention, the higher the specificity of a particular cell surface molecule, the better the therapeutic window is understood to be when considering the activity of effector molecules inside the cell. For example, suitable targets for the first conjugate to target are, among other tumor cell-specific receptors, HER2, EGFR, such as EGFR1, and CD71.

[0085] In one embodiment, the sdAb contained in the first conjugate is a single sdAb or at least two, preferably two, sdAbs, and the sdAb is selected from 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-angiogenic 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-HIVgp41, and the sdAb is preferably V HH and more preferably V of camelidae H It is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0086] In one embodiment, the first conjugate contains an sdAb that can bind to HER2, CD71, HIVgp41 and / or EGFR, and the sdAb is preferably V HH and more preferably V of camelidae H It is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0087] One embodiment is that the first 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 HIVgp41 produced by clone anti-HIVgp41 Q8-C tag; or an sdAb encoded by any one of the cDNAs of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; or any one of the sdAbs having the amino acid sequences 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 one or more of the covalently bound sdAbs for binding to albumin, for example, any one of the sdAbs having the amino acid sequences of SEQ ID NOs: 33, 34 and 35. Preferably, the sdAb is V HH and more preferably camelid V H This is the pharmaceutical combination or pharmaceutical composition of the present invention. V suitable for incorporation into the first conjugate of the present invention HH is found, for example, in the single domain antibody database (Wilton, E.E. et al. (2018)), US Patent Application Publication No. 2016 / 0251440 (anti-CD123, anti-CEACAM), US Patent No. 9,683,045 (anti-c-Met), US Patent Application Publication No. 2009 / 0252681 (anti-EGFR, anti-IGF-1R), US Patent No. 9,969,805 (anti-HER2), US Patent Application Publication No. 20190023796A1 (anti-HER3), and Kijanka et al. (2013) (for anti-HER2) and Mercier et al. (2019) (for anti-HER2). A series of V 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 The amino acid sequences and / or cDNA sequences of are also provided below in the present specification as SEQ ID NOs: 1 to 32 and 36 to 72, taking into account their ability to bind to tumor cell-specific receptors. In particular, V that can bind to the binding site of any of the tumor cell-specific receptors HER2, VEGFR, and CD71 HH is suitable for incorporation into the first conjugate of the present invention. The inventors have shown that an ADC comprising V targeting any one of such receptors HH is effective for delivering an effector molecule conjugated to an sdAb. See, for example, the Examples section and FIGS. 4 to 7.

[0088] One embodiment is a pharmaceutical combination or composition of the present invention, wherein the effector molecule comprises or consists of at least one of small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, xeno nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof.

[0089] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein the effector molecule is selected from any one or more of a vector, a gene, a cell suicide-inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), anti-microRNA (anti-miRNA), a DNA aptamer, an RNA aptamer, mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), www.biosyn.com / literaturevault / superior-silencing-by-2-4-bnanc-based-short-antisense-oligonucleotides.aspx, 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA), and a threose nucleic acid (TNA), or a derivative thereof, more preferably BNA, for example, BNA for silencing HSP27 protein expression or BNA for silencing apolipoprotein B expression.

[0090] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein the effector molecule is an oligonucleotide selected from any one or more of short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin type 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 NC ), BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-AON).

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

[0092] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein the effector molecule is an oligonucleotide capable of silencing a gene (when present in a cell containing such a gene), and the gene is 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), glycolic acid oxidase (GO), complement component C5 (CC5), hepatitis B virus X gene (HBV), HBV S gene, alpha-1 antitrypsin (AAT), and lactate dehydrogenase (LDH), and / or is capable of targeting an abnormal miRNA (when present in a cell containing such an miRNA).

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

[0094] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present 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 when an sdAb that binds to any of HER2, VEGFR, CD71 (this sdAb is combined with a toxin, such as a protein toxin, such as dianthin or saporin, in the first conjugate) is selected, very effective tumor cell killing is achieved. Examples demonstrating the high efficacy of such first conjugates of the present invention are provided in the Examples section and shown in FIGS. 4-7.

[0095] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein 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 a viral toxin such as apoptin; bacterial toxins such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins such as α-sarcin; plant toxins such as ribosome-inactivating proteins, and the A chain of type 2 ribosome-inactivating proteins, such as dianthin (e.g., dianthin-30 or dianthin-32), saporin (e.g., saporin-S3 or saporin-S6), bouganin or a deimmunized derivative debouganin of bouganin, Shiga-like toxin A, yamabogobu antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin A chain, volkensin, volkensin A chain, viscumine, viscumine A chain; or an animal or human toxin such as frog RNase, or granzyme B, or human angiogenin, or any one or more of any toxic fragments or toxic derivatives thereof; preferably, the protein toxin is dianthin and / or saporin.

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

[0097] One embodiment is a pharmaceutical combination or composition of the invention, wherein the effector molecule is at least one of a toxin targeting ribosome, a toxin targeting elongation factor, a toxin targeting tubulin, a toxin targeting DNA, and a toxin targeting RNA, more preferably, emtansine, pastotoxin, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolobenzodiazepine, AZ13599185, cryptophycin, lysocine, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivative, amanitin, α-amanitin, spliceostatin, tyranstatin, ozogamicin, tesirine, ambrastatin 269, and sorabtansine, or any one or more of derivatives thereof, or consists of these.

[0098] One embodiment includes or consists of a pharmaceutical combination or composition of the invention, comprising an ADC comprising at least one sdAb and a toxin, wherein the first conjugate is an antibody-drug conjugate (ADC), for example, 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 H domain may require some improvement with respect to domain stability (the "camelization" of the human V H domain) known in the art.

[0099] Effector moieties useful as part of an ADC or AOC (i.e., the first conjugate of the present invention) in the present invention preferably rely on late endosomal escape to exert their effects. For example, some effector molecules such as Pseudomonas exotoxin are rerouted to other organelles before the "late endosome" and thus generally do not benefit from incorporation into an ADC for combination with a second conjugate according to the present invention. However, such toxins can be made suitable for use in the present invention, for example, by deleting the signal peptide involved in rerouting. In particular, toxins that are highly toxic and require only one molecule to escape the endosome to kill cells can be modified to have reduced 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). The first conjugate of the present invention, included in a pharmaceutical composition of the present invention or included in a pharmaceutical combination, is a functionalized scaffold covalently conjugated and targets a scaffold containing one or more effector moieties covalently bound for targeting one or more effector moieties bound in a target cell, e.g., a tumor cell or an autoimmune cell, more preferably includes a scaffold such as an oligomeric or polymeric molecule or a trifunctional linker. Further, to reduce off-target toxicity, cell membrane-impermeable low molecular weight toxins are preferred effector molecules over cell membrane-permeable toxins.

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

[0101] The inventors have shown that when a monoclonal antibody or sdAb targeting tumor cells (i.e., the first conjugate of the present invention) comprising a covalently linked antisense BNA such as BNA(HSP27) and, the same or different, a covalently linked saponin is contacted with tumor cells in combination with a monoclonal antibody or sdAb targeting tumor cells (i.e., the second conjugate of the present invention), both the BNA (i.e., the first conjugate) linked (by a cleavable bond) to the cell-targeting ligand and the saponin linked (by a cleavable bond) to the antibody (e.g., the sdAb of the second conjugate of the present invention) are able to silence HSP27 in in vivo tumors as compared to controls and an AOC having BNA in the absence of the second conjugate of the present invention containing saponin (SO1861). Thus, the ADC (the first conjugate of the present invention) is administered together with the second conjugate containing the saponin of the present invention, or an antibody-oligonucleotide conjugate such as the antibody-BNA conjugate of the present invention, wherein the antibody is VV HHBy administering it together with a second conjugate comprising the saponin of the present invention, the ADC or AOC is given antitumor cell activity that is not seen with only the ADC or AOC at the same dose in the absence of the second conjugate of the present invention. Antisense BNA (HSP27) was a BNA having the oligonucleotide 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]. Notably, as far as the inventors know, BNA is designed for application as a free nucleic acid. The inventors HH demonstrated for the first time in a way that the gene silencing activity is retained in vitro and, more importantly, in tumor cells of animals with tumors in vivo that the antisense BNA can be covalently linked via a (non)-cleavable linker to, for example, a ligand or an antibody such as an mAb or an sdAb, such as V

[0102] One embodiment is that the saponin is 2α-hydroxyoleanolic acid; 16α-hydroxyoleanolic acid; hederagenin (23-hydroxyoleanolic acid); 16α,23-dihydroxyoleanolic acid; gypsogenin; kierraic acid; protoescigenin 21(2-methylbut-2-enoate)-22-acetate; 23 - Oxo - valligenol C - 21,22 - bis(2 - methylbut - 2 - enoate); 23 - Oxo - valligenol C - 21(2 - methylbut - 2 - enoate) - 16,22 - diacetate; Digitogenin; 3,16,28 - Trihydroxyolean - 12 - ene; and Gypogenin acid comprising an aglycone core structure selected from, or from which a saponin derivative is derived, preferably, the saponin comprises an aglycone core structure selected from gypogenic acid and gypogenin or a saponin derivative is derived therefrom, more preferably the saponin comprises an aglycone core structure of gypogenic acid or a saponin derivative is derived therefrom, a pharmaceutical combination or pharmaceutical composition of the present invention.

[0103] While not wishing to be bound by any theory, when such a saponin co - localizes with these effector molecules in the cell, within the endosome of said cell, as part of the second conjugate of the present invention, or (for example, when the second conjugate is delivered inside the endosome or lysosome of the target cell and is separated from the second conjugate) is in a free form inside the endosome, and the cell is exposed to the first conjugate having an sdAb targeting the effector molecule and the cell, the presence of an aldehyde group (or its derivative) in the aglycone core structure of the saponin (also referred to herein as "aglycone") 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. Thus, a second conjugate of the present invention comprising a saponin having an aglycone having an aldehyde group is preferred. In gypogenic acid and gypogenin, the aldehyde group is at the C 23 atom.

[0104] One embodiment is that the saponin or saponin derivative preferably contains a first sugar chain bonded to the C3 atom of the aglycone core structure of the saponin or saponin derivative, and / or the saponin or saponin derivative optionally contains a second sugar chain bonded to the C 28 atom, and preferably at least one saponin contains the first and second sugar chains, which is the pharmaceutical combination or pharmaceutical composition of the present invention. Therefore, when the saponin contained in the second conjugate of the present invention has two glycans (sugar chains), the first sugar chain is bonded to the C3 position of the aglycone core structure, and the second sugar chain is bonded to the C 28 atom. 28 position.

[0105] One embodiment is · The saponin or saponin derivative 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-, and a first sugar chain selected from these derivatives, and / or · a saponin or 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- (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- (where 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-(where 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-(where 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-(where 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)) The pharmaceutical combination or pharmaceutical composition of the present invention comprising a second sugar chain selected from Glc-(1→3)-[Glc-(1→6)]-Gal-, and derivatives thereof.

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

[0107] One embodiment is that the saponin or saponin derivative in the second conjugate contains a first sugar chain and the second sugar chain described in claim 24 or 25, the first sugar chain contains two or more sugar moieties, the second sugar chain contains two or more sugar moieties, the aglycone core structure is kiraic acid or gypogenin or a derivative thereof, and satisfies one, two or three of the following, preferably one or two: i. The aldehyde group in the aglycone core structure is derivatized. ii. The carboxyl group of the glucuronic acid moiety in the first sugar chain is derivatized, and iii. At least one acetoxy (Me(CO)O-) group in the second sugar chain is derivatized.

[0108] One embodiment is the pharmaceutical combination or pharmaceutical composition of the present invention, wherein the saponin or saponin derivative in the second conjugate contains the following: 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 -An aglycone core structure containing an aldehyde group derivatized by 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); ii. A first sugar chain containing a carboxyl group, preferably the carboxyl group of a glucuronic acid moiety, 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 sugar chain containing an acetoxy group (Me(CO)O-) derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Two or three derivatives of any combination of i., ii. and / or iii., preferably any combination of two derivatives of i., ii. and / or iii.

[0109] One embodiment is that the saponin or saponin derivative is selected from quillaja bark saponin, dipsacoside B, psychotria saponin A, psychotria saponin D, macranthoidin A, esculentoside A, phytolaccagenin, escin, AS6.2, NP-005236, AMA-1, AMR, α-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-017705, NP-017773, 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-apio, QS-21A-xylo, QS-21B-apio, QS-21B-xylo, β-escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primulic 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 QS-21 derivative, SO1861 or SO1861 derivative, SA1641 or SA1641 derivative, and GE1741 or GE1741 derivative, more preferably QS-21 derivative or SO1861 derivative, most preferably SO1861 derivative, wherein preferably such saponin derivative is the saponin derivative according to the present invention, which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0110] One embodiment is that the saponin or saponin derivative is selected from 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, Kiryasaponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypenoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, or derivatives thereof, or stereoisomers thereof, and / or any combination thereof, preferably the saponin derivative is an SO1861 derivative, and / or a GE1741 derivative, and / or a SA1641 derivative, and / or a QS-21 derivative, more preferably the saponin derivative is an SO1861 derivative or a QS-21 derivative, and most preferably the saponin derivative is the SO1861 derivative according to the present invention.

