Conjugate of single domain antibody, saponin and effector molecule, pharmaceutical composition comprising the same, therapeutic use of said pharmaceutical composition

Abstract

To provide a conjugate for transferring an effector molecule from outside a cell into said cell.SOLUTION: Provided is a conjugate comprising an effector molecule, a cell surface molecule targeting antibody, and a saponin. Therein: the effector molecule, the antibody, and the saponin are covalently bound to each other; the effector molecule is an oligonucleotide; the saponin is a bi-desmosidic triterpene glycoside comprising an aglycone core structure selected from quillaic acid or gypsogenin and comprising a first glycan bound to a C3 atom of the aglycone core structure and a second glycan bound to a C28 atom of the aglycone core structure; the first glycan is branched and comprising a glucuronic acid unit; the second glycan is branched and comprising a carbohydrate unit selected from any one or more of fucose (Fuc), rhamnose (Rha), and quinovose (Qui); and the antibody is capable of binding to a cell surface molecule of the cell.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a conjugate for transferring an effector molecule from outside a cell into the cell, comprising at least one effector molecule to be transferred into the cell, which is covalently bonded to each other, at least one saponin of the mono-dodesmoside triterpenoid glycoside type or the bi-dodesmoside triterpenoid glycoside type, and at least one single domain antibody (sdAb), wherein the sdAb can bind to a cell surface molecule of the cell. The present invention also relates to a pharmaceutical composition comprising the conjugate of the present invention. Furthermore, the present invention relates to the pharmaceutical composition of the present invention for use as a medicament. In addition, the present invention relates to the pharmaceutical composition of the present invention for use in any one or more of the following treatments or prevention of onset: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, diseases related to enzyme deficiency, gene deficiency, diseases related to gene deficiency, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, amyloidosis and transthyretin-mediated amyloidosis. The present invention also relates to an in vitro or in vivo method for transferring the conjugate from outside the cell into the interior of the cell or for transferring the effector molecule contained by the conjugate of the present invention from outside the cell into the interior of the cell, preferably into the cytosol of the cell.

Background Art

[0002] Molecules with therapeutic bioactivity are often, in theory, suitable for application as effective therapeutic agents for the treatment of diseases such as cancer in human patients in need. A typical example is a small molecule bioactive moiety. However, many of the promising drug-like molecules and therapeutic agents currently used in the clinic have at least one of many drawbacks and deficiencies, if not all. When administered to the human body, therapeutic active molecules may exhibit off-target effects in addition to the desired bioactivity related to the treatment of a disease or health problem. Such off-target effects are undesirable and carry the risk of inducing side effects that can endanger the health or life of the administered molecule. The occurrence of such adverse events causes many drug-like compounds and therapeutic moieties to fail in Phase III or Phase IV clinical trials (post-marketing investigational surveillance). Therefore, it is highly desirable to provide drug molecules such as small molecule therapeutic agents, and the therapeutic effects of the drug molecules should, inter alia, (1) be highly specific for a disease-promoting biological factor or biological process, (2) be sufficiently safe, (3) be sufficiently effective, (4) be sufficiently induced in diseased cells with little or no off-target activity against non-diseased cells, (5) have a well-timed mechanism of action (e.g., the administered drug molecule needs to reach the targeted site in a human patient within a certain time frame and remain at the targeted site within a certain time frame), and / or (6) have a sufficiently persistent therapeutic activity in the patient's body. Unfortunately, to date, "ideal" therapeutic agents having many or even all of the advantageous features outlined above herein are not available to patients, despite extensive and intensive investigations over a long period of time and impressive progress made in several areas of the difficulties and deficiencies addressed individually.

[0003] Chemotherapy is one of the most important treatment options for cancer treatment. However, it often has a narrow therapeutic window because it lacks specificity for cancer cells over dividing cells in healthy tissues. The invention of monoclonal antibodies has provided the possibility as a mechanism for targeting and delivering cytotoxic agents to cancer cells while sparing normal cells by utilizing their specific binding properties. 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). Usually, very potent payloads such as emtansine (DM1), which has a limited therapeutic index (the ratio comparing toxic dose to effective dose) in its unconjugated form, are used. Conjugation of DM1 to trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla, increases the tolerated dose of DM1 by at least twofold in monkeys. Enormous efforts and investments have been made in the past decades to develop therapeutic ADCs. However, despite promising preclinical data, it has remained difficult to bring ADCs to the clinic. The first ADC approved for clinical use was gemtuzumab ozogamicin (Mylotarg, CD33-targeted, Pfizer / Wyeth) for relapsed acute myeloid leukemia (AML) in 2000. However, Mylotarg was withdrawn from the market at the request of the Federal Drug Administration (FDA) due to several concerns including its safety profile. Patients treated with Mylotarg were found to die more often than those treated with conventional chemotherapy. Mylotarg was re-approved in 2017 with a reduced recommended dose, different schedules in combination with chemotherapy or as a single agent, and in a new patient population. To date, only five ADCs have been approved for clinical use, while the clinical development of approximately 55 ADCs has been discontinued. However, the interest remains high, and currently approximately 80 ADCs are still in clinical development in nearly 600 clinical trials.

[0004] Despite the potential to use toxic payloads that are not normally tolerated by patients, the low therapeutic index is a major problem accounting for 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 premature release of drugs in the bloodstream. The systematic re-evaluation of ADCs by the FDA found that the toxicity profiles of most ADCs can be classified according to the payload used rather than the antibody used, suggesting that toxicity is largely determined by the premature release of the payload. Of the approximately 55 ADCs that have been discontinued, at least 23 are presumed to have been due to an inadequate therapeutic index. For example, the development of trastuzumab tesirine conjugate (ADCT-502, HER2-targeted, ADC therapeutic) was recently discontinued due to a low therapeutic index, presumably resulting from an on-target, off-tissue effect in lung tissue expressing significant levels of HER2. In addition, several ADCs in phase 3 trials have been discontinued due to the lack of a primary evaluation item. For example, the phase 3 trials of depatuxizumab mafodotin conjugate (ABT-414, EGFR-targeted, AbbVie) tested in patients with newly diagnosed glioblastoma, and mirvetuximab soravtansine conjugate (IMGN853, folate receptor alpha (FRα)-targeted, ImmunoGen) tested in patients with platinum-resistant ovarian cancer were recently discontinued, indicating no survival benefit. It is important to recognize that some clinically available doses of 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 mainly consists of an antibody, a cytotoxic moiety such as a payload, and a linker. Several novel strategies have been proposed and implemented in the design and development of new ADCs to overcome the existing problems targeting each of the ADC components. For example, by identifying and validating sufficient antigen 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 to be accessible to circulating ADCs, and antigens that enable internalization of the ADC into cells after binding, and by selecting alternative mechanisms of activity, designing and optimizing linkers that enhance the solubility and drug-to-antibody ratio (DAR) of the ADC, overcoming resistance induced by proteins capable of transporting chemotherapeutic agents extracellularly, increasing the DAR ratio by inclusion of additional payloads, and selecting and optimizing antibodies to improve antibody homogeneity and developability. In addition to the technical development of ADCs, new clinical and translational strategies such as changing the dosing schedule by fractionated administration, conducting biodistribution studies, capturing response signals early, monitoring the duration and depth of response, and including biomarkers to optimize patient selection for informing combination trials are also being adopted to maximize the therapeutic index.

[0006] Examples of ADCs with clinical potential are ADCs such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, which are 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, have been tested in clinical trials, and the ADCs have been combined, respectively, with rituximab, a monoclonal antibody that binds to CD20, which is co-administered and not provided with the payload [B. Yu and D. Liu, Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12:94]. Combinations of monoclonal antibodies such as these examples are further approaches and attempts to reach the "magic bullet" that combines many or all of the aforementioned desirable features 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 deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) for approaches such as gene therapy, RNA interference (RNAi), antisense oligonucleotides (ASO, AON), and short interfering RNA (siRNA), microRNA, as well as those based on DNA and RNA aptamers. Many of them share the same basic basis of action by inhibiting either DNA or RNA expression, thereby preventing the expression of abnormal proteins associated with diseases. The most clinical trials have been conducted in the field of gene therapy, and nearly 2,600 clinical trials are ongoing or completed worldwide, but only about 4% have entered Phase 3. This is followed by clinical trials with ASO. Similar to ADC, despite numerous approaches being explored, therapeutic nucleic acids share two major problems during clinical development: delivery to cells and off-target effects. For example, ASOs such as peptide nucleic acid (PNA), phosphoramidate morpholino oligomers (PMO), locked nucleic acid (LNA), and bridged nucleic acid (BNA) have been investigated as attractive strategies for specifically inhibiting target genes and inhibiting genes that are particularly difficult to target with small molecule inhibitors or neutralizing antibodies. Currently, the effectiveness of different ASOs is also being tested in many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, as well as in some cancer stages. The application of ASO as a potential therapeutic agent requires a safe and effective method for their delivery to the cytoplasm and / or nucleus of target cells and tissues. Although the clinical significance of ASO has been demonstrated, inefficient cellular uptake both in vitro and in vivo limits the effectiveness of ASO and poses a barrier to therapeutic drug development. Cellular uptake is less than 2% of the dose, and the ASO concentration at the active site is too low to obtain effective and sustained outcomes. This consequently requires an increase in the administered dose to induce off-target effects. The most common side effects are activation of the complement cascade, inhibition of the coagulation cascade, and toll-like receptor-mediated stimulation of the immune system.

[0008] Chemotherapy is the most common small molecule, but their effectiveness is hampered by severe off-target side effect toxicities, as well as their poor solubility, rapid clearance and limited tumor exposure. Scaffold-small molecule drug conjugates, such as polymer-drug conjugates (PDCs), are macromolecular constructs with pharmacological activity and contain one or more molecules of a small molecule drug conjugated to a carrier scaffold (e.g., polyethylene glycol (PEG)).

[0009] Such conjugate principles have been under attention and investigation for decades. Most of the conjugates of small molecule drugs under preclinical or clinical development are for oncology efficacy. However, to date, only one drug not related to cancer has been approved in 2014 for opioid-induced constipation in patients with chronic pain (Movantik, a PEG oligomer conjugate of the opioid antagonist naloxone, AstraZeneca), which is a non-oncology efficacy. The translational application of drug-scaffold conjugates to the treatment of human subjects has been largely clinically unsuccessful to date. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) showed excellent anti-cancer activity in both solid tumors and leukemia in a mouse model and was in clinical trials for oncology efficacy. Despite demonstrating a significant reduction in non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer activity in patients was found to be minimal, and as a result, further development of PK1 was discontinued.

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

[0011] A potential solution to the aforementioned problem is the application of nanoparticle systems for drug delivery, such as liposomes. Liposomes are spherical vesicles composed of one or more phospholipid bilayers and are spontaneously formed when phospholipids are dispersed in water. The amphiphilic nature of phospholipids confers upon them self - organizing properties, as well as emulsifying and wetting properties, which can be exploited in the design of new drugs and new drug delivery systems. Encapsulation of drugs in liposome delivery systems can offer several advantages compared to the direct administration of drugs, such as improvements and control over pharmacokinetics and pharmacodynamics, tissue - targeting properties, reduced toxicity, and enhanced drug activity. Such success stories are the liposome - encapsulated forms of the small - molecule chemotherapeutic agent doxorubicin (Doxil: pegylated liposome - encapsulated form of doxorubicin; Myocet: non - pegylated liposome doxorubicin), which are approved for clinical use.

[0012] Therefore, there remains a need to find solutions that enable drug therapies, such as anti - tumor therapies, which are applicable for non - systemic use when desired, and the drugs have, for example, an acceptable safety profile, low off - target activity, sufficient efficacy, a sufficiently low clearance rate from the patient's body, and a sufficiently broad therapeutic window.

[0013] European Patent No. 1623715 B1 describes a composition comprising a pharmacologically active agent coupled to a target cell - specific binding molecule combined with a saponin. The pharmacologically active agent is, for example, a poison. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0014] Regarding embodiments of the present invention, a first objective is to provide an improved bioactive compound or a composition comprising such an improved bioactive compound.

[0015] One of several objectives of embodiments of the present invention is to provide a solution to the problem of non-specificity encountered when administering a (small molecule) therapeutically active compound to a human patient in need thereof. One of several objectives of embodiments of the present invention is to provide a solution to the problem of drugs having specificity that is not optimized for a biological factor or biological process that promotes a disease. One of several objectives of embodiments of the present invention is to provide a solution to the problem of the inadequate safety profile of current drugs when administered to a human patient in need thereof. One of several objectives of embodiments of the present invention is to provide a solution to the problem of current drugs being less effective than desired when administered to a human patient in need thereof. One of several objectives of embodiments of the present invention is to provide a solution to the problem of current drugs not being sufficiently induced in diseased cells with little or no off-target activity against non-diseased cells when administered to a human patient in need thereof. One of several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs do not have a well-timed mechanism of action when administered to a human patient in need thereof (e.g., the administered drug molecule needs to reach the targeted site in a human patient within a certain time frame and remain at the targeted site within a certain time frame). One of several objectives of embodiments of the present invention is to provide a solution to the problem that current drugs do not have sufficient sustained therapeutic activity in the body of a patient when administered to a human patient in need thereof.

[0016] At least one of the above objectives of embodiments of the present invention is, in accordance with the present invention, V HHA cell-targeting moiety that is a single-domain antibody (sdAb) such as, and at least one effector moiety such as at least one saponin and a proteinaceous toxin and / or an oligonucleotide such as BNA, an antibody-drug conjugate (ADC) or an antibody-oligonucleotide (AOC) such as an antibody-BNA covalent conjugate provided with a covalently linked saponin, including an AOC provided with a covalently linked saponin that is also suitable for use as an ADC and / or a medicament provided with a covalently linked saponin. This is achieved by providing an

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

[0018] A first aspect of the present invention is a conjugate for transferring an effector molecule from outside the cell into the cell, comprising at least one effector molecule to be transferred into the cell, at least one single-domain antibody (sdAb), and at least one saponin, which are covalently bonded to each other directly or via at least one linker, wherein at least one saponin is a mono-desposide triterpenoid glycoside or a bi-desposide triterpenoid glycoside, and the sdAb is capable of binding to a cell surface molecule of the cell.

[0019] A second aspect of the present invention relates to a pharmaceutical composition comprising the conjugate of the present invention, and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0020] A third aspect of the present invention relates to the pharmaceutical composition of the present invention for use as a medicament.

[0021] The fourth aspect of the present invention relates to the pharmaceutical composition of the present invention for use in any one or more of the following treatments or prevention of onset: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency disorders, diseases associated with enzyme deficiency disorders, gene deficiencies, diseases associated with gene deficiencies, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, amyloidosis, and transthyretin-mediated amyloidosis.

[0022] The fifth aspect of the present invention is a method in vitro or ex vivo for transferring an effector molecule of the present invention (the effector molecule contained by the conjugate of the present invention) from outside the cell into the interior of the cell, preferably into the cytosol of the cell, comprising: a) providing, on the cell surface, a binding site for at least one sdAb contained by the conjugate of the present invention, wherein the binding site is preferably present on a cell surface molecule of the cell as described herein, and the cell preferably expresses a binding site selected from hepatocytes, abnormal cells such as virus-infected cells, autoimmune cells, cells containing gene deficiencies, cells containing enzyme deficiencies, and tumor cells; b) providing the conjugate of the present invention, which contains an effector molecule to be transferred to the cell provided in step a); and c) contacting the cell of step a) with the conjugate of step b) in vitro or ex vivo, thereby including the step of transferring the conjugate containing the effector molecule from outside the cell into the interior of the cell, and the step of transferring the effector molecule from outside the cell into the interior of the cell, preferably into the cytosol of the cell, by the transfer of the conjugate.

[0023] The sixth aspect of the present invention is a method in vitro or ex vivo for transferring the conjugate of the present invention from outside the cell into the interior of the cell, comprising: a) On the cell surface, a binding site for at least one sdAb comprised by the conjugate of the present invention, wherein the binding site is preferably present on a cell surface molecule of the cell as described herein, and the cell preferably expresses a binding site selected from liver cells, abnormal cells such as virus-infected cells, autoimmune cells, cells containing gene defects, cells containing enzyme defects, and tumor cells; b) Providing any one conjugate of the present invention; and c) A method comprising contacting the cells of step a) with the conjugate of step b) in vitro or ex vivo, thereby including the step of transferring the conjugate from outside the cell into the interior of the cell.

[0024] Aspects of the present invention relate to an instruction for use of the conjugate or the pharmaceutical composition of the present invention for the treatment or prevention of onset of any one or more of the following: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency disorders, diseases associated with enzyme deficiency disorders, gene defects, diseases associated with gene defects, infections such as virus infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver diseases, acute hepatic porphyria, amyloidosis, and transthyretin-mediated amyloidosis, or an instruction for the application of the in vitro or ex vivo method according to the present invention, or a kit of parts including the same.

[0025] Aspects of the present invention include conjugates such as ADCs or AOCs of structure C, or semi-finished ADC conjugates or semi-finished AOC conjugates, including cell surface molecule targeting molecules such as sdAbs of the present invention, and including at least one effector portion of the present invention and / or including at least one saponin of the present invention: A(-S) b (-E) c (Structure C) (wherein A is a cell surface molecule targeting molecule such as sdAb; S is saponin; E is an effector moiety; b = 0 to 64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64 or any integer (or fractional part) therebetween; c = 0 to 8, preferably 0, 1, 2, 3, 4, 6, 8 or any integer (or fractional part) therebetween; S is coupled to A and / or E, and E is coupled to A and / or S, preferably S is coupled to A and E is coupled to A).

[0026] Definition The term "proteinaceous" has its ordinary scientific meaning and, as used herein, refers to a molecule that is protein-like, meaning that the molecule has to some extent the physicochemical properties characteristic of a protein, is of a protein, is related to a protein, contains a protein, belongs to a protein, consists of a protein, is similar to a protein, or is a protein. For example, the term "proteinaceous" when used in "proteinaceous molecule" refers to the presence of at least a portion of a molecule that is similar to or is a protein, and "protein" is 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 assemblies of proteins or protein domains. In a proteinaceous molecule, at least two amino acid residues are joined via 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 2000 amino acid residues. In one embodiment, a proteinaceous molecule is a molecule that contains from 2 to 20 (typical for a peptide) amino acids. In one embodiment, a proteinaceous molecule is a molecule that contains from 21 to 1000 (typical for polypeptides, proteins, protein domains, e.g., ligands for receptors such as antibodies, Fab, scFv, EGF, etc.) amino acids. Preferably, the amino acid residues are (typically) joined via peptide bonds. According to the present invention, the amino acid residues are (modified) (non)natural amino acid residues or include them.

[0027] For example, when referring to an "effector molecule" or "effector moiety" in the context of being part of a covalently conjugated conjugate, the term has its ordinary scientific meaning and herein refers to a molecule that can selectively bind to any one or more of target molecules such as proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, and enzymes, and regulates the biological activity of such one or more target molecules. Effector molecules are molecules selected from any one or more of, for example, small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, foreign nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof. Thus, for example, an effector molecule or effector moiety can selectively bind to any one or more of target molecules such as proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, and enzymes, and when bound to the target molecule, 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, foreign nucleic acids or siRNA, enzymes, peptides, proteins, or any combination thereof that regulates the biological activity of such one or more target molecules. Usually, effector molecules can exert a biological effect inside cells such as mammalian cells such as human cells, for example, in the cytosol of the cells. Thus, the effector molecule or moiety of the present invention is any substance that affects cell metabolism by interaction with an intracellular effector molecule target, and this effector molecule target is any molecule or structure inside the cell excluding the compartments of the endocytosis and recycling pathways and the lumen of the vesicles, but including the membranes of these compartments and vesicles. Thus, the aforementioned structures inside the cell include the inside of the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, cell membrane, and cytosol. Thus, typical effector molecules are drug molecules, enzymes, plasmid DNA, toxins such as toxins included by antibody-drug conjugates (ADCs), siRNA, BNA, oligonucleotides such as nucleic acids included by antibody-oligonucleotide conjugates (AOCs).For example, an effector molecule can be a molecule that can act as a ligand that increases or decreases (intracellular) enzyme activity, gene expression, or cell signaling.

[0028] The term "saponin" has its ordinary scientific meaning and herein refers to the group of amphiphilic glycosides containing one or more hydrophilic glycon moieties combined with a lipophilic aglycone core that is a sapogenin. Saponins may be naturally occurring or synthetic (i.e., not naturally occurring). The term "saponin" includes naturally occurring saponins, derivatives of naturally occurring saponins, and saponins newly synthesized via chemical and / or biotechnological synthetic routes.

[0029] The term "saponin derivative" (also known as "modified saponin") has its ordinary scientific meaning and herein refers to a compound corresponding to a naturally occurring saponin derivatized by one or more chemical modifications such as oxidation of a functional group, reduction of a functional group, and / or formation of a covalent bond with another molecule (also referred to as "conjugation" or "covalent conjugation"). Preferred modifications include derivatization of the aldehyde group of the aglycone; the carboxyl group of the sugar chain or the acetoxy group of the sugar chain. Usually, saponin derivatives do not have natural counterparts, i.e., saponin derivatives are not naturally produced, for example, by plants or trees. The term "saponin derivative" includes derivatives obtained by derivatization of naturally occurring saponins and derivatives newly synthesized via chemical and / or biotechnological synthetic routes that result in compounds corresponding to naturally occurring saponins derivatized by one or more chemical modifications.

[0030] The term "aglycone core structure" has its ordinary scientific meaning and herein refers to the aglycone core of a saponin that does not have one or two carbohydrate antennae or sugar chains (glycans) attached thereto. For example, kierraic acid is the aglycone core structure for SO1861, QS-7, and QS21. Usually, the glycans of saponins are monosaccharides or oligosaccharides such as linear or branched glycans.

[0031] The term "sugar chain" has its ordinary scientific meaning and, as used herein, refers to any of a glycan, a carbohydrate antenna, a monosaccharide moiety (monosaccharide), or a chain containing a plurality of sugar moieties (oligosaccharide, polysaccharide). The sugar chain may consist only of sugar moieties or may contain additional moieties such as, for example, 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, such as those present in OS-21.

[0032] In relation to the name of a sugar chain, the term "Api / Xyl-" or "Api- or Xyl-" has its ordinary scientific meaning and, as used herein, refers to a sugar chain containing an apiose (Api) moiety or a xylose (Xyl) moiety.

[0033] The term "saponin-based modified saponin" has its ordinary scientific meaning and, as used herein, refers to a saponin that has been modified to yield a modified saponin. Typically, a saponin-based modified saponin is a naturally occurring saponin that is subjected to chemical modification to yield a modified saponin.

[0034] The term "modified saponin based on saponin" has its ordinary scientific meaning and, as used herein, refers to a saponin that has been subjected to a chemical modification process such that a modified saponin is yielded, and the saponin from which the modified saponin is produced is typically a naturally occurring saponin.

[0035] The term "oligonucleotide" has its ordinary scientific meaning and, as used herein, refers to any natural or synthetic sequence of nucleic acids presented, inter alia, as single-stranded or double-stranded molecules such as BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNA), antisense DNA, antisense RNA, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids.

[0036] 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, and any molecule that can exert a therapeutic effect when contacted with cells of a subject such as a human patient, including active pharmaceutical ingredients, toxins, oligonucleotides, enzymes, small molecule drug compounds, etc.

