Conjugates of single domain antibodies, saponins and effector molecules, pharmaceutical compositions comprising same, and therapeutic uses of said pharmaceutical compositions

Abstract

The present invention relates to a conjugate for translocating an effector molecule from the outside of a cell into the cell, the conjugate comprising at least one effector molecule to be translocated into the cell, at least one saponin of the mono-desmosidic triterpene glycoside type or the bi-desmosidic triterpene glycoside type, and at least one single domain antibody (sdAb), all covalently linked to each other, wherein the sdAb is capable of binding 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 a pharmaceutical composition of the present invention for use as a medicament. Additionally, the present invention relates to a pharmaceutical composition of the present invention for use in treating or preventing any one or more of the following: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, diseases associated with enzyme deficiency, gene defects, diseases associated with gene defects, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-associated 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 a conjugate from the outside of a cell to the inside of said cell or for transferring an effector molecule comprised by a conjugate of the invention from the outside of a cell to the inside of said cell, preferably to the cytosol of said cell.

Description

[Technical Field]

[0001] The present invention relates to a conjugate for translocating an effector molecule from the outside of a cell into the cell, the conjugate comprising at least one effector molecule to be translocated into the cell, at least one saponin of the mono-desmosidic triterpene glycoside type or the bi-desmosidic triterpene glycoside type, and at least one single domain antibody (sdAb), all covalently linked to each other, wherein the sdAb is capable of binding 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 a pharmaceutical composition of the present invention for use as a medicament. Additionally, the present invention relates to a pharmaceutical composition of the present invention for use in treating or preventing any one or more of the following: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, diseases associated with enzyme deficiency, gene defects, diseases associated with gene defects, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-associated 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 a conjugate from the outside of a cell to the inside of said cell or for transferring an effector molecule comprised by a conjugate of the invention from the outside of a cell to the inside of said cell, preferably to the cytosol of said cell. [Background technology]

[0002] Molecules with therapeutic biological activity are often theoretically 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 biologically active moiety. However, many, if not all, of the promising drug-like molecules and therapeutic agents currently used in the clinic suffer from at least one of numerous drawbacks and deficiencies. When administered to the human body, therapeutically active molecules may exert off-target effects in addition to the desired biological activity for the treatment of a disease or health problem. Such off-target effects are undesirable and carry the risk of inducing health- or even life-threatening side effects of the administered molecule. The occurrence of such adverse events causes many drug-like compounds and therapeutic moieties to fail Phase III or Phase IV clinical trials (post-marketing surveillance). Thus, there is a strong desire to provide drug molecules, such as small molecule therapeutics, whose therapeutic effect must, among other things, (1) be highly specific for disease-promoting biological factors or biological processes, (2) be sufficiently safe, (3) be sufficiently effective, (4) be sufficiently targeted to diseased cells with little or no off-target activity against non-diseased cells, (5) have a sufficiently timely mechanism of action (e.g., the administered drug molecule must reach and remain at the targeted site in a human patient within a certain time frame), and / or (6) have a sufficiently long-lasting therapeutic activity in the patient's body. Unfortunately, to date, the "ideal" therapeutic possessing many or even all of the advantageous characteristics outlined herein above has not been available to patients, despite impressive progress already made in several areas of long-term intensive research and the challenges and deficiencies encountered that have been individually addressed.

[0003] Chemotherapy is one of the most important therapeutic options for cancer treatment. However, due to a lack of specificity for cancer cells beyond dividing cells in healthy tissues, chemotherapy is often associated with a narrow therapeutic window. The invention of monoclonal antibodies (mAbs) has opened up the possibility of leveraging their specific binding properties as a mechanism for targeting and delivering cytotoxic drugs to cancer cells while sparing normal cells. This can be achieved by chemically conjugating a cytotoxic effector (also known as a payload or warhead) to the antibody to create an antibody-drug conjugate (ADC). Typically, highly potent payloads such as emtansine (DM1), which has a limited therapeutic index (the ratio comparing the toxic dose to the effective dose) in its unconjugated form, are used. Conjugation of DM1 to trastuzumab (ado-trastuzumab emtansine), also known as Kadcycla, increases the tolerable dose of DM1 by at least two-fold in monkeys. Tremendous effort and investment have been devoted over the past few decades to developing therapeutic ADCs. However, despite promising preclinical data, translating ADCs into the clinic remains challenging. The first ADC approved for clinical use was gemtuzumab ozogamicin (Mylotarg, CD33-targeting, 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 patients treated with conventional chemotherapy. Mylotarg was relaunched in 2017 with a reduced recommended dose, a different schedule in combination with chemotherapy or as a single agent, and a new patient population. To date, only five ADCs have been approved for clinical use, while approximately 55 ADCs have ceased clinical development. However, interest remains high, and approximately 80 ADCs are currently in clinical development in nearly 600 clinical trials.