[0111] Such triterpenoid glycoside-type saponins are included in the first conjugate and can enhance the endosomal escape of effector molecules present in the endosomes (or lysosomes) of cells when the saponin co-localizes intracellularly with such effector molecules as part of the second conjugate or in free form. The inventors have demonstrated that the endosomal escape enhancing activity of these saponins is about 100 to 1000 times more potent when the saponin is brought into contact with cells as part of the second conjugate of the present invention. Free saponin stimulates the delivery of effector molecules in the cytosol of cells at a concentration 100 to 1000 times higher compared to the concentration of the same saponin included in the second conjugate of the present invention required to achieve a comparable delivery of effector molecules from outside the target cells into the interior of the endosomes and ultimately into the cytosol of said cells when such cells are brought into contact with the effector molecule and saponin as part of the first conjugate. Saponins exhibiting such endosomal escape enhancing activity, as well as saponins having a high structural similarity to saponins whose ability to enhance the cytosolic delivery of effector molecules included in the first conjugate has been demonstrated, are listed in Table A1. When the saponin is part of the second conjugate of the present invention, the targeted delivery of the saponin upon binding of the binding molecule of the second conjugate, e.g., an antibody or sdAb, to the second cell surface binding site on the target cell and after endocytosis of the saponin into the endosomes of said cells is thus about 100 to 1000 times more effective compared to contacting the same cells with free non-targeted saponin (derivatives) not equipped with a binding molecule such as an antibody or sdAb for binding to the second cell surface molecule of the target cell. In certain embodiments, the binding molecule included in the second conjugate is an sdAb. For example, the small size of the sdAb of the second conjugate of the present invention compared to an IgG-type antibody or a fragment thereof, e.g., Fab, scFv, contributes to the efficient uptake by the target cell that exposes the second binding site for binding of the sdAb included in the second conjugate, e.g., uptake by endocytosis.Generally, the sdAb in the second conjugate of the present invention can bind to a cell surface receptor of a target cell that is the same as or different from the cell surface receptor targeted by the sdAb contained in the first conjugate of the present invention, for example, a tumor cell-specific cell surface receptor. Thus, the first and second conjugates of the present invention are particularly suitable for endocytosis into tumor cells expressing, for example, the first and second cell surface receptors, and these first and second cell surface receptors may be the same or different.

[0112] One embodiment is that the second conjugate comprises a binding molecule for binding to a second cell surface molecule, and the binding molecule is a proteinaceous ligand for binding to a second cell surface molecule, such as EGF or a cytokine, or an immunoglobulin capable of binding to a second cell surface molecule or at least one binding fragment or binding domain of said immunoglobulin, etc., any one or more of the ligands, 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 domain-containing or consisting of molecule, variable heavy chain novel antigen receptor (V NAR ) domain, Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, etc., any one or more of the antibodies, which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0113] One embodiment is that the second conjugate comprises a binding molecule for binding to a second cell surface molecule, and the second cell surface molecule is a cell surface receptor present on abnormal cells such as tumor cells, autoreactive cells, infected cells such as virus-infected cells, etc., which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0114] One embodiment includes a binding molecule for a second conjugate to bind to an antibody, or a binding fragment or domain thereof, such as an sdAb, or a second cell surface molecule such as a receptor ligand, wherein the second cell surface receptor is 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, integrin, 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, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, 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), and most preferably a cell surface receptor selected from HER2, c-Met, VEGFR2, CXCR7, CD71, EGFR, and EGFR1, which is a pharmaceutical combination or pharmaceutical composition of the present invention.

[0115] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein the second conjugate comprises a binding molecule for binding to a second cell surface molecule, and the binding molecule is an immunoglobulin selected from the following, preferably an sdAb derived from or based on the following: anti-CD71 antibodies such as IgG-type 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, anti-EGFR antibodies such as cetuximab, matuzumab, panitumumab, nimotuzumab, anti-CD30 antibodies such as brentuximab, anti-CD33 antibodies such as gemtuzumab, huMy9-6, anti-vascular integrin αvβ3 antibodies such as etaracizumab, anti-CD52 antibodies such as alemtuzumab, anti-CD22 antibodies such as epratuzumab, pinatuzumab, binding fragment (Fv) of the anti-CD22 antibody moxetumomab, humanized monoclonal antibody inotuzumab, anti-CEA antibodies such as labelizumab, anti-CD44v6 antibodies such as bevacizumab, 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, anti-CD38 monoclonal antibody OKT-10, anti-CA6 antibodies such as DS6, anti-IGF-1R antibodies such as cixutumumab, anti-integrin antibodies such as 3B7, CNTO 95, anti-syndecan-1 antibodies such as B-B4, anti-CD79b such as polatuzumab, anti-HIVgp41 antibody, preferably anti-HIVgp41 antibody, any one of anti-CD71 antibody, anti-HER2 antibody and anti-EGFR antibody, more preferably any one of trastuzumab, pertuzumab, cetuximab, matuzumab, anti-CD71 antibody, OKT-9, most preferably trastuzumab, cetuximab, anti-CD71 antibody OKT-9.

[0116] One embodiment is that the second conjugate comprises an sdAb capable of binding to HER2, CD71, HIVgp41 and / or EGFR, and the sdAb is preferably V HH and more preferably camelid VH It is a pharmaceutical combination or pharmaceutical composition of the present invention.

[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 HIVgp41 produced by clone anti-HIVgp41 Q8-C tag; or an sdAb encoded by any one of the cDNAs 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 sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36 to 72. Optionally, the first conjugate further comprises any one or more of the covalently bound sdAbs for binding to albumin, for example, sdAbs having the amino acid sequences of SEQ ID NOs: 33, 34 and 35. Preferably, the sdAb is V HH and more preferably camelid V H It is a pharmaceutical combination or pharmaceutical composition of the present invention. V suitable for incorporation into the second conjugate of the present invention HHis found, for example, in the single domain antibody database (Wilton, E.E. et al. (2018)), U.S. Patent Application Publication No. 2016 / 0251440 (anti-CD123, anti-CEACAM), U.S. Patent No. 9,683,045 (anti-c-Met), U.S. Patent Application Publication No. 2009 / 0252681 (anti-EGFR, anti-IGF-1R), U.S. Patent No. 9,969,805 (anti-HER2), U.S. Patent Application Publication No. 20190023796A1 (anti-HER3), and Kijanka et al. (2013) (for anti-HER2) and Mercier et al. (2019) (for anti-HER2). A series of V HH amino acid sequences and / or cDNA sequences are also provided below in this specification as SEQ ID NOs: 1 to 32 and 36 to 72, considering their ability to bind to tumor cell-specific receptors. In particular, V HH that can bind to the binding site of any of the tumor cell-specific receptors HER2, VEGFR, and CD71 is suitable for incorporation into the second conjugate of the present invention. The inventors have shown that a conjugate of saponin and a binding molecule comprising V HH targeting any one of such receptors is effective for the delivery of saponin conjugated to sdAb. See, for example, the Examples section and FIGS. 2 to 4.

[0118] These cell surface molecules are typically present on tumor cells that have at least some degree of tumor cell specificity. The tumor cell specificity makes these receptors suitable targets for the first and second conjugates of the present invention. Thus, the sdAb in the first conjugate can bind to such cell surface receptors, and accordingly, the binding molecules included in the second conjugate of the present invention, such as antibodies or sdAbs, can bind to such cell surface receptors. Tumor cells generally contain binding molecules that target tumor cell receptors, such as antibodies or their binding fragments or domains, and are targeted by a second conjugate that contains saponin and is delivered inside the target tumor cells, more specifically into the cytosol of said tumor cells. The saponin included in the second conjugate of the present invention can stimulate the release and delivery of the effector molecule included in the first conjugate of the present invention in the cytosol of cells, such as (tumor) cells targeted by the binding molecule of the second conjugate, such as an sdAb included in the second conjugate of the present invention. Therefore, it is particularly preferred to select cell surface receptors known to be suitable for functioning as target (tumor) cell surface molecules of binding molecules such as sdAbs, for example as targets of ADCs and AOCs. Accordingly, the first and second conjugates of the present invention are suitable for the co-delivery of the effector molecule, which is part of the first conjugate, an ADC or AOC containing an sdAb, together with the saponin included in the second conjugate of the present invention. By targeting tumor cell-specific receptors with the second conjugate of the present invention, the endocytosis and delivery of saponin, which is part of the second conjugate, to the target cell endosome and / or lysosome are promoted. When tumor cells are also contacted with an ADC or AOC, for example the first conjugate of the present invention, such an ADC or AOC or the effector molecule included in the first conjugate of the present invention is co-delivered to the endosome or lysosome and subsequently delivered to the cytosol of the target cell under the influence of saponin.As described above, the application of the targeted saponin as part of the second conjugate of the present invention results in an enhancement of the stimulating effect of saponin by about 100 to 1000 times compared to the application of free saponin lacking a cell-targeting binding molecule such as a receptor ligand, antibody or sdAb, when considering the biological activity of the effector molecule contained in the first conjugate of the present invention. Camelidae animal V. in the second conjugate of the present 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 whom the second conjugate has been administered, compared to the clearance rate generally observed with antibody-based ADCs. This is also evident for the first conjugate containing sdAb, as described above. In addition, because of the relatively small size of the sdAb, there is a limited risk of restricting or interfering with saponin activity inside the target cell due to the presence of the linked protein domain, for example, compared to the larger size of an antibody conjugated to saponin. Generally, the smaller the size of the molecule linked to saponin, the more likely it is that the linked molecule, for example V HHThe risk of interference with intracellular saponin activity due to the presence of antibody domains such as this is reduced. Furthermore, the relatively small size of the sdAb results in their rapid distribution in tissues such as tumor tissue, improving access to target cells by the second conjugate of the present invention, and at the same time improving the binding (degree) to target cells compared to the relatively large-sized IgG commonly applied in, for example, ADCs and OACs. One of the many advantages of applying sdAb in the second conjugate of the present invention (which also applies to applying sdAb in, for example, ADCs and AOCs, i.e., the first conjugate of the present invention) is that there is no Fc tail common to normal antibodies of the IgG type. The absence of an Fc tail in the sdAb of the first and second conjugates of the present invention prevents Fcγ receptor-mediated off-target effects when the first and second conjugates are administered to patients who require it, for example, the occurrence of undesirable side effects associated with Fcγ receptor activation. The absence of the Fc tail eliminates the risk of side effects caused by, for example, the binding of Fc to the cells of patients to whom an antibody-based ADC is administered. The sdAb containing the conjugate of the present invention does not have this risk of Fc-mediated undesirable side effects, but when tumor cells are in contact with both the ADC (at a relatively low dose; see also the Examples section) and the second conjugate of the present invention (at a relatively low dose; see also the Examples section), the inventors have revealed that such first and second conjugates are very efficient and effective in enhancing the effector molecules contained in the first conjugate.

[0119] One embodiment is a pharmaceutical combination or composition of the present invention, wherein the second conjugate comprises a saponin or saponin derivative covalently bound via a thio-ether bond to a sulfhydryl group in a binding molecule, and the covalent bond via the linker N-ε-maleimidocaproic acid hydrazide (EMCH) is to the aldehyde group at the C 23 position and is covalently bound to a sulfhydryl group in a binding molecule such as the sulfhydryl group of cysteine.

[0120] One embodiment is that the second conjugate is a bidesmosidic triterpenoid saponin or a derivative thereof belonging to the type of 12,13-dehydrooleanane having an optionally aldehyde functional group at the C 23 position, and includes a saponin or a saponin derivative containing a glucuronic acid unit in the first sugar chain of the C3β-OH group of the aglycone core structure of the saponin. The saponin or its derivative is covalently bonded to the amino acid residue of the binding molecule via the carboxyl group of the glucuronic acid unit of the first sugar chain, preferably via at least one linker. The amino acid residue is preferably selected from cysteine and lysine, which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0121] One embodiment is that the second conjugate includes a saponin or a derivative thereof containing a glucuronic acid unit in the first sugar chain of the C3β-OH group of the aglycone core structure of at least one saponin or a derivative thereof. This glucuronic acid unit is covalently bonded to a linker, and this linker is preferably covalently bonded via an amide bond to an amine group in the binding molecule, such as the amine group of lysine or the N-terminus of the binding molecule. Preferably, the glucuronic acid unit is covalently bonded to the linker, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0122] One embodiment is that the second conjugate includes two or more covalently bonded saponins or derivatives thereof, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1 to 100, or any number in between, such as 7, 9, 12 saponins or derivatives thereof, which is the pharmaceutical combination or pharmaceutical composition of the present invention.

[0123] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein two or more covalently linked saponins or saponin derivatives are directly covalently bound to an amino acid residue of a binding molecule, preferably cysteine and / or lysine, and / or are covalently bound via at least one linker and / or at least one cleavable linker. The binding of 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 sdAb and 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 the endosomal escape of the effector molecule contained in the first conjugate (when the binding molecule such as 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, wherein two or more covalently linked saponins or saponin derivatives comprise at least one oligomeric or polymeric molecule and are part of a covalently linked saponin conjugate in which two or more saponins or saponin derivatives are covalently linked, and the covalently linked saponin conjugate is covalently linked to a binding molecule. Such a covalently linked saponin conjugate can bind to a binding molecule contained in a second conjugate, such as an antibody or sdAb, via a single bond, preferably via a (cleavable) linker, and functions as a carrier for multiple saponin moieties. The covalently linked saponin conjugate can have any selected number of covalently linked saponin moieties, for example 1 to 200 saponin moieties, in relation to a selected type of oligomeric or polymeric structure containing binding sites for covalently linking these saponins, thus providing a degree of freedom in the application of such covalently linked saponin conjugates when considering the number of saponin moieties in the second conjugate of the invention. For example, for cytosolic delivery of a selected effector molecule contained 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 covalently linked saponin conjugate having a number of saponin moieties sufficient to stimulate cytosolic delivery of the effector molecule (when the covalently linked saponin conjugate is part of the second conjugate of the invention and the effector molecule co-localizes with the second conjugate in the endosome or lysosome of the target cell in which it is to exert its biological activity).

[0125] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, in which 1 to 8, more preferably 2 to 4, such covalent saponin conjugates are bound to a binding molecule, and at least one covalent saponin conjugate is optionally dendron-based, and optionally 1 to 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 bound, directly or via a linker, to an oligomeric or polymeric molecule of at least one covalent saponin conjugate. Preferably, one or two of the covalent saponin conjugates are bound to a single binding molecule such as an sdAb in the second conjugate of the present 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 the second conjugate is sufficient to efficiently stimulate the delivery of effector molecules into target cells and into the cytosol of said cells, where the effector molecules are 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 contains a saponin or a single antibody or single sdAb to which a saponin or covalent saponin conjugate is bound, preferably a single saponin or a single covalent saponin conjugate is part of the second conjugate.