[0037] 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 H and the like, and any oligonucleotide molecule that can exert a therapeutic effect when contacted with cells of a subject such as a human patient, including oligonucleotides selected from the natural or synthetic sequences of nucleic acids including single-stranded or double-stranded molecules such as BNA, antisense oligonucleotide (ASO), short or small interfering RNA (siRNA; silencing RNA), antisense DNA, antisense RNA, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids.

[0038] The term "bridged nucleic acid", or simply "BNA", or "locked nucleic acid" or simply "LNA", has its ordinary scientific meaning and refers herein to modified RNA nucleotides. BNA is also referred to as a "constrained RNA molecule" or a "sterically inaccessible RNA molecule". A BNA monomer can contain a 5-, 6- or 7-membered bridged structure having a "locked" C3'-end sugar puckering. The bridge is synthetically incorporated at the 2',4'-positions of ribose, resulting in 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 having increased binding affinity and stability.

[0039] The term "S", when used in an antibody-saponin conjugate containing a linker, etc., represents a "stable linker" that remains intact under the weak acidic conditions (pH < 6.6, e.g., pH 4.0 - 5.5) in the endosomes and lysosomes of mammalian cells such as human cells such as human tumor cells.

[0040] The term "L", when used in an antibody-saponin conjugate containing a linker, etc., represents an "unstable linker" that is cleaved under the weak acidic conditions (pH < 6.6, e.g., pH 4.0 - 5.5) in the endosomes and lysosomes of mammalian cells such as human cells such as human tumor cells.

[0041] Terms such as first, second, third, etc. in the specification and claims are used, for example, to distinguish between similar elements, compositions, components, or separate method steps in a composition and are not necessarily for describing a sequential or temporal order. The terms are interchangeable under appropriate circumstances and embodiments of the invention can function in orders other than those described or illustrated herein unless otherwise specified.

[0042] Embodiments of the invention described herein can function in combination and synergy unless otherwise specified.

[0043] Furthermore, although various embodiments are referred to as "preferred" or "e.g." or "for example" or "in particular", etc., they are not intended to limit the scope of the present invention and are to be construed as exemplary manners in which the present invention may be implemented.

[0044] The term "comprising" as used in the claims should not be construed as being limited to the elements, method steps or components listed thereafter; it does not exclude other elements, method steps or components in a particular composition. It should be construed as specifying the presence of the recited features, integers, (method) steps or components as referred to, but not excluding 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; rather, with respect to the present invention, the recited steps of the method are only A and B, and furthermore the claims should be construed as including 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; rather, with respect to the present invention, the recited components of the composition are only A and B, and furthermore the claims should be construed as including equivalents of those components.

[0045] In addition, a reference to an element or component by the indefinite article "a" or "an" does not exclude the possibility that there are two or more elements or components, unless the context clearly requires that there be only one element or component. Thus, the indefinite article "a" or "an" usually means "at least one".

[0046] The term "Saponinum album" has its usual scientific meaning and herein refers to a mixture of saponins manufactured by Merck KGaA (Darmstadt, Germany) containing SA1657 and mainly SA1641 and derived from Gypsophila paniculata and Gypsophila arostii.

[0047] The term "Quillaja saponin" has its usual scientific meaning and herein refers to the source for the saponin fraction of Quillaja saponaria and thus all other QS saponins mainly containing QS-18 and QS-21.

[0048] "QS-21" or "QS21" has its usual scientific meaning and 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%).

[0049] Similarly, "QS-21A" has its usual scientific meaning and herein refers to a mixture of QS-21 A-apio (about 65%) and QS-21 A-xylo (about 35%).

[0050] Similarly, "QS-21B" has its usual scientific meaning and herein refers to a mixture of QS-21 B-apio (about 65%) and QS-21 B-xylo (about 35%).

[0051] The term "Quil-A" refers to a commercially available semi-purified extract derived from Quillaja saponaria, containing a variable amount of over 50 different saponins, many of which incorporate the partial structure of triterpenoid-trisaccharide Gal-(1→2)-[Xyl-(1→3)]-GlcA- found in QS-7, QS-17, QS18, and QS-21 with a C-3β-OH group. The saponins found in Quil-A are listed in van Setten (1995), Table 2 [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer and Gideon 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 quillajasaponin are also fractions of saponins derived from Quillaja saponaria, both containing a wide variety of different saponins with mostly overlapping contents. The two fractions have different specific compositions because they are obtained by different purification procedures.

[0052] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. QS1861 has a molecular mass of 1861 daltons, and QS1862 has a molecular mass of 1862 daltons. QS1862 is described in Fleck et al. (2019), Table 1, column number 28 [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171]. The described structure is the api-variant QS1862 of QS-7. The molecular mass is 1862 daltons because this mass is the formal mass including protons with glucuronic acid. At neutral pH, the molecule is deprotonated. When measured in mass spectrometry in the anion mode, the measured mass is 1861 daltons.

Brief Description of Drawings

[0053]

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Mode for Carrying Out the Invention

[0054] For a bioactive molecule (e.g., an effector molecule) to function, the molecule needs to be able to bind to its target, which is, for example, outside the cell surface or inside the cell or organelle, in, for example, serum. The active moiety of almost all protein-based targeted toxins needs to enter the cytosol of the target cell or mediate its target regulatory effect, for example. In many instances, the toxin remains ineffective because (1) the targeting moiety has insufficient internalization and remains bound to the outside of the cell, (2) is recycled back to the cell surface after internalization, or (3) is transported to endosomes where it is degraded. These fundamental problems have been known for decades, over 500 targeted toxins have been investigated in the past decades, the problems remain unsolved, and only a pair of antibody-targeted protein toxins are market-approved with warning labels regarding severe toxicity. Moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP) has been approved by the FDA to date for relapsed or refractory hairy cell leukemia. Other such approved ADCs are Elzonris, Ontak.

[0055] To overcome these problems, many strategies have been described, including approaches that direct the toxin to the intracellular membrane trafficking complex of the biosynthetic pathway in the endoplasmic reticulum and techniques that disrupt or weaken the integrity of the endosome membrane, i.e., a compartment of the intracellular endocytosis pathway, thereby promoting endosomal escape. This includes the use of lysosomotropic amines, carboxy ionophores, calcium channel antagonists, viruses, bacteria, plants, animals, human and synthetic source various cell-penetrating peptides, other organic molecules, as well as photoinduced approaches. The efficacy of targeted toxins is usually increased 100- or 1000-fold, and in exceptional cases, more than a million-fold, in cell culture, but the requirements for co-administering other substances with endosome escape enhancers pose new problems, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell type-dependent variability.

[0056] All strategies, including physicochemical approaches, interact more or less directly with membranes and require enhancer molecules, which 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 distribute in a different kinetics from targeted toxins. This is a major drawback of existing approaches.

[0057] A first objective of the present invention is to provide improved ADCs and AOCs having an increased therapeutic window and, for example, improved ADCs and AOCs for the delivery of an effective amount or dose of an effector molecule, when delivery from outside the target cell to said cell is considered, or more specifically when delivery of the effector molecule in the cytosol of said target cell is considered. A second objective of the present invention is to provide an improved method of treating (human) patients suffering from a disease to be treated with a conjugate comprising an effector molecule and a ligand, i.e., for example, to improve the therapeutic window of an ADC or AOC comprising an effector molecule to be delivered in the cytosol of a target tumor cell.

[0058] An object of the present invention is to provide a conjugate which is a combination of an effector molecule activity enhancing molecule and an ADC or AOC for use in therapies such as anti-cancer therapy. By providing such a conjugate of the present invention, the therapeutic window of the effector molecule, which is part of a conjugate such as an ADC or AOC, is efficiently widened.

[0059] At least one of the above objectives is achieved by providing improved ADCs and improved AOCs which are conjugates further comprising an effector molecule activity enhancing molecule.

[0060] While the present invention will be described with respect to particular embodiments, the invention is not limited thereto and is limited only by the claims. The invention is described in terms of several embodiments, but alternatives, modifications, permutations, and equivalents will be apparent to those skilled in the art upon reading the specification and upon investigation of the drawings and graphs. The invention is in no way limited to the illustrated embodiments. Modifications can be made without departing from the scope defined by the appended claims.

[0061] The inventors have established that the therapeutic area of conjugates such as antibody-drug conjugates or antibody-oligonucleotide conjugates is increased when the conjugate contains at least one covalently bound saponin when administered to a tumor-bearing mammal (mouse) to which the conjugate is administered (see, for example, FIGS. 8-14 in the Examples section for a series of in vitro tumor cell and in vivo tumor model examples of the effects of saponin covalently bound to effector molecules and antibodies). Saponins are conjugated to effector molecules such as antibodies and oligonucleotides such as protein toxins and BNAs. The inventors first established and determined that conjugating a ligand for binding to a cell surface molecule such as an antibody, or an sdAb such as V HH with the saponin of the present invention provides a conjugate for the cell-specific delivery of saponin on the cell surface of a target cell that exposes the cell surface molecule on its cell surface, and then for the delivery of saponin into the cell interior such as cell endosomes, endolysosomes, lysosomes, and ultimately into the cytosol of the cell. Examples of such cell-targeted saponin conjugates are provided, for example, for saponin-V HH conjugates in FIGS. 2-5 and FIGS. 5-7, 15-23, and 25-34. Saponins are conjugated to ligands such as EGF, Her2-targeted V HH or IgG, EGFR-targeted IgG.

[0062] The conjugate of the present invention, which contains an sdAb or a full-length antibody or different immunoglobulin (Ig) formats as a cell surface molecule binding molecule, preferably and in all exemplary examples, covalently, more preferably, has at least one glycoside such as the saponin of the present invention covalently bound thereto via a (cleavable) linker. Without wishing to be bound by any theory, saponin probably enhances the therapeutic efficacy of the effector moiety bound to the cell surface molecule targeting molecule (antibody, sdAb) by enhancing endosomal escape into the cytosol where the activity of the effector moiety is desired. In this way, the therapeutic effect has been established under the influence of the presence of the conjugate containing saponin at a lower dose of the effector molecule than the conventional dose of ADC or AOC, i.e., at a lower dose of the conjugate of the present invention, whereby the saponin is carried to the vicinity of the cell to be targeted, the cell to be targeted and / or inside the cell to be targeted. The cells to be targeted are, for example, diseased cells such as tumor cells or autoimmune cells or B cells associated with B cell diseases. The effector moiety is, for example, a toxin as part of an ADC or an oligonucleotide such as an antisense BNA as part of an AOC according to the present invention.

[0063] A first aspect of the present invention relates to a conjugate for transferring an effector molecule from outside the cell into the cell, comprising at least one effector molecule to be transferred into the cell, at least one single domain antibody (sdAb), and at least one saponin, which are covalently bound to each other directly or via at least one linker, wherein at least one saponin is a mono-despimoside triterpenoid glycoside or a bi-despimoside triterpenoid glycoside, and the sdAb is capable of binding to a cell surface molecule of the cell.

[0064] The inventors of the present invention have found that saponins such as saponin in the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, and SO1861, can be covalently coupled to cell surface molecule targeting molecules, such as antibodies, sdAbs, via, for example, a trifunctional linker, such as the trifunctional linker of Structure A (shown below in the present specification), or via an oligomeric or polymeric structure of a scaffold containing covalently bound saponin, which results in an improvement in the cytotoxicity exerted by effector moieties such as toxins contained in the conjugate of the present invention under the influence of the covalently coupled saponin in the conjugate. This is disclosed herein.

[0065] Accordingly, one aspect of the present invention relates to a conjugate comprising an endosome escape enhancing molecule, i.e., a saponin, an effector moiety, and a binding molecule, such as an sdAb, wherein, for example, a glycoside molecule and an effector molecule are bound to one and the same binding molecule in the endosome escape enhancing conjugate, and the endosome escape enhancing conjugate can specifically bind to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of a complex of the conjugate and the target cell-specific surface molecule (following binding of the conjugate to the receptor, internalization of the conjugate / receptor complex follows). Preferably, the combination of the saponin and the effector moiety in the endosome escape enhancing conjugate enables enhancement of endosome escape of the effector moiety by the saponin. By doing so, the conjugate preferably improves the effect of the effector molecule as compared to an ADC comprising a binding molecule and an effector moiety without a saponin.

[0066] To explain the present invention in more detail, the process of uptake of substances into cells and the terminology used in the present invention will first be described. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. The vesicle budding can be characterized by (1) receptor-dependent ligand uptake mediated by the cytoplasmic protein clathrin, (2) lipid raft uptake mediated by the cholesterol-binding protein caveolin, (3) non-specific fluid uptake (pinocytosis), or (4) non-specific particle uptake (phagocytosis). All types of endocytosis lead to the following cellular processes of vesicular transport and substance sorting called the endocytic pathway. The endocytic pathway is complex and not fully understood. Previously, it was thought that organelles were newly formed and matured into the next organelle via the endocytic pathway. Currently, a hypothesis has been proposed that the endocytic pathway includes stable compartments connected by vesicular transport. The compartments are complex multifunctional membrane organelles specialized for a specific set of essential functions for the cell. Vesicles are considered to be more simple transient organelles in composition and are defined as containers enclosed in membranes newly formed by budding from existing compartments. In contrast to compartments, vesicles may undergo maturation, a physiologically irreversible series of biochemical changes. Early endosomes and late endosomes become stable compartments in the endocytic pathway, but even early endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and lysosomes are vesicles. Endocytic vesicles most prominently arise from the cell membrane 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 back to the cell membrane via recycling vesicles (the recycling pathway). Components to be degraded are transported via multivesicular bodies to acidic late endosomes (pH less than 6). Lysosomes are vesicles that can build mature lysosomal enzymes and deliver them to the late endosomal compartment if necessary. The resulting organelle is called a hybrid organelle or endolysosome.Lysosomes bud off hybrid organelles in a process called lysosome reorganization. Late endosomes, lysosomes, and hybrid organelles are very dynamic organelles, and the distinction between them is often difficult. The degradation of molecules trapped in the plasma membrane occurs inside the endolysosome. Endosomal escape is the active or passive release of substances from the lumen of any type of compartment or vesicle derived from the endocytic pathway, preferably clathrin-mediated endocytosis, or the recycling pathway, into the cytoplasm. Thus, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes or lysosomes, including intermediates and hybrid organelles. After entering the cytosol, the substance may move to other cellular units such as the nucleus. In the context of the present invention, a glycoside molecule (saponin) is a compound that can enhance the effect of an effector molecule, particularly by promoting endosomal escape. The glycoside molecule interacts with the membranes of compartments and vesicles of the endocytic pathway and the recycling pathway, causing them to leak the effector molecule, resulting in enhanced endosomal escape.

[0067] The term "improving the effect of an effector molecule" means that saponin preferably increases the functional effectiveness of an effector molecule (e.g., the therapeutic index of a toxin or drug; the metabolic effectiveness of a modifying factor in a biotechnological process; the effectiveness of gene transfection in cell culture assays) by enabling or enhancing its target binding. Preferably, it does not include accelerating, prolonging, or enhancing an antigen-specific immune response. Therapeutic effectiveness includes, but is not limited to, a more potent therapeutic effect with fewer administrations and / or fewer side effects. "Improving the effect of an effector molecule" may also mean that an effector molecule that could not be used due to lack of effect (e.g., was not known to be an effector molecule) becomes effective when used in combination with the present invention. Any other beneficial or desirable effects that may result from the combination of an effector moiety and saponin in one conjugate, as provided by the present invention, are considered "improved effects". In the context of the present invention, the saponins of the present invention are "enhancers" of the functional effectiveness of effector molecules in the conjugates of the present invention.

[0068] One of the major drawbacks in the enhancement of targeted toxins by glycosides, such as saponins, up to the present invention is that while the targeted toxin is internalized by receptor-mediated endocytosis (followed by the internal translocation of the conjugate / receptor complex after the binding of the conjugate to the receptor), the glycoside diffuses passively through the cell membrane and reaches the endosomal membrane, presumably through interaction with cholesterol. In principle, the glycoside can enter any cell, non-target cell (off-target cell), causing inefficient utilization of enhancers in target cells and in some cases side effects in non-target cells for the effective release of the targeted toxin. One major problem is that the translocation of the targeted toxin and the glycoside proceeds in different kinetics and these kinetics vary from cell (line) to cell and from tissue to tissue, so that the exact time difference for the application of the two substances (ADC, free saponin) can vary widely for each tumor (cell (line)). Furthermore, in organisms, the release, absorption, distribution, metabolism and excretion of these substances also differ. Additionally, the non-specific uptake of the glycoside by non-targeted cells can induce unwanted effects in these cells. This can be, for example, the delivery of a compound that was supposed to be delivered to the lysosome to the cytosol, the interference with antigen presentation, etc. The off-target administration of glycosides and targeted drugs can also be a problem in drug development and can prevent or at least delay the marketing approval by the relevant authorities (e.g., FDA or EMA). A targeted toxin or a targeted drug in the context of the present invention means a toxin or a drug in which the toxin or the drug is specifically targeted to a membrane-bound molecule on the target cell, for example, a toxin or a drug that binds to a ligand of a membrane receptor or binds to an antibody that specifically recognizes a structure on the cell membrane of the target cell.

[0069] Thus, it is highly useful to direct the glycoside via the same route as the effector molecule, for example, via targeting a ligand to the target cell such that the enhancer becomes available at an effective concentration inside the acidic compartment of the endocytosis pathway of the target cell and exhibits a synergistic action with the toxin. Accordingly, the present invention provides a novel approach for redirecting both the effector molecule and the endosome escape enhancer (i.e., the saponin of the present invention) via targeting a ligand (binding molecule) to the acidic compartment of the endocytosis pathway of the target cell.

[0070] The inventors have established that an effector molecule, which is part of a conjugate containing an sdAb, is delivered inside the cell with high efficiency under the influence of the saponin also contained by the conjugate when the effect of the effector molecule inside the cell is considered. Surprisingly, despite the relatively small size of sdAbs such as V HH the binding of a conjugate containing such an sdAb to the cell surface receptor still occurs when both the effector molecule and the saponin are contained by a conjugate containing an sdAb such as V HH The combined binding of the saponin and the effector molecule to sdAbs such as V HH does not cause steric hindrance when the ability to bind to cell surface molecules such as V HH is considered. That is, for example, contacting tumor cells with a sub-optimal dose of, for example, an ADC does not result in intracellular effector molecule activity in the absence of the saponin covalently coupled to said ADC (the target cells are not efficiently killed when exposed to the biological activity of the effector molecule). However, when the target tumor cells are contacted with a conjugate of the present invention containing an effector molecule, containing a saponin, and further containing a target cell-binding sdAb, efficient tumor cell death is achieved.

[0071] By targeting a single cell surface molecule with the conjugate of the present invention, the delivery of saponin and effector moieties bound to a cell surface molecule-targeting antibody such as sdAb in the conjugate of the present invention into the cytoplasm and interior of the targeted cell exposing the cell surface molecule on the cell surface is improved and more specific, for example, compared to contacting the cell only with a normal ADC lacking the saponin of the present invention and thus without the presence of the cell-targeting saponin (the conjugate of the present invention). The abnormal cells selected for targeting by the cell surface molecule-targeting sdAb of the conjugate preferably have a high degree of epitope on the cell surface molecule to which the cell surface molecule-targeting molecule can bind (i.e., for example, the expression of the targeted cell surface molecule on the targeted cells such as tumor cells or autoimmune cells is relatively higher than the expression on non-targeted cells such as normal cells), and / or expose the epitope in the cell surface molecule targeted for binding by the cell surface molecule-targeting sdAb of the conjugate, especially when normal (adjacent) cells within the patient are considered. Preferably, the cell surface molecule targeted by the cell surface molecule-targeting sdAb of the conjugate of the present invention is expressed relatively highly and / or specifically on the targeted (disease, tumor) cells compared to normal cells. One embodiment is the conjugate of the present invention, wherein the target cell surface molecule for the cell surface molecule-targeting sdAb of the conjugate, such as a tumor cell receptor, is expressed specifically or at a relatively high level compared to the expression of the cell surface molecule on the surface of normal (adjacent) cells. Thus, the epitope on the targeted cell surface molecule is preferably unique to the targeted disease cells and is present and exposed specifically, at least, on the surface of the targeted cells. After binding of the conjugate of the present invention to the epitope on the cell surface molecule on the targeted cell, endocytosis of the conjugate and cell surface molecule complex follows (after binding of the conjugate to the receptor, internalization of the conjugate / receptor complex follows).The conjugate can enter target cells only through binding interactions with cell surface molecules that are expressed specifically or intrinsically to a sufficient degree on the cells to be targeted as compared to healthy cells that should not be targeted. Therefore, the accumulation of a therapeutically effective amount of the effector moiety and saponin contained by the conjugate inside the target cells is possible and occurs only when the expression level of the cell surface molecule to be targeted exceeds a certain minimum expression threshold. At the same time, the fact that the effector moiety bound to the cell surface molecule-targeted sdAb of the conjugate can exert its intracellular (e.g., cytotoxic or gene silencing) activity only in the presence of the exact same conjugate having a covalently bound saponin also provides protection against negative unwanted side effects of the effector moiety on healthy cells and healthy tissues that are targeted and affected by the effector moiety, as compared to exposure of cells to ADCs that do not have a covalently bound saponin. That is, a sufficiently low expression or absence of the exposed cell surface molecule to which the conjugate can bind ideally does not allow the conjugate to enter (non-targeted) healthy cells to an amount that would result in endosomal escape of the effector moiety under the influence of the saponin contained by the conjugate. The ADC having a coupled saponin or the AOC having a covalently coupled saponin according to the present invention can be used at a lower dose as compared to when an ADC or AOC not having a coupled saponin is applied in a treatment regimen. Therefore, the entry of an ADC having a low level of coupled saponin or an AOC having a coupled saponin into healthy cells already has a lower risk with respect to the occurrence of unwanted side effects when the targeting and killing of target disease cells such as tumor cells and autoimmune cells are considered, for example.

[0072] Thus, the inclusion of sdAb in the conjugate has a number of advantages compared to the inclusion of an antibody such as IgG, or a binding fragment or domain thereof. Importantly, since sdAb does not contain the Fc tail present in IgG, there is no risk of off-target side effects due to the binding of the conjugate to Fc receptors on cells such as endothelial cells of the host to which the conjugate is administered. Thus, the risk profile of the 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 conjugate of the present invention cannot be bound by Fc receptors, the conjugate is already less likely or not at all likely to be undesirably captured by cell surface receptors different from the target cell surface molecule of interest, and thus is effective at a lower dose than the dose required to achieve the same effector molecule activity by ADCs and AOCs based on full-length antibodies. Furthermore, for example, due to the relatively small size of sdAb compared to Fab, scFv, IgG, tissue penetration is improved, which is advantageous for reaching target cells when the conjugate is administered to a patient in need of therapy. All of these advantages of the application of sdAb in the conjugate of the present invention, when compared to the application of larger antibodies such as IgG containing an Fc tail or fragments thereof, as well as when included by the conjugate of the present invention, result in an improvement in the therapeutic window for effector molecules, similar to ADCs or AOCs. For example, an ADC based on sdAb may achieve an improvement in the killing of target cells in the case of targeted tumor cells when the effector molecule is, for example, a toxin, and at the same dose, an ADC based on IgG and containing the same effector molecule may be ineffective or only sub-optimally effective. For aspects of the present invention, here, a conjugate containing sdAb, i.e., a part of the conjugate of the present invention, treats a patient with a lower dose of the effector molecule compared to the higher dose required when using an ADC or AOC based on an antibody containing the same effector molecule, and at the same time it is possible to reach the same or improved effector molecule-mediated effect in target cells.Administration of such conjugates of the invention at lower dosages reduces the patient's risk with respect to the occurrence of side effects, for example, by non-targeting, non-specific entry into healthy cells. This is important, for example, when cell surface molecules targeted by the sdAb comprised by the conjugate are more highly expressed on target (tumor) cells, but not inherently expressed on such target cells. Lower dosages of the conjugate reduce the risk regarding binding of the conjugate to such low expressors as non-tumor healthy cells.