[0004] Despite the possibility of using toxic payloads that are not usually tolerated by patients, a low therapeutic index is a major issue that accounts for the discontinuation of many ADCs in clinical development. This can be caused by several mechanisms, such as off-target toxicity against normal cells, the development of resistance to cytotoxic drugs, and premature drug release in the circulation. A systematic review 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 premature payload release. It is estimated that at least 23 of the approximately 55 discontinued ADCs were due to a poor therapeutic index. For example, development of a trastuzumab tesirin conjugate (ADCT-502, a HER2-targeted ADC therapeutic) was recently halted due to a low therapeutic index, likely due to on-target, off-tissue effects in lung tissue, which expresses significant levels of HER2. Additionally, several ADCs in Phase 3 trials have been discontinued due to missing primary endpoints. For example, phase 3 trials of the depatuxizumab mafodotin conjugate (ABT-414, EGFR-targeting, AbbVie) in patients with newly diagnosed glioblastoma and the mirvetuximab soravtansine conjugate (IMGN853, folate receptor alpha (FRα)-targeting, ImmunoGen) in patients with platinum-resistant ovarian cancer were recently halted, demonstrating no survival benefit. It is important to acknowledge that clinically available doses of some ADCs may not be sufficient for their full anticancer activity. For example, ado-trastuzumab emtansine has an MTD of 3.6 mg / kg in humans. In preclinical models of breast cancer, ado-trastuzumab emtansine induced tumor regression at dose levels of 3 mg / kg or higher, although more potent efficacy was observed at 15 mg / kg. This suggests that at clinically administered doses, ado-trastuzumab emtansine may not exert its maximum potential antitumor effect.

[0005] ADCs are primarily composed 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 novel ADCs to overcome the existing problems of targeting each of the ADC components. For example, by identifying and validating sufficient antigen targets for antibody components, linkers that increase the solubility and drug-antibody ratio (DAR) of ADCs can be designed and optimized by selecting antigens with high expression levels in tumors and little or no expression in normal tissues, antigens present on the cell surface so as to be accessible to circulating ADCs, and antigens that allow the ADC to be internalized into cells after binding, as well as alternative mechanisms of activity, overcoming resistance induced by proteins that can transport chemotherapeutic agents outside cells, increasing the DAR ratio by inclusion of additional payloads, and selecting and optimizing antibodies to improve antibody uniformity and developability. In addition to technological developments in ADCs, new clinical and translational strategies, such as altering dosing schedules through fractionated dosing, performing biodistribution studies, and including biomarkers to capture response signals early, monitor duration and depth of response, and optimize patient selection to inform combination trials, are also being employed to maximize the therapeutic index.

[0006] Examples of ADCs with clinical potential include brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, which are being evaluated as treatment options for lymphoid malignancies and multiple myeloma. Polatuzumab vedotin, which binds to CD79b on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, are being tested in clinical trials. These ADCs are combined with co-administered rituximab and monoclonal antibodies that bind to CD20, respectively, and are not provided with a payload [B. Yu and D. Liu, Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12:94]. Combinations of monoclonal antibodies, such as these examples, represent a further approach and attempt to arrive at a "magic bullet" that combines many or all of the desirable features of the aforementioned ADCs.