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

[0127] In one embodiment, the cleavable linker is cleaved under acidic conditions, reducing conditions, enzymatic conditions, and / or photoinduced conditions. Preferably, the cleavable linker is a cleavable bond selected from hydrazone bonds and hydrazide bonds that are cleaved under acidic conditions, and / or a bond that is susceptible to proteolysis, for example, proteolysis by cathepsin B, and / or a bond that is susceptible to cleavage under reducing conditions such as a disulfide bond. It is a pharmaceutical combination or pharmaceutical composition of the present invention.

[0128] In one embodiment, the cleavable linker is cleaved in vivo under acidic conditions, preferably pH 4.0 to 6.5, more preferably pH ≤ 5.5, such as those present in the endosomes and / or lysosomes of mammalian cells, preferably human cells. Such a cleavable linker that is cleavable under conditions such as those found in endosomes and lysosomes facilitates the delivery of free saponin into the endosome or lysosome when cleaving saponin from the remainder of the second conjugate of the present invention. Thus, the second conjugate of the present invention combines the targeted delivery of saponin to cells upon specific binding of a binding molecule such as an antibody or sdAb to a second cell surface molecule on the target cell, and the benefit of the presence of free saponin within the cell, i.e., within the endosome (or lysosome), which contributes to the ability of free saponin to stimulate and / or facilitate the delivery of the effector molecule contained in the first conjugate of the present invention from the endosome (or lysosome) to the cytosol of the target cell.

[0129] In one embodiment, the oligomeric or polymeric molecule of the covalent saponin conjugate is covalently bound to the binding molecule, preferably to an amino acid residue of the binding molecule. It is a pharmaceutical combination or pharmaceutical composition of the present invention.

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

[0131] One embodiment is a pharmaceutical combination or pharmaceutical composition of the present invention, wherein a saponin or saponin derivative is covalently bound to an oligomeric or polymeric molecule of a 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] One embodiment is that the saponin or saponin derivative is C 23It contains an aglycone core structure with an aldehyde functional group at the position, and the saponin optionally contains a glucuronic acid functional group in the first sugar chain of the C3β-OH group of the aglycone core structure of the saponin. The aldehyde functional group is involved in the covalent bond to the oligomer molecule or polymer molecule of the covalent saponin conjugate, and / or the glucuronic acid functional group (when present) is involved in the covalent bond to the oligomer molecule or polymer molecule of the covalent saponin conjugate. The bond of the saponin or saponin derivative is either directly through a covalent bond or through a linker, and the linker is a cleavable linker or a stable linker. This is the pharmaceutical combination or pharmaceutical composition of the present invention. Here, "stable" refers to the bond between the saponin and the binding molecule such as sdAb, or the bond between the saponin and the oligomer or polymer structure, and this bond remains intact (not cleaved) under acidic conditions within the cell, especially under acidic conditions within the endosome or lysosome of such cells. Furthermore, such a stable bond remains intact (i.e., not cleaved) within the circulation and organs of the human subject to whom the second conjugate of the present invention containing the covalent saponin conjugate is administered. In contrast, a cleavable linker related to the binding of the saponin to the binding molecule contained in the second conjugate such as an antibody or sdAb, or to the oligomer structure or polymer structure, refers to a bond that is cleaved under acidic conditions found inside the endosomes and lysosomes of mammalian cells such as human cells, for example, tumor cells. On the one hand, such a cleavable linker remains intact (not cleaved) when the second conjugate containing such a cleavable bond is present in the circulation or organs, that is, for example, in the extracellular space of the human subject to whom the second conjugate of the present invention is administered.

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

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

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

[0136] One embodiment includes a polymeric or oligomeric molecule of a covalently linked saponin conjugate, which is a linear polymer, branched polymer and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, poly-lysine, poly-ethylene glycol, oligo-ethylene glycol (OEG), such as OEG3, OEG4 and OEG5, selected polymer structures and / or oligomer structures, or aggregates of these polymer structures and / or oligomer structures. The aggregate is preferably constructed by covalent cross-linking. Preferably, the polymeric or oligomeric molecule of the covalently linked saponin conjugate is a dendron, such as a poly-amidoamine (PAMAM) dendrimer, and is a pharmaceutical combination or pharmaceutical composition of the present invention. Driven by the number of selected saponins incorporated into the second conjugate of the present invention, the type and size or length of the oligomeric or polymeric structure are selected. That is, to form the second conjugate of the present invention, the number of saponins that bind to the binding molecule contained in the second conjugate, such as an sdAb, allows selection of a suitable oligomeric or polymeric structure having a sufficient number of binding sites to bind the desired number of saponins, thereby providing a covalently linked saponin conjugate having a selected number of saponin moieties that bind to a binding molecule such as an antibody, receptor ligand or sdAb to provide the second conjugate of the present invention. For example, the length of the OEG or the size of the dendron or polylysine molecule determines the maximum number of saponins that can be covalently bound to such an oligomeric or polymeric structure.

[0137] Accordingly, the second conjugate according to the invention comprises at least one saponin. "At least one" in this context means that the second conjugate contains one saponin molecule, but may also contain several (e.g., 2, 3 or 4) saponins or a large number (e.g., 10, 20 or 100) of saponins. Depending on the use, the second conjugate can include a covalently bound scaffold (covalent saponin conjugate) containing covalently bound saponins, and the scaffold can be designed to contain a defined number of saponins. Preferably, the second conjugate according to the invention contains a defined number or range of saponins rather than a random number. This is particularly advantageous for pharmaceutical development related to manufacturing and marketing approvals. In this regard, a defined number means that the second conjugate preferably contains a previously defined number of saponins. This is achieved, for example, by designing a scaffold that includes a polymeric structure having a specific number of possible moieties for (covalently) binding saponins. Under ideal circumstances, all of these moieties bind to saponins and the scaffold contains a previously defined number of saponins. It is envisioned to provide a standard set of scaffolds containing, for example, 2, 4, 8, 16, 32, 64 saponins, etc., so that the user can easily test the optimal number as needed. One embodiment is the second conjugate of the invention comprising the scaffold of the invention (the covalent saponin conjugate of the invention), wherein the saponins are present within a defined range, for example, under non-ideal circumstances, not all of the moieties present in the polymeric structure bind to saponins. Such ranges can be, for example, 2 to 4 saponin molecules per scaffold, 3 to 6 saponin molecules per scaffold, 4 to 8 saponin molecules per scaffold, 6 to 8 saponin molecules per scaffold, 6 to 12 saponin molecules per scaffold, etc. In such cases, the second conjugate comprising the scaffold of the invention (the covalent saponin conjugate of the invention) thus contains 2, 3 or 4 saponins when the range is defined as 2 to 4.

[0138] The scaffold is basically independent of the type of saponin covalently attached to the scaffold, and the scaffold is subsequently (in sequence) covalently attached to cell surface molecule binding molecules such as receptor ligands, antibodies such as sdAbs, in the second conjugate of the present invention. Thus, the second conjugate of the present invention containing the scaffold (the covalent saponin conjugate of the present invention) is a product that serves as the basis for a platform technology. At least one covalently attached saponin in the second conjugate of the present invention mediates the intracellular delivery of the effector moiety bound to the sdAb targeting the cell surface molecule, which is included in the first conjugate of the present invention, such as an ADC or AOC containing sdAb according to the present invention. Therefore, the scaffold technology according to the present invention combines an ADC or AOC (i.e., the first conjugate of the present invention) with a second conjugate of the present invention containing a saponin and an antibody such as an sdAb that binds to a target cell surface molecule, such as, for example, and preferably, a receptor and a receptor involved in the endocytosis of the conjugate bound thereto, to mediate a system for controlled intracellular effector moiety delivery by the saponin. The scaffold provides an optimized functionally active unit that can be linked to the saponin and a ligand targeting a cell surface molecule such as an antibody such as sdAb in the second conjugate at a single and predetermined position in the ligand such as sdAb.

[0139] One embodiment is a second conjugate of the present invention comprising a scaffold (the covalent saponin conjugate of the present invention), wherein the number of monomers of the polymer or oligomer structure is an exactly defined number or range. Preferably, the polymer or oligomer structure has a structure such as poly(amine), e.g., polyethyleneimine and poly(amidoamine), or a structure such as polyethylene glycol, poly(ester), e.g., poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, poly(dextrin), or a structure such as a peptide or protein, or a structure such as natural and / or artificial polyamino acids, e.g., poly-lysine, DNA polymer, stabilized RNA polymer or PNA (peptide nucleic acid) polymer, which may be in the form of linear, branched or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates (single or mixture) of these structures. Preferably, the polymer or oligomer structure is biocompatible, where biocompatible means that the polymer or oligomer structure does not exhibit substantial acute or chronic toxicity in an organism and is either excreted as such or can be excreted by the body's metabolism and / or completely degraded into physiological compounds. The aggregates can be constructed by covalent or non-covalent bonds and / or attractive forces. Thus, they can also form nanogels, microgels or hydrogels, or they can bind to carriers such as inorganic nanoparticles, colloids, liposomes, micelles, or particulate structures containing cholesterol and / or phospholipids. The polymer or oligomer structure preferably has an exactly 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 sdAb (where sdAb is preferred in the present invention)).Preferably, at least about 50%, more preferably at least about 75%, more preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, more preferably at least about 98%, more preferably at least about 99%, and most preferably (about) 100% of the precisely defined number or range of linking moieties (chemical groups) in the polymer or oligomer structure are occupied by the glycoside molecules (saponins of the present invention) of the scaffold (covalent saponin conjugate of the present invention).

[0140] Preferably, a dendron is a branched, well-defined tree-like polymer having a single chemically addressable group at the origin of the tree, called the focal point. A dendrimer is an assembly in which two or more dendrons are connected at their focal points. A dendronized polymer is an assembly in which the focal point of one or more dendrons is connected to a polymer. In a preferred embodiment, the scaffold according to the present invention, wherein the polymer or oligomer structure includes a linear, branched or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or an assembly (single or mixture) of these structures, and the assembly can be constructed by covalent cross-linking or non-covalent attractive forces and can form a nanogel, microgel, or hydrogel. Preferably, the polymer is a poly(amine), such as derivatives of polyethyleneimine and poly(amidoamine), and polyethylene glycol, poly(ester), such as poly(lactide), poly(lactam), poly(lactide-co-glycolide) copolymer, and poly(dextrin) and other structures, and natural and / or artificial polyamino acids, such as poly-lysine, or peptides or proteins, or DNA polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers and other structures are provided. Preferably, the polymer or oligomer structure is biocompatible.

[0141] When binding molecules such as sdAbs in the second conjugate of the present invention and sdAbs in the first conjugate of the present invention, i.e., in an ADC or AOC, target different cell surface molecules such as different tumor cell-specific receptors, it will be understood that both of the two different targets are present on the surface of the target cells to which the effector molecule contained in the first conjugate, i.e., the ADC or AOC, and the saponin contained in the second conjugate of the present invention should be co-delivered. When the ADC or AOC (the first conjugate of the present invention) and the second conjugate of the present invention target different (tumor) cell receptors, the advantage is that the doses of the first conjugate (ADC or AOC) of the present invention and the second conjugate of the present invention are such that when contacting a mixture of different cell types containing the target cells, for example, when (simultaneously) administered to a patient in need thereof, the risk of off-target effects, such as on cells having one of the two cell surface molecules on their surface, is reduced. By administering a dose of the first conjugate (ADC or AOC) of the present invention that is too low to deliver a therapeutically effective amount of the effector molecule into the target cells, if the ADC or AOC binds to such target cells in the absence of the targeted saponin in the form of the second conjugate of the present invention, the ADC or AOC (the first conjugate of the present invention) may bind to cell surface molecules that the cell may express, but the invasion of cells that do not express the target of the binding molecule such as sdAb in the second conjugate of the present invention does not, for example, induce cytotoxicity when considering the ADC. Endosomal escape occurs only in target cells having both a receptor for binding the ADC or AOC (the first conjugate of the present invention) and a receptor for binding the second conjugate of the present invention, under the influence of the saponin co-localized inside the target cells expressing both receptors, then the dose of the effector molecule is sufficient to exert its biological effect in the cytosol of the cells.

[0142] In some embodiments, the binding molecule such as sdAb included in the second conjugate of the present invention and the sdAb included in the first conjugate of the present invention (i.e., ADC or AOC) target the same cell surface molecule such as the same tumor cell-specific receptor. Thus, when only a single tumor cell-specific receptor exists, it is possible to simultaneously deliver both the ADC or AOC (the first conjugate of the present invention) and the second conjugate of the present invention into the interior of target cells having such a single type of target receptor. The advantage of such simultaneous delivery into target cells using a single cell-specific surface molecule such as a tumor cell-specific receptor, for example, HER2, EGFR, CD71, etc., is the possibility of targeting tumor cells that express only a single receptor that is sufficiently specific for the specific targeting of such target tumor cells by the second conjugate of the present invention having saponin and the ADC or AOC (i.e., the first conjugate of the present 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 cells, and as a result, an effective dose of the effector molecule can still be effectively provided to such therapeutically difficult tumor cells.

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

[0144] One aspect of the present invention relates to the pharmaceutical combination of the present invention or the pharmaceutical composition according to 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, α1-antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis.