[0073] The inventors have also found that the therapeutic range of the conjugates of the present invention is broadened due to the incorporation of the covalently bound saponin in the conjugates of the present invention. That is, when the ADC or AOC provided with saponin (i.e., the conjugate of the present invention) is contacted with the target cell upon binding of the sdAb to its binding partner on the surface of the target cell, the saponin contained in the conjugate of the present invention is also carried proximally with the effector molecule of the conjugate, i.e., to the surface of the target cell. When a target cell having a cell surface molecule, i.e., a target for the sdAb contained in the conjugate, is contacted with the conjugate of the present invention, both the effective amount of the effector molecule and the effective amount of the saponin are lower than when the target cell is contacted with the ADC or AOC in the absence of saponin or in the presence of free (non-targeted) saponin. The presence of the targeted saponin as part of the conjugate of the present invention enhances the activity of the effector molecule in the target cell, and as a result, the therapeutic range of the conjugate, and therewith the therapeutic range of the effector molecule, is broadened. The efficiency of the sufficient effector molecule is achieved at a lower dose when the target cell is contacted with the conjugate of the present invention. A similar effect is observed when the ADC or AOC is contacted with the target cell in the presence of saponin or its functional derivative when the effector molecule enhancing activity of the saponin is considered, but is 100 to 1000 times higher than the effective dose established when the conjugate of the present invention containing both the effector molecule and the effector molecule activity enhancing saponin together with the sdAb for the targeted binding of the conjugate to the target (tumor) m cell is applied, as found by the inventors at a concentration of free saponin (derivative). Thus, providing a binding molecule (i.e., the sdAb contained in the conjugate of the present invention) to saponin or its derivative and also providing an effector molecule (i.e., the effector molecule contained in the conjugate of the present invention) to the very same conjugate results in an improvement in the effector molecule activity enhancing effect when the conjugate of the present invention is contacted with a target cell expressing a cell surface molecule on its surface, i.e., a binding target for the sdAb.Targeted saponin is already effective at lower doses than free saponin in the delivery of effector molecules into target cells and the delivery of said cells from endosomes or lysosomes into the cytosol, and the effector molecule should bind to its target binding partner and exert its biological activity (e.g., cell death when the target cell is a tumor cell and the effector molecule is, for example, a toxin), but the inventors have found that the combination of saponin, targeting moiety (sdAb) and effector molecule (e.g., toxin, AON) in a single molecule is even more effective.

[0074] Accordingly, the inventors provide a pharmaceutical composition comprising a conjugate comprising a saponin (derivative), an effector molecule and an sdAb for the targeted delivery of the conjugate in target cells, and the pharmaceutical composition has an improved therapeutic window when compared to existing Fc-based ADCs comprising full-length antibodies or constructs thereof not provided together with covalently linked saponin, a reduced risk of inducing side effects when an effective dose of the effector molecule comprised by the conjugate is administered to patients in need of effector molecule-based therapy, and improved effector molecule activity resulting from improved delivery of the conjugate into the interior of target cells, more specifically, into the cytosol of such target cells, under the influence of the targeted saponin as part of the conjugate of the invention. It is part of the invention that such a conjugate of the invention is administered to patients in need of effector molecule-based therapy together with a dose of free saponin (derivative), but application of the conjugate alone is preferred.

[0075] One embodiment is a V domain derived from an antibody, preferably a heavy chain of immunoglobulin G origin, preferably of human origin; a V domain derived from an antibody, preferably a light chain of immunoglobulin G origin, preferably of human origin; a V domain derived from an antibody consisting only of a heavy chain (HCAb) such as an Ig-NAR origin such as from Camelidae or a novel antigen receptor (V H domain; a V domain derived from an antibody consisting only of a heavy chain (HCAb) such as an Ig-NAR origin such as from Camelidae or a novel antigen receptor (V L domain; a V domain derived from an antibody consisting only of a heavy chain (HCAb) such as an Ig-NAR origin such as from Camelidae or a novel antigen receptor (V NAR ) domain, etc.HH comprises at least one sdAb which is any one or more of domains (preferably, HCAb is derived from Camelidae origin); preferably, at least one sdAb is derived from HCAb from camel, llama, alpaca, dromedary, vicuña, guanaco and guanaco, such as those derived from HCAb of Camelidae origin (camelids V H ) derived from HCAb of V HH domain, which is a conjugate of the present invention.

[0076] In particular, V HH domain is suitable for application in the conjugate of the present invention. Such V HH domains are generally well-known for their high stability, i.e., resistance to unfolding, e.g., their ability to bind to a binding partner without the need for the presence of a second V domain required for IgG to bind to its binding partner via two V domains, their ease of production by techniques known in the art (such as immunization of camelids, phage display technology, etc.), and their ability to penetrate tissues to a greater extent than that seen in full-length IgG, which is beneficial when the target (tumor) cells are located inside or as part of such (organ) tissue.

[0077] One embodiment is a conjugate of the present invention comprising at least two sdAbs together with all of at least one sdAb covalently bound to one of at least one effector molecule and / or one of at least one saponin, or at least two sdAbs wherein at least one sdAb is bound to at least one effector molecule and / or at least one sdAb is bound to at least one saponin, or at least two sdAbs each separately bound to an effector molecule of at least one effector molecule or a saponin of at least one saponin, or both.

[0078] One embodiment is a conjugate of the invention in which at least one sdAb comprises at least two sdAbs, which are the same sdAb, preferably 2 to 8 sdAbs, more preferably 2 to 4 sdAbs.

[0079] One embodiment comprises 1 to 8 sdAbs capable of binding to the same binding site on a cell surface molecule, and at least one effector molecule and / or at least one saponin is bound to a single first sdAb of the 1 to 8 sdAbs or at least one effector molecule and / or at least one saponin, if present, is bound to more than one of the sdAbs, and at least one effector molecule and at least one saponin are bound to the same sdAb or to different sdAbs, preferably each of the at least one effector molecules is bound to a separate sdAb and / or each of the at least one saponins is bound to a separate sdAb, and the effector molecule and the saponin are bound to the same sdAb or to separate sdAbs, which is a conjugate of the invention.

[0080] By providing a conjugate of the invention comprising a (linear) array of a plurality of sdAbs covalently linked to each other, the advantage of the ability of the conjugate to bind to target cells with higher avidity can be provided, which can lead to improved uptake (endocytosis) of the conjugate by the target cells (after binding of the conjugate to the receptor, internalization of the conjugate / receptor complex follows).

[0081] Synchronization is the missing link between a successful delivery strategy for mice and its application to humans when considering the enhanced endosomal escape effect of saponin on effector molecules. Indeed, the inventors have established that in a series of in vivo mouse tumor models, separately administering to mice a certain dose of free saponin and an ADC having a certain dose, for example, without a coupled saponin, did not result in any desired anti-tumor activity such as delay of tumor growth, tumor regression, decrease in tumor growth, and slowdown, compared to control animals not treated with the ADC in the presence of free saponin. See also Examples 4 to 18 below in this specification. The free saponin was administered using various routes of administration and various time points of administering the free saponin (administering the free saponin before, during, and after administration of the ADC) compared to the moment of administering the ADC. The ADCs tested in the in vivo tumor models were cetuximab-diantin (with free SO1861), or trastuzumab-saporin (with free SO1861). Varying the dose of the free saponin did not result in effective anti-tumor activity. The ADCs referred to were administered at doses that did not themselves confer any beneficial anti-tumor effect on tumor-bearing animals. Surprisingly, the inventors have now established that advantageous anti-tumor activity in various in vitro mammalian cell-based assays using human tumor cells and / or in various in vivo animal tumor models can be achieved by treating cells or animals with the conjugates according to the invention. The conjugates optionally include a scaffold according to the invention (see below; a covalent saponin conjugate comprising an oligomeric or polymeric structure having one or more saponin moieties covalently attached thereto).The scaffold is a trifunctional linker having a covalently linked saponin (e.g., SO1861, QS-21) via a cleavable or non-cleavable bond and / or an effector moiety (e.g., dianthin, gene silencing antisense BNA (HSP27)) covalently linked via a non-cleavable or cleavable bond. The scaffold is covalently linked to a cell surface molecule targeting molecule of a conjugate such as a monoclonal antibody such as cetuximab, trastuzumab, OKT-9, etc., or the scaffold is a dendron, e.g., a G4-dendron (e.g., four moieties such as four saponin molecules can be attached), or a dendron for attaching, e.g., two saponins and two effector molecules, a dendron containing a chemical group for (covalently) coupling to a cell surface molecule targeting antibody such as an sdAb of the conjugate. Reference is made to the sections on further embodiments and examples, which illustrate some of these scaffolds according to the invention that exhibit in vivo and / or in vitro anti-tumor cell activity when cytotoxicity exerted by a proteinaceous toxin is considered or when gene silencing in tumor cells is considered.

[0082] While not wishing to be bound by any theory, in view of the failures observed when considering the treatment of cancer-bearing animals with an ADC combined with free saponin, 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. For ADCs and free saponins, synchronizing the presence of the molecules in late endosomes to obtain a synergistic effect in vivo was not advantageously obtained according to the attempts of the inventors. In one aspect, the present invention relates to solving at least the following problems with respect to combining an effector moiety and a saponin, preferably in a single conjugate molecule: While not wishing to be bound by any theory, for example, the only suitable chemical group within a saponin that can be used for a (covalent), particularly a single cleavable and retainable coupling, is required for endosomal escape activity. For example, the significant endosomal escape enhancer effect of the saponins of the present invention exemplified herein has been known for over 10 years, but saponins have not been used in combination with pharmaceutically active substances in clinical trials, most likely due to known limitations, except for the application of saponins in vaccination regimens where their use as an immune enhancing adjuvant substance has been implied. For example, providing the conjugates of the present invention with covalently bound saponins solves at least in part these difficulties, for example, in the context of scaffolds having several saponins. Surprisingly, saponins previously applied for their immune enhancing activity in the context of vaccination containing saponins as an adjuvant component are still suitable at present for (covalently) coupling to cell surface molecule-targeting antibodies, such as sdAbs, contained in the conjugates of the present invention for anti-tumor activity in vitro or in vivo.

[0083] One embodiment is that at least one sdAb is a single sdAb or at least two, preferably two sdAbs, and the sdAb can bind to a cell surface molecule of a cell such as HIV gp41, or the sdAb is a cell surface receptor of a cell such as a tumor cell surface receptor of a cell, preferably a tumor cell-specific receptor, more preferably CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate-specific membrane antigen (PSMA), CanAg, integrin alpha V, 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 the conjugate of the present invention can bind to at least one receptor selected from the group consisting of, most preferably, HER2, c-Met, VEGFR2, CXCR7, CD71, and EGFR1.

[0084] It is part of the present invention that the sdAb contained by the conjugate of the present invention has binding specificity for cell surface molecules specifically expressed on target cells. "Specifically expressed" should be understood herein as the exclusive expression of cell surface molecules only on target cells (e.g., healthy cells that should not bind to the conjugate are not targeted due to the lack of cell surface exposure of the molecule to be targeted), or, for example, as the upregulated or relatively high expression of target cell surface molecules on target cells as compared to the low expression of cell surface molecules on healthy cells that should not bind to the conjugate or should bind at least to a much lower extent. These listed cell receptors are such cell surface molecules that are sufficiently specific for the cells that are the target of the conjugate and are thus preferred candidates for binding by the conjugate. The higher the specificity of a particular cell surface molecule when compared to the expression on other cells where the expression of the cell surface molecule on the target cells is not targeted by the conjugate of the present invention, the better the therapeutic window will be when the activity of effector molecules inside the cell is considered. For example, suitable targets for targeting by the conjugate are among other tumor cell-specific receptors, HER2, EGFR, e.g., EGFR1, and CD71.

[0085] One embodiment is that at least one sdAb is a single sdAb or at least two, preferably two, 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-vascular integrin alpha-v beta-3 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, anti-HIVgp41, and the sdAb is preferably V HH , more preferably camelid V H is the conjugate of the present invention.

[0086] One embodiment is that at least one sdAb includes an sdAb that can bind to HER2, CD71, HIVgp41 and / or EGFR, and the sdAb is preferably V HH , more preferably camelid V H is the conjugate of the present invention.

[0087] One embodiment includes an sdAb that binds to HER2, where the at least one sdAb is selected from sdAbs generated by clone 11A4, clone 18C3, clone 22G12, clone Q17, or clone Q17-C-tag; or includes an sdAb that binds to EGFR and is generated by clone anti-EGFR Q86-C-tag; or includes an sdAb that binds to CD71 and is generated by clone anti-CD71 Q52-C-tag; or includes an sdAb that binds to HIV gp41 and is generated by clone anti-HIV gp41 Q8-C-tag; or includes 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 includes 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 conjugate further includes at least one additional sdAb different from the at least one sdAb, such as any one or more of the additional sdAbs having the amino acid sequences of SEQ ID NOs: 33, 34, and 35, which are additional sdAbs for binding to albumin, and preferably, the additional sdAb is V HH , more preferably, camelid V H is the conjugate of the present invention.

[0088] The V suitable for incorporation in the conjugate of the present invention HH is found, for example, in the single domain antibody database (Wilton, E.E. et al. (2018)), U.S. Patent Application Publication No. 20160251440 (anti-CD123, anti-CEACAM), U.S. Patent No. 9683045 (anti-c-Met), U.S. Patent Application Publication No. 20090252681 (anti-EGFR, anti-IGF-1R), U.S. Patent No. 9969805 (anti-HER2), U.S. Patent Application Publication No. 20190023796A1 (anti-HER3), and for anti-HER2 in Kijanka et al. (2013), and for anti-HER2 in Mercier et al. (2019). A series of suitable VHH The amino acid sequences and / or cDNA sequences of HH are also provided herein below with respect to anti-HER2, anti-HER3, anti-CD123, anti-CEACAM, anti-c-Met, anti-EGFR, anti-IGF-1R, anti-PD-L1, anti-CTLA-4, anti-CD19, anti-HER1 and anti-VGFR2 as SEQ ID NOs: 1-32 and 36-72, in view of their ability to bind to tumor cell-specific receptors. In particular, V that can bind to the binding site of any one of the tumor cell-specific receptors HER2, VEGFR and CD71 HH is suitable for incorporation in the conjugates of the present invention. The inventors have demonstrated that ADCs comprising V targeting any one of such receptors HH are effective in the delivery of effector molecules conjugated to sdAb. See, for example, the Examples section, and FIGS. 4-7.

[0089] One embodiment comprises or consists of at least one of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as BNA, a foreign nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof.

[0090] One embodiment is a conjugate according to the invention wherein the effector molecule is a pharmaceutically active substance such as a toxin, a drug, a polypeptide or a polynucleotide, such as a proteinaceous toxin. The pharmaceutically active substances in the present invention are effector molecules used to achieve beneficial outcomes in organisms, preferably vertebrates, more preferably humans. The advantages include the diagnosis, prognosis, treatment, cure and prevention of diseases and / or symptoms. Pharmaceutically active substances can also cause unwanted harmful side effects. In this case, the advantages and disadvantages must be compared to determine whether a pharmaceutically active substance is suitable for a particular case. If the effect of the pharmaceutically active substance inside the cell is predominantly beneficial for the whole organism, e.g., a human patient, the cell is called a target cell. If the effect inside the cell is predominantly harmful for the whole organism, the cell is called an off-target cell. In artificial systems such as cell cultures and bioreactors, target cells and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules include drugs, toxins, polypeptides (such as enzymes), and polynucleotides, including polypeptides and polynucleotides containing unnatural amino acids or nucleic acids. Effector molecules include, inter alia, DNA: single-stranded DNA (e.g., DNA for adenine phosphoribosyltransferase); linear double-stranded DNA; circular double-stranded DNA (e.g., plasmids); RNA: -mRNA (e.g., TLA effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, asRNA, LNA and BNA; proteins and peptides: Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)boganine, agrostin, ricin (toxin A chain); yamabogoe antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin) metabolic enzymes (argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; enzymes for cell signaling; cell cycle regulators; gene regulators (transcription factors such as NF-κB or gene repressors such as methionine repressor).The toxins as used in the present invention are defined as pharmaceutically active substances capable of killing or inactivating cells. Preferably, the targeted toxin is a toxin that is toxic only to the target cells, or at least predominantly toxic, but not toxic to off-target cells. The net effect of the targeted toxin is preferably beneficial to the whole organism.

[0091] One embodiment is a conjugate of the present invention, wherein at least one effector molecule is selected from 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 small circular DNA, a peptide nucleic acid (PNA), a phosphoramidite morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA), and a threose nucleic acid (TNA), or a derivative thereof, more preferably any one or more of BNA, for example, BNA for silencing HSP27 protein expression or BNA for silencing apolipoprotein B expression.

[0092] One embodiment is a conjugate of the present invention, wherein at least one effector molecule is selected from 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 ), siRNA based on BNA, and antisense oligonucleotide based on BNA (BNA-AON).

[0093] In one embodiment, at least one effector molecule is an oligonucleotide selected from any one of anti-miRNA, BNA-AON such as BNA-based siRNA or siRNA, preferably a chemically modified siRNA, a metabolically stable siRNA, and a chemically modified and metabolically stable siRNA, which is a conjugate of the present invention.

[0094] In one embodiment, at least one 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 gene: 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), X gene of hepatitis B virus (HBV), S gene of HBV, alpha-1 antitrypsin (AAT) and lactate dehydrogenase (LDH), and / or an abnormal miRNA, which is a conjugate of the present invention capable of targeting when present in a cell containing such an abnormal miRNA.

[0095] In one embodiment, 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 proteins: apoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, the expression product of the X gene of HBV, the expression product of the S gene of HBV, AAT and LDH, or weakens or restores miRNA functions such as inhibition of oncogenic miRNA (onco-miR) or suppression of onco-miR expression when present in a cell containing such a miRNA, which is a conjugate of the present invention.

[0096] The inventors have shown that tumor cell-targeted monoclonal antibodies provided together with covalently coupled antisense BNA such as BNA(HSP27) and the covalently coupled saponin of the present invention (both BNA and saponin are coupled to an antibody (e.g., cetuximab) via a cleavable bond) can silence HSP27 in vivo in tumors as compared to a control and as compared to AOCs that have only BNA and do not have saponin (SO1861, Quil-A). Thus, by administering an ADC-saponin conjugate of the present invention or an antibody-oligonucleotide conjugate-saponin conjugate (AOC-saponin) of the present invention, e.g., an antibody-BNA-saponin conjugate, the ADC-saponin or AOC-saponin confers antitumor cell activity not seen with only an ADC or only an AOC that do not have saponin covalently bound to the monoclonal antibody at the same dose. Notably, AOCs as a combination of two separate conjugates and separate monoclonal antibodies having covalently coupled saponin, when administered separately to tumor-bearing mice in separate groups of mice, increase HSP27 expression in tumor cells as compared to a control group (to which only the vehicle is administered). Only the administration of the AOC-saponin conjugate of the present invention containing an effector portion of the present invention shows a decrease in HSP27 expression when compared to a control. The antisense BNA(HSP27) was a BNA having an oligonucleic acid sequence according to Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333]. Notably, to the best of the inventors' knowledge, BNA is designed for application as a free nucleic acid.The inventors have now for the first time demonstrated that antisense BNA can be covalently coupled via a (non)cleavable linker to a ligand or antibody such that the gene silencing activity is retained in tumor cells of tumor-bearing animals in vitro and, more importantly, in vivo. This approach of providing BNA-based AOCs has opened up a new way of administering targeted BNA to human (cancer) patients in need thereof.

[0097] One embodiment is a conjugate of the invention, wherein at least one 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 very effective tumor cell killing is achieved when an sdAb that binds to any of HER2, VEGFR, CD71 is selected, the sdAb is combined with a toxin such as a protein toxin such as dianthin or saporin, and the sdAb is combined with saponin. Examples demonstrating the high efficacy of certain conjugates comprising sdAb and effector molecules are provided in the Examples section and shown in FIGS. 4-7.

[0098] In one embodiment, at least one effector molecule comprises or consists of at least one of urease and Cre-recombinase, proteinaceous toxins, ribosome-inactivating proteins, protein toxins, bacterial toxins, plant toxins, more preferably, viral toxins such as apoptin; bacterial toxins such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; mycotoxins such as alpha-sarcin; ribosome-inactivating proteins and dianthins, for example, dianthin-30 or dianthin-32, saporin, for example, saporin-S3 or saporin-S6, borganin or deimmunized derivative deborganin of borganin, Shiga-like toxin A, yamabog anti-viral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumine, viscumine A chain and other plant toxins containing the A chain of type 2 ribosome-inactivating proteins; or animal or human toxins such as frog RNase, or granzyme B or human angiogenin, or any one or more of their toxic fragments or toxic derivatives; preferably, the protein toxin is a conjugate of the present invention which is dianthin and / or saporin.

[0099] In one embodiment, the conjugate of the present invention is such that at least one effector molecule comprises or consists of at least one payload.

[0100] One embodiment is a conjugate of the present invention comprising one or more of at least one effector molecule, which 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, pseudotoxin, 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, indolinobenzodiazepine, AZ13599185, cryptophycin, lysocine, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, a shortened form of Pseudomonas aeruginosa exotoxin (PE38), duocarmycin derivative, amanitin, α-amanitin, spliceostatin, tyranstatin, ozogamicin, tesirine, ambrastatin 269, and sorabtansine, or a derivative thereof, or consisting of any one or more thereof.

[0101] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effects. For example, some effector molecules such as Pseudomonas exotoxin change their route to other organelles before the "late endosomal stage" and thus usually do not benefit from incorporation in conjugates according to the present invention. However, such toxins can be adapted for use according to the present invention, for example, by deleting the signal peptide involved in the route change. In particular, toxins that are highly toxic and require only one molecule to escape the endosome and kill the cell can be modified to be less potent. It is preferred to use toxins that kill cells when at least 2, more preferably at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and most preferably at least 100 toxin molecules escape the endosome (and enter the cytosol). The conjugate of the present invention is a scaffold having a covalently conjugated function, comprising one or more covalently conjugated effector moieties for targeting a scaffold comprising one or more effector moieties bound to a target cell such as a tumor cell or an autoimmune cell, i.e., a scaffold such as an oligomeric or polymeric scaffold or a trifunctional linker. Furthermore, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0102] Preferably, the effector moiety comprised by the conjugate of the present invention, the effect of which is enhanced by the saponin comprised by the conjugate, detaches from the conjugate and, for example, from an antibody such as an sdAb present in the conjugate as the cell surface molecular targeting portion of the conjugate when invaginating into the plasma membrane. This can be achieved, for example, by a cleavable bond that cleaves under acidic, reducing, enzymatic or photoinductive conditions.