[0007] Meanwhile, over the past few decades, nucleic acid-based therapeutics have been under development. Therapeutic nucleic acids can be based on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), antisense oligonucleotides (ASOs, AONs), and short interfering RNA (siRNA) for approaches such as gene therapy, RNA interference (RNAi), microRNA, and DNA and RNA aptamers. Many of them share the same basic basis of action: inhibiting either DNA or RNA expression, thereby preventing the expression of abnormal proteins associated with disease. Gene therapy has the most clinical trials, with nearly 2,600 clinical trials underway or completed worldwide, although only about 4% have entered phase 3. This is followed by clinical trials using ASOs. Similar to ADCs, despite numerous approaches being explored, therapeutic nucleic acids share two major problems during clinical development: cellular delivery and off-target effects. ASOs, such as peptide nucleic acids (PNAs), phosphoramidate morpholino oligomers (PMOs), locked nucleic acids (LNAs), and bridged nucleic acids (BNAs), are being investigated as an attractive strategy to specifically inhibit target genes, particularly those that are difficult to target with small molecule inhibitors or neutralizing antibodies. The efficacy of different ASOs is currently 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 several cancer stages. The application of ASOs as potential therapeutic agents requires safe and effective methods for their delivery to the cytoplasm and / or nucleus of target cells and tissues. Although the clinical significance of ASOs has been demonstrated, inefficient cellular uptake both in vitro and in vivo limits their efficacy and poses a barrier to their therapeutic development. Cellular uptake is less than 2% of the dose, resulting in ASO concentrations at the active site that are too low to achieve effective and sustained outcomes. This ultimately necessitates increased doses, which can 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] Chemotherapeutics are most commonly small molecules, but their effectiveness is hindered by severe off-target side effects and toxicity, as well as their poor solubility, rapid clearance, and limited tumor exposure. Scaffold-small molecule drug conjugates, such as polymer-drug conjugates (PDCs), are pharmacologically active macromolecular constructs that contain one or more molecules of a small molecule drug bound to a carrier scaffold (e.g., polyethylene glycol (PEG)).

[0009] Such conjugate principles have been attracting attention and investigation for decades. The majority of small molecule drug conjugates under preclinical or clinical development are for oncology purposes. However, to date, only one non-cancer-related drug 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), a non-oncology indication. The translational application of drug-scaffold conjugates to the treatment of human subjects has met with little clinical success to date. For example, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer doxorubicin; developed by Pharmacia, Pfizer) has shown excellent anticancer activity in both solid tumors and leukemia in mouse models and is under clinical investigation for oncology purposes. Although it demonstrated significantly reduced non-specific toxicity and improved pharmacokinetics in humans, the improvement in anti-cancer activity in patients was found to be modest, and as a result, further development of PK1 was discontinued.

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

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

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

[0013] In European Patent No. 1623715B1, compositions are described that comprise a pharmacologically active agent coupled to a target cell-specific binding molecule in combination with a saponin. The pharmacologically active agent is, for example, a toxin. Summary of the Invention [Means for solving the problem]

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

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

[0016] At least one of the above objects of the embodiments of the present invention is achieved by the present invention, HHThis is achieved by providing an antibody-drug conjugate (ADC) or an antibody-oligonucleotide (AOC), such as an antibody-BNA covalent complex, of the invention comprising a cell-targeting moiety which 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 a BNA, an ADC provided with a covalently linked saponin and / or an AOC provided with a covalently linked saponin that is also suitable for use as a pharmaceutical.

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

[0018] A first aspect of the present invention relates to a conjugate for transferring an effector molecule from the outside of a cell into said 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, covalently linked to each other either directly or via at least one linker, wherein the at least one saponin is a mono-desmosidic triterpene glycoside or a bi-desmosidic triterpene glycoside, and wherein the sdAb is capable of binding to a cell surface molecule of said cell.

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

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

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

[0022] A fifth aspect of the present invention relates to an in vitro or ex vivo method for translocating an effector molecule of the present invention (an effector molecule comprised by a conjugate of the present invention) from the outside of a cell to the inside of said cell, preferably to the cytosol of said cell, comprising: a) providing a cell which expresses on its cell surface a binding site for at least one sdAb comprised by a conjugate of the invention, said binding site preferably being present on a cell surface molecule of the cell as described herein, said cell preferably being selected from liver cells, abnormal cells such as virus-infected cells, autoimmune cells, cells comprising a genetic defect, cells comprising an enzyme defect and tumor cells; b) providing a conjugate of the invention, said conjugate comprising an effector molecule to be transferred to the cells provided in step a); and c) contacting the cells of step a) in vitro or ex vivo with the conjugate of step b) Including, Thus, the present invention relates to a method comprising the steps of translocating the conjugate comprising an effector molecule from the outside of a cell to the inside of the cell, and translocating the effector molecule from the outside of a cell to the inside of the cell, preferably to the cytosol of the cell, by translocation of the conjugate.