[0145] Surprisingly, the inventors have found that the dose of the first conjugate of the present invention, i.e., the ADC, which does not cause any tumor cell death, is sufficient for efficient tumor cell death when the first conjugate of the present invention containing the effector molecule is brought into contact with tumor cells in the presence of the second conjugate of the present 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 -toxin and other doses of the first conjugate of the present invention do not exert a toxic effect on tumor cells when brought into contact with tumor cells. Typical toxins are protein toxins, for example, dianthin and saporin. However, when the first conjugate of the present invention is co-administered together with the second conjugate of the present invention, efficient tumor cell death is achieved. The second conjugate of the present invention is, for example, anti-HER2 V HH -SO1861, anti-CD71 V HH -SO1861, anti-EGFR V HH -SO1861, cetuximab-SO1861, trastuzumab-SO1861. The first conjugate of the present invention can bind, for example, to HER2, CD71 or EGFR V HHAn ADC such as an ADC containing, for example, a protein toxin such as dianthin or saporin. As described above, by applying the second conjugate of the present invention, the amount of saponin required to achieve efficient biological activity of the effector molecule contained in the first conjugate of the present invention, i.e., the ADC or AOC, within the target cell is about 100 to 1000 times lower compared to the amount of free saponin required when the second conjugate containing saponin is co-administered with the first conjugate of the present invention, i.e., the ADC or AOC, to the target cell, or when the first conjugate of the present invention is co-administered with free saponin to the target cell. Thus, the second conjugate of the present invention enhances the ADC or AOC, which contains an sdAb of the first conjugate of the present invention, i.e., the ADC or AOC, at a dose of the ADC or AOC that would be ineffective against tumor cells when administered to a patient in need thereof in the absence of the targeted saponin (the second conjugate of the present invention). Furthermore, the second conjugate of the present invention is already sufficiently effective at a relatively low dose, i.e., at a dose where free saponin, which is not provided together with a binding molecule targeting tumor cells such as an sdAb, is not sufficiently effective for enhancing the effector molecule, considering the enhancement of the effector molecule of the first conjugate of the present invention, i.e., the ADC or AOC. Additionally, when the ADC or AOC is co-administered to a patient in the presence of the second conjugate of the present invention, it is already effective at a low dose, thus providing an improved method for treating a human patient in need of treatment using a therapeutically effective amount of the first conjugate of the present invention, i.e., an ADC or AOC containing an sdAb targeting tumor cells. 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 absence of the Fc tail prevents unwanted binding of the conjugate to off-target cells of patients having Fc receptors, which otherwise could result in side effects as seen in many conventional ADCs and AOCs when such an Fc tail is present.Antibody-based ADCs containing an Fc tail and AOCs containing an Fc tail suffer from a decrease in efficacy due to the undesirable binding of such IgG-based ADCs and 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 absence of an Fc tail provides several advantages in this regard. For the first conjugate, preferably also for the second conjugate, off-target and undesirable binding to Fc receptors cannot occur. Thereby, considering target receptor-mediated endocytosis and delivery of the first and second conjugates of the present invention into endosomes, the conjugates of the present invention have a lower effective dose because the conjugate is not "lost" due to Fc receptor binding, which is 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 were replaced with a conventional IgG containing an Fc tail. Similarly, as a result, the therapeutic window of the second conjugate of the present invention is wider than the therapeutic window that 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, for example, a conventional IgG containing 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 an SO1861 derivative or a QS-21 derivative, preferably a saponin derivative of the present invention.

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

[0148] A further aspect of the invention is an in vivo or ex vivo method of transferring the first conjugate of the invention from outside the cell into the cell interior, preferably into the cytosol of the cell, comprising a) providing a cell that expresses on its surface the first cell surface molecule according to the invention and optionally expresses on its surface the second cell surface molecule according to the invention, said cell preferably being selected from abnormal cells such as hepatocytes, virus-infected cells, autoimmune cells and tumor cells; b) providing the first conjugate of the invention to be transferred to the cell provided in step a); c) providing the saponin or saponin derivative of the invention and / or, when the cell provided in step a) expresses the second cell surface molecule according to the invention on its surface, providing the second conjugate of the invention; d) contacting the cell 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 transfer of the first conjugate from outside the cell into the cell interior, preferably into the cytosol of the cell.

[0149] One embodiment is an in vitro or ex vivo method for transferring a first conjugate of the present invention from outside the cell to inside the cell according to the present invention. If the first conjugate is transferred into the cell, in order to further transfer the effector molecule contained in the first conjugate from the endosome, endolysosome or lysosome of the cell into the cytosol of the cell, a) providing a cell that expresses a first cell surface molecule according to the present invention on its surface and, optionally, expresses a second cell surface molecule according to the present invention on its surface, wherein the cell is preferably selected from abnormal cells such as hepatocytes, virus-infected cells, autoimmune cells and tumor cells; b) providing a first conjugate of the present invention to be transferred to the cell provided in step a); c) providing any one saponin or saponin derivative of the present invention, or preferably, when the cell provided in step a) expresses a second cell surface molecule according to the present invention on its surface, providing a second conjugate of the present invention; d) contacting the cell 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), comprising thereby establishing transfer of the first conjugate from outside the cell to inside the cell and thereby establishing delivery of the effector molecule contained by the first conjugate to the cytosol of the cell, optionally as part of the first conjugate or as a free effector molecule cleaved from the first conjugate in the endosome, endolysosome or lysosome.

[0150] In formal terms, a glycoside is any molecule in which a sugar group is glycosidically linked via its anomeric carbon to another group. Without wishing to be bound by any theory, glycoside molecules such as saponins in the context of the present invention are molecules that can further enhance the effect of an effector moiety, particularly by promoting endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (the saponins of the present invention, such as those exemplified herein and in the claims) interact with the membranes of the compartments and vesicles of the endocytosis and recycling pathways, making them more permeable to said effector moiety and increasing endosomal escape. The term "scaffold can increase the endosomal escape of the effector moiety" means that in a second conjugate of the present invention, at least one saponin (glycoside molecule) that is bound to an antibody targeting a cell surface molecule such as an sdAb, either via a linker or directly, or via the polymeric or oligomeric structure of the scaffold (the covalent saponin conjugate of the present invention), can increase the endosomal escape of the effector moiety contained in a first conjugate of the present invention when both molecules are within an endosome, such as a late endosome, and optionally, and preferably, after at least one saponin is released from the second conjugate of the present invention, for example, by cleavage of a cleavable bond between the at least one glycoside (saponin) and the second conjugate, such as via the polymeric or oligomeric structure of the scaffold and / or via the linker. The optional linker or scaffold-mediated bond between at least one saponin according to the present invention and a ligand targeting a cell surface molecule such as an antibody, such as an sdAb, of a second conjugate of the present invention may be a "stable bond", but this does not mean that such a bond cannot be cleaved within an endosome, for example by an enzyme.For example, saponin may optionally be cleaved from the remaining linker fragment or oligomeric or polymeric structure, together with a linker or scaffold oligomeric or polymeric structure. For example, a protease can cleave a (proteinaceous) linker or proteinaceous polymeric structure, such as albumin, thereby releasing at least one saponin. However, the glycoside molecule (preferably saponin) is released in its original form before it binds (is prepared to bind) via an optionally linker and / or oligomeric or polymeric scaffold (the covalent saponin conjugate of the present invention) to a molecule that targets a cell surface molecule, such as an sdAb of the second conjugate of the present invention in its active form. Thus, after such cleavage, the glycoside (saponin) either has its native structure or has a chemical group or linker (part thereof) attached thereto. On the other hand, glycoside biological activity (saponin biological activity), such as endosome / lysosome escape enhancing activity against an effector moiety present in the same endosome or lysosome, is preferably maintained or restored upon said cleavage of the bond between the glycoside (saponin) and an antibody targeting a cell surface molecule, such as an sdAb, optionally including a linker and / or scaffold of the present invention. For the purposes of the present invention, the term "stable" with respect to, for example, the bond between saponin and an amino acid residue of an sdAb targeting a cell surface molecule in a second conjugate, a linker, a polymeric or oligomeric structure (scaffold, i.e., of the covalent saponin conjugate of the present invention), a ligand, a (monoclonal) immunoglobulin or its binding domain or fragment, and / or an effector (effector moiety, effector molecule) means that the bond is not readily disrupted, or is at least designed not to be readily disrupted, by, for example, a pH difference, salt concentration, or UV light, reducing conditions.Regarding the present invention, for example, with respect to the binding between 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, the term "cleavable" means that the binding is designed to be readily disrupted, for example, by a pH difference, salt concentration, reducing conditions, etc. Those skilled in the art are well aware of such cleavable bonds and methods for preparing them.

[0151] Prior to the present invention, one of the major hurdles in introducing ADCs and AOCs to the market was the small therapeutic window: the therapeutic effective dose of an ADC or AOC was accompanied by (unacceptable) side effects, hindering the progress and significance of the treatment of patients with ADCs. By applying the sdAbs targeting effector molecules and cell surface molecules and the first conjugate of the present invention, the second conjugate, for example, an sdAb-saponin conjugate of the present invention, it has become possible to introduce one or more glycoside molecules (saponins) into (target) cells together with an ADC carrying a payload, or together with an sdAb conjugated to an oligonucleotide such as BNA according to the present invention. In particular, it was previously impossible to specifically and simultaneously introduce a specific number or range of glycoside molecules (saponins) per effector moiety (a predetermined, controllable) into the cytosol of a cell via, for example, the endocytosis pathway of the cell, for the effector portion of an ADC or AOC, or any other conjugate of a payload with a molecule targeting a (proteinaceous) cell surface molecule. As described above, the ADC or AOC of the present invention (i.e., the first conjugate of the present invention) and the second conjugate can target the same cell surface molecule, or different cell surface molecules, and 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 solutions provided by the present invention include the covalent attachment of at least one saponin to a molecule that targets the cell surface molecule of the second conjugate of the present invention, such as an antibody, preferably an sdAb. Further solutions provided by the present invention include (firstly) using an oligomer or polymer scaffold to polymerize glycoside molecules (saponins), and providing a cluster of covalently attached saponins to a molecule that targets the cell surface molecule contained in the second conjugate of the present invention, such that, for example, after endocytosis, the mode of action of the saponin is enabled to re-monomerize one or more saponins at an intracellular location where the mode of action of the saponin is desired. In this context, "polymerize" means the reversible and / or irreversible multiple conjugation of saponin molecules to a cell surface molecule-binding molecule of the second conjugate, such as an antibody like sdAb, for example via a linker, or directly, or via a polymer or oligomer structure that forms a scaffold (the covalent saponin conjugate of the present invention), or the reversible and / or irreversible multiple conjugation of (modified) saponins that form a polymer or oligomer structure to form a scaffold (the covalent saponin conjugate of the present invention). "Re-monomerize" in this context means cleaving the saponin from the linker that links the saponin to a ligand that targets a cell surface molecule such as the sdAb of the second conjugate, or from the scaffold, for example from the scaffold after endocytosis, and returning the saponin to its (natural) chemical state of an unbound saponin, and the unbound saponin may or may not contain additional chemical groups such as a chemical linker that binds to a chemical group of the saponin for linking the saponin to the linker, an amino acid residue of the second conjugate, or the scaffold, and / or a chemical group of the saponin such as an aldehyde group or carboxylic acid group. Due to the complex chemical nature of saponins, for example, the "polymerization" of saponins in a scaffold or other linking linker, and their "re-monomerization" at a desired location such as intracellularly after endocytosis, have been difficult problems.In particular, the chemical reactions used to provide linkers and scaffolds, such as triterpenoid saponins (polymerization of glycosides), which contain covalent glycosides for covalently attaching to cell surface molecule-binding molecules to provide a second conjugate, typically occur in water-free organic solvents. However, saponins and, for example, biocompatible polymers applied as scaffolds for carrying the attached saponins are water-soluble molecules. The chemical properties of unmodified saponins themselves prevent polymerization, and another solution possible for binding multiple saponins (directly) to cell surface molecule-binding molecules such as antibodies, e.g., sdAbs, is that antibodies such as sdAbs typically do not provide sufficient binding sites, and also the binding products are quite heterogeneous, and / or there is a risk of affecting and inhibiting the activity of saponins when binding a biologically active molecule such as a saponin to an antibody targeting a cell surface molecule such as an sdAb, and / or, for example, in such saponin-containing second conjugates of the present invention, there is a risk of affecting and inhibiting the binding of the sdAb to the cell surface molecule, so it was presumed to be less promising. Embodiments of the present invention address at least one of these drawbacks.

[0153] Whether the second conjugate of the present invention containing saponin, with or without one or more (cleavable) linkers and / or an optional scaffold (the covalent saponin conjugate of the present invention), interferes with the acidic environment and may inhibit the endosomal escape function of at least one glycoside (saponin) can be determined readily in assays described in the Examples section and known in the art. Inhibition is described as the "doubling dose of glycoside (saponin of the present invention) required to induce 50% cell death". The scaffold or second conjugate of the present invention preferably does not result in an increase that at least increases the glycoside molecules (saponin) required to obtain 50% cell death as observed when using chloroquine as a positive control. Alternatively, and also preferably, with or without one or more (cleavable) linkers and / or an optional scaffold (the covalent saponin conjugate of the present invention), the second conjugate containing saponin does not result in an increase of at least 4-fold, more preferably at least 2-fold, of the glycoside molecules to induce 50% cell death. The fold increase needs to be measured in the assay, and chloroquine as a positive control induces a 2-fold increase in the glycoside amount, preferably the saponin amount, where the saponin is any one or more of the saponins of the present invention for observing 50% cell death (previous embodiment).

[0154] As described above, at least one saponin comprised in the second conjugate according to the invention increases the effectiveness of at least the current effector part and the new effector part as defined in the invention. In order to reduce the dosage of the effector part comprised in the first conjugate of the invention, which further comprises a cell surface molecule-binding sdAb such as an ADC or an AOC, without reducing the effectiveness, potential side effects are reduced. Accordingly, the invention provides a combination of a second conjugate according to the invention and a first conjugate according to the invention, and the invention also provides a first conjugate according to the invention for use in medicine or for use as a medicament. One aspect of the invention relates to a pharmaceutical composition according to the invention for use as a medicament, the pharmaceutical composition comprising a first conjugate according to the invention. One aspect of the invention relates to a pharmaceutical composition according to the invention for use as a medicament, the pharmaceutical composition comprising a first conjugate according to the invention and comprising a second conjugate according to the invention.