[0103] One embodiment is an antibody-drug conjugate (ADC) comprising a conjugate that includes at least one sdAb derived from gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and / or an ADC comprising a toxin present in any one or more of gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin and / or at least one effector molecule selected from dianthin and saporin. When the sdAb is derived from such a human antibody, the V H domains of such human antibodies may require some improvement (the "camelization" of the human V H domains) with respect to domain stability known in the art.

[0104] One embodiment is that at least one saponin is (Group C): 2 alpha-hydroxyoleanolic acid; 16 alpha-hydroxyoleanolic acid; hederagenin (23-hydroxyoleanolic acid); 16 alpha,23-dihydroxyoleanolic acid; gypsogenin; kila acid; protoescigenin-21(2-methylbut-2-enoate)-22-acetate; 23-oxo-valingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-oxo-valingtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; digitogenin; 3,16,28-trihydroxyolean-12-ene; gypsogenic acid; or an aglycone core structure selected from its derivatives, Preferably, at least one saponin comprises an aglycone core structure selected from kierraic acid and gypogenin, and more preferably, at least one saponin is the conjugate of the present invention comprising the aglycone core structure kierraic acid.

[0105] While not wishing to be bound by any theory, the presence of an aldehyde group (or a derivative thereof) in the aglycone core structure of the saponin (also referred to herein as "aglycone") is such that the endosomal escape of the effector molecule contained by the conjugate of the present invention is stimulated and / or enhanced when such saponin coexists with these effector molecules as part of the conjugate of the present invention in the cell, in the endosome of said cell, or is in the endosome in free form (e.g., separates from the conjugate when the conjugate is delivered inside the target cell endosome or lysosome). Thus, the conjugate of the present invention containing a saponin having an aglycone with an aldehyde group is preferred. In kierraic acid and gypogenin, the aldehyde group is at the C 23 atom.

[0106] One embodiment is that at least one saponin comprises a first sugar chain attached to the C3 atom or the C 28 atom, preferably attached to the C3 atom, of the aglycone core structure of at least one saponin, and one or both of a second sugar chain attached to the C 28 atom of the aglycone core structure of at least one saponin, and preferably at least one saponin is the conjugate of the present invention comprising the first sugar chain and the second sugar chain. Thus, when the saponin contained by the conjugate of the present invention has two glycans (sugar chains), the first sugar chain is attached at the C3 position of the aglycone core structure, and the second sugar chain is attached at the C 28 position of the aglycone core structure of the saponin.

[0107] One embodiment is · at least one saponin is (Group A): 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-、 including a first sugar chain selected from Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA- and any derivative thereof, and / or · at least one saponin being (Group B): Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc- (wherein R1 is 4E-methoxycinnamic acid), Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc- (wherein R2 is 4Z-methoxycinnamic acid), Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc- (wherein R3 is 4E-methoxycinnamic acid), Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-、 (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-)、 Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-、 Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-、 Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-、 Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-、 Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-、 Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc- (wherein, R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)、 Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc- (wherein, R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)、 Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-、 Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-、 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-、 Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- (wherein R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- (wherein R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- (wherein R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- (wherein R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- (wherein R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- (wherein R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- (wherein R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Glc-(1→3)-[Glc-(1→6)]-Gal-, and any derivative thereof The conjugate of the present invention comprising a second sugar chain selected from

[0108] Thus, when the saponin contained by the conjugate of the present invention has two glycans (sugar chains), the first sugar chain is bonded at the C3 position of the aglycone core structure of the saponin, and the second sugar chain is bonded at the C 28 position of the saponin. Preferably, the saponin has an aldehyde group in the aglycone.

[0109] One embodiment is that at least one saponin contains a first sugar chain and a second sugar chain according to Group A and Group B respectively, the first sugar chain contains two or more sugar moieties, and the second sugar chain contains two or more sugar moieties, and the aglycone core structure is preferably kierraic acid or gypsogenin, more preferably kierraic acid, 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. One, two or three, preferably one or two of the at least one acetoxy (Me(CO)O-) groups in the second sugar chain are derivatized, which is the conjugate of the present invention.

[0110] One embodiment is that at least one saponin i. - Reduction to alcohol; - 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 and converted to a hydrazone bond via the reaction with EMCH; - 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 and converted to a hydrazone bond via the reaction with BMPH; or - N-[κ-maleimidoundecanoic acid] hydrazide (KMUH), wherein the maleimide group of KMUH is derivatized by conversion to a hydrazone bond via reaction with KMUH optionally derivatized by formation of a thioether bond with mercaptoethanol; an aglycone core structure containing an aldehyde group; ii. A first sugar chain containing a carboxyl group derivatized by conversion to an amide bond via reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl) maleimide (AEM), preferably the carboxyl group of a glucuronic acid moiety; iii. A second sugar chain containing an acetoxy group (Me(CO)O-) derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. A conjugate of the invention comprising any combination of two or three derivatizations i., ii. and / or iii., preferably any combination of two derivatizations i., ii. and / or iii.

[0111] One embodiment is that at least one saponin is Quillaja bark saponin, dipsacoside B, psychotrine A, psychotrine D, macranthoidin A, esculentoside A, phytolaccagenin, escin salt, AS6.2, NP-005236, AMA-1, AMR, alpha-hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, SO1862, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primulic acid 1 and AS64R, or a derivative thereof, or a stereoisomer thereof, and / or any combination thereof, preferably any one or more of QS-21 or a QS-21 derivative, SO1861 or a SO1861 derivative, SA1641 or a SA1641 derivative and GE1741 or a GE1741 derivative, more preferably a QS-21 derivative or a SO1861 derivative, most preferably a SO1861 derivative, for example, any one or more of the saponin derivatives according to the present invention, which is a conjugate of the present invention.

[0112] One embodiment is that at least one saponin is any one or more of SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypenoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, or a derivative thereof, or a stereoisomer 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 QS21 derivative, most preferably, the saponin derivative is an SO1861 derivative according to the present invention, which is a conjugate of the present invention.

[0113] Such triterpenoid glycoside-type saponins are contained by the conjugate and can enhance the endosomal escape of effector molecules present in the endosomes (or lysosomes) of cells when the saponin as part of the conjugate or in free form coexists with such effector molecules inside the cell. The inventors have established that the endosomal escape enhancing activity of these saponins is more than about 100 to 1000 times more potent when the saponin is contacted with cells as part of the conjugate of the present invention. Free saponin stimulates the delivery of effector molecules in the cytosolic of cells at a saponin concentration 100 to 1000 times higher compared to the concentration of the same saponin contained by the conjugate of the present invention required to achieve the same degree of delivery of effector molecules from outside the target cell into the endosome and ultimately into the cytosolic of said cell when such cells are contacted with effector molecules and saponin as part of a certain cell targeting conjugate such as an ADC or AOC. Saponins having a high structural similarity to saponins whose endosomal escape enhancing activity and ability to enhance the cytosolic delivery of effector molecules contained by the conjugate have been established are listed in Table A1. Thus, when the saponin is part of the conjugate of the present invention, upon binding of the sdAb of the conjugate, upon binding to the targeted cell surface binding site on the target cell, upon binding to said cell, and after endocytosis, the targeted delivery of the saponin into the endosome of said cell is about 100 to 1000 times more effective compared to contacting the same cell with free untargeted saponin (derivative) not provided together with a binding molecule such as an antibody or sdAb for binding to the cell surface molecule of the target cell.

[0114] For example, the sdAb of the conjugate of the present invention, which is small in size compared to an IgG-type antibody or its fragments such as Fab or scFv, contributes to efficient uptake by target cells that expose a binding site for the binding of the sdAb contained by the conjugate, for example, uptake by endocytosis. Usually, the sdAb in the conjugate of the present invention can bind to the cell surface receptor of target cells such as tumor cell-specific cell surface receptors. In this way, the conjugate of the present invention is particularly suitable for endocytosis into, for example, tumor cells expressing cell surface receptors.

[0115] One embodiment is a conjugate of the present invention in which at least one sdAb comprises an sdAb for binding to a cell surface molecule of a cell, and the cell is an abnormal cell such as a tumor cell, an autoimmune cell, an infected cell such as a virus-infected cell, or a cell containing a gene deficiency or an enzyme deficiency.

[0116] One embodiment is an sdAb for binding to a cell surface molecule of a cell, which is an sdAb, such as an anti-CD71 antibody like immunoglobulin: IgG-type OKT-9, an anti-HER2 antibody such as trastuzumab (Herceptin), pertuzumab, an anti-CD20 antibody such as rituximab, ofatumumab, tositumomab, obinutuzumab ibritumomab, an anti-CA125 antibody such as oregovomab, an anti-EpCAM (17-1A) antibody such as edrecolomab, an anti-EGFR antibody such as cetuximab, matuzumab, panitumumab, nimotuzumab, an anti-CD30 antibody such as brentuximab, an anti-CD33 antibody such as gemtuzumab, huMy9-6, an anti-vascular integrin alpha-v beta-3 antibody such as etaracizumab, an anti-CD52 antibody such as alemtuzumab, an anti-CD22 antibody such as epratuzumab, pinatuzumab, a binding fragment (Fv) of an anti-CD22 antibody moxetumomab, a humanized monoclonal antibody inotuzumab, an anti-CEA antibody such as labelizumab, an anti-CD44v6 antibody such as bevacizumab, an anti-FAP antibody such as sibrotuzumab, an anti-CD19 antibody such as huB4, an anti-CanAg antibody such as huC242, an anti-CD56 antibody such as huN901, an anti-CD38 antibody such as daratumumab, an anti-CD38 monoclonal antibody OKT-10, an anti-CA6 antibody such as DS6, an anti-IGF-1R antibody such as sizatumumab, an anti-integrin antibody such as 3B7, CNTO 95, an anti-syndecan-1 antibody such as B-B4, an anti-CD79b such as polatuzumab, any one or more of anti-HIVgp41 antibodies, preferably any one of anti-HIVgp41 antibody, anti-CD71 antibody, anti-HER2 antibody and anti-EGFR antibody, more preferably trastuzumab, pertuzumab, cetuximab, matuzumab, anti-CD71 antibody, OKT-9, most preferably any one of trastuzumab, cetuximab, anti-CD71 antibody OKT-9, or a conjugate of the present invention comprising an sdAb derived from or based on any one of them.

[0117] These cell surface molecules are typically present on tumor cells that have at least a certain degree of tumor cell specificity. The tumor cell specificity makes these receptors suitable targets for the conjugates of the present invention, and thus the sdAb in the conjugate can bind to such cell surface receptors. The saponin included by the conjugate of the present invention can stimulate the release and delivery of the effector molecule included by the conjugate of the present invention in the cytosol of cells such as (tumor) cells targeted by the sdAb included by the conjugate of the present invention. Therefore, it is particularly suitable to select as a target (tumor) cell surface molecule for sdAb, a cell surface receptor known for its suitability for functioning as a target for, for example, ADC and AOC. At the same time, the conjugate of the present invention is suitable for the co-delivery of the effector molecule, which is part of the conjugate, together with the saponin included by the same conjugate of the present invention. The conjugate is an improved ADC or improved AOC that includes sdAb and includes saponin. By targeting tumor cell-specific receptors with the conjugate of the present invention, it promotes the endocytosis and delivery of saponin as part of the conjugate to the target cell endosome and / or lysosome. When the tumor cell is contacted with the conjugate of the present invention, the effector molecule included by the conjugate of the present invention is co-delivered to the endosome or lysosome and, under the influence of the co-existing saponin, the effector molecule is subsequently transferred to the cytosol of the target cell. As described earlier herein, the application of the targeted saponin as part of the conjugate of the present invention provides an improvement of about 100-fold to 1000-fold in the enhancing effect of the saponin when considering the biological activity of the effector molecule included by the conjugate of the present invention as compared to the application of free saponin lacking a cell-targeting binding molecule such as a receptor ligand, antibody or sdAb.

[0118] Camels V in the conjugate of the present invention HThe application of small sdAbs such as those described above impedes or slows the clearance of the conjugates of the present invention from the bloodstream and body of a human subject to whom the conjugate is administered, as compared to the clearance rates generally observed for antibody-based ADCs. Additionally, due to the relatively small size of the sdAbs, the risk of limiting or interfering with the saponin activity inside target cells due to the presence of the linked protein domain is limited, as compared to larger-sized antibodies, such as when such antibodies are conjugated to saponin. Generally, the smaller the size of the molecule conjugated to saponin, the lower the risk of interference with the saponin activity inside cells due to the presence of sdAbs such as V HH etc. Furthermore, the relatively small size of the sdAbs results in rapid distribution in tissues such as tumor tissue, enabling improved access to target cells by the conjugates of the present invention, as compared to the relatively large-sized IgG generally applied in ADCs, OACs, etc., and enabling improved binding (degree) to target cells. One of the many advantages of applying sdAbs in the conjugates of the present invention is the absence of an Fc tail, which is common to normal antibodies of the IgG type. The absence of an Fc tail in the sdAbs of the conjugates of the present invention prevents the occurrence of Fcγ-receptor-mediated off-target effects, such as unwanted side effects associated with Fcγ-receptor activation, when the conjugate is administered to a patient in need thereof. The absence of an Fc tail eliminates the risk of side effects generated by the binding of Fc to the cells of a patient to whom an antibody-based ADC is administered. The conjugates containing the sdAbs of the present invention do not have this risk of unwanted side effects mediated by Fc.

[0119] In one embodiment, at least one effector molecule is covalently attached via a linker to at least one sdAb, preferably to one and / or at least one of at least one saponin, preferably one, of at least one sdAb or is directly covalently attached to the sdAb and / or saponin, and / or at least one saponin is covalently attached via a linker to at least one sdAb, preferably to one and / or at least one of at least one effector molecule, preferably one, of at least one sdAb or is directly covalently attached to the sdAb and / or effector molecule, which is a conjugate of the present invention.

[0120] In one embodiment, the conjugate comprises a trifunctional linker to which each of at least one sdAb, at least one saponin, and at least one effector molecule is preferably covalently attached separately, directly, or via a linker, preferably the conjugate comprises a trifunctional linker to which one sdAb, at least one saponin, and at least one, preferably one, effector molecule are covalently attached separately, directly, or via a linker, which is a conjugate of the present invention.

[0121] Coupling of saponin to sdAb and / or effector molecule via a linker provides flexibility when the binding sites for coupling of saponin to sdAb and / or effector molecule are considered. Further, such a linker may act as a spacer between the sdAb, saponin, and effector molecule, such that the sdAb maintains its ability to bind to the binding site on the cell surface molecule, the saponin maintains its ability to enhance endosomal escape of the effector molecule comprised by the conjugate, and the effector molecule maintains its biological activity against its intracellular binding partner.

[0122] In one embodiment, at least one saponin is covalently linked via a thioether bond to a sulfhydryl group in one of at least one sdAb and / or one of at least one effector molecule, and the covalent bond is preferably to an aldehyde group at the C 23 position and is via a linker N-ε-maleimidocaproic acid hydrazide (EMCH) that is covalently linked to an aldehyde group at the C position of the aglycone core structure of the saponin and to a sulfhydryl group in an sdAb and / or a sulfhydryl group in an effector molecule such as a cysteine sulfhydryl group. It is a conjugate of the present invention.

[0123] In one embodiment, at least one saponin belongs to the type of 12,13-dehydrooleanane optionally having an aldehyde functional group at the C 23 position and is a bi-desmoside triterpene saponin or a derivative thereof containing a glucuronic acid unit in a first sugar chain linked by a C3 beta-OH group of the aglycone core structure of the saponin, and the saponin is preferably covalently linked via a linker to an amino acid residue of at least one sdAb and / or at least one effector molecule via a carboxyl group of the glucuronic acid unit in the first sugar chain, and the amino acid residue is preferably selected from cysteine and lysine. It is a conjugate of the present invention.

[0124] In one embodiment, at least one saponin contains a glucuronic acid unit in a first sugar chain at the C3 beta-OH group of the aglycone core structure of the saponin, the glucuronic acid unit is covalently linked to a linker, and the linker is preferably covalently linked via an amide bond to an amine group in at least one sdAb and / or at least one effector molecule such as a lysine or an N-terminal amine group of the sdAb and / or the effector molecule. Preferably, the linker is 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). It is a conjugate of the present invention.

[0125] One embodiment is a conjugate of the invention comprising two or more covalently linked saponin moieties of at least one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1 to 100 such moieties, or any number in between such as 7, 9, 12 saponin moieties.

[0126] One embodiment is a conjugate of the invention wherein two or more covalently linked saponin moieties are covalently linked directly to amino acid residues of at least one sdAb and / or at least one effector molecule, preferably to cysteine and / or lysine, and / or via a linker and / or a cleavable linker.

[0127] One embodiment is molecular structure (II): (Saponin-linker-) a Immunoglobulin-effector portion (Structure (II)) (wherein a = 1 to 4, preferably 1, 2, 4, and the immunoglobulin is preferably an sdAb) or molecular structure (III): (Saponin-dendron(-saponin) x ) b -Immunoglobulin-effector portion (Structure (III)) (wherein x = 1 to 100, preferably 1 to 63, 1 to 31, 1 to 15, 1 to 7, or 3; b = 1 to 4, preferably 1, 2, 4, and the immunoglobulin is preferably an sdAb) or molecular structure (IV): (Saponin-trifunctional linker(-effector portion)) c -Immunoglobulin (Structure (IV)) (wherein c = 1 to 4, preferably 1, 2, 4, and the immunoglobulin is preferably an sdAb) or molecular structure (V): ((Saponin-dendron(-saponin) y)-Trifunctional linker (-effector portion)) d -Ig (Structure (V)) (In the formula, y = 1 to 100, preferably 1 to 63, 1 to 31, 1 to 15, 1 to 7, or 3; d = 1 to 4, preferably 1, 2, 4, and immunoglobulin ("Ig") is preferably a sdAb) is an endosome and / or lysosome escape-enhancing conjugate according to the present invention that essentially has a molecular form of

[0128] Preferably, x or y is 3, 7 or 15. Preferably, b or d is 1, 2 or 4, but in some embodiments, b or d is 3 and the immunoglobulin is V HH When it is a sdAb such as, a is preferably 1, b is preferably 1, c is preferably 1, and d is preferably 1. The dendron is, for example, a G4 dendron or a G5 dendron. Preferably, the saponin is bound to the linker via a cleavable bond such as a hydrazone bond that is cleaved intracellularly under pH conditions of <6.5 (i.e., the pH in endosomes, endolysosomes, lysosomes). Preferably, the linker is EMCH. Preferably, the trifunctional linker is a linker having structure A as shown below in this specification. Regarding structures II and III, preferably, the effector portion is bound to Ig via a linker such as a cleavable linker.

[0129] One embodiment is a covalent saponin conjugate in which two or more covalently linked saponin moieties are part of a covalent saponin conjugate comprising at least one oligomeric or polymeric molecule and two or more saponins covalently linked thereto, the covalent saponin conjugate being covalently linked to at least one of at least one sdAb and / or at least one of at least one effector molecule, which is a conjugate of the present invention.

[0130] Such covalently linked saponin conjugates can function as carriers for multiple saponin moieties, which can be linked to the sdAb included by the conjugate via a single bond, preferably via a (cleavable) linker. The covalently linked saponin conjugate can have any selected number of covalently linked saponin moieties, such as 1 to 200 saponin moieties associated with a selected oligomeric or polymeric structure type containing binding sites for covalently linking these saponins. Thus, the application of such covalently linked saponins provides freedom when the number of saponin moieties in the conjugate of the present invention is considered. For example, with respect to the cytosolic delivery of effector molecules included by the conjugate of the present invention, the number of saponins present in the conjugate of the present invention can be adapted by providing a covalently linked saponin conjugate having several saponin moieties sufficient and adequate to stimulate the cytosolic delivery of the effector molecule when the covalently linked saponin conjugate is part of the conjugate of the present invention and the effector molecule coexists with the saponin as a complex part of the same conjugate in the endosome or lysosome of the target cell where the effector molecule should exert its biological activity.

[0131] Preferably, 1 to 8 covalently linked saponin conjugates, more preferably 2 to 4 such covalently linked saponin conjugates, are linked to the sdAb and / or effector molecule, and at least one covalently linked saponin conjugate is optionally dendron-based and optionally has 1 to 32 saponin moieties, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 such moieties, or any number of such moieties in between, such as 7, 9, 12 saponin moieties, which are covalently linked directly or via a linker to the oligomeric or polymeric molecule of at least one covalently linked saponin conjugate.

[0132] Preferably, one or two of the covalently linked saponin conjugates are attached to a single sdAb in the conjugate of the invention. For many purposes, the coupling of a single saponin to a single sdAb contained by the conjugate or the coupling of a single covalently linked saponin conjugate is sufficient for the efficient stimulation of the delivery of effector molecules to the target cell and the cytosol of said cell, and the effector molecules are contained by the conjugate of the invention. Usually, 4, 8 or 16 saponins are contained by the conjugate of the invention, such as 4 or 8 saponins contained by a single covalently linked saponin conjugate coupled to the sdAb in the conjugate of the invention. Usually, such a conjugate of the invention comprises a single sdAb to which one or more saponins or covalently linked saponin conjugates are attached, and preferably, a single saponin or a single covalently linked saponin conjugate is part of the conjugate.

[0133] One embodiment is a conjugate of the invention in which at least one saponin is covalently attached to at least one of at least one sdAb and / or at least one of at least one effector molecule via a cleavable linker.

[0134] One embodiment is a conjugate of the invention in which the cleavable linker comprises a cleavable bond selected from hydrazone and hydrazide bonds that are cleaved under acidic conditions, and / or bonds that are susceptible to proteolysis, such as proteolysis by cathepsin B, and / or bonds that are readily cleaved under reducing conditions, such as disulfide bonds, where the cleavable linker is subjected to cleavage under acidic conditions, reducing conditions, enzymatic conditions and / or photoinductive conditions, preferably.

[0135] One embodiment is a conjugate of the invention in which a cleavable linker is cleaved in vivo under acidic conditions such as those found in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at pH 4.0 to 6.5, more preferably at pH ≤ 5.5.

[0136] Such a cleavable linker, which is cleavable under conditions such as those found in endosomes and lysosomes, facilitates the delivery of free saponin into endosomes or lysosomes upon cleavage (separation) of the saponin from the remainder of the conjugate of the invention. Thus, the conjugate of the invention contributes to the ability of free saponin to stimulate and / or facilitate the delivery of effector molecules contained by the conjugate of the invention from endosomes (or lysosomes) to the cytosolic compartment of target cells, the advantage of cell-targeted delivery of saponin upon specific binding of the sdAb to cell surface molecules on the target cell, and the advantage of the presence of free saponin within cells, i.e., within endosomes (or lysosomes).

[0137] One embodiment is a conjugate of the invention in which an oligomeric or polymeric molecule of a covalent saponin conjugate is covalently bound to at least one of at least one sdAb and / or at least one of at least one effector molecule, preferably to an amino acid residue of the sdAb and / or effector molecule.

[0138] One embodiment is a conjugate of the invention in which at least one saponin is covalently bound to an oligomeric or polymeric molecule of a covalent saponin conjugate via a cleavable linker according to the invention.

[0139] One embodiment is a conjugate of the invention in which at least one saponin 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.