[0023] A sixth aspect of the present invention provides an in vitro or ex vivo method for transferring a conjugate of the present invention from the exterior of a cell to the interior of said cell, comprising: a) providing a cell which expresses on its cell surface a binding site for at least one sdAb comprised by a conjugate of the invention, said binding site preferably being present on a cell surface molecule of the cell as described herein, said cell preferably being selected from liver cells, abnormal cells such as virus-infected cells, autoimmune cells, cells comprising a genetic defect, cells comprising an enzyme defect and tumor cells; b) providing any one of the conjugates of the present invention; and c) contacting the cells of step a) in vitro or ex vivo with the conjugate of step b), thereby translocating the conjugate from the outside of the cells to the inside of said cells.

[0024] An aspect of the present invention relates to a kit-of-parts comprising a conjugate of the invention or a pharmaceutical composition of the invention and instructions for use of said conjugate or said pharmaceutical composition for use in the treatment or prevention of the onset of any one or more of the following: cancer, autoimmune diseases such as rheumatoid arthritis, enzyme deficiency, diseases associated with enzyme deficiency, gene defect, diseases associated with gene defect, infections such as viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-associated liver disease, acute hepatic porphyria, amyloidosis and transthyretin-mediated amyloidosis, or instructions for applying an in vitro or ex vivo method according to the present invention.

[0025] An embodiment of the invention is a conjugate such as an ADC or AOC of structure C, or a semi-completed ADC conjugate or semi-completed AOC conjugate, comprising a cell surface molecule targeting molecule such as an sdAb of the invention and comprising at least one effector moiety of the invention and / or comprising at least one saponin of the invention: A(-S) b (-E) c (Structure C) where A is a cell surface molecule targeting molecule, such as an 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 decimal point) therebetween; c=0 to 8, preferably 0, 1, 2, 3, 4, 6, 8 or any integer (or decimal point) 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 proteinaceous, meaning that the molecule has, to some extent, physicochemical properties characteristic of a protein, is of a protein, relates to a protein, contains a protein, belongs to a protein, consists of a protein, resembles 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 resembles or is a protein, and "protein" is to be understood to include chains of amino acid residues 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 linked via an amide bond, e.g., 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 comprising at least two amino acid residues, preferably from 2 to about 2,000 amino acid residues. In one embodiment, the proteinaceous molecule is a molecule comprising 2 to 20 amino acids (typical for peptides). In one embodiment, the proteinaceous molecule is a molecule comprising 21 to 1000 amino acids (typical for polypeptides, proteins, protein domains, e.g. antibodies, Fabs, scFvs, ligands for receptors such as EGF). Preferably, the amino acid residues are (typically) linked via peptide bonds. According to the present invention, said amino acid residues are or comprise (modified) (non-)natural amino acid residues.

[0027] The term "effector molecule," or "effector moiety," when referring to an effector molecule, for example, as part of a covalent conjugate, has its ordinary scientific meaning and, as used herein, refers to a molecule that can selectively bind to one or more target molecules, such as proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA and RNA, and enzymes, thereby modulating the biological activity of one or more such target molecules. The effector molecule is, for example, a molecule selected from one or more of a small molecule, such as a drug molecule, a toxin, such as a protein toxin, an oligonucleotide, such as a BNA, a xenobiotic nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof. Thus, for example, an effector molecule or effector moiety is a molecule or moiety selected from one or more of the following: a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a xenobiotic nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof, that can selectively bind to one or more target molecules: proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA and RNA, or enzymes, and that, upon binding to the target molecule, modulates the biological activity of such one or more target molecules. Typically, an effector molecule can exert a biological effect inside a cell, such as a mammalian cell, such as a human cell, e.g., in the cytosol of the cell. Thus, an effector molecule or moiety of the present invention is any substance that affects cellular metabolism by interacting with an intracellular effector molecule target, which is any molecule or structure inside the cell, including, but not limited to, compartments of the endocytic and recycling pathways and the lumen of vesicles. Thus, such structures within a cell include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the cell membrane, and the interior of the cytosol. Typical effector molecules are therefore drug molecules, enzymes, plasmid DNA, toxins such as those contained by antibody-drug conjugates (ADCs), oligonucleotides such as siRNAs, BNAs, and nucleic acids contained by antibody-oligonucleotide conjugates (AOCs).For example, an effector molecule is a molecule that can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling.

[0028] The term "saponin" has its ordinary scientific meaning and, as used herein, refers to a group of amphiphilic glycosides comprising one or more hydrophilic glycone 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 synthesized de novo via chemical and / or biotechnological synthetic routes.