[0155] For the endosome escape enhancer comprised in the second conjugate of the invention, i.e. the saponin of the invention, 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 part into the cytosol of off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector part, (4) they are preferably biodegradable or excretable, and (5) they preferably do not substantially interfere with biological processes of organisms that are unrelated to the biological activity of the effector molecule with which the endosome escape enhancer is combined, for example, they do not interact with hormones. Examples of saponins of the invention that meet at least to some extent the foregoing criteria are the bidesmoside triterpenes, preferably bidesmoside triterpene saponins, such as SO1861, SA1641, QS-21, GE1741, and further saponins according to the invention, which are listed throughout the present specification.

[0156] Also provided is the use of a first conjugate according to the invention for the manufacture of a medicament. Also provided is the use of a first conjugate according to the invention and a second conjugate of the invention for the manufacture of a medicament. In particular, cancer therapeutics, especially classical chemotherapeutic agents, are well-known for their side effects. For the time and place targeting and synchronization of both the pharmaceutically active substance contained in a first conjugate such as an 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 of the invention is particularly useful for use as a medicament, especially in a method for treating cancer, where the medicament is a single composition comprising an ADC or AOC (the first conjugate of the invention) and the second conjugate of the invention according to the invention, or a therapeutic combination of a first pharmaceutical composition comprising the first conjugate of the invention, such as an ADC or AOC, and a second pharmaceutical composition comprising the second conjugate of the invention. Accordingly, the invention provides a first conjugate according to the invention for use in a method for treating cancer. The invention also provides a first conjugate according to the invention for use in a method for treating an acquired or genetic disorder, especially a single gene deficiency disorder. Accordingly, 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 invention is combined with at least one first conjugate of the invention comprising an effector moiety and an sdAb, such as an ADC or AOC of the invention. Accordingly, one aspect of the invention relates to a second conjugate according to the invention, which second conjugate comprises a covalently linked saponin and a cell surface molecule-binding antibody such as an sdAb for use in a method for the treatment of cancer or an autoimmune disease, the method further comprising administering a first conjugate according to the invention to a subject such as a cancer patient in need thereof.

[0157] Further uses of the first and second conjugates of the present invention in medicine are for use in a method for replacing intracellular enzymes in target cells where the amount or functionality of the enzyme produced is insufficient. The second conjugate of the present invention is administered, for example, in combination with the first conjugate of the present invention containing the enzyme or oligonucleotide for gene therapy to be replaced, to a patient in need thereof, for replacement of the target cell 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 shortening of the lifespan of the patients involved. An example of such a disease is phenylketonuria, which is an inborn error of metabolism that results in reduced metabolism of the amino acid phenylalanine. This disease is characterized by mutations in the gene for the liver enzyme phenylalanine hydroxylase. Phenylketonuria has been incurable to date. The incidence is approximately 1:10,000, and in Turkey, where the highest incidence is known, it is 1:2,600. Together with the conjugated saponin, an antibody targeting the cell surface molecule contained in the second conjugate of the present invention, preferably V HH such as sdAb, and a first conjugate containing the same or different cell surface molecule-binding sdAb and phenylalanine hydroxylase, or the same or different cell surface molecule-binding sdAb, together with a conjugated polynucleotide encoding phenylalanine hydroxylase, can be used to target hepatocytes and replace the defective enzyme in hepatocytes by using a suitable specific antibody or sdAb in the second conjugate of the present invention and a suitable specific sdAb in the first conjugate according to the present invention. This is an example of the use of the second conjugate of the present invention containing the saponin conjugated thereto and, in this example, the first conjugate of the present invention containing an effector molecule such as the enzyme or oligonucleotide conjugated thereto according to the present invention, for replacement or gene therapy. In a preferred embodiment, the second conjugate according to the present invention for use in a gene therapy or replacement therapy method is provided in combination with the first conjugate of the present invention containing a cell surface molecule-binding sdAb and an enzyme or nucleic acid encoding the enzyme.

[0158] Using the first and second conjugates of the present invention, it is now possible to design and manufacture a two-component non-viral clinically applicable gene delivery technology. For example, the second conjugate of the present invention enables the development of a non-viral-based combination of the first and second conjugates for gene delivery technology, which enhances therapeutic efficacy at lower therapeutic doses, thereby improving the health of patients. The second conjugate of the present invention particularly includes an antibody targeting a covalently bound cell surface molecule such as a monoclonal antibody or sdAb for binding to (tumor, autoimmune) cell surface-specific molecules, an effector portion such as an oligonucleotide such as BNA, and V HH When combined with a first conjugate containing an sdAb targeting cell surface molecules such as, it overcomes a major bottleneck in the field of gene delivery over the years, namely, enabling the efficient, safe, and cost-effective transfer of gene therapy products into the cytosol / nucleosol via the endosomal membrane. In fact, gene therapy is one of the most promising treatment options for advanced future treatments in a wide range of diseases. The success of gene delivery requires the recognition of target cells and the uptake of genes into the cytosol and nucleosol. One of the major problems in the field of non-viral gene therapy is the inefficient and inadequate safe delivery of gene materials for therapeutic use in patients.

[0159] Therefore, when applying a second conjugate of the present invention in combination with a first conjugate of the present invention, such as an AOC or an sdAb, which comprises a cell-targeting molecule targeting a cell, such as a ligand or preferably an antibody (fragment, domain, preferably an sdAb) targeting a cell surface molecule, comprises an oligonucleotide such as an antisense BNA, and comprises an sdAb targeting a cell surface molecule, the inventors have now been able 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 present invention represents a technology designed to enable targeting of any addressable cell type with all known gene agents and subsequent efficient delivery of the gene into the cytosol, thereby ensuring better patient treatment, which is important not only for genetic disorders but also for cancer treatment and thus for a large patient population. The technology based on the first and second conjugates of the present invention comprises a targeting ligand, or a (monoclonal) (tumor cell-specific) antibody or fragment thereof, or preferably a V HHA polymer or oligomeric scaffold (the covalent saponin conjugate of the invention) that functions as a carrier for an endosome escape enhancer (EEE) such as saponin as exemplified herein for molecules targeting cell surface molecules such as sdAb, and a second conjugate of the invention comprising a saponin of an embodiment of the invention may be included. In this case, the first conjugate of the invention comprises a first cell surface molecule binding molecule such as sdAb, and an effector moiety, here an effector gene such as LNA or BNA. The use of the first conjugate of the invention in combination with the second conjugate of the invention, which comprises an oligonucleotide such as sdAb and BNA targeting the same or different cell surfaces, for example an antibody (fragment) or sdAb targeting cells, has the potential to deliver any kind of biological macromolecule to 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 aimed at delivery into the cytosol of diseased cells such as cancer cells. Thus, the first and second conjugates of the invention can also be used with current and future targeting sdAbs and antibodies by click chemistry, and exist as molecular tools that enable customized drug application and future development in the field of tissue and cell targeting technologies. Thus, the first conjugate according to the invention can also link current and future therapeutic oligonucleotides (as well as payloads such as protein toxins) to, for example, sdAbs by click chemistry, or can be linked, and exist as molecular tools that enable customized drug application and future development in the field of tissue and cell targeting technologies. The first and second conjugates of the invention can include antibodies and ligands as molecules targeting cell surface molecules, but sdAbs are preferred. The global market for gene therapy drugs is growing rapidly and is targeted at potential treatments for a wide range of disease areas such as cancer, cardiovascular disease, Parkinson's disease, Alzheimer's disease, HIV and many rare (single gene) diseases.Current virus vector-based gene therapy technologies have significant issues 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 containing the gene to be delivered and the molecule targeting the cell surface, i.e., sdAb, enables use in a technology platform that is an alternative to current viral gene delivery technologies. Thus, the first and second conjugates of the present invention are suitable for incorporation into approaches for developing non-viral gene therapies for diseases such as cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, HIV infection, and many rare (single gene) diseases. The first and second conjugates of the present invention are suitable for the development of novel therapies for transforming the fields of antibody-drug conjugates (ADCs) and oligonucleotide-based therapeutics by creating non-viral vector-based gene therapeutics such as those based on target antisense BNA. The application of the second conjugate of the present invention, particularly the covalent conjugate containing an antibody such as sdAb and saponin, in combination with the first conjugate containing cell surface molecule-binding molecules, i.e., oligonucleotides such as sdAb and BNA, is one of many beneficial approaches enabled by the present invention. For example, the use of the first and second conjugates of the present invention enables utilization of the endocytosis pathway of mammalian cells. Endocytosis is utilized for the delivery of therapeutics, and the second conjugate of the present invention contributes to the uptake of, for example, siRNA contained in the first conjugate and improvement of endosomal escape. Preferably, the first and second conjugates of the present invention are preferably used together with small molecules that act as delivery enhancers for, for example, payloads, oligonucleotides. Thereby, the second conjugate of the present invention having saponin and a covalently bound cell-targeting moiety such as a ligand, preferably an antibody (domain or fragment, preferably V. HH ) having, in combination with the second conjugate of the present invention, a covalently bound oligonucleotide such as BNA and a covalently bound cell-targeting moiety, i.e., sdAb, preferably V HHThe first conjugate of the present invention having [content in Japanese] applies them as two components including free untargeted saponin, and thus provides a solution to the current problems seen in current endosomal escape enhancers and gene therapy products regarding the complication of therapeutic approval and clinical applicability. Such a second conjugate of the present invention containing saponin is combined with a gene product such as BNA and a (tumor) cell targeting moiety, i.e., the first conjugate of the present invention containing sdAb. Therefore, the present invention provides that an endosomal escape enhancer (e.g., the glycoside of the embodiments and examples provided by the present invention) and a second targeting ligand or antibody (e.g., sdAb according to the embodiments of the present invention and exemplified in the following example section of the present specification) are included in the second conjugate of the present invention, and a gene therapy product (an oligonucleotide according to the present invention such as BNA), and the first conjugate of the present invention containing an sdAb targeting a first cell surface molecule such as an sdAb targeting the same or different cell surface molecules included in the second conjugate of the present invention, and the sdAb targeting the first cell surface molecule binds to the same or different cell surface molecules as the molecule targeting the cell surface molecule of the second conjugate of the present invention, providing a non-viral gene delivery technology. Therefore, such first and second conjugates of the present invention provide current and future therapeutic opportunities for macromolecular drugs for a wide range of diseases and a large patient population. By applying such a second conjugate of the present invention containing at least one saponin and at least one specific cell targeting moiety such as an immunoglobulin or sdAb in combination with the first conjugate of the present invention containing at least one oligonucleotide and at least one specific cell targeting moiety, i.e., sdAb, it is clear for the current method of separately applying a non-cell surface molecule-targeted free endosomal escape enhancer (e.g., saponin not conjugated to a cell surface molecule-binding antibody) and a gene therapy product, which does not guarantee the simultaneous presence of both compounds at the interaction site in this current method, addressing the problem.This problem was solved by using the second conjugate of the invention in combination with the first conjugate of the invention containing a nucleic acid. Here, both the second conjugate and the first conjugate of the invention are molecules targeting cell surface molecules, for example, considering the second conjugate, it contains an antibody such as an sdAb according to the invention, and considering the first conjugate, it contains an sdAb. That is, such a combination of the second conjugate of the invention and the first conjugate containing an oligonucleotide provides a non-viral gene delivery technique with increased synchronization (in terms of time and location) of both compounds, i.e., gene products such as saponin and BNA.

[0160] Gene therapy can help hereditary diseases that were previously incurable, such as cystic fibrosis, chorea, Huntington's disease or hemophilia. However, currently, some problems have not been solved. For example, the therapeutic gene must reach specific target cells in the body accurately. On the other hand, the therapeutic gene must be absorbed by the target cells, but the therapeutic gene must not be destroyed. Current gene therapy is a method that uses viruses as ferries for genes. However, these procedures are associated with significant risks and cannot be diverted to the introduction of other biomolecules. One embodiment is a second conjugate of the present invention comprising a glycoside (e.g., any one of the saponins of the present invention) (derived from plants), which not only enables the delivery of such genes when the gene is included in the first conjugate of the present invention as a carrier molecule, but also enables the delivery of different therapeutic biomolecules to be introduced into target cells. Therefore, the second conjugate of the present invention is used to develop nucleic acid-based therapies for cystic fibrosis, chorea, Huntington's disease or hemophilia. Thereby, a new gene therapy strategy for improving the health of patients with hereditary diseases, including patients with cystic fibrosis, Huntington's disease, and hemophilia, is available using the second conjugate of the present invention. As part of the present invention, a plant-derived endosome escape enhancer (glycoside; i.e., the saponin of the present invention) and a targeting ligand (e.g., an antibody such as sdAb) included in a single second conjugate of the present invention are V HHA non-viral gene delivery technology is developed for combination with the first conjugate of the present invention, which includes cell surface molecule-binding sdAbs and gene therapy products. The non-viral gene therapy obtained based on the first and second conjugates of the present invention shows a delivery efficiency about 40 times higher with the first conjugate containing a lower dose of gene than currently available strategies. Thereby, the second conjugate of the present invention is for clinical use, for example, for the repair or replacement of defective genes such as in patients with cystic fibrosis, and for the targeted delivery of specific genes for destroying cancer cells, etc. In fact, the first and second conjugates of the present invention are suitable for application in the treatment regimens of any diseases caused by genetic defects such as cystic fibrosis, Huntington's disease, and hemophilia, which cannot be currently cured. Gene therapy using the first and second conjugates of the present invention helps to solve the following two current problems. First, the second conjugate of the present invention enables the delivery of the first conjugate containing a therapeutic gene to specific target cells in the body. Second, the therapeutic gene enters the interior of the target cell but is not destroyed because, for example, by using the oligomer or polymer scaffold (the covalent effector molecule conjugate of the present invention) of the present invention as part of the first and second conjugates of the present invention, there are targeting moieties (the second conjugate of the present invention) such as saponin and antibodies or sdAbs for binding to the target cell, and there are targeting sdAbs (the first conjugate of the present invention) for binding to the oligonucleotide product and the target cell.