[0140] One embodiment is that at least one saponin is C 23It includes an aglycone core structure containing an aldehyde functional group at a position, and at least one saponin optionally contains a glucuronic acid functional group in the first sugar chain at the C3 beta-OH group of the aglycone core structure of the saponin. The aldehyde functional group is involved in a covalent bond to an oligomeric or polymeric molecule of a covalent saponin conjugate, and / or when present, the glucuronic acid functional group is involved in a covalent bond to an oligomeric or polymeric molecule of a covalent saponin conjugate that is a bond of the saponin via a direct covalent bond or via a linker (the linker is a cleavable linker or a stable linker). As used herein, stable refers to a bond between a saponin and an sdAb or an effector molecule, or a bond between a saponin and an oligomeric or polymeric structure, and this bond remains unchanged (not cleaved) under acidic conditions within a cell, particularly under acidic conditions within an endosome or lysosome of such a cell. Additionally, such a stable bond remains unchanged (i.e., not cleaved) in the bloodstream and organs of a human subject to whom a conjugate of the present invention containing a covalent saponin conjugate is administered. In contrast, a cleavable linker associated with the binding of a saponin to an sdAb or an effector molecule contained by the conjugate, or an oligomeric or polymeric structure, refers to a bond that is cleaved under acidic conditions as seen within endosomes and lysosomes of mammalian cells such as tumor cells in human cells, but such a cleavable linker remains unchanged (not cleaved) when a conjugate containing such a cleavable bond is present outside the bloodstream or organs, i.e., outside the cells of a human subject to whom a conjugate of the present invention is administered.

[0141] One embodiment is the C of the aglycone core structure of at least one saponin 23The aldehyde functional group at the position is covalently bonded to the linker EMCH, and EMCH is covalently bonded via a thioether bond to a sulfhydryl group in an oligomeric or polymeric molecule of a covalent saponin conjugate such as the sulfhydryl group of cysteine, which is the conjugate of the present invention. The binding of the EMCH linker to the aldehyde group of the sapogenin 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. The sdAb contained by the conjugate of the present invention or the effector molecule contained by the conjugate of the present invention, or a saponin coupled to an oligomeric or polymeric structure of a covalent saponin conjugate, such a covalent saponin conjugate is coupled to the sdAb or effector molecule of the conjugate and is released from the conjugate of the present invention when delivered to the endosome or lysosome of a target cell that exposes a cell surface molecule to which the sdAb of the conjugate can bind. Thus, the saponin coupled to the sdAb or effector molecule in the conjugate of the present invention is transferred from the outside of the cell to the endosome (or lysosome), and in the endosome (or lysosome), the saponin is released from the rest of the conjugate upon cleavage driven by the pH of the hydrazone bond. In the endosome (or lysosome), the free saponin can exert its stimulatory activity when the delivery of the effector molecule contained by the conjugate of the present invention to the cytosol is considered. Surprisingly, the inventors have established that for saponin, the presence of saponin in free form in endosomes or lysosomes is not essential for the endosome escape enhancing activity of saponin. Also, for example, the saponin contained by a particular conjugate enhances the delivery of the effector molecule from the endosome / lysosome to the cytosol of the targeted cell when both the effector molecule and the saponin as part of a particular conjugate are contacted with the same target cell.

[0142] In one embodiment, the glucuronic acid functional group in the first sugar chain at the C3 beta-OH group of the aglycone core structure of saponin is covalently bonded to the linker HATU, and HATU is covalently bonded via an amide bond to the amine group in the oligomer molecule or polymer molecule of the covalent saponin conjugate such as the lysine or N-terminal amine group of the protein. It is the conjugate of the present invention. When the HATU linker is coupled to the saponin and sdAb or effector molecule of the conjugate of the present invention, the saponin is, for example, the N-terminus of the sdAb or effector molecule (when such effector molecule is a proteinaceous effector molecule such as a protein toxin) or is present in the sdAb or is bound to the amine group of lysine present in the effector molecule.

[0143] In one embodiment, the polymer molecule or oligomer molecule of the covalent saponin conjugate is bound to at least one, preferably one, of at least one sdAb and / or at least one, preferably one, of at least one effector molecule, preferably to the amino acid residue of the sdAb and / or the amino acid residue of the effector molecule, and contains a click chemistry group on the polymer molecule or oligomer molecule of the covalent saponin conjugate, 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. It is the conjugate of the present invention.

[0144] In one embodiment, the polymeric or oligomeric molecule of the covalent saponin conjugate comprises a polymeric structure and / or an oligomeric structure selected from linear polymers, branched polymers and / or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, DNA, polypeptides, poly-lysine, poly-ethylene glycol, oligo-ethylene glycol (OEG) such as OEG3, OEG4 and OEG5, or aggregates, preferably constructed by covalent cross-linking, and preferably, the polymeric or oligomeric molecule of the covalent saponin conjugate is a dendron such as a poly-amidoamine (PAMAM) dendrimer, which is a conjugate of the present invention. Since it is driven by the number of selected saponins to be incorporated in the conjugate of the present invention, the type and size or length of the oligomeric or polymeric structure are selected. That is, the number of saponins to be coupled to the sdAb or effector molecule included by the conjugate for the formation of the conjugate of the present invention can determine the selection of a suitable oligomeric or polymeric structure having a sufficient amount of binding sites for coupling the desired number of saponins, and at the same time, provide a covalent saponin conjugate having the selected number of saponin moieties to be coupled to the sdAb or effector molecule for the provision of the conjugate of the present invention. For example, the length of the OEG or the size of the dendron or poly-lysine molecule determines the maximum number of saponins that can be covalently linked to such an oligomeric or polymeric structure.

[0145] Accordingly, the conjugate according to the invention comprises at least one saponin. In this context, "at least one" means that the conjugate may contain one saponin molecule, but may also contain a pair (e.g., 2, 3 or 4) of saponins or a plurality (e.g., 10, 20 or 100) of saponins. Depending on the application, the conjugate may comprise a covalently bound scaffold having covalently bound saponins (covalent saponin conjugate), and the scaffold may be designed to contain a defined number of saponins as defined for it. Preferably, the conjugate according to the invention contains a defined number or range of saponins, not a random number. This is particularly advantageous for drug development related to marketing approval. The defined number in this regard means that the conjugate preferably contains the previously defined number of saponins. This is achieved, for example, by designing a scaffold comprising a polymeric structure having a certain number of possible moieties for saponin binding. Under ideal circumstances, all of these moieties are coupled to the saponin and the scaffold contains the previously defined number of saponins. For example, it is envisaged that a standard set of scaffolds containing 2, 4, 8, 16, 32, 64, etc. saponins can be provided and the optimal number can be easily tested by the user according to the user's needs. One embodiment is the conjugate of the invention comprising the scaffold of the invention (the covalent saponin conjugate of the invention), where the saponin is present in a defined range, for example, because not all moieties present in the polymeric structure bind to the saponin under non-ideal circumstances. 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. Thus, in such a case, the conjugate comprising the scaffold according to the invention contains 2, 3 or 4 saponins when the range is defined as 2 to 4.

[0146] The scaffold does not basically depend on the type of saponin covalently attached to the scaffold, and the scaffold is then (sequentially) covalently coupled to the conjugate. Thus, the conjugate of the present invention containing the scaffold (the covalent saponin conjugate of the present invention) is a basic product for platform technology. Since at least one covalently attached saponin mediates the intracellular delivery of the effector molecule bound to the cell surface molecule-targeted sdAb contained by the conjugate of the present invention, the scaffold technology according to the present invention is a system that mediates the controlled delivery of the effector moiety into cells by saponin. The scaffold provides an optimized functionally active unit that can be linked to the saponin and the cell surface molecule-targeted sdAb contained by the conjugate at a single defined position in the sdAb.

[0147] One embodiment is a conjugate of the present invention (a covalent saponin conjugate of the present invention) comprising a scaffold according to the present invention, wherein the number of monomers in the polymer or oligomer structure is an exactly defined number or range. Preferably, the polymer or oligomer structure is a poly(amine), such as a structure of polyethyleneimine and poly(amidoamine), or a poly(ester) such as polyethylene glycol, poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, poly(dextrin), or a structure such as a peptide or protein, or a natural and / or artificial polyamino acid, such as poly-lysine, DNA polymer, stabilized RNA polymer or PNA (peptide nucleic acid) polymer, and appears as a linear, branched or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer or an aggregate of these pure or mixed structures. Preferably, the polymer or oligomer structure is biocompatible, and biocompatibility means that the polymer or oligomer structure does not exhibit substantial acute or chronic toxicity in an organism and can be either excreted as such or completely decomposed into compounds that can be excreted by the body's metabolism and / or physiological compounds. The aggregates can be constructed by covalent cross-linking or non-covalent and / or attractive forces. Thus, they can also form nanogels, microgels, or hydrogels, or bind to carriers such as inorganic nanoparticles, colloids, liposomes, micelles or particle-like structures containing cholesterol and / or phospholipids. The polymer or oligomer structure preferably has an exactly defined number or range of coupling moieties (chemical groups) for the coupling of glycoside molecules (and / or effector molecules and / or ligands, fragments thereof such as monoclonal antibodies or sdAb, etc., carrier molecules).Preferably, at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably (about) 100% of the precisely defined number or range of coupling moieties (chemical groups) in the polymer or oligomer structure are occupied by the glycoside molecules (the saponins of the present invention) in the scaffold (the covalent saponin conjugate of the present invention) according to the present invention.

[0148] Preferably, a dendron is a branched, precisely defined dendritic polymer having a single chemically addressable group at the origin of the tree called the focal point. A dendrimer is one in which two or more dendrons are linked at their focal points. A dendronized polymer is one in which the focal points of one or more dendrons are linked to the polymer. In a preferred embodiment, a scaffold according to the present invention is provided, and the polymer or oligomer structure includes a linear, branched or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer or an aggregate of these structures, pure or mixed, the aggregate can be constructed by covalent cross-linking or non-covalent attraction, and can form a nanogel, microgel, or hydrogel, and preferably, the polymer is a derivative of poly(amine), such as polyethyleneimine and poly(amidoamine), and structures such as poly(ester) such as polyethylene glycol, poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), and poly-lysine, or natural and / or artificial polyamino acids such as peptides or proteins or structures such as DNA polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers. Preferably, the polymer or oligomer structure is biocompatible.

[0149] One embodiment has at least one saponin with a trifunctional linker, preferably, Structure A:

[0150] [Chem.]

[0151] covalently bound to at least one, preferably one, of at least one sdAb via a trifunctional linker represented by, and covalently bound to at least one, preferably one, of at least one effector molecule, the conjugate preferably comprises a trifunctional linker of structure A and has structure B:

[0152] [Chem.]

[0153] having a molecular structure represented by, wherein S is at least one saponin or covalent saponin conjugate according to the invention, E is at least one, preferably one, effector molecule, A is at least one sdAb such as a single sdAb, and L1, L2 and L3 are each individually either a bond between a trifunctional linker and a saponin or covalent saponin conjugate, an effector molecule and an sdAb, or L1, L2 and L3 are linkers (L1, L2 and L3 may be the same or different), a conjugate of the invention.

[0154] Unless otherwise indicated, particularly when referring to the endosomal escape mechanism of the saponins of the invention, the terms "endosome" or "endosomal escape" are always used herein to include endolysosomes and lysosomes, and escape from endolysosomes and lysosomes, respectively. After entering the cytosol, the substance may move to other cellular units such as the nucleus.

[0155] In formal terms, a glycoside is any molecule in which a sugar group is attached via its anomeric carbon to another group by a glycosidic bond. Glycoside molecules such as saponins in the context of the present invention do not wish to be bound by any theory, but in particular are molecules that can further enhance the effect of an effector moiety by promoting endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (such as the saponins of the present invention exemplified herein and in the claims) interact with the membranes of the compartments and vesicles of the endocytosis pathway and recycling pathway, causing them to leak such that the effector moiety, resulting in enhanced endosomal escape. The term "scaffold can enhance endosomal escape of the effector moiety" means that at least one saponin (glycoside molecule) coupled to a cell surface molecule-targeting antibody such as an sdAb via a linker or directly or via a polymer or oligomeric structure of the scaffold (the covalent saponin conjugate of the present invention) is, optionally and preferably, when both molecules are within an endosome, e.g., a late endosome, at least one saponin is, for example, released from the conjugate, such as by cleavage of a cleavable bond between at least one glycoside (saponin) and the conjugate (e.g., via a polymer or oligomeric structure of the scaffold and / or via a linker), and can enhance endosomal escape of the effector moiety. Even if the bond between at least one saponin according to the present invention and the conjugate cell surface molecule-targeting sdAb of the present invention via a linker or scaffold is an "stable bond" optionally, such a bond does not necessarily mean that it cannot be cleaved in the endosome, for example, by an enzyme. For example, a saponin optionally combined with a part of the oligomeric or polymeric structure of the linker or scaffold can be cleaved from the remaining linker fragment or oligomeric or polymeric structure.For example, proteases may cleave a (proteinaceous) linker or proteinaceous polymer structure, such as albumin, thereby releasing at least one saponin. However, the glycoside molecule (preferably a saponin) is in its native form before being released and conjugated (prepared to be conjugated) to the cell surface molecule-targeting sdAb of the conjugate of the present invention via an active form, preferably, optionally, a linker and / or an oligomer or polymer scaffold (the covalent saponin conjugate of the present invention). Thus, whether the glycoside (saponin) has its natural structure after such cleavage or the glycoside (saponin) has a chemical group or a linker (part of it) attached thereto, while the glycoside bioactivity (saponin bioactivity), for example, the endosome / lysosome escape enhancing activity against an effector moiety present in the same endosome or lysosome, is preferably maintained or restored at the time of such cleavage of the bond between the glycoside (saponin) containing the linker and / or scaffold of the present invention and a cell surface molecule-targeting antibody such as sdAb. For the present invention, for example, the term "stable" with respect to the bond between a saponin and an amino acid residue of a cell surface molecule-targeting sdAb, a linker, a polymer or oligomer structure (scaffold also known as the covalent saponin conjugate of the present invention), a ligand, a (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or an effector (effector moiety, effector molecule) means that the bond is not readily cleaved or at least is not designed to be readily cleaved, for example, by a difference in pH, salt concentration, or UV light, reducing conditions. For the present invention, for example, the term "cleavable" with respect to the bond between a saponin and a cell surface molecule-targeting sdAb, a linker, an amino acid residue, a polymer or oligomer structure of a covalent saponin conjugate, a ligand, an antibody and / or an effector molecule means that the bond is designed to be readily cleaved, for example, by a difference in pH, salt concentration, under reducing conditions, etc. Those skilled in the art are well aware of such cleavable bonds and methods for preparing them.

[0156] Prior to the present invention, one of the major hurdles to introducing ADCs and AOCs to the market was their narrow therapeutic window: the therapeutically effective doses of ADCs or AOCs were associated with (unacceptable) side effects that interfered with (precluded) the development and significance of treating patients with ADCs. The application of conjugates of the present invention, such as ADC-saponin conjugates and AOC-saponin conjugates, now enables the delivery of one or more glycoside molecules (saponins) to (target) cells, together with an ADC having a payload or together with (monoclonal) antibodies (sdAbs) conjugated to oligonucleotides such as BNAs according to the present invention. In particular, previously it was not possible to specifically deliver simultaneously to the cytosol of a cell, via, for example, the endocytosis pathway of the cell, an effector portion of an ADC or AOC or any other conjugate of a payload, and a (proteinaceous) cell surface molecular targeting molecule, and a specific number or range of glycoside molecules (saponins) per effector portion.

[0157] The solution provided by the present invention involves the covalent attachment of at least one saponin to a cell surface molecule targeting molecule of the conjugate of the present invention, i.e., an sdAb. A further solution provided by the present invention involves polymerizing (initially) glycoside molecules (saponins) using an oligomer or polymer scaffold and resulting in a cell surface molecule targeting molecule comprised by a conjugate of the present invention having a group of covalently attached saponins, for example, enabling the re-monomerization of one or more saponins at an intracellular site where the mechanism of action of the saponin is desired after endocytosis. In this context, "polymerizing" means forming a scaffold (the covalent saponin conjugate of the present invention) by reversible and / or irreversible multiple conjugation of saponin molecules to an sdAb via a linker or directly or via a polymeric or oligomeric structure, or forming a polymeric or oligomeric structure by reversible and / or irreversible multiple conjugation of (modified) saponins to form a scaffold (the covalent saponin conjugate of the present invention). In this context, "re-monomerization" means, for example, after endocytosis, cleaving the saponin from the conjugate, from the linker that links the saponin to the cell surface molecule targeting sdAb of the conjugate, or from the scaffold, to restore the (natural) chemical state of the unbound saponin, which may or may not include additional chemical groups such as chemical groups for attaching the saponin to the linker, amino acid residues of the conjugate or scaffold, and / or chemical linkers attached to chemical groups of the saponin such as aldehyde groups or carboxylic acid groups. Due to the complex chemical action of saponins, for example, the "polymerization" of saponins at a scaffold or other linking linker and their "re-monomerization" at a desired location such as intracellularly after endocytosis have been difficult challenges. In particular, the chemical reactions used to provide a covalently linked glycoside for covalently attaching to a linker and conjugate, for example, a triterpenoid saponin (polymerization of glycosides), typically occur in a water-free organic solvent, while saponins and, for example, biocompatible polymers applied as scaffolds for having bound saponins are water-soluble molecules.The chemical properties of unmodified saponin further prohibit polymerization by itself, and one other possible solution for binding multiple saponins (directly) to an effector molecule was presumed to be less promising because the effector molecule (drug, toxin, polypeptide or polynucleotide) usually does not provide sufficient binding sites, and the coupling product becomes highly heterogeneous and / or when coupling a bioactive molecule such as saponin together with, for example, a peptide, toxin, nucleic acid, there is a risk of affecting and interfering with the activity of one or both of the molecules conjugated to the conjugate containing such saponin. Further, when saponin is coupled to, for example, an ADC or an antibody-oligonucleotide conjugate (AOC), there was a high risk that the effector site contained by the conjugate of the present invention would lose its function. Embodiments of the present invention address at least one of these deficiencies.

[0158] A second aspect of the present invention relates to a pharmaceutical composition comprising the conjugate of the present invention and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0159] Whether the conjugate of the present invention comprising saponin, further comprising one or more (cleavable) linkers and / or optionally a scaffold (the covalent saponin conjugate of the present invention) or not, interferes with the acidic environment and can inhibit the endosomal escape function of at least one glycoside (saponin) can be readily determined in the art by an assay as described in the Examples section. The inhibition is described as "the doubling rate of the amount of glycoside (saponin of the present invention) required to induce cell death of 50% of the cells". The scaffold preferably does not result in an increase in the amount of glycoside molecules (saponin) required to obtain cell death of 50% of the cells as observed when using chloroquine as a positive control. Alternatively, and preferably, the conjugate comprising saponin, further comprising one or more (cleavable) linkers and / or optionally a scaffold or not, does not result in an increase of at least 4-fold of the glycoside molecules to induce cell death of 50% of the cells, more preferably, does not result in an increase of at least 2-fold. The doubling rate will be measured in the assay, and chloroquine as a positive control induces an increase of 2-fold of the amount of glycoside, preferably, the amount of saponin, and the saponin is any one or more of the saponins of the present invention (previous embodiments) that observe cell death of 50% of the cells.

[0160] As described above, at least one saponin comprised by the conjugate according to the present invention increases the effectiveness of at least the existing and new effector moieties as defined in the present invention. Potential side effects will be reduced without a decrease in effectiveness because the administration of the effector moiety comprised by the conjugate is reduced. Accordingly, the present invention provides a conjugate according to the present invention for use in medicine or for use as a medicament. A third aspect of the present invention relates to a pharmaceutical composition of the present invention for use as a medicament.

[0161] Several preferred features can be incorporated for the endosome escape enhancer comprised by the conjugate of the present invention, i.e., for the saponin of the present invention: (1) They are preferably not toxic and do not elicit an immune response. (2) They preferably do not mediate cytosolic uptake of the effector moiety into off-target cells. (3) Their presence at the site of action preferably coincides with the presence of the effector moiety. (4) They are preferably biodegradable or excretable. And (5) they preferably do not substantially interfere with biological processes of the organism that are not related to the biological activity of the effector molecule to which the endosome escape enhancer is conjugated, e.g., do not interact with hormones. Examples of saponins of the present invention that meet at least to some extent the foregoing criteria are bidesmoside triterpenes, preferably bidesmoside triterpenoid saponins such as SO1861, SA1641, QS-21, GE1741, and additional saponins recited throughout this specification.

[0162] Also provided is the use of the conjugate according to the invention for the manufacture of a medicament. In particular, cancer medicaments, and in particular conventional chemotherapeutic medicaments, are well known for their side effects. For the targeting and synchronization of both the pharmaceutically active substance comprised by the conjugate and the saponin comprised by the very same conjugate molecule in terms of timing and location, the therapeutic conjugate according to the invention is particularly useful for use as a medicament, in particular in a method for treating cancer. Accordingly, the invention provides a therapeutic conjugate according to the invention for use in a method for treating cancer. The invention also provides a therapeutic conjugate according to the invention for use in a method for treating an acquired or genetic disorder, in particular a monogenic deficiency disorder. Thus, the therapeutic conjugate comprises at least one saponin and at least one effector moiety, and an sdAb for targeting the conjugate to abnormal target cells such as tumor cells or autoreactive cells. Thus, one aspect of the invention relates to a therapeutic conjugate according to the invention, the conjugate comprising a covalently bound effector moiety for use in a method for the treatment of cancer or an autoimmune disease, and a covalently bound saponin, and a cell surface molecule-binding antibody such as an sdAb.

[0163] A further application of the conjugates of the invention in medicine is the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting diseases can be genetic or acquired. In many cases, only symptomatic treatment is possible, and for some rare diseases, the insufficient treatment options lead to a shortening of the patient's lifespan. An example of such a disease is phenylketonuria, which is a congenital disorder of metabolism that causes a decrease in the metabolism of the amino acid phenylalanine. The disease is characterized by mutations in the gene for the liver enzyme phenylalanine hydroxylase. Phenylketonuria is not curable to date. The incidence is approximately 1:10,000, and the highest known incidence is 1:2,600 in Turkey. The cell surface molecule-targeted antibody, preferably a V HH such as sdAb, of the conjugate of the invention comprising a conjugated phenylalanine hydroxylase or a conjugated polynucleotide encoding phenylalanine hydroxylase can be used to target liver cells and replace the deficient enzyme in hepatocytes by the use of a suitable specific antibody or sdAb. This is an example of the use of the therapeutic conjugate of the invention comprising a conjugated saponin and a conjugated enzyme or oligonucleotide for replacement or gene therapy. In a preferred embodiment, a therapeutic conjugate according to the invention for use in a method of gene therapy or replacement therapy is provided.

[0164] The conjugates of the present invention now enable the design and manufacture of a non-viral clinically applicable gene delivery technology for a single component. For example, the conjugates of the present invention enable the development of a non-viral gene delivery technology that enhances the therapeutic efficacy with lower therapeutic doses, thereby improving the health of patients. The conjugates of the present invention, in particular, overcome the long-standing major bottleneck in the field of gene delivery, namely, the efficient, safe, and cost-effective translocation to the cytosol / nucleosol through the endosomal membrane of a gene therapy product, when they contain a covalently bound cell surface molecule targeting antibody, such as a monoclonal antibody or sdAb, for binding to (tumor, autoimmune) cell surface specific molecules, and when they are bound to an effector moiety, such as an oligonucleotide, for example, a BNA. In fact, gene therapy is one of the most promising therapeutic options for future advanced therapies 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 insufficiently safe delivery of gene materials for therapeutic use in patients.