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

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

[0163] Suitable dosage forms depend on the use or route of administration, such as transdermal or injection. Such dosage forms must allow the compound to reach the target cells, whether the target cells are present in a multicellular host or not. Other factors are known in the art and include considerations such as toxicity and dosage forms that delay 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 Exemplary Embodiments Example 1.V HH -SO1861 + mAb - saporin (1T2C and 2T2C) The 1 - target 2 - component system (1T2C) is V HH -SO1861 and a combination therapy of mAb - protein toxin, VHH and the mAb recognizes and binds to the same cell surface receptor (Figure 1A). The two-target two-component system (2T2C) is a combination therapy of V HH -SO1861 and mAb-protein toxin, and V HH recognizes and binds to a cell surface receptor different from the mAb (Figure 1B). SO1861-EMCH was conjugated to anti-HER2V HH with DAR1 via the terminal cysteine residue (Cys) (unstable) (HER2V HH -SO1861). HER2V HH -SO1861 was titrated against fixed concentrations of 10 pM CD71mab-saporin (CD71 monoclonal antibody conjugated to the protein toxin saporin with DAR4) or 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin saporin with DAR4). Targeted protein toxin-mediated cell death against SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. This revealed enhanced cell death with low concentrations of HER2V HH -SO1861 in combination with both 10 pM CD71mab-saporin or 50 pM trastuzumab-saporin in SK-BR-3 (IC50 = 300 nM; Figure 2A). Equivalent concentrations of HER2V HH -SO1861 alone induced cell death at high concentrations (IC50 = 4,000 nM), but equivalent concentrations of HER2V HH , HER2V HH +CD71mab-saporin, or HER2V HH +trastuzumab-saporin were unable to induce cell death activity (IC50 > 5000 nM; Figure 2A). In MDA-MB-468 (HER2 - / CD71 + ), the combination of HER2V HH -SO1861 + 10 pM CD71mab-saporin showed cell death activity at high concentrations (IC50 = 2,000 nM; Figure 2B), while HER2VHH - The combination of trastuzumab-saporin at -SO1861 + 50 pM showed cell killing activity at much higher concentrations (IC50 > 5,000 nM; Figure 2B). HER2V HH , HER2V HH + CD71mab-saporin or HER2V HH + equivalent concentrations of trastuzumab-saporin were unable to induce cell killing activity in MDA-MB-468 cells (IC50 > 5,000 nM; Figure 2B).

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

[0171] Next, trastuzumab-saporin or CD71mab-saporin was titrated against HER2V HH -SO1861 at a fixed concentration of 900 nM, and targeted protein toxin-mediated cell killing against SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. This revealed that 900 nM HER2V HH -SO1861 combined with low concentrations of trastuzumab-saporin or CD71mab-saporin already induced efficient cell killing of SK-BR-3 (IC50 = 0.0001 pM; Figure 3A), while on the other hand, CD71mab-saporin + 900 nM HER2V HH or trastuzumab-saporin + 900 nM HER2V HH were found to be able to induce cell killing at high concentrations (IC50 = 50 pM; IC50 = 400 pM; Figure 3). In MDA-MB-468 cells (HER2 - / CD71 + ), CD71mab-saporin + 900 nM HER2V HH-SO1861 showed cell killing at IC50 = 0.01 pM, while trastuzumab-saporin + 900 nM HER2V HH -SO1861 showed activity at IC50 = 2,000 pM. Trastuzumab-saporin + 900 nM of HER2V HH or CD71mab-saporin + 900 nM HER2V HH showed cell killing only at (IC50 > 10,000 pM and IC50 = 20 pM, Figure 3B), respectively. All of this indicates that in HER2 ++ / CD71 + expressing cells, relatively low concentrations of trastuzumab-saporin or CD71mab-saporin are effective and can induce cell killing in combination with a relatively low concentration of HER2V HH -SO1861 (DAR1).

[0172] Note: Throughout the description including the examples, and in the claims, "V 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 -dianthin (2T2C) The two-target two-component system (2T2C) is a combination therapy of V HH 1-SO1861 and V HH 2 protein toxins, where each V HH recognizes a different cell surface receptor (Figure 1C). SO1861-EMCH was conjugated to the terminal cysteine residue of V HH targeting HER2 to generate HER2V HH -SO1861 (DAR1). HER2V HH -SO1861 was titrated against CD71V HH -dianthin at a fixed concentration of 50 pM, and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 +) Measured targeted protein toxin-mediated cell death against. As a result, enhanced cell death in HER2-L-SO1861 was revealed (SK-BR-3: IC50 = 300 nM; Figure 4A). The equivalent concentration of HER2V HH -SO1861 alone induced cell death at a high concentration (IC50 = 4,000 nM), but the equivalent concentration of HER2V HH -SO1861, HER2V HH HER2V HH +50 pM CD71V HH -dianthin could not induce cell death (IC50 > 5,000 nM; Figure 4A). In MDA-MB-468 (HER2 - / CD71 + ), the combination of HER2V HH -SO1861 + 50 pM CD71V HH -dianthin showed cell death activity at a high concentration (IC50 = 600 nM; Figure 4B), but the equivalent concentration of HER2V HH HER2V HH -SO1861 or HER2V HH +50 pM CD71V HH -dianthin could not induce cell death activity (IC50 > 5,000 nM; Figure 4B).

[0174] Next, CD71V HH -dianthin was titrated against a fixed concentration of 900 nM HER2V HH -SO1861, and targeted protein toxin-mediated cell death against SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was measured. As a result, 900 nM HER2V HH -SO1861 combined with a low concentration of CD71V HH -dianthin induced efficient cell death of SK-BR-3 cells (IC50 = 0.05 pM; Figure 5A), while on the other hand, CD71V HH dianthin or CD71VH HH -dianthin + 900 nM HER2V HHIt was revealed that it could induce cell death at high concentrations (IC50 > 10,000 pM); Figure 5A). Furthermore, CD71V HH -dianthin was also titrated against trastuzumab-SO1861 (DAR4) at a fixed concentration of 77 nM, which also revealed a strong enhancement of cell death activity in SK-BR-3 (HER2 ++ / CD71 + ) cells ((IC50 < 0.0001 pM)). In MDA-MB-468 cells (HER - / CD71 + ), CD71V HH -dianthin + 900 nM HER2V HH -SO1861 showed cell death at very high concentrations (IC50 = 10 pM, Figure 5B), while CD71V HH -dianthin, CD71V HH -dianthin + 900 nM HER2V HH , or CD71V HH -dianthin + trastuzumab-SO1861 (DAR4) showed cell death at IC50 = 2,000 pM; Figure 5B).

[0175] All of this indicates that at relatively low concentrations, V HH CD71-dianthin is effective in high HER2 / CD71-expressing cells and can induce cell death in combination with low concentrations of V HH HER2-SO1861 conjugate.

[0176] The combination according to the invention showed no cell death activity in MDA-MB-468 cells (HER2 - / CD71 + ). This indicates that without sufficient receptor expression, the SO1861 delivered into the cell does not reach the effective concentration (threshold) that induces endosomal escape and cytoplasmic delivery of the protein toxin.

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

[0178] Dianthin-C (dianthin with a terminal cysteine) was conjugated to the terminal cysteine residues of V HH targeting HER2, V HH targeting CD71, and V HH targeting EGFR to generate HER2V HH -dianthin (DAR1), CD71V HH -dianthin (DAR1), and EGFRV HH -dianthin (DAR1).

[0179] CD71V HH -dianthin, HER2V HH -dianthin, or EGFRV HH -dianthin was titrated against a fixed concentration of cetuximab-SO1861 (DAR4), and target or protein toxin-mediated cell death against A431 (EGFR ++ / HER2 + / - / CD71 + ) and A2058 (EGFR - / HER2 + / - / CD71 + ) was measured. As a result, a very low concentration of CD71V HH -dianthin in combination with 77 nM cetuximab-SO1861 induced efficient cell death of A431 cells (IC50 < 0.0001 pM; Figure 6A), while CD71V HH -dianthin alone showed activity with an IC50 = 2000 pM. EGFRV HH -dianthin + 77 nM cetuximab-SO1861, and HER2V HH-Giantin + 77 nM cetuximab-SO1861 and two other combinations showed efficient cell death with IC50 = 20 pM and IC50 = 50 pM, respectively, but EGFRV HH -Giantin or HER2V HH -Giantin alone was unable to induce efficient cell death in A431 cells (IC50 > 10,000 pM; Figure 6A). In A2058 cells (EGFR - / HER2 + / - / CD71 + ), CD71V HH -Giantin, and CD71V HH -Giantin + 77 nM cetuximab-SO1861 showed cell death activity with IC50 = 3,000 pM and IC50 = 1,000 pM, respectively, but in A2058 cells, none of the other treatments or combinations showed cell death up to an IC50 of 10,000 pM of V HH -toxin (Figure 6B).

[0180] This indicates that cetuximab-SO1861 (DAR4) can efficiently induce endosomal escape of three different V HH -Giantin conjugates, thereby enhancing the induction of cell death in A431 cells.

[0181] Next, CD71V HH -Giantin, HER2V HH -Giantin or EGFRV HH -Giantin was titrated against a fixed concentration of trastuzumab-SO1861 (DAR4), and targeted protein toxin-mediated cell death against SK-BR-3 (HER2 ++ / EGFR = / CD71 + ) and MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + ) was measured. This showed that very low concentrations of CD71V in combination with 77 nM cetuximab-SO1861 HH-Giantin induced efficient cell death of SK-BR-3 cells (IC50 < 0.0001 pM; Figure 7A), whereas CD71V HH -Giantin alone was revealed to be active with an IC50 = 10,000 pM. EGFRV HH -Giantin + 77 nM trastuzumab-SO1861, and HER2V HH -Two other combinations of giantin + 77 nM trastuzumab-SO1861 showed efficient cell death with IC50 = 400 pM and IC50 = 6 pM, respectively, whereas EGFRV HH -Giantin or HER2V HH -Giantin alone was unable to induce efficient cell death in SK-BR-3 cells (IC50 > 10,000 pM; Figure 7A). In MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + ), CD71V HH -Giantin, and CD71V HH -Giantin + 77 nM cetuximab-SO1861 showed cell death activity with IC50 = 3000 pM and IC50 = 2000 pM, respectively, but in MDA-MB-468 cells, all other treatments or combinations showed no cell death up to an IC50 = 10,000 pM of V HH -Giantin (Figure 7B). This indicates that trastuzumab-SO1861 (DAR4) can efficiently induce endosomal escape of three different V HH -Giantin conjugates, thereby enhancing the induction of cell death in SK-BR-3 cells.

[0182] Materials and Methods Materials SO1861 was isolated and purified from a crude plant extract obtained from Saponaria officinalis by Analyticon Discovery GmbH. V 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 (registered trademark), Roche), cetuximab (Cet, Erbitux (registered trademark), Merck KGaA) were purchased from the pharmacy (Charite, Berlin). The CD71 monoclonal antibody was purchased from BioCell (Okt9, #BE0023). Custom trastuzumab-saporin and anti-CD71 mab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA). The dianthin mutant with a single C-terminal cysteine, dianthin-Cys (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 Column (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris Protein Gel (Thermo-Fisher), NuPAGE™ MES SDS Running Buffer (Thermo-Fisher), Novex™ Sharp Pre-Stained Protein Standard (Thermo-Fisher), PageBlue™ Protein Staining Solution (Thermo-Fisher), Pierce™ BCA Protein Assay Kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), 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 dihydrate (99%, Sigma-Aldrich), polyoxyethylene sorbitan monolaurate (TWEEN® 20, Sigma-Aldrich), Dulbecco's Phosphate Buffered Saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetate dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filter 0.2 μm and 0.45 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200 PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio) propionate (PEG4-SPDP, Thermo-Fisher), HSP27 BNA disulfide oligonucleotide (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), deionized water (DI) was freshly taken from Ultrapure Lab Water Systems (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), Sephadex G-25 resin (GE Healthcare) packed PD MiniTrap desalting column, PD10 G25 desalting column (GE Healthcare), Zeba spin desalting column 0.5, 2, 5 and 10 mL (Thermo-Fisher), Vivaspin centrifugal filters T4 10 kDa MWCO, T4 100 kDa MWCO, and T15 (Sartorius), Biosep s3000 aSEC column (Phenomenex), Vivacell ultrafiltration units 10 and 30 kDa MWCO (Sartorius), Nalgene Rapid-Flow filter (Thermo-Fisher).

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

[0185] Cell Viability Assay After treatment, the cells were incubated at 37 °C for 72 h, and then cell viability was determined by MTS assay performed according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Briefly, the MTS solution was diluted 20× with phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS. The cells were washed once with 200 μL of PBS per well and then 100 μL of diluted MTS solution was added per well. The plates were incubated at 37 °C for approximately 20 - 30 min. Subsequently, the OD 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 then the cell viability of treated / untreated cells was calculated (×100) by dividing the background-corrected signal of the treated wells by the background-corrected signal of the untreated wells.