[0165] Therefore, when applying the conjugates of the present invention comprising a cell-targeting cell surface molecular targeting molecule such as a ligand or preferably an antibody (its fragment, domain, preferably sdAb) and an oligonucleotide such as antisense BNA, the inventors have now enabled the overcoming of a long-standing major bottleneck in the field of gene delivery: the safe transfer of gene therapy products into the cytosol / nucleosol through the endosomal membrane. The conjugates of the present invention enable the targeting of any addressable cell type by all known genetic factors, thereby becoming a technology designed not only for genetic disorders but also for ensuring good patient therapy for cancer therapy, and thus being important for a large patient population. The technology based on the conjugates of the present invention is due to the endosome escape enhancer (EEE) such as saponin as exemplified herein and the saponin of the embodiments according to the present invention, a targeting ligand or a (monoclonal) (tumor cell-specific) antibody, or its fragment, or preferably V HHCell surface molecule targeting molecules such as sdAbs, and effector moieties, polymers or oligomeric scaffolds (the covalent saponin conjugates of the present invention) that function as carriers for effector genes such as LNAs or BNAs of the present specification may be included. For example, the use of the conjugates of the present invention containing cell-targeting antibodies (fragments) or sdAbs and oligonucleotides such as BNAs has the potential to carry all kinds of biopolymers into the cytosol and nucleus. The development of novel targeting ligands, sdAbs and monoclonal (human, humanized) antibodies is being continuously investigated by numerous research groups and companies around the world. The same is true for oligonucleotides aimed at delivery in the cytosol of diseased cells such as cancer cells. Thus, the conjugates of the present invention serve as molecular interfaces through which current and future targeting sdAbs and antibodies as well as current and future therapeutic oligonucleotides (as well as payloads such as protein toxins) can be or can be conjugated by click chemistry to the oligomeric or polymeric scaffold modules (the covalent saponin conjugates of the present invention) of the present invention, enabling future developments in the fields of personalized drug application and tissue and cell targeting technologies. The conjugates of the present invention may contain antibodies and ligands as cell surface molecule targeting molecules, but sdAbs are preferred. The global market for gene therapy drugs is growing rapidly and encompasses possible treatments for a wide range of disease areas such as cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, HIV and many rare (monogenic) diseases. Gene therapy drug technologies based on existing viral vectors have important issues such as safety, manufacturing logistics, and the associated high costs. The conjugates of the present invention enable use in a technology platform that serves as an alternative to existing viral gene delivery technologies. Thus, the conjugates of the present invention are suitable for practice in an approach for developing non-viral gene therapy for diseases such as cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, HIV infection and many rare (monogenic) diseases.The conjugate of the present invention is suitable for developing a novel therapy for revolutionizing the fields of antibody-drug conjugates (ADCs) and therapeutics based on oligonucleotides by producing gene therapy agents based on non-viral vectors such as those based on targeted antisense BNA. In particular, the application of the conjugate of the present invention in covalently conjugated conjugates having antibodies such as sdAbs and oligonucleotides such as BNA and at least one saponin is one of many advantageous approaches made possible by the present invention. For example, the use of the conjugate of the present invention currently enables the exploitation of the endocytosis pathway of mammalian cells. Endocytosis is exploited for the delivery of therapeutic agents, and the conjugate of the present invention contributes, for example, to the uptake of siRNA contained by the conjugate and the improvement of endosomal escape. The conjugate of the present invention is preferably used together with, for example, a small molecule that acts as a delivery enhancer for the payload, oligonucleotide. In addition to this, it has a covalently coupled oligonucleotide such as BNA and a ligand and preferably an antibody (domain or fragment, preferably V. HH) having a covalently coupled cell targeting moiety such as, the conjugate of the present invention having the saponin of the present invention is related to their application as two components, thereby complicating therapeutic approval and clinical applicability. It provides a solution to the existing problems found in existing endosome escape enhancers and gene therapy products, which is because such conjugates of the present invention are single conjugate therapeutic molecules comprising a saponin, a gene product such as BNA, and a (tumor) cell targeting moiety such as a (monoclonal) antibody or sdAb. Thus, the present invention provides a non-viral gene delivery technology, an endosome escape enhancer (e.g., the glycoside of the embodiments and examples provided of the present invention), a gene therapy product (an oligonucleotide according to the present invention such as BNA), and a targeting ligand or antibody (e.g., those according to the embodiments of the present invention and the sdAb exemplified below in the Examples section of the present specification) are all included by the conjugate of the present invention. Thus, such conjugates of the present invention provide therapeutic opportunities for existing and future macromolecular drugs for a wide range of diseases and large patient populations. By the application of such conjugates of the present invention comprising at least one saponin, at least one oligonucleotide, and at least one specific cell targeting moiety such as an immunoglobulin or sdAb, the obvious problems with existing methods of applying endosome escape enhancers and gene therapy products separately (existing methods do not guarantee that both compounds are present simultaneously at the interaction site) are addressed. This problem is now overcome by using the conjugates of the present invention. That is, such conjugates of the present invention provide a non-viral gene delivery technology having an increased synchronization (timing and location) of both compounds, i.e., a saponin and a gene product such as BNA.

[0166] Gene therapy may be useful for hereditary diseases such as cystic fibrosis, cholera, Huntington's disease or hemophilia that were previously incurable. However, several current problems have not been overcome: for example, the therapeutic gene must reach specific target cells in the body precisely. On the other hand, the therapeutic gene should be absorbed by the targeted cells, but the therapeutic gene must not be destroyed. Existing gene therapy approaches use viruses as shuttles for the gene. However, these procedures involve significant risks and cannot be diverted to the introduction of other biomolecules. Certain embodiments involve the use of a (plant-derived) glycoside (e.g., any one of the saponins of the present invention) for a platform technology that not only enables gene delivery when included in a conjugate as a carrier molecule, but also enables the delivery of different therapeutic biomolecules that will be introduced into target cells. Thus, the conjugates of the present invention are used to develop nucleic acid-based therapies for cystic fibrosis, cholera, Huntington's disease or hemophilia. In addition, with the conjugates of the present invention, novel gene therapy strategies are available to improve the health of patients with genetic diseases, including those patients with cystic fibrosis, Huntington's disease, and hemophilia. As part of the present invention, a non-viral gene delivery technology is developed that combines a plant-derived endosome escape enhancer (glycoside; i.e., the saponin of the present invention), a gene therapy product, and a targeting ligand (i.e., sdAb), all included in a single conjugate. The resulting non-viral gene therapy based on the conjugates of the present invention shows an approximately 40-fold increase in delivery efficiency at lower doses than currently available strategies. In addition, the conjugates of the present invention are for use in clinical applications such as for the repair or replacement of defective genes as in patients with cystic fibrosis and for the targeted delivery of specific genes, for example, to destroy cancer cells. Indeed, the conjugates of the present invention are suitable for application in treatment regimens for any disease caused by defective genes such as cystic fibrosis, Huntington's disease and hemophilia that are currently incurable.Gene therapy using the conjugates of the present invention is useful in overcoming two existing problems: First, the conjugates of the present invention enable the delivery of therapeutic genes to specific target cells in the body; Second, the therapeutic genes enter but are not destroyed inside these cells due to the presence of, for example, saponin, oligonucleotide products, and targeting moieties such as antibodies or sdAbs that bind to target cells, all of which are covalently linked together in the conjugates of the present invention using, for example, the oligomer or polymer scaffolds (covalent saponin conjugates of the present invention) of the present invention.

[0167] The present invention also provides a method for treating cancer, which comprises administering a medicament comprising a therapeutic conjugate according to the present invention to a patient in need thereof, preferably administering an effective dose of said medicament to a patient in need thereof, preferably a human cancer patient.

[0168] Considerations regarding suitable forms of administration are known in the art and include toxic effects, solubility, route of administration, and maintenance of activity. For example, a pharmacological composition to be injected into the bloodstream needs to be soluble.

[0169] Suitable dosage forms depend in part on the use or route of entry, for example, transdermal or by injection. Such dosage forms must deliver the compound to the target cells regardless of whether the target cells are present in a multicellular host. Other factors are known in the art and include considerations such as toxicity and dosage forms that delay the exertion of the action of the compound or composition.

[0170] The fourth aspect of the present invention relates to the pharmaceutical composition of the present invention for use in any one or more of the following treatments or prevention of onset: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency diseases, diseases related to enzyme deficiency, gene deficiency, diseases related to gene deficiency, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, amyloidosis and transthyretin-mediated amyloidosis.

[0171] Surprisingly, the inventors have found that a certain dose of an ADC that does not result in the death of any tumor cells is sufficient and adequate for efficient tumor cell death when such an ADC is contacted with tumor cells in the presence of a saponin coupled to a tumor cell-targeting antibody or V HH Examples are provided in Figures 2-7. For example, a certain dose of an ADC anti-CD71 V HH -toxin, anti-HER2 V HH -toxin or anti-EGFR V HH -toxin and other ADCs do not exert a toxic effect on tumor cells when contacted with tumor cells. Typical toxins are protein toxins such as dianthin and saporin. However, when such an ADC is co-administered with a conjugate containing saponin, efficient tumor cell death is achieved. The conjugate containing saponin is, for example, anti-HER2 V HH -SO1861, anti-CD71 V HH -SO1861, anti-EGFR V HH -SO1861, cetuximab-SO1861, trastuzumab-SO1861. The ADC is, for example, an ADC containing a V HH that can bind to HER2, CD71 or EGFR. For example, the ADC contains a protein toxin such as dianthin or saporin. Saponin and a tumor cell-targeting antibody or V HHBy applying a conjugate containing saponin, the saponin dosage required to achieve efficient biological activity of the effector molecule contained in the ADC inside the target cell, i.e., the ADC or AOC, is about 100 to 1000 times lower compared to the dosage of free saponin required when the conjugate containing saponin is co-administered with the ADC, i.e., the ADC or AOC, to the target cell and when the ADC is co-administered with free saponin to the target cell. In addition to this, the conjugate containing saponin enhances the ADC, i.e., the sdAb containing the ADC or AOC, at a dosage of the ADC or AOC that would otherwise have no effect on tumor cells when administered to patients who require it in the absence of the targeted saponin. In addition to this, the conjugate containing saponin is already sufficiently effective at a relatively low dosage when enhancement of the effector molecule of the ADC or AOC is considered, i.e., at a dosage at which free saponin provided without a tumor cell-targeting binding molecule such as an sdAb is not sufficiently effective for enhancement of the effector molecule. Taken together, the inventors have found that the ADC or AOC already has a tumor cell-targeting antibody or V HHProvided is an improved method for treating a human patient in need of treatment with an ADC or AOC comprising a therapeutically effective amount of a tumor cell-targeted sdAb, which is effective at a lower dose when co-administered to the patient in the presence of a conjugate comprising and saponin. One of the many advantages of this combination is the absence of an Fc tail in the sdAb portion of the ADC, and preferably the conjugate comprising saponin. The absence of the Fc tail prevents unwanted binding to off-target patient cells having an Fc receptor of the conjugate, which otherwise could result in side effects such as those seen for many conventional ADCs, AOCs where such Fc tails are present. ADCs and AOCs based on antibodies containing an Fc tail have the drawback of reduced efficacy due to unwanted binding of such IgG-based ADCs, AOCs to Fc receptors. As a result of Fc receptor binding, the effective amount of such IgG-based ADCs and AOCs is decreased. In addition, the absence of the Fc tail provides several advantages in this regard. Off-target and unwanted binding of the ADCs, AOCs and improved ADCs and improved AOCs of the present invention (i.e., the conjugates of the present invention) to Fc receptors cannot occur. In addition, the ADCs, AOCs and conjugates of the present invention have a lower effective dose when considering target receptor-mediated endocytosis and delivery of the ADCs, AOCs and conjugates of the present invention into endosomes because the conjugate is not "lost" due to Fc receptor binding, which is a defect seen in IgG-based conjugates. As a result, the therapeutic window of the ADCs, AOCs and conjugates of the present invention comprising cell-targeted sdAb instead of antibodies all containing Fc is wider than the therapeutic window that would have been achieved when the sdAb was replaced by a conventional IgG containing an Fc tail. Similarly, as a result, the therapeutic window of the conjugate comprising saponin is wider than the therapeutic window that would have been achieved when the sdAb in such conjugate of tumor cell-targeted sdAb and saponin was replaced by a binding molecule for binding to cell surface molecules on target cells such as conventional IgG containing an Fc tail.

[0172] One embodiment is a pharmaceutical composition for use of the present invention, wherein the saponin is SO1861, an SO1861 derivative, QS-21, or a QS-21 derivative, preferably an SO1861 derivative or a QS-21 derivative, more preferably an SO1861 derivative according to the present invention.

[0173] One embodiment is a pharmaceutical composition for use of the present invention, - said use is for the treatment or prevention of cancer in a human subject; and / or - said use is for the treatment or prevention of onset of cancer in a patient in need thereof, wherein at least one sdAb binds to a cell surface molecule of the cell, preferably a tumor cell surface molecule of the cell, more preferably a tumor cell-specific surface molecule of the cell; and / or - the pharmaceutical composition, preferably a therapeutically effective amount of the pharmaceutical composition, is administered to a patient in need thereof, preferably a human patient.

[0174] A fifth aspect of the present invention is an in vitro or ex vivo method for translocating an effector molecule of the present invention from outside the cell into the cell, preferably into the cytosol of the cell, comprising: a) providing a cell expressing a binding site for at least one sdAb comprised by the conjugate of the present invention on its cell surface, said binding site preferably being present on a cell surface molecule of the cell, said cell preferably being selected from liver cells, abnormal cells such as virus-infected cells, autoimmune cells, cells containing gene defects, cells containing enzyme defects, and tumor cells; b) providing the conjugate of the present invention comprising an effector molecule that is to be translocated to the cell provided in step a); and c) contacting the cell of step a) with the conjugate of step b) in vitro or ex vivo, Accordingly, the method includes a step of transferring the conjugate containing the effector molecule from the outside of the cell to the inside of the cell, and a step of transferring the effector molecule from the outside of the cell to the inside of the cell, preferably to the cytosol of the cell, by the transfer of the conjugate.

[0175] A sixth aspect of the present invention is an in vitro or ex vivo method for transferring the conjugate of the present invention from the outside of the cell to the inside of the cell, comprising: a) providing a cell expressing a binding site for at least one sdAb contained in the conjugate of the present invention on its cell surface, wherein the binding site is preferably present on a cell surface molecule of the cell, and the cell is preferably selected from abnormal cells such as liver cells and virus-infected cells, autoimmune cells, cells containing gene defects, cells containing enzyme defects, and tumor cells; b) providing the conjugate of the present invention; and c) contacting the cell of step a) with the conjugate of step b) in vitro or ex vivo, thereby transferring the conjugate from the outside of the cell to the inside of the cell.

[0176] [Table 1]

[0177] [Table 2]

[0178] [Table 3]

[0179] [Table 4]

[0180] Aspects of the invention relate to kits comprising a container containing an endosome escape-enhancing conjugate according to the invention, the kit further comprising instructions for using the conjugate.

[0181] Certain aspects of the invention relate to any of the following ADCs provided with at least one covalently linked saponin, and the following AOCs provided with at least one covalently linked saponin, and their semi-finished conjugates, comprising a cell surface molecular targeting molecule (i.e., sdAb) of the invention and comprising at least one effector portion of the invention that results in an ADC or AOC, or comprising at least one saponin of the invention: The following antibodies are sdAbs: Anti-EGFR antibody-saponin; Anti-EGFR antibody - belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at C-23, and optionally comprising a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, a triterpenoid saponin and / or a bidesmoside triterpenoid saponin; Anti-EGFR antibody-SO1861; Anti-EGFR antibody-GE1741; Anti-EGFR antibody-SA1641; Anti-EGFR antibody-Quil-A; Anti-EGFR antibody-QS-21; Anti-EGFR antibody - saponin in the water-soluble saponin fraction of Quillaja saponaria; sdAb-saponin derived from cetuximab; sdAb - belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at C-23 and optionally comprising a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, derived from cetuximab, a triterpenoid saponin and / or a bidesmoside triterpenoid saponin; sdAb-SO1861 derived from V H or V L of cetuximab; V of cetuximabH or V L derived sdAb-GE1741; V of cetuximab H or V L derived sdAb-SA1641; V of cetuximab H or V L derived sdAb-Quil-A; V of cetuximab H or V L derived sdAb-QS-21; V of cetuximab H or V L derived sdAb-saponin in the water-soluble saponin fraction of Quillaja saponaria; anti-HER2 antibody-saponin; anti-HER2 antibody-triterpenoid saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at C-23 and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin and / or bidesmoside triterpenoid saponin; anti-HER2 antibody-SO1861; anti-HER2 antibody-GE1741; anti-HER2 antibody-SA1641; anti-HER2 antibody-Quil-A; anti-HER2 antibody-QS-21; anti-HER2 antibody-saponin in the water-soluble saponin fraction of Quillaja saponaria; V of trastuzumab H or V L derived sdAb-saponin; V of trastuzumab H or V L derived sdAb-triterpenoid saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at C-23 and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin and / or bidesmoside triterpenoid saponin; V of trastuzumab H or V L derived sdAb-SO1861; V of trastuzumab H or V L derived sdAb-GE1741; V of trastuzumab H or V L derived sdAb-SA1641; V of trastuzumab H or V L derived sdAb-Quil-A; V of trastuzumab H or V L derived sdAb-QS-21; V of trastuzumab H or V L derived sdAb-saponin in the water-soluble saponin fraction of Quillaja saponaria; Anti-CD71 antibody-saponin; Anti-CD71 antibody-12,13-dehydrooleanane type having an aldehyde functional group at C-23 and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of saponin and / or bidesmoside triterpene saponin; Anti-CD71 antibody-SO1861; Anti-CD71 antibody-GE1741; Anti-CD71 antibody-SA1641; Anti-CD71 antibody-Quil-A; Anti-CD71 antibody-QS-21; Anti-CD71 antibody-saponin in the water-soluble saponin fraction of Quillaja saponaria; V of OKT-9 H or V L derived sdAb-saponin; V of OKT-9 H or V LTriterpenoid saponins and / or bidesmoside triterpen saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at the sdAb-C-23 position derived therefrom and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin; V of OKT-9 H or V L sdAb-SO1861 derived therefrom; V of OKT-9 H or V L sdAb-GE1741 derived therefrom; V of OKT-9 H or V L sdAb-SA1641 derived therefrom; V of OKT-9 H or V L sdAb-Quil-A derived therefrom; V of OKT-9 H or V L sdAb-QS-21 derived therefrom; V of OKT-9 H or V L saponins in the water-soluble saponin fraction of sdAb-Quillaja saponaria derived therefrom ; Anti-EGFR antibody-oligonucleotide; Anti-EGFR antibody-antisense oligonucleotide; Anti-EGFR antibody-siRNA; Anti-EGFR antibody-antisense BNA; Anti-EGFR antibody-antisense BNA (HSP27); Anti-EGFR antibody-proteinaceous toxin; Anti-EGFR antibody-ribosome-inactivating protein; Anti-EGFR antibody-diantin; Anti-EGFR antibody-saporin; V of cetuximab H or V L sdAb-oligonucleotide derived therefrom; V of cetuximab H or V LsdAb-antisense oligonucleotides derived from; V of cetuximab H or V L sdAb-siRNAs derived from; V of cetuximab H or V L sdAb-antisense BNA derived from; V of cetuximab H or V L sdAb-antisense BNA (HSP27) derived from; V of cetuximab H or V L sdAb-proteinaceous toxin derived from; V of cetuximab H or V L sdAb-ribosome-inactivating protein derived from; V of cetuximab H or V L sdAb-dianthin derived from; V of cetuximab H or V L sdAb-saporin derived from; anti-HER2 antibody-oligonucleotide; anti-HER2 antibody-antisense oligonucleotide; anti-HER2 antibody-siRNA; anti-HER2 antibody-antisense BNA; anti-HER2 antibody-antisense BNA (HSP27); anti-HER2 antibody-proteinaceous toxin; anti-HER2 antibody-ribosome-inactivating protein; anti-HER2 antibody-dianthin; anti-HER2 antibody-saporin; V of trastuzumab H or V L sdAb-oligonucleotides derived from; V of trastuzumab H or V L sdAb-antisense oligonucleotides derived from; V of trastuzumab H or V LsdAb-siRNA derived from; V of trastuzumab H or V L sdAb-antisense BNA derived from; V of trastuzumab H or V L sdAb-antisense BNA (HSP27) derived from; V of trastuzumab H or V L sdAb-proteinaceous toxin derived from; V of trastuzumab H or V L sdAb-ribosome-inactivating protein derived from; V of trastuzumab H or V L sdAb-dianthin derived from; V of trastuzumab H or V L sdAb-saporin derived from; Anti-CD71 antibody-oligonucleotide; Anti-CD71 antibody-antisense oligonucleotide; Anti-CD71 antibody-siRNA; Anti-CD71 antibody-antisense BNA; Anti-CD71 antibody-antisense BNA (HSP27); Anti-CD71 antibody-proteinaceous toxin; Anti-CD71 antibody-ribosome-inactivating protein; Anti-CD71 antibody-dianthin; Anti-CD71 antibody-saporin; V of OKT-9 H or V L sdAb-oligonucleotide derived from; V of OKT-9 H or V L sdAb-antisense oligonucleotide derived from; V of OKT-9 H or V L sdAb-siRNA derived from; V of OKT-9 H or V LsdAb-antisense BNA derived from; V of OKT-9 H or V L sdAb-antisense BNA (HSP27) derived from; V of OKT-9 H or V L sdAb-proteinaceous toxin derived from; V of OKT-9 H or V L sdAb-ribosome-inactivating protein derived from; V of OKT-9 H or V L sdAb-diantin derived from; V of OKT-9 H or V L sdAb-saporin derived from; Anti-EGFR antibody (-oligonucleotide) (-saponin) (the oligonucleotide is any one or more of an antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab); Anti-EGFR antibody (-proteinaceous toxin) (-saponin) (the proteinaceous toxin is any one or more of ribosome-inactivating protein, dianthin, and saporin, and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab); Anti-HER2 antibody (-oligonucleotide) (-saponin) (the oligonucleotide is any one or more of antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab); Anti-HER2 antibody (-proteinaceous toxin) (-saponin) (the proteinaceous toxin is any one or more of ribosome-inactivating protein, dianthin, and saporin, and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab); Anti-CD71 antibody (-oligonucleotide) (-saponin) (the oligonucleotide is any one or more of antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9); and anti-CD71 antibody (-proteinaceous toxin) (-saponin) (the proteinaceous toxin is any one or more of ribosome-inactivating protein, dianthin and saporin, and the saponin belongs to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally contains a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, triterpenoid saponin and / or bidesmoside triterpenoid saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9).

[0182] One embodiment is that the cell surface molecule targeting molecule is cetuximab, trastuzumab, sdAb derived from the V H or V L of OKT-9 (i.e., the sdAb is based on the V H or V L of such monoclonal antibodies and can specifically bind to a target receptor on the cell surface of target cells), and / or the effector part is selected from dianthin, saporin and antisense BNA (HSP27), and / or the saponin is selected from SO1861, GE1741, SA1641, Quil-A, QS-21 and the saponin or its derivative in the water-soluble saponin fraction of Quillaja saponaria, the semi-finished conjugate (sdAb-saponin or sdAb-effector part) of the present invention or the conjugate of the present invention.

[0183] One embodiment is that the cell surface molecule targeting molecule is cetuximab, trastuzumab, sdAb derived from the V H or V L of OKT-9 (i.e., the sdAb is based on the V H or V LSelected based on and capable of specifically binding to a target receptor on the cell surface of a target cell), and / or the effector moiety is selected from dianthin, saporin and antisense BNA (HSP27) and / or the saponin is selected from saponin or a derivative thereof in the water-soluble saponin fraction of SO1861, GE1741, SA1641, Quil-A, QS-21 and Quillaja saponaria, which is a conjugate according to the present invention.

[0184] One aspect of the present invention is an ADC or AOC of structure C, or a semi-finished ADC conjugate or a semi-finished AOC conjugate, comprising the cell surface molecule targeting molecule sdAb of the present invention and at least one effector moiety of the present invention and / or at least one saponin of the present invention: A(-S) b (-E) c (Structure C) (wherein A is a cell surface molecule targeting sdAb; S is a saponin; E is an effector moiety; b = 0 to 64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64 or any integer or decimal part therebetween; c = 0 to 8, preferably 0, 1, 2, 3, 4, 6, 8 or any integer or decimal part therebetween; S is coupled to A and / or E, and E is coupled to A and / or S, preferably S is coupled to A and E is coupled to A).

[0185] One embodiment is that A is an sdAb derived from an anti-EGFR antibody such as cetuximab, an anti-HER2 antibody such as trastuzumab, an anti-CD71 antibody such as OKT-9, and / or S is a saponin, belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at the C-23 position and optionally containing a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin, and / or a triterpenoid saponin and / or a bidesmoside triterpenoid saponin such as videosside, SO1861, GE1741, SA1641, Quil-A, QS-21, and any one or more of the saponins in the water-soluble saponin fraction of Quillaja saponaria, and / or E is any one or more of an oligonucleotide, an antisense oligonucleotide, siRNA, an antisense BNA, and an antisense BNA (HSP27), and / or any one or more of a proteinaceous toxin, a ribosome-inactivating protein, dianthin, and saporin, which is the structural C of the present invention.

[0186] One embodiment is that the saponin (if present), and / or the effector moiety (if present) is covalently coupled via at least one linker such as a cleavable linker and / or a covalent saponin conjugate (scaffold) such as a covalent saponin conjugate based on N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate or N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP), and a linker based on 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and dendrons such as G4-dendron or a trifunctional linker such as the trifunctional linker of structural A (i.e., at least one oligomeric or polymeric scaffold), and / or a cell surface molecule targeting antibody, preferably V according to the present invention HHAt least the lysine side chain and / or cysteine side chain of sdAb such as etc. are involved in the covalent bond with saponin and / or effector moiety and / or linker and / or cleavable linker and / or covalent saponin conjugate, preferably, saponin and / or effector moiety are covalently linked to cell surface molecular targeting molecules, preferably, antibodies such as sdAb, and the covalent linkage comprises or consists of amide bond, hydrazone bond, disulfide bond, which is the structure C of the present invention, the conjugate of the present invention or the semi-finished conjugate of the present invention.

[0187] One aspect of the present invention relates to the use of any of the aforementioned conjugate as a medicine, an ADC containing covalently linked saponin, an AOC containing covalently linked saponin, a semi-finished ADC, and a semi-finished AOC.

[0188] One aspect of the present invention relates to the use of any of the aforementioned conjugate as a medicine, an ADC containing covalently linked saponin, an AOC containing covalently linked saponin, a semi-finished ADC, and a semi-finished AOC for the treatment or prevention of cancer or autoimmune diseases.

Examples

[0189] 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 the combined treatment of mAb-protein toxin, V HH and mAb recognize and bind to the same cell surface receptor (Figure 1A). The 2-target 2-component system (2T2C) is V HH -SO1861 and the combined treatment of mAb-protein toxin, V HH recognizes and binds to a cell surface receptor different from mAb (Figure 1B). SO1861-EMCH was conjugated to anti-HER2V at DAR1 via (labile) terminal cysteine residue (Cys) HHconjugated to (HER2V HH -SO1861). HER2V HH -SO1861 was titrated against fixed concentrations of 10 pM CD71 mab-saporin (a protein toxin with DAR4, a CD71 monoclonal antibody conjugated to saporin) or 50 pM trastuzumab-saporin (trastuzumab conjugated to saporin with DAR4). Targeted protein toxin-mediated cell death against SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was determined. This revealed enhanced cell death at low concentrations of HER2V HH -SO1861 in SK-BR-3 for both combinations with 10 pM CD71 mab-saporin or 50 pM trastuzumab-saporin (IC50 = 300 nM; Figure 2A). The same concentration of HER2V HH -SO1861 alone induced cell death at high concentrations (IC50 = 4,000 nM), while the same concentration of HER2V HH , HER2V HH + CD71 mab-saporin or HER2V HH + trastuzumab-saporin could not induce cell death activity (IC50 > 5000 nM; Figure 2A). In MDA-MB-468 (HER2 - / CD71 + ), the combination of HER2V HH -SO1861 + 10 pM CD71 mab-saporin revealed cell death activity at high concentrations (IC50 = 2,000 nM; Figure 2B), while the combination of HER2V HH -SO1861 + 50 pM trastuzumab-saporin showed cell death activity at much higher concentrations (IC50 > 5,000 nM; Figure 2B). The same concentration of HER2V HH , HER2V HH + CD71 mab-saporin or HER2V HHTrastuzumab-saporin was unable to induce cytotoxic activity in MDA-MB-468 cells (IC50 > 5,000 nM; Figure 2B).

[0190] All of these indicate that conjugation to HER2-targeted V of SO1861-EMCH enhances endosomal escape and cytoplasmic delivery of targeted protein toxins (targeting the same or different cell surface receptors), resulting in cell death of HER2-expressing cells. HH Next, trastuzumab-saporin or CD71mab-saporin was titrated against a fixed concentration of 900 nM HER2V

[0191] -SO1861, and the targeted protein toxin-mediated cytotoxicity against SK-BR-3 (HER2 HH / CD71 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was determined. This showed that 900 nM HER2V HH -SO1861 already induced efficient cell death of SK-BR-3 when combined with low concentrations of trastuzumab-saporin or CD71mab-saporin (IC50 = 0.0001 pM; Figure 3A), while CD71mab-saporin + 900 nM HER2V HH or trastuzumab-saporin + 900 nM HER2V HH was able to induce cell death only 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 death with an IC50 = 0.01 pM, while trastuzumab-saporin + 900 nM HER2V HH -SO1861 showed activity with an IC50 = 2,000 pM. Trastuzumab-saporin + 900 nM HER2V HH or CD71mab-saporin + 900 nM HER2V HHshowed cell killing only at (IC50>10,000 pM and IC50 = 20 pM, Figure 3B). This indicates that relatively low concentrations of trastuzumab-saporin or CD71mab-saporin are effective and can induce cell killing in combination with relatively low HER2V ++ / CD71 + expression cells and relatively low HER2V HH -SO1861 (DAR1) concentrations.

[0192] 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 that targets HER2 to generate HER2V HH -SO1861 (DAR1). HER2V HH -SO1861 was titrated against a fixed concentration of 50 nM CD71V HH -dianthin, and targeted protein toxin-mediated cell killing against SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was determined. This revealed enhanced cell killing with relatively low concentrations of V HH -HER2-L-SO1861 (SK-BR-3; IC50 = 300 nM; Figure 4A). The same concentration of HER2V HH -SO1861 alone induced cell killing at high concentrations (IC50 = 4,000 nM), while the same concentration of HER2V HH , HER2V HH + 50 pM CD71VHH-dianthin could not induce cell killing (IC50>5,000 nM; Figure 4A). MDA-MB-468 (HER2 - / CD71 +) In this case, HER2V HH -SO1861 + 50 pM CD71V HH - The combination of dianthin revealed cell-killing activity at higher concentrations (IC50 = 600 nM; Figure 4B), while the same concentration of HER2V HH , HER2V HH -SO1861 or HER2V HH + 50 pM CD71V HH - Dianthin was unable to induce cell-killing activity (IC50 > 5,000 nM; Figure 4B).

[0193] 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 determined. This showed that 900 nM HER2V HH -SO1861 combined with low concentrations of CD71V HH - Dianthin induced efficient cell death in SK-BR-3 cells (IC50 = 0.05 pM; Figure 5A), while CD71V HH Dianthin or CD71V HH - Dianthin + 900 nM HER2V HH was only able to induce cell death at high concentrations (IC50 > 10,000 pM); Figure 5A). Additionally, CD71V HH - Dianthin was also titrated against a fixed concentration of 77 nM trastuzumab-SO1861 (DAR4), which also revealed a strong enhancement of cell-killing activity in SK-BR-3 (HER2 ++ / CD71 + ) cells ((IC50 < 0.0001 pM). In MDA-MB-468 cells (HER2 - / CD71 + ), CD71V HH - Dianthin + 900 nM HER2V HH-SO1861 only demonstrated cell killing at much higher concentrations (IC50 = 10 pM, Figure 5B), whereas CD71V HH - Jianqing, CD71V HH -Giantin + 900nM HER2V HH or CD71V HH -Gianthin + trastuzumab-SO1861 (DAR4) showed cell killing only with IC50 = 2,000 pM (Figure 5B).

[0194] All of these are at relatively low concentrations of V HH CD71-gianthin exhibits low V in high HER2 / CD71 expressing cells HH It is shown that in combination with HER2-SO1861 conjugate concentrations it is effective and can induce cell death.

[0195] MDA-MB-468 cells (HER2 - / CD71 + ) did not reveal any cell killing activity, indicating that in the absence of sufficient receptor expression, the effective intracellular delivered SO1861 concentration does not reach the (threshold) for inducing endosomal escape and intracytoplasmic delivery of the protein toxin.

[0196] Example 3.V HH -Giantin + mAb-SO1861 (1T2C and 2T2C) The one-target two-component system (1T2C) is a combination of mAb-SO1861 and V HH - A combination therapy of a protein toxin, a mAb and a V HH The two-target two-component system (2T2C) also binds to the mAb-SO1861 and V (Figure 1E). HH - A combination therapy of a protein toxin, a mAb and a V HH recognizes a different cell surface receptor (Figure 1D).

[0197] Gianthin-C (giantin with a terminal cysteine) is a V-cell targeting HER2. HH , V targeting CD71HH or conjugated to the terminal cysteine residue of V targeting EGFR to form HER2V HH -dianthin (DAR1), CD71V HH -dianthin (DAR1), and EGFRV HH -dianthin (DAR1) were generated. HH

[0198] CD71V HH -dianthin, HRE2V HH -dianthin, or EGFRV HH -dianthin was titrated against a fixed concentration of cetuximab-SO1861 (DAR4), and target protein toxin-mediated cell killing against A431 (EGFR ++ / HER2 + / - / CD71 + ) and A2058 (EGFR - / HER2 + / - / CD71 + ) was determined. This revealed that very low concentrations of CD71V HH -dianthin in combination with 77 nM cetuximab-SO1861 induced efficient cell killing of A431 cells (IC50 < 0.0001 pM; Figure 6A), while CD71V HH -dianthin alone showed activity with an IC50 = 2000 pM. The other two combinations, EGFRV HH -dianthin + 77 nM cetuximab-SO1861 and HER2V HH -dianthin + 77 nM cetuximab-SO1861 showed efficient cell killing with IC50 = 20 pM and IC50 = 50 pM, respectively, while EGFRV HH -dianthin or HER2V HH -dianthin alone could not induce efficient cell killing in A431 cells (IC50 > 10,000 pM; Figure 6A). In A2058 cells (EGFR - / HER2 + / - / CD71 + ), CD71V HH -dianthin and CD71V HH ​-Giantin + 77 nM cetuximab-SO1861 showed cytotoxic activity with IC50 = 3,000 pM and IC50 = 1,000 pM respectively, while all other treatments or combinations showed no cytotoxicity up to IC50 = 10,000 pM V HH -toxin (Figure 6B).

[0199] This indicates that cetuximab-SO1861 (DAR4) can induce enhanced cytotoxicity in A431 cells by efficiently inducing endosomal escape of three different V HH -giantin conjugates.

[0200] Next, CD71V HH -giantin, HRE2V HH -giantin or EGFRV HH -giantin was titrated against a fixed concentration of trastuzumab-SO1861 (DAR4), and targeted protein toxin-mediated cytotoxicity against SK-BR-3 (HER2 ++ / EGFR = / CD71 + ) and MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + ) was determined. This revealed that very low concentrations of CD71V HH -giantin in combination with 77 nM trastuzumab-SO1861 induced efficient cytotoxicity in SK-BR-3 cells (IC50 < 0.0001 pM; Figure 7A), while CD71V HH -giantin alone showed activity with an IC50 = 10,000 pM. The other two combinations, EGFRV HH -giantin + 77 nM trastuzumab-SO1861 and HER2V HH -giantin + 77 nM trastuzumab-SO1861 showed efficient cytotoxicity with IC50 = 400 pM and IC50 = 6 pM respectively, while EGFRV HH -giantin or HER2V HH-Giantin alone could not 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, while all other treatments or combinations showed no cell death up to IC50 = 10,000 pM V HH -Giantin in MDA-MB-468 cells (Figure 7B). This indicates that trastuzumab-SO1861 (DAR4) can induce enhanced cell death in SK-BR-3 cells by efficiently inducing endosomal escape of three different V HH -Giantin conjugates.

[0201] Materials and Methods Materials SO1861 was isolated and purified by Analyticon Discovery GmbH from a raw plant extract obtained from Saponaria officinalis. V HH was obtained from QVQ, Utrecht, The Netherlands (HER2V HH : clone name: Q17c; CD71V HH : clone name: Q52c EGFRV HH: Clone name: Purchased from Q86c). Trastuzumab (Tras, Herceptin®, Roche), Cetuximab (Cet, Erbitux®, Merck KGaA) were purchased from the pharmacy (Charite, Berlin). The CD71 monoclonal antibody was purchased from BioCell (Okt9, #BE0023). The ordered Trastuzumab-saporin and anti-CD71 mab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA). Diantin-Cys (Dia-Cys, a diantin mutant with a single C-terminal cysteine) was manufactured by Proteogenix, France.

[0202] 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-Fischer), Pierce(™) BCA Protein Assay Kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50 M (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), anhydrous D-(+)-trehalose (99%, Sigma-Aldrich), polyethyleneglycol sorbitan monolaurate (TWEEN(®) 20, Sigma-Aldrich), Dulbecco's Phosphate Buffered Saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetate dihydrate (EDTA-N a2, 99%, Sigma - Aldrich), sterile filters 0.2μm and 0.45μm (Sartorius), N - succinimidyl 4-(N - maleimidomethyl) cyclohexane - 1 - carboxylate (SMCC, Thermo - Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), N - succinimidyl 3 - (2 - pyridyldithio) propionate tetra(ethylene glycol) (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 salt (AEM, 98%, Sigma - Aldrich), L - cysteine (98.5%, Sigma - Aldrich), deionized water (DI) freshly obtained 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 hydrogen carbonate (99.7%, Sigma - Aldrich), sodium carbonate (99.9%, Sigma - Aldrich), PD MiniTrap desalting column with Sephadex G - 25 resin (GE Healthcare), PD10 G25 desalting column (GE Healthcare), 0.Zeba Spin Desalting Columns (Thermo-Fisher) at 5, 2, 5, and 10 mL, 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 Filters (Thermo-Fisher).

[0203] Method SO1861-EMCH Synthesis To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) were added methanol (over-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 combined, pooled, 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

[0204] Cell Viability Assay After the treatment, the cells were incubated at 37 °C for 72 hours, and then the cell viability was determined by an MTS-assay carried out according to the manufacturer's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega). Briefly, the MTS solution was diluted 20-fold in DMEM (PAN-Biotech GmbH) without phenol red (PAN-Biotech GmbH) supplemented with 10% FBS. The cells were washed once with 200 μL / PBS 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. Thereafter, the OD at 492 nm was measured on 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 percentage of cell viability of treated / untreated cells was calculated (x100) by dividing the background-corrected signal of the treated wells by the background-corrected signal of the untreated wells.

[0205] FACS analysis Cells were seeded at 500,000 c / plate in a 10 cm dish in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) and incubated for 48 hours (5% CO2, 37 °C) until a 90% confluence density was reached. Next, the cells were trypsinized (TryplE Express, Gibco Thermo Scientific) to make single cells. 0.75x10 6 cells were transferred to a 15 mL Falcon tube and centrifuged (1,400 rpm, 3 minutes). The supernatant was discarded while keeping the cell pellet immersed. 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+ dissociated, resuspended in 2% FBS), and equally divided into three round-bottom FACS tubes (1 mL / tube). The cells were centrifuged again, and 200 μL of cold PBS (Mg 2+ and Ca 2+ dissociated, 2% FBS) or 195 μL of cold PBS (Mg 2+ and Ca 2+ dissociated, 2% FBS) and resuspended in 200 μL of antibody solution containing 5 μL of antibody. APC mouse IgG1, κ APC anti-human EGFR (#352906, Biolegend) was used to stain the EGFR receptor. PE anti-human HER2 APC anti-human CD340 (erbB2 / HER-2) (#324408 Biolegend) was used to stain the HER2 receptor, and PE mouse IgG2a, κ isotype Ctrl FC (#400212, Biolegend) was used as the corresponding isotype control. PE anti-human CD71 (#334106, Biolegend) was used to stain the CD71 receptor, and PE mouse IgG2a, κ isotype Ctrl FC (#400212, Biolegend) was used as the 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 (Mg 2+ and Ca 2+ dissociated, 2% FBS), 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 results of the analysis of cell surface expression of EGFR, HER2, and CD71 on various cells are summarized in Table A2.

[0206]

Table 5

[0207] V HH Procedure for the conjugation of -SO1861 A certain amount of V HH was added with a certain amount of freshly prepared TCEP solution (10.0 mg / ml), the mixture was vortexed briefly, and then incubated at 20 °C for 30 minutes by roller mixing. After incubation, the resulting V HH -SH was purified to TBS pH 7.5 by gel filtration using a zebaspin desalting column. The resulting V HH -SH was added with a freshly prepared SPT-EMCH solution, the mixture was vortexed briefly, and then incubated at 20 °C overnight.

[0208] After incubation, a certain amount of V HH -SO1861 mixture was removed, characterized by the Ellman assay, and the incorporation of SO1861 was confirmed. The conjugate was purified by a 1.6 × 35 cm Superdex 200PG column eluting with DPBS pH 7.5 to obtain purified V HH -SO1861. A certain amount was filtered through 0.2 μm, concentrated, and standardized to 1.0 mg / ml to obtain V HH -SO1861.

[0209] V HH Procedure for the conjugation of V-dianthin Dianthin-Cys was concentrated by ultrafiltration using a Vivaspin T15 10KDa MWCO centrifugal filter and buffer-exchanged into TBS pH 7.5. To the concentrated dianthin-Cys, a certain volume of freshly prepared TCEP solution (10.0 mg / ml) was added, the mixture was vortexed briefly, and then incubated at 20 °C for 60 minutes by roller mixing. After incubation, the resulting dianthin-SH was purified by gel filtration using a Zeba spin desalting column and then the centrifugal washing cycle was repeated using a Vivaspin T15 10KDa MWCO centrifugal filter to bring it to TBS pH 7.5. 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. Subsequently, dianthin-DTME was obtained after purification by gel filtration using a Zeba spin desalting column to bring it to TBS pH 7.5. Dianthin-DTME was stored at 20 °C until conjugation. At the same time, a certain volume of V HH was concentrated by ultrafiltration using a Vivaspin T15 10KDa MWCO centrifugal filter and buffer-exchanged into TBS pH 7.5. The concentrated V HH a certain volume 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 by roller mixing. After incubation, the resulting V HH was purified by gel filtration using a Zeba spin desalting column and then the centrifugal washing cycle was repeated using a Vivaspin T4 5KDa MWCO centrifugal filter to bring it to TBS pH 7.5. A certain volume of the resulting V HH -SH was reacted with dianthin-DTME, the mixture was vortexed briefly, and then incubated at 20 °C overnight. Subsequently, 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 eluting with DPBS pH 7.5.

[0210] Antibody-(L-SO1861)4 Trastuzumab and cetuximab are hereinafter referred to as "Ab". Ab was conjugated to saponin SO18161-EMCH via a Michael-type thiol-ene conjugation reaction with DARs of 1, 2, 3, 4, 5, and 6. The SO1861-EMCH molecule obtained 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.

[0211] To a solution of cetuximab (40 mg, 8.0 ml), 10 μl / ml each of tris concentrate (127 mg / ml, 1.05 M), tris.HCl concentrate (623 mg / ml, 3.95 M), and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain a 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0212] To 4 aliquots of cetuximab (9.73 mg each, 4.864 mg / ml, 65 nmol) were added an aliquot of freshly prepared TCEP solution (0.5 - 2.0 mg / ml, 1.15 - 7.02 molar equivalents, 75 - 455 nmol). The mixture was vortexed briefly and then incubated at 20 °C for 300 min by roller mixing. After incubation (before addition of SO1861-EMCH), an aliquot of ~1 mg (0.210 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman assay (each thiol to Ab ratio = 2.0, 4.2, 5.9 and 6.8). To each of the native 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 min. 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 0.25 mg / ml solution) or TBS pH 7.5 buffer (2.2 - 8.7 μl) at 20 °C for 120 min as positive and negative controls, respectively. After incubation (before addition of NEM), an aliquot of 0.200 ml of the Ab-SO1861-EMCH mixture was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This aliquot was characterized by UV-vis and the positive and negative controls were characterized side-by-side by Ellman assay to obtain incorporation of SO1861-EMCH. To the native 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 purified cetuximab-(L-SO1861) conjugate. The 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.

[0213]

Table 6

[0214]

Table 7

[0215] Materials Throughout the specification, claims and drawings, "VHH", "Vhh", "V hh " and "V HH " should be understood to refer to the same type of single domain antibody. The same applies to single domain antibodies of the type referred to as either "VH", "Vh", "V h " or "V H ".

[0216] HER2-V HH 、EGFR-V HH 、CD71-V HH(Purchased), 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-Fischer), Pierce™ BCA Protein Assay Kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50 M (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), anhydrous D-(+)-trehalose (99%, Sigma-Aldrich), polyethyleneglycol sorbitan monolaurate (TWEEN® 20, Sigma-Aldrich), Dulbecco's Phosphate Buffered Saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium ethylenediaminetetraacetate dihydrate (EDTA-N a2, 99%, Sigma - Aldrich), sterile filters 0.2μm and 0.45μm (Sartorius), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), 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) freshly obtained from Ultrapure Lab Water Systems (MilliQ, Merck), nickel-nitrilo triacetic acid agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5'-dithiobis(2-nitrobenzoic acid (Ellman's reagent, DTNB, 98%, Sigma - Aldrich), sodium bicarbonate (99.7%, Sigma - Aldrich), sodium carbonate (99.9%, Sigma - Aldrich), PD MiniTrap desalting column with Sephadex G-25 resin (GE Healthcare), PD10 G25 desalting column (GE Healthcare), Zeba spin desalting columns at 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), acrylamide (99.9%, Sigma-Aldrich), sodium dodecyl sulfate (98%, Sigma-Aldrich), ammonium persulfate (APS, 98%, Sigma-Aldrich), glycerol (99%, Sigma-Aldrich), bromophenol blue (Sigma-Aldrich), polyethylene glycol dodecyl ether (Brij-35, Sigma-Aldrich). All SO1861 derivatives (SO1861-EMCH, SO1861-AEM, dendron-[L-SO1861]n), all QS21 derivatives (QS21-EMCH, QS21-AEM, dendron-[L-QS21]n), and trifunctional linker derivatives were prepared in-house.