[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% confluence was reached. Next, the cells were trypsinized (TryplE Express, Gibco Thermo Scientific) to single cells. 0.75×10 6 cells were transferred to a 15 mL Falcon tube and centrifuged (1,400 rpm, 3 min). With the cell pellet remaining at the bottom, the supernatant was discarded. The pellet was dissociated by gently tapping the Falcon tube on a vortex shaker, and the cells were washed with 4 mL of cold PBS (Mg 2+ and Ca 2+ free, 2% FBS). After washing, the cells were resuspended in 3 mL of cold PBS (Mg 2+and Ca 2+ resuspended in 2% FBS without Mg and Ca, 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+ (2% FBS), or resuspended in 200 μL of antibody solution containing 5 μL of antibody in 195 μL of cold PBS without Mg 2+ and Ca 2+ (2% FBS). The EGFR receptor was stained using APC mouse IgG1, κ APC anti-human EGFR (#352906, Biolegend). The HER2 receptor was stained using PE anti-human HER2 APC anti-human CD340 (erbB2 / HER-2) (#324408 Biolegend), and PE mouse IgG2a, κ isotype Ctrl FC (#400212, Biolegend) was used as its corresponding isotype control. The CD71 receptor was stained using PE anti-human CD71 (#334106, Biolegend), and PE mouse IgG2a, κ isotype Ctrl FC (#400212, Biolegend) was used as its corresponding isotype control. The samples were incubated at 4 °C for 30 minutes on a tube roller mixer. Then, the cells were washed three times with cold PBS without Mg 2+ and Ca 2+ (2% FBS) and fixed with 2% PFA solution in PBS at room temperature for 20 minutes. The cells were washed twice with cold PBS and resuspended in 250 - 350 μL of cold PBS for FACS analysis. The samples were analyzed using a BD FACSCanto II flow cytometry system (BD Biosciences) and FlowJo software. The analysis results of cell surface expression of EGFR, HER2, and CD71 in various cells are summarized in Table A2.

[0187]

Table 6

[0188] V HH Conjugation procedure of V-SO1861 VHH An aliquot of the sample was added to an aliquot of freshly prepared TCEP solution (10.0 mg / ml), and the mixture was vortexed briefly and then incubated at 20 °C for 30 minutes 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. The resulting V HH -SH was added to freshly prepared SPT-EMCH solution, the mixture was vortexed briefly, and then incubated at 20 °C overnight.

[0189] After incubation, an aliquot of the V HH -SO1861 mixture was taken out, characterized by Ellman assay, and the incorporation of SO1861 was confirmed. This conjugate was purified by a 1.6 × 35 cm Superdex 200 PG column eluting with DPBS (pH 7.5) to obtain purified V HH -SO1861. The aliquot was filtered through 0.2 μm, concentrated, and standardized to 1.0 mg / ml to obtain V HH -SO1861.

[0190] V HH Conjugation procedure of V Dianthin-Cys was concentrated by ultrafiltration using a Vivaspin T15 10KDa MWCO centrifugal filter, and the buffer was exchanged to TBS (pH 7.5). An aliquot of freshly prepared TCEP solution (10.0 mg / ml) was added to the concentrated dianthin-Cys, and the mixture was vortexed briefly and then incubated at 20 °C for 60 minutes with roller mixing. After incubation, the resulting dianthin-SH was purified by gel filtration using a Zeba spin desalting column, and then the centrifugation-washing cycle with TBS (pH 7.5) was repeated using a Vivaspin T15 10KDa MWCO centrifugal filter. The resulting dianthin-SH was reacted with a freshly prepared DTME solution (10 mg / ml) in DMSO, the mixture was vortexed briefly, and then incubated at 20 °C for 60 minutes. Thereafter, it was purified by gel filtration into TBS (pH 7.5) using a Zeba spin desalting column to obtain dianthin-DTME. Dianthin-DTME was stored at 20 °C until conjugation. At the same time, HH an aliquot of V HH was concentrated by ultrafiltration using a Vivaspin T15 10KDa MWCO centrifugal filter, and the buffer was exchanged to TBS (pH 7.5). To the concentrated V HH , an aliquot of freshly prepared TCEP solution (10.0 mg / ml) was added, the mixture was vortexed briefly, and then incubated at 37 °C for 60 minutes with roller mixing. After incubation, the resulting V HH -SH was purified by gel filtration using a Zeba spin desalting column, and then the centrifugation-washing cycle with TBS (pH 7.5) was repeated using a Vivaspin T4 5KDa MWCO centrifugal filter. An aliquot of the resulting V HH -SH was reacted with dianthin-DTME, the mixture was vortexed briefly, and then incubated at 20 °C overnight. Thereafter, the reaction mixture was concentrated using a Vivaspin T4 10KDa MWCO centrifugal tube and purified by gel filtration using a 1.6 × 35 cm Superdex 200PG column and eluted with DPBS (pH 7.5).

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

[0192] To a solution of cetuximab (40 mg, 8.0 ml), 10 μl / ml 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) were added respectively 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 each), an aliquot of freshly prepared TCEP solution (0.5 - 2.0 mg / ml, 1.15 - 7.02 molar equivalents, 75 - 455 nmol) was added, the mixture was vortexed briefly and then incubated at 20 °C for 300 minutes with roller mixing. After incubation (before addition of SO1861-EMCH), an aliquot of approximately 1 mg (0.210 ml) of Ab-SH was taken from each mixture and purified by gel filtration into TBS (pH 7.5) using a zeba spin desalting column. The properties of these aliquots were characterized by UV-vis analysis and Ellman assay (thiol to Ab ratio = 2.0, 4.2, 5.9, and 6.8, respectively). To each of the bulk Ab-SH, 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 minutes. In addition to each conjugation reaction, two aliquots of desalted Ab-SH (0.25 mg, 1.67 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 4.3 - 17.4 nmol, 2.2 - 8.7 μl of a 0.25 mg / ml solution) or TBS (pH 7.5) buffer (2.2 - 8.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), an aliquot of 0.200 ml of Ab-SO1861-EMCH was removed and purified by gel filtration into TBS (pH 7.5) using a Zeba spin desalting column. The properties of this aliquot were characterized by UV-vis, and in parallel, the properties of the positive and negative controls were characterized by Ellman 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 a Zeba spin desalting column eluting with DPBS (pH 7.5) to obtain the purified cetuximab-(L-SO1861) conjugate. This product was standardized to 2.5 mg / ml, filtered through 0.2 μm, and then aliquoted for biological evaluation. The reaction conditions and results for the trastuzumab-L-SO1861 conjugate, and the reaction conditions and results for the cetuximab-L-SO1861 conjugate are summarized in Tables A3 and A4.

[0194]

Table 7

[0195]

Table 8

[0196] Overview of numerous embodiments related to antibodies and receptor ligands covalently linked to protein toxins or saponins mAb: Trastuzumab (HER2) or Cetuximab (EGFR) Ligand: EGF Protein toxin: ribosome-inactivating protein, saporin, dianthin Endosome escape-enhancing conjugate of saponin and ligand: mAb-SO1861 endosome escape-enhancing conjugate · Containing a cleavable hydrazone linker · Trastuzumab-SO1861 DAR 4.0 · Cetuximab-SO1861 DAR 3.7; In in vitro or in vivo test models, the endosome escape-enhancing conjugate of saponin and ligand is combined with the following: mAb / ligand-protein toxin conjugate · Containing a non-cleavable chemical linker or being a recombinant fusion protein · Trastuzumab-saporin DAR 3.0 · Cetuximab-saporin DAR 2.6 · Trastuzumab-dianthin DAR 1.0 · EGF-dianthin (fusion protein) DAR 1.0 · IgG-saporin DAR 2.2

[0197] For Examples 4 - 9: Materials: Trastuzumab and Cetuximab were purchased from the pharmacy (Charite, Berlin). SO1861 was isolated and purified from a crude plant extract obtained from Saponaria officinalis L by Analyticon Discovery GmbH. Methods 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 performed by FleetBioprocessing (UK). SO1861-EMCH was conjugated to the cysteine of the antibody. Conjugation of saporin to trastuzumab and cetuximab Custom trastuzumab-saporin and cetuximab-saporin conjugates were manufactured 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 analyzed by FlowJo V10 software. The FACS antibodies were as follows: 1) Isotype: APC mouse IgG1, κ isotype Ctrl (FC) (400122, Biolegend). EGFR: APC anti-human EGFR (352906, Biolegend) HER2: APC anti-human CD340 (erbB2 / HER-2) (324408, Biolegend). Gianthin production Gianthin was expressed in bacterial cultures and the protein purified according to conventional cell culture and protein purification procedures known in the art. Conjugation of antibodies to gianthin Conjugation of antibodies to gianthin followed general procedures known in the art. Cell culture Cells were cultured at 37 °C and 5% CO2 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 at 100 μL / well in a 96-well plate and incubated overnight at 37°C. The next day, a 10× concentrated treatment mixture sample in PBS containing both SO1861 conjugated to an antibody (i.e., the "binding molecule" or "endosome escape enhancing conjugate" of the present invention) and a targeted toxin (i.e., the "binding molecule") at a 10× final concentration was prepared. 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 the treatment mixture per well. For the control, 10× treatment mixture samples containing only the antibody-conjugated SO1861 at the corresponding concentration, only the antibody, only SO1861, only the targeted toxin, or PBS without compound as a vehicle control were prepared. When using an endosome acidification inhibitor (chloroquine (Sigma Aldrich) or bafilomycin A1 (Enzo Life Sciences)), 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. After incubating the plate at 37°C for 1 hour, the 10× treatment mixture sample was added. The remaining incubation and treatment steps were performed according to standard procedures known in the art.

[0198] After treatment, the cells were incubated at 37 °C for 72 hours, and then cell viability was determined by an MTS assay performed according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Briefly, the MTS solution was diluted 20× with phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS. The cells were washed once with 200 μL of PBS per well and then 100 μL of the diluted MTS solution was added per well. The plate was incubated at 37 °C for approximately 20 - 30 minutes. Subsequently, the 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 then the ratio of treated / untreated cells was calculated by dividing the background-corrected signal of the treated wells by the background-corrected signal of the untreated wells.

[0199] Results Example 4.1 Target two-component system The one-target two-component system is a combination treatment of mAb1-protein toxin and mAb1-SO1861 (see FIGS. 1A, E), and the two-target two-component system is a combination of mAb1-protein toxin and mAb2-SO1861, or mAb2-protein toxin and mAb1-SO1861 (FIGS. 1B - D).

[0200] Cetuximab-SO1861 (a monoclonal antibody that recognizes and binds to EGFR conjugated to the saponin molecule SO1861; an endosome escape-enhanced conjugate) was titrated against a fixed concentration of 10 pM cetuximab-saporin (a protein toxin, a monoclonal antibody that recognizes and binds to EGFR conjugated to 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 a high concentration 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; Figures 8A, 8B; Table A6). This indicates that the targeted conjugate SO1861 is more effective in inducing endosome escape compared to non-targeted unconjugated SO1861. Next, cetuximab-saporin was titrated against a fixed concentration of 300 nM cetuximab-SO1861, and targeted protein toxin-mediated cell killing against EGFR-expressing cells was measured. High EGFR-expressing cells (A431 or CaSKi) showed cell killing only when combined with non-targeted unconjugated 300 nM SO1861 at high concentrations of cetuximab-saporin (A431: [toxin] IC50 = 40 pM; CaSki: [toxin] IC50 = 40 pM; Figures 8C, 8D; Table A7), while 300 nM cetuximab-SO1861 combined with low concentrations of cetuximab-saporin concentration already induced efficient cell killing (A431: [toxin] IC50 = 0.4 pM; CaSKi: [toxin] IC50 = 2 pM; Figures 8C and 8D; Table A7).When high concentrations of non-targeted unconjugated SO1861 (1500 nM) are combined with low concentrations of cetuximab-saporin, the highest cell killing efficiency is achieved (A431: [toxin] IC50 = 0.03 pM; CaSki: [toxin] IC50 = 0.02 pM; FIGS. 8C, 8D; Table A7). All of this indicates that when relatively low concentrations of SO1861 are conjugated to cetuximab, in combination with relatively low concentrations of cetuximab-saporin, it is possible to efficiently kill high EGFR-expressing cells. High concentrations (1500 nM) of non-targeted unconjugated SO1861 combined with low concentrations of cetuximab-saporin are the most effective in a one-target two-component system because in this case, the two conjugates compete for the same EGFR receptor, and receptor competition does not play a role in the entry of SO1861 into cells. The receptor competition principle is also clearly shown by titration of cetuximab-toxin, with or without 75 nM cetuximab (A431: [toxin] IC50 = 40 pM; CaSki: [toxin] IC50 = 40 pM) or (A431: [toxin] IC50 = 1000 pM; Caski: IC50 = 1000 pM; FIGS. 8C, 8D).

[0201] Next, cetuximab-SO1861 was titrated against cetuximab-saporin at a fixed concentration of 10 pM (calculated at the concentration of SO1861), and cell killing against low / non-EGFR expressing cells was measured. Low EGFR-expressing cells (HeLa) showed cell killing only when 10 pM of cetuximab-saporin was combined with a high concentration of non-targeted unconjugated SO1861, while A2058 cells that do not express EGFR (A2058) were completely insensitive (HeLa: [SO1861] IC50 = 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figures 9A, 9B; Table A6). The combination of 10 pM of cetuximab-saporin and cetuximab-SO1861 with increasing concentrations did not induce significant cell killing in both cell lines (HeLa: [SO1861] IC50 = 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figures 9A, 9B; Table A6). This indicates that in the absence of sufficient receptor expression, intracellular SO1861 does not reach the effective concentration (threshold) that induces endosomal escape of the protein toxin and toxin-mediated cell killing. Next, cetuximab-saporin was titrated against cetuximab-SO1861 at a fixed concentration of 300 nM, and targeted protein toxin-mediated cell killing against low / non-EGFR expressing cells was measured. Low EGFR-expressing cells (HeLa) showed cell killing only when a very high concentration of cetuximab-saporin was combined with 278 nM of cetuximab-SO1861 or 300 nM of unconjugated SO1861, while A2058 cells (EGFR) were insensitive at the tested concentrations (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) combined with low concentrations of cetuximab-saporin showed efficient cell death, while in A2058 cells, only very high concentrations of cetuximab-saporin combined with 1500 nM of non-targeted SO1861 induced specific cell death (Hela: [toxin] IC50 = 0.03 pM; A2058: [toxin] IC50 = 20 pM; Figures 9C, 9D; Table A6). All of this indicates that cells with low or no EGFR receptor expression are not sensitive to the combination of cetuximab-SO1861 + cetuximab-saporin because they lack sufficient EGFR receptors to facilitate sufficient entry of SO1861 and the toxin into the cell.