[0217] Synthesis 1.V HH -[S-trifunctional linker-(L-SO1861)-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(L-SO1861)-(L-HSP27)]4, HER2-V HH -[S-tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-tri-(L-SO1861)-(L-HSP27)]4, EGFR-VHH-[S-tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(L-SO1861)-(L-HSP27)]4, CD71-V HH -[S-tri-(blocking)-(L-HSP27)]4, HER2-V HH , EGFR-V HH , and CD71-V HH will later be referred to as "Ab". Ab was conjugated to two different maleimides (Mal) having an HSP27 BNA derivative later referred to as "HSP27-Mal" via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were namely: 1) Mal-trifunctional linker-(L-SO1861)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was for HER2-VHH -[S-trifunctional linker-(L-SO1861)-(L-HSP27 BNA)]4 is exemplified as follows: The Ab was reconstituted to 21 mg / ml with deionized water (DI) and then diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0218] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 min by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to ab ratio = 4.0). The native Ab-SH was divided into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the HSP27 BNA-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 min. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 min as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of HSP27 BNA derivative 2.To each protomer Ab-construct mixture, a fixed amount of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and the mixture was purified by gel filtration using 1.6×30 cm Sephadex G50M eluted with DPBS pH 7.5. After that, centrifugal filtration and washing were repeated using a 100KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate. After filtering the product through a 0.2 μm filter, it was aliquoted for biological evaluation.

[0219] 2.V HH -[S-trifunctional linker-(S-SO1861)-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(S-SO1861)-(L-HSP27)]4, HER2-V HH -[S-tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-tri-(S-SO1861)-(L-HSP27)]4, EGFR-VHH-[S-tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(S-SO1861)-(L-HSP27)]4, CD71-V HH -[S-tri-(blocking)-(L-HSP27)]4, HER2-V HH 、EGFR-V HH 、and CD71-V HH will later be referred to as "Ab". The Ab was conjugated to two different maleimides (Mal) having an HSP27 BNA derivative, later referred to as "HSP27-Mal", via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were namely: 1) Mal-trifunctional linker-(S-SO1861)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was for HER2-V HH-[S-trifunctional linker-(S-SO1861)-(L-HSP27 BNA)]4 is exemplified as follows: The Ab was reconstituted to 21 mg / ml with deionized water (DI), and then diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0220] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 min by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to ab ratio = 4.0). The native Ab-SH was divided into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the HSP27 BNA-Mal derivative 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 min. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 min as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of HSP27 BNA derivative 2.To each bulk Ab-construct mixture, an aliquot of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added and the mixture was purified by gel filtration using 1.6 x 30 cm Sephadex G50M eluted with DPBS pH 7.5, followed by repeated centrifugal filtration and washing using a 100 KDa MWCO concentrator to obtain purified Ab-construct 1-2 conjugate. The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0221] 3.V HH -[S-trifunctional linker-(S-dendron-(L-SO1861) n )-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-HSP27)]4, HER2-V HH -[S-Tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-HSP27)]4, EGFR-V HH -[S-Tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-HSP27)]4, CD71-V HH -[S-Tri-(blocking)-(L-HSP27)]4, HER2-V HH , EGFR-V HH , and CD71-V HHis hereinafter referred to as "Ab". Ab is conjugated to two different maleimides (Mal) having an HSP27 BNA derivative hereinafter referred to as "HSP27-Mal" via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives are, namely: 1) Mal-trifunctional linker-(S-dendron-(L-SO1861)n)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). "n" refers to the number of SO1861 molecules which is 4, 8, or more than 8. The procedure is HER2-V HH -[S-trifunctional linker-(S-dendron-(L-SO1861)4)-(L-HSP27 BNA)]4 is exemplarily described: Ab was reconstituted to 21 mg / ml with deionized water (DI) and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0222] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a fixed amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added. The mixture was vortexed briefly and then incubated at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a fixed amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These fixed amounts were characterized by UV-vis analysis and Ellman assay (thiol-to-Ab ratio = 4.0). The native Ab-SH was divided into two fixed amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each fixed amount, a fixed amount of each of the HSP27 BNA-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added. The mixture was vortexed briefly and then incubated at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two fixed amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a fixed amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This fixed amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of HSP27 BNA derivative 2.To each bulk Ab-construct mixture, an aliquot of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added and the mixture was purified by gel filtration using 1.6 x 30 cm Sephadex G50M eluted with DPBS pH 7.5, followed by repeated centrifugal filtration and washing using a 100 KDa MWCO concentrator to obtain purified Ab-construct 1-2 conjugate. The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0223] 4.V HH -[S-trifunctional linker-(L-QS21)-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(L-QS21)-(L-HSP27)]4, HER2-V HH -[S-Tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-Tri-(L-QS21)-(L-HSP27)]4, EGFR-VHH-[S-Tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(L-QS21)-(L-HSP27)]4, CD71-V HH -[S-Tri-(blocking)-(L-HSP27)]4, HER2-V HH , EGFR-V HH , and CD71-V HH The Ab was conjugated to two different maleimides (Mal) with HSP27 BNA derivatives, later referred to as "HSP27-Mal", via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were: 1) Mal-trifunctional linker-(L-QS21)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was carried out to obtain the HER2-V HH-[S-trifunctional linker-(L-QS21)-(L-HSP27 BNA)]4 is exemplified as follows: The Ab was reconstituted to 21 mg / ml with deionized water (DI), and then diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0224] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to Ab ratio = 4.0). The native Ab-SH was divided into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the HSP27 BNA-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis, and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of HSP27 BNA derivative 2.To each bulk Ab-construct mixture, an aliquot of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added and the mixture was purified by gel filtration using 1.6 x 30 cm Sephadex G50M eluted with DPBS pH 7.5, followed by repeated centrifugal filtration and washing using a 100 KDa MWCO concentrator to obtain purified Ab-construct 1-2 conjugate. The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0225] 5.V HH -[S-trifunctional linker-(S-QS21)-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(S-QS21)-(L-HSP27)]4, HER2-V HH -[S-Tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-Tri-(S-QS21)-(L-HSP27)]4, EGFR-VHH-[S-Tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(S-QS21)-(L-HSP27)]4, CD71-V HH -[S-Tri-(blocking)-(L-HSP27)]4, HER2-V HH , EGFR-V HH , and CD71-V HH The Ab was conjugated to two different maleimides (Mal) with HSP27 BNA derivatives, later referred to as "HSP27-Mal", via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were: 1) Mal-trifunctional linker-(S-QS21)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was carried out to obtain the HER2-V HH-[S-trifunctional linker-(S-QS21)-(L-HSP27 BNA)]4 is exemplified as follows: The Ab was reconstituted to 21 mg / ml with deionized water (DI) and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0226] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly and then incubated at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to ab ratio = 4.0). The native Ab-SH was divided into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, a defined amount of each of the HSP27 BNA-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly and then incubated at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of HSP27 BNA derivative 2.To each protomer Ab-construct mixture, a fixed amount of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and the mixture was purified by gel filtration using 1.6×30 cm Sephadex G50M eluted with DPBS pH 7.5. Then, centrifugal filtration and washing were repeated using a 100KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate. After filtering the product through a 0.2 μm filter, it was aliquoted for biological evaluation.

[0227] 6.V HH -[S-trifunctional linker-(S-dendron-(L-QS21) n )-(L-HSP27 BNA)]4 HER2-V HH -[S-tri-(S-dendron-(L-QS21)n)-(L-HSP27)]4, HER2-V HH -[S-tri-(blocking)-(L-HSP27)]4, EGFR-V HH -[S-tri-(S-dendron-(L-QS21)n)-(L-HSP27)]4, EGFR-V HH -[S-tri-(blocking)-(L-HSP27)]4, CD71-V HH -[S-tri-(S-dendron-(L-QS21)n)-(L-HSP27)]4, CD71-V HH -[S-tri-(blocking)-(L-HSP27)]4, HER2-V HH , EGFR-V HH , and CD71-V HHis hereinafter referred to as "Ab". Ab is conjugated via a Michael-type thiol-ene reaction to two different maleimides (Mal) having an HSP27 BNA derivative hereinafter referred to as "HSP27-Mal". These HSP27-Mal derivatives were, namely: 1) Mal-trifunctional linker-(S-dendron-(L-QS21)n)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). "n" refers to the number of QS21 molecules which is 4, 8, or more than 8. The procedure is HER2-V HH -[S-trifunctional linker-(S-dendron-(L-QS21)4)-(L-HSP27 BNA)]4 is illustratively described: Ab was reconstituted in deionized water (DI) to 21 mg / ml and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of tris concentrate (127 mg / ml, 1.05 M), tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0228] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly and then incubated at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol-to-Ab ratio = 4.0). The native Ab-SH was divided into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the HSP27 BNA-Mal derivative 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly and then incubated at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of the HSP27 BNA derivative 2.To each bulk Ab-construct mixture, an aliquot of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added and the mixture was purified by gel filtration using 1.6 x 30 cm Sephadex G50M eluted with DPBS pH 7.5, followed by repeated centrifugal filtration and washing using a 100 KDa MWCO concentrator to obtain purified Ab-construct 1-2 conjugate. The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0229] 7.V HH -[S-trifunctional linker-(L-SO1861)-(L-dianthine)]4 HER2-V HH -[S-tri-(L-SO1861)-(L-dianthine)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(L-SO1861)-(L-dianthine)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-V HH -[S-tri-(L-SO1861)-(L-dianthine)]4, CD71-V HH -[S-tri-(blocking)-(L-dianthine)]4, HER2-V HH , EGFR-V HH , and CD71-V HH The Ab was conjugated to two different maleimides (Mal) with Gianthine derivatives, later referred to as "Gianthine-Mal", via a Michael-type thiol-ene reaction. These Gianthine-Mal derivatives were: 1) Mal-trifunctional linker-(L-SO1861)-(L-Gianthine), 2) Mal-trifunctional linker-(blocking)-(L-Gianthine). The procedure was carried out to obtain the HER2-V HH-[S-trifunctional linker-(L-SO1861)-(L-diantin)]4 is described illustratively: The Ab was reconstituted to 21 mg / ml with deionized water (DI) and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0230] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to Ab ratio = 4.0). The native Ab-SH was split into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the dithiane-Mal derivative 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-dithiane derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of the dithiane derivative 2. To each native Ab-construct mixture, a defined amount of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and the mixture was purified by gel filtration using a 1.6 × 30 cm Sephadex G50M eluting with DPBS pH 7.5, followed by repeated centrifugal filtration and washing using a 100 KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate.After filtering the product through a 0.2 μm filter, it was aliquoted for biological evaluation.

[0231] 8.V HH -[S-trifunctional linker-(S-SO1861)-(L-diantin)]4 HER2-V HH -[S-tri-(S-SO1861)-(L-diantin)]4, HER2-V HH -[S-tri-(blocking)-(L-diantin)]4, EGFR-V HH -[S-tri-(S-SO1861)-(L-diantin)]4, EGFR-V HH -[S-tri-(blocking)-(L-diantin)]4, CD71-V HH -[S-tri-(S-SO1861)-(L-diantin)]4, CD71-V HH -[S-tri-(blocking)-(L-diantin)]4, HER2-V HH , EGFR-V HH , and CD71-V HH are hereinafter referred to as "Ab". Ab was conjugated to two different maleimides (Mal) having a diantin derivative hereinafter referred to as "diantin-Mal" via a Michael-type thiol-ene reaction. These diantin-Mal derivatives were, namely: 1) Mal-trifunctional linker-(S-SO1861)-(L-diantin), 2) Mal-trifunctional linker-(blocking)-(L-diantin). The procedure is exemplified for HH -[S-trifunctional linker-(S-SO1861)-(L-diantin)]4: Ab was reconstituted to 21 mg / ml with deionized water (DI) and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0232] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 minutes by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to ab ratio = 4.0). The native Ab-SH was split into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the dithiol-Mal derivative 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 minutes. In addition to the conjugation reaction of Ab-dithiol derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 minutes as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis, and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of the dithiol derivative 2. To each native Ab-construct mixture, a defined amount of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and after the mixture was purified by gel filtration using a 1.6 × 30 cm Sephadex G50M eluting with DPBS pH 7.5, centrifugal filtration and washing were repeated using a 100 KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate.The product was filtered through a 0.2 μm filter and then aliquoted for biological evaluation.

[0233] 9.V HH -[S-trifunctional linker-(S-dendron-(L-SO1861) n )-(L-diantin)]4 HER2-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-diantin)]4, HER2-V HH -[S-tri-(block)-(L-diantin)]4, EGFR-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-diantin)]4, EGFR-V HH -[S-tri-(block)-(L-diantin)]4, CD71-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-diantin)]4, CD71-V HH -[S-tri-(block)-(L-diantin)]4, HER2-V HH , EGFR-V HH , and CD71-V HH are hereinafter referred to as "Ab". Ab was conjugated to two different maleimides (Mal) having a diantin derivative hereinafter referred to as "diantin-Mal" via a Michael-type thiol-ene reaction. These diantin-Mal derivatives were, namely: 1) Mal-trifunctional linker-(S-dendron-(L-SO1861)n)-(L-diantin), 2) Mal-trifunctional linker-(block)-(L-diantin). "n" refers to the number of SO1861 molecules that is 4, 8, or more than 8. The procedure was for HER2-V HH-Exemplary description of [S-trifunctional linker-(S-dendron-(L-SO1861)4)-(L-diantin)]4: Ab was reconstituted to 21 mg / ml with deionized water (DI) and subsequently diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0234] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined aliquot of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, the mixture was vortexed briefly, and then incubated at 20 °C for 90 min by roller mixing. After incubation (before addition of the construct), a defined aliquot of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined aliquots were characterized by UV-vis analysis and Ellman assay (thiol to ab ratio = 4.0). The native Ab-SH was split into two defined aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined aliquot, a defined aliquot of each of the dithiane-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, the mixture was vortexed briefly, and then incubated at 20 °C for 120 min. In addition to the conjugation reaction of Ab-dithiane derivative 2, two defined aliquots of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 min as positive and negative controls, respectively. After incubation (before addition of NEM), a 0.100 ml defined aliquot of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined aliquot was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of dithiane derivative 2. To each native Ab-construct mixture, a defined aliquot of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and after the mixture was purified by gel filtration using a 1.6 × 30 cm Sephadex G50M eluting with DPBS pH 7.5, centrifugal filtration and washing were repeated using a 100 KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate.The product was filtered through a 0.2 μm filter and then aliquoted for biological evaluation.

[0235] 10.V HH -[S-trifunctional linker-(L-QS21)-(L-diantin)]4 HER2-V HH -[S-tri-(L-QS21)-(L-diantin)]4, HER2-V HH -[S-tri-(blocking)-(L-diantin)]4, EGFR-V HH -[S-tri-(L-QS21)-(L-diantin)]4, EGFR-V HH -[S-tri-(blocking)-(L-diantin)]4, CD71-V HH -[S-tri-(L-QS21)-(L-diantin)]4, CD71-V HH -[S-tri-(blocking)-(L-diantin)]4, HER2-V HH , EGFR-V HH , and CD71-V HH are hereinafter referred to as "Ab". Ab was conjugated to two different maleimides (Mal) having a diantin derivative hereinafter referred to as "diantin-Mal" via a Michael-type thiol-ene reaction. These diantin-Mal derivatives were, namely: 1) Mal-trifunctional linker-(L-QS21)-(L-diantin), 2) Mal-trifunctional linker-(blocking)-(L-diantin). The procedure is exemplified for HER2-V HH -[S-trifunctional linker-(L-QS21)-(L-diantin)]4: Ab was reconstituted in deionized water (DI) to 21 mg / ml and then diluted to 5 mg / ml using histidine buffer pH 6. To a 20 mg (4.0 ml) aliquot, 10 μl / ml each of Tris concentrate (127 mg / ml, 1.05 M), Tris.HCl concentrate (623 mg / ml, 3.95 M) and EDTA-Na2 concentrate (95 mg / ml, 0.26 M) were added to obtain 50 mM TBS, 2.5 mM EDTA buffer pH 7.5.

[0236] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), a defined amount of freshly prepared TCEP solution (1.00 mg / ml, 2.35 molar equivalents, 0.32 μmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 90 min by roller mixing. After incubation (before addition of the construct), a defined amount of approximately 0.2 mg (0.044 ml) of Ab-SH was removed from each mixture and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. These defined amounts were characterized by UV-vis analysis and Ellman assay (thiol to Ab ratio = 4.0). The native Ab-SH was split into two defined amounts (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol), and to each defined amount, each of a defined amount of the dithiane-Mal derivatives 1-2 (freshly prepared in TBS pH 7.5, 2 mg / ml, 1.3 molar equivalents per "thiol", 40 nmol and 43 nmol) was added, and the mixture was vortexed briefly, followed by incubation at 20 °C for 120 min. In addition to the conjugation reaction of Ab-dithiane derivative 2, two defined amounts of desalted Ab-SH (50 μg, 3.3 nmol) were reacted with NEM (1.3 molar equivalents per "thiol", 17.3 nmol, 6.7 μl of 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) at 20 °C for 120 min as positive and negative controls, respectively. After incubation (before addition of NEM), a defined amount of 0.100 ml of the mixture of Ab-construct 2 was removed and purified by gel filtration using a Zeba spin desalting column equilibrated with TBS pH 7.5. This defined amount was characterized by UV-vis and the positive and negative controls were characterized side by side by Ellman assay to obtain the incorporation of dithiane derivative 2. To each native Ab-construct mixture, a defined amount of freshly prepared NEM solution (0.25 mg / ml, 2.5 molar equivalents, 19 and 21 nmol) was added, and after purification of the mixture by gel filtration using a 1.6 × 30 cm Sephadex G50M eluting with DPBS pH 7.5, centrifugal filtration and washing were repeated using a 100 KDa MWCO concentrator to obtain the purified Ab-construct 1-2 conjugate.The product was filtered through a 0.2 μm filter and then aliquoted for biological evaluation.

[0237] 11.V HH -[S-trifunctional linker-(S-QS21)-(L-diantin)]4 HER2-V HH -[S-tri-(S-QS21)-(L-diantin)]4, HER2-V HH -[S-tri-(blocking)-(L-diantin)]4, EGFR-V HH -[S-tri-(S-QS21)-(L-diantin)]4, EGFR-V HH -[S-tri-(blocking)-(L-diantin)]4, CD71-VHH-[S-tri-(S-QS21)-(L-diantin)]4, CD71-VHH-[S-tri-(blocking)-(L-diantin)]4, HER2-V HH , EGFR-V HH , and CD71-V HH are hereinafter referred to as "Ab". Ab was con...

Claims

1. A conjugate for transferring an effector molecule from the exterior of a cell into said cell, comprising: - an effector molecule to be transferred to said cell, - Cell surface molecule targeting antibodies, and - contains saponin, the effector molecule, the antibody and the saponin are covalently linked; the effector molecule is an oligonucleotide; The saponin comprises an aglycone core structure selected from quillic acid or gypsogenin, and the C of the aglycone core structure ３ a first glycan bonded to an atom, and a C ２８ a bi-desmoside triterpene glycoside comprising a second glycan bonded to an atom, the first glycan being branched and comprising a glucuronic acid unit, the second glycan being branched and comprising a carbohydrate unit selected from any one or more of fucose (Fuc), rhamnose (Rha) and quinovose (Qui); The conjugate, wherein said antibody is capable of binding to a cell surface molecule of said cell.

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

3. The cell surface molecule targeting antibody is 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 conjugate of claim 1 or 2 which is a domain.

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

5. 5. The conjugate of any one of claims 1 to 4, wherein the 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 conjugate according to any one of claims 1 to 5, wherein said 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 conjugate of any one of claims 1 to 6, wherein the saponin comprises an aglycone core structure that is quinoline.

8. 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 conjugate according to any one of claims 1 to 7, which is selected from

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

10. The conjugate of any one of claims 1 to 9, wherein the cell surface molecule targeting antibody binds to CD71.

11. The conjugate according to any one of claims 1 to 10, wherein the cell surface molecule targeting antibody is capable of binding to a cell surface receptor of a target cell.

12. The conjugate of claim 11, wherein the cell surface receptor of the target cell is CD71.

13. The conjugate according to any one of claims 1 to 12, wherein the effector molecule is covalently attached to the cell surface molecule-targeting antibody either directly or via a linker.

14. The conjugate according to any one of claims 1 to 13, wherein the saponin is covalently attached to the cell surface molecule targeting antibody and / or the effector molecule, either directly or via a linker.

15. The saponin is a C ２３ 15. The conjugate of any one of claims 1 to 14, wherein the conjugate is covalently attached via the linker N-ε-maleimidocaproic acid hydrazide (EMCH), which is covalently attached to the aldehyde group at position 1. Njugate.

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

17. The conjugate of claim 16 , wherein the cleavable linker is subject to cleavage under acidic conditions.

18. 18. The conjugate of claim 16 or 17, wherein the cleavable linker is subject to cleavage at a pH of ≦5.

5.

19. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 18 as a medically active ingredient.

20. 20. The pharmaceutical composition of claim 19 for the treatment of a human disease selected from any one or more of cancer, autoimmune diseases, diseases associated with enzyme deficiencies, diseases associated with gene defects, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin associated liver disease, acute hepatic porphyria, amyloidosis and transthyretin mediated amyloidosis.

21. 21. The pharmaceutical composition of claim 19 or 20, wherein the saponin is SO1861.

22. Kit includes: a) a first pharmaceutical composition comprising a first conjugate comprising a first cell surface molecule targeting antibody, the cell surface molecule targeting antibody according to any one of claims 1 to 3 and 10 to 12, covalently linked according to claim 13 to the oligonucleotide according to any one of claims 1, 4 to 6, 13 and 14 directly or via a linker, and b) a second pharmaceutical composition comprising: i. a second conjugate comprising the saponin of any one of claims 1 and 7 to 9, covalently linked directly or via a linker to a second cell surface molecule targeting antibody according to any one of claims 14 to 18, the second cell surface molecule targeting antibody being the cell surface molecule targeting antibody of any one of claims 1 to 3 and 10 to 18, the second cell surface molecule targeting antibody being the same or different from the first cell surface molecule targeting antibody; ii. A free saponin or a saponin derivative, wherein the saponin comprises an aglycone core structure selected from quillaric acid or gypsogenin, and the C of the aglycone core structure ３ a first glycan bonded to an atom, and a C ２８ A free saponin or saponin derivative which is a bi-desmoside triterpene glycoside comprising a second glycan bonded to an atom, said first glycan being branched and comprising a glucuronic acid unit, and said second glycan being branched and comprising a carbohydrate unit selected from any one or more of fucose (Fuc), rhamnose (Rha) and quinovose (Qui).

23. 23. The kit of claim 22, comprising the first conjugate and the second conjugate as medically active ingredients.

24. 24. The kit of claim 22 or 23 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.

25. The kit according to any one of claims 22 to 24, wherein the saponin is SO1861.

26. The kit of any one of claims 22 to 25, wherein the cell surface molecule targeting antibody binds to CD71.

27. The kit according to any one of claims 22 to 26, wherein the oligonucleotide is selected from short interfering RNA (siRNA) and antisense oligonucleotide (ASO).

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