[0202] Trastuzumab-SO1861 (a monoclonal antibody that recognizes and binds to HER2 conjugated to the saponin molecule SO1861; an endosome escape-enhanced conjugate according to the present invention) was titrated against trastuzumab-saporin (a protein toxin, a monoclonal antibody that recognizes and binds to HER2 conjugated to saporin) at a fixed concentration of 50 pM (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 of trastuzumab-saporin was combined with a high concentration of non-targeted unconjugated SO1861 (SKBR3; Figures 10A, 10B; Table A6). However, when trastuzumab-saporin was combined with trastuzumab-SO1861, potent cell killing was already induced at low concentrations of SO1861 (SKBR3; Figures 10A, 10B; Table A6). This indicates that the targeted conjugate SO1861 is more effective in inducing endosome escape compared to non-targeted unconjugated SO1861. Next, trastuzumab-saporin was titrated against trastuzumab-SO1861 at a fixed concentration of 50 nM, and targeted protein toxin-mediated cell killing against HER2-expressing cells was measured. High HER2-expressing cells (SKBR3 or BT474) showed cell killing only when a high concentration of trastuzumab-saporin was combined with non-targeted unconjugated 10 nM SO1861 (Table A7), while 10 nM trastuzumab-SO1861 combined with a low concentration of trastuzumab-saporin already induced efficient cell killing (Table A7). The highest cell killing efficiency was achieved when a high concentration of non-targeted unconjugated SO1861 (1500 nM) was combined with a low concentration of trastuzumab-saporin (Table A7). All of this indicates that when low concentrations of SO1861 are conjugated to trastuzumab, it can efficiently kill high HER2-expressing cells in combination with a relatively low concentration of trastuzumab-saporin.High concentration (1500 nM) of non-targeted unconjugated SO1861 in combination with low concentration of trastuzumab-saporin, in a one-target two-component system, the receptor competition does not play a role in the entry of SO1861 into cells because both conjugates compete for the same EGFR receptor, and thus it remains the most effective. The receptor competition principle is also clearly shown by the titration of trastuzumab toxin regardless of the presence or absence of 2.5 nM trastuzumab (SKBR3: [toxin] IC50 = 1000 nM).

[0203] Next, trastuzumab-SO1861 was titrated against trastuzumab-saporin at a fixed concentration of 50 pM (calculated at the concentration of SO1861), and cell killing against low / non-EGFR-expressing cells was measured. Low EGFR-expressing cells (JIMT1; A431) showed cell killing only when 50 pM of trastuzumab-saporin was combined with a high concentration of non-targeted unconjugated SO1861 (JIMT1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figures 11A, 11B; Table A6). The combination of 50 pM of trastuzumab-saporin and increasing concentrations of trastuzumab-SO1861 did not induce significant cell killing in either cell line (JIMT1: [SO1861] IC50 > 1000 nM; A431: [SO1861] IC50 > 1000 nM; Figures 11A, 11B; Table A6). This indicates that in the absence of sufficient receptor expression, intracellular SO1861 does not reach the effective concentration (threshold) to induce endosomal escape of the protein toxin and toxin-mediated cell killing. Next, trastuzumab-saporin was titrated against 10 nM of trastuzumab-SO1861 at a fixed concentration, and targeted protein toxin-mediated cell killing against low / non-HER2-expressing cells was measured. Low HER2-expressing cells (JIMT1; A431) did not show significant cell killing with high concentrations of trastuzumab-saporin combined with 10 nM of 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 (1500 nM) combined with low concentrations of trastuzumab-saporin showed efficient cell killing (JIMT1: [toxin] IC50 = 0.1 pM; A431: [toxin] IC50 = 0.8 pM; Figures 11C, 11D; Table A5). All of this indicates that cells with low HER2 receptor expression are not sensitive to the combination of trastuzumab-SO1861 + trastuzumab-saporin because they lack sufficient HER2 receptors to promote sufficient entry of SO1861 and the toxin into the cell.

[0204] Example 5.2 Target two-component system Cetuximab-SO1861 was titrated against trastuzumab-saporin at a fixed concentration of 50 pM (calculated at the concentration of SO1861), and cell killing against high EGFR / low HER2-expressing cells was measured. A431 and CaSki cells showed efficient cell killing when 50 pM of 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 than the non-targeted unconjugated SO1861 in inducing endosomal escape. Next, trastuzumab-saporin was titrated against cetuximab-SO1861 at a fixed concentration of 300 nM, and targeted protein toxin-mediated cell killing against high EGFR / low HER2-expressing cells (A431 and CaSki) was measured. No efficient cell killing was observed with high concentrations of trastuzumab-saporin combined with 300 nM of non-targeted unconjugated SO1861 (A431 and Caski:: [toxin]IC50 > 10,000 pM; Figures 12C, 12D; Table A7), while 300 nM of cetuximab-SO1861 combined 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 was achieved when high concentrations (1500 nM) of non-targeted unconjugated SO1861 were combined with low concentrations of cetuximab-saporin (A431: [toxin]IC50 = 1 pM; Figure 12C; Table A7).In Caski cells, the EGFR expression in these cells was significantly lower compared to A431, and thus, due to the fact that the targeted delivery of SO1861 to Caski cells was not very sufficient, the response was slightly stronger compared to the combination of cetuximab-SO1861 and trastuzumab-saporin (Caski: [toxin] IC50 = 0.2 pM; see Figure 12D; Table A7). All of this indicates that when low concentrations of SO1861 are conjugated to cetuximab, in combination with relatively low concentrations of trastuzumab-saporin, it can efficiently kill high EGFR-expressing cells. High concentrations (1500 nM) of non-targeted non-conjugated SO1861 in combination with low concentrations of trastuzumab-saporin, in a two-target two-component system, where both conjugates are delivered by different receptors, i.e., SO1861 is delivered by EGFR and the toxin is delivered by the HER2 receptor, receptor competition does not play a role in the entry of SO1861 into cells, and thus, it has equivalent activity.

[0205] Next, cetuximab-SO1861 was titrated against trastuzumab-saporin at a fixed concentration of 50 pM (calculated at the concentration of SO1861), and cell killing against low / non-EGFR / HER2-expressing cells was measured. Low EGFR / HER2-expressing cells showed cell killing only when 50 pM of trastuzumab-saporin was combined with high concentrations of non-targeted unconjugated SO1861 (HeLa: [SO1861] IC50 > 1000 nM; A2058: [SO1861] IC50 > 1000 nM; Figure 13A, 13B; Table A6). The combination of 50 pM of trastuzumab-saporin and 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 in the absence of sufficient receptor expression, intracellular SO1861 does not reach the effective concentration (threshold) that induces endosomal escape of the protein toxin and toxin-mediated cell killing. Next, trastuzumab-saporin was titrated against a fixed concentration of cetuximab-SO1861, and targeted protein toxin-mediated cell killing against low / non-EGFR / HER2-expressing cells was measured. Low / non-EGFR / HER2-expressing cells (HeLa and A2058) did not show significant cell killing at high concentrations of trastuzumab-saporin combined with 278 nM of cetuximab-SO1861 (HeLa: [toxin] IC50 > 10,000 pM; A2058: [toxin] IC50 > 10,000 pM; Figures 13C, 13D; Table A7). High concentrations of unconjugated SO1861 (1500 nM) combined with low concentrations of trastuzumab-saporin showed efficient cell killing (HeLa: [toxin] IC50 = 0.4 pM; A2058: [toxin] IC50 = 0.5 pM; Figures 13C, 13D; Table A5). All of this indicates that cells with low / non-EGFR / low HER2 expression are not sensitive to the combination of cetuximab-SO1861 + trastuzumab-saporin because they lack sufficient EGFR receptors to promote sufficient entry of SO1861 to ensure efficient cytoplasmic delivery of the toxin intracellularly.

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

[0207] Next, trastuzumab-SO1861 was tit...

Claims

1. Kit includes: a) a first pharmaceutical composition comprising a first conjugate comprising at least one effector molecule and an antibody 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, and optionally comprising a pharma- ceutically acceptable excipient and / or a pharma-ceutically acceptable diluent; and b) a second pharmaceutical composition comprising: i. a second conjugate comprising an antibody for binding to a second cell surface molecule and a saponin, said antibody and said saponin being covalently bound to each other directly or via a linker, said second cell surface molecule being the same or different from said first cell surface molecule, and when said first and second cell surface molecules are different, said first and second cell surface molecules are present on the same target cell, and optionally comprising a pharma- ceutically acceptable excipient or / and a pharma-ceutically acceptable diluent; or ii. Free saponin or saponin derivatives.

2. The antibody may be an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V Ｈ Domains, Single Domain Antibodies, V ＨＨ , or Camelidae V Ｈ The kit of claim 1 , comprising:

3. The antibody has a V derived from a heavy chain of an antibody or of immunoglobulin G origin or of human origin. Ｈ Domain; V derived from the light chain of an antibody or of immunoglobulin G origin or of human origin Ｌ Domain; V from heavy chain only antibody (HCAb) ＨＨ domain, or a novel antigen receptor of Camelidae origin, or of Ig-NAR origin, or of variable heavy chain (V ＮＡＲ ) domain derived from a heavy chain-only antibody (HCAb) of Ig-NAR origin ＨＨ domain, or any one or more of said HCAbs from Camelidae origin, and / or said at least one sdAb is any one or more of said HCAbs from Camelidae origin (Camelidae V Ｈ ) V derived from HCAb ＨＨ Domains or V derived from HCAbs from camel, llama, alpaca, dromedary, vicuna, guanaco and Bactrian camel ＨＨ The kit according to claim 1 or 2, which is a domain.

4. The kit according to any one of claims 1 to 3, wherein the natural, synthetic or modified oligonucleotide is selected from short interfering RNA (siRNA) and antisense oligonucleotide (ASO).

5. 5. The kit of any one of claims 1 to 4, wherein the natural, synthetic or modified oligonucleotide is capable of silencing a gene selected from 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), Hepatitis B virus (HBV) X gene, HBV S gene, alpha-1 antitrypsin (AAT) and lactate dehydrogenase (LDH).

6. The kit according to any one of claims 1 to 5, wherein the natural, synthetic or modified oligonucleotide is capable of targeting 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.

7. The kit of any one of claims 1 to 6, wherein the saponin comprises an aglycone core structure that is quillaric acid or an aglycone core structure selected from quillaric acid and gypsogenin.

8. The saponin comprises an aglycone core structure which is quillaric acid or an aglycone core structure selected from quillaric acid and gypsogenin, and the saponin comprises a C of the aglycone core structure of the saponin. ３ Atom or C ２８ and optionally a first glycan bonded to an atom C of said aglycone core structure. ２８ a second glycan linked to position A; The first sugar chain is 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-, and Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA- is selected from and / or The second sugar chain is Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, 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-, wherein R3 is 4E-methoxycinnamic acid; Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-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-, where 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-, where 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-, where 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-, where R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and 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. The kit according to any one of claims 1 to 7, wherein the kit is selected from the group consisting of

9. The kit according to any one of claims 1 to 8, wherein the saponin is SO1861.

10. The kit according to any one of claims 1 to 9, wherein the antibody is capable of binding to a cell surface receptor of a target cell.

11. The first cell surface molecule and / or the second cell surface molecule of the target cell are CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, 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, CD30, CD239, CD70, CD123, CD35 2, 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), and glucocorticoid-inducible TNFR-related protein (GITR).

12. The kit of any one of claims 1 to 11, wherein the first cell surface molecule and / or the second cell surface molecule of the target cell is selected from HER2, c-Met, VEGFR2, CXCR7, CD71, and EGFR1.

13. The kit of any one of claims 1 to 12, wherein the antibody binds to CD71.

14. The saponin is a C-type saponin comprising the aglycone core structure of the saponin. ２３ 8. The kit of claim 7, wherein the carboxyl group is covalently attached via a linker N-ε-maleimidocaproic acid hydrazide (EMCH) which is covalently attached to the aldehyde group at position 1.

15. The kit of any one of claims 1 to 14, wherein the saponin is covalently attached via a cleavable linker.

16. The kit of claim 15 , wherein the cleavable linker is cleaved under acidic, reducing, enzymatic and / or light-induced conditions.

17. 17. The kit of claim 15 or 16, 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.

18. The kit according to any one of claims 15 to 17, wherein the cleavable linker is cleaved in vivo under acidic conditions.

19. 19. The kit of claim 17 or 18, wherein the cleavable linker is cleaved at a pH of ≦5.

5.

20. A kit according to any one of claims 1 to 19, comprising said first and second conjugates as medically active ingredients.

21. 21. The kit of claim 20 for the treatment of a human disease selected from any one or more of cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, gene deficiency, diseases associated with gene deficiency, amyloidosis, diseases associated with enzyme deficiency, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin associated liver disease, acute hepatic porphyria and transthyretin mediated amyloidosis.

22. 22. The kit of claim 20 or 21, wherein the saponin is SO1861.

23. The kit of any one of claims 20 to 22, wherein the antibody binds to CD71.

24. The kit according to any one of claims 20 to 23, wherein the natural, synthetic or modified oligonucleotide is selected from short interfering RNA (siRNA) and antisense oligonucleotide (ASO).

25. An in vitro method for transferring the first conjugate according to any one of claims 1 to 19 from outside a cell to inside the cell, comprising: a) providing a cell expressing on its surface the first cell surface molecule of any one of claims 1 to 19 and optionally expressing on its surface the second cell surface molecule of any one of claims 1 to 19; b) providing said first conjugate according to any one of claims 1 to 19 for transfer to said cells provided in step a); c) providing the second conjugate of any one of claims 1 to 19; d) contacting the cells of step a) in vitro with the first conjugate of step b) and with the second conjugate of step c); thereby establishing the transfer of the first conjugate from outside the cell into the cell.

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