[0029] The term "saponin derivative" (also known as "modified saponin") has its ordinary scientific meaning and, as used herein, refers to a compound corresponding to a naturally occurring saponin that has been 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 an aldehyde group on the aglycone; of a carboxyl group on the sugar chain; or of an acetoxy group on the sugar chain. Typically, a saponin derivative has no natural counterpart, i.e., it is not naturally produced, for example, by a plant or tree. The term "saponin derivative" includes derivatives obtained by derivatization of a naturally occurring saponin as well as derivatives synthesized de novo via chemical and / or biotechnological synthetic routes that result in a compound corresponding to a naturally occurring saponin that has been derivatized by one or more chemical modifications.

[0030] The term "aglycone core structure" has its usual scientific meaning and, as used herein, refers to the aglycone core of a saponin without one or two carbohydrate antennae or sugar chains (glycans) attached thereto. For example, quinoline is the aglycone core structure for SO1861, QS-7, and QS21. Typically, the glycans of a saponin are monosaccharides or oligosaccharides, such as linear or branched glycans.

[0031] The term "glycan" has its usual scientific meaning and refers herein to either a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide) or a chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycan may consist of only sugar moieties or may include additional moieties such as, for example, any one of 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid, and 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, such as those present in OS-21.

[0032] The term "Api / Xyl-" or "Api- or Xyl-" in relation to the name of a glycan has its ordinary scientific meaning and refers herein to a glycan that contains an apiose (Api) moiety or that contains a xylose (Xyl) moiety.

[0033] The term "saponin on which the modified saponin is based" has its ordinary scientific meaning and, as used herein, refers to a saponin that has been modified to produce the modified saponin. Typically, the saponin on which the modified saponin is based is a naturally occurring saponin that has been subjected to chemical modification to produce the 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 to yield a modified saponin; the saponin from which the modified saponin is made is typically a naturally occurring saponin.

[0035] The term "oligonucleotide" has its ordinary scientific meaning and refers herein to any natural or synthetic stretch of nucleic acid, including DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, presented as single- or double-stranded molecules such as BNAs, antisense oligonucleotides (ASOs), short or small interfering RNAs (siRNAs; silencing RNAs), antisense DNA, antisense RNA, among others.

[0036] The term "antibody-drug conjugate" or "ADC" has its ordinary scientific meaning and is used herein to refer to an antibody or drug conjugate comprising an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H Domain, Single Domain Antibody, V HH , Camelidae V H and any conjugate of an antibody, such as a mAb, or antibody-binding protein, as well as any molecule that can exert a therapeutic effect when contacted with cells of a subject, such as a human patient, such as an active pharmaceutical ingredient, a toxin, an oligonucleotide, an enzyme, a small molecule drug compound, etc.

[0037] The term "antibody-oligonucleotide conjugate" or "AOC" has its ordinary scientific meaning and is used herein to refer to an antibody or oligonucleotide conjugate comprising an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H Domain, Single Domain Antibody, V HH , Camelidae V H and any conjugate of an antibody, such as an antibody fragment, or a fragment thereof, as well as an oligonucleotide selected from natural or synthetic stretches of nucleic acids, including BNAs, antisense oligonucleotides (ASOs), short or small interfering RNAs (siRNAs; silencing RNAs), antisense DNA, DNA presented as single- or double-stranded molecules such as antisense RNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acids, etc., which are capable of exerting a therapeutic effect when contacted with cells of a subject, such as a human patient.

[0038] The terms "bridged nucleic acid," or "BNA" for short, or "locked nucleic acid," or "LNA" for short, have their usual scientific meaning and, as used herein, refer to modified RNA nucleotides. BNAs are also referred to as "tethered RNA molecules" or "inaccessible RNA molecules." BNA monomers can contain 5-, 6-, or 7-membered bridge structures with "locked" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2',4'-position of the ribose to yield a 2',4'-BNA monomer. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNAs are structurally rigid oligonucleotides with increased binding affinity and stability.

[0039] The term "S" when used in reference to antibody-saponin conjugates and the like comprising a linker denotes a "stable linker" that remains intact in the endosomes and lysosomes of mammalian cells, such as human cells, such as human tumor cells, and therefore under mildly acidic conditions (pH<6.6, e.g., pH 4.0-5.5).

[0040] The term "L" when used in linker-containing antibody-saponin conjugates and the like refers to a "labile linker" that is cleaved under mildly acidic conditions (pH < 6.6, e.g., pH 4.0 to 5.5) in the endosomes and lysosomes of mammalian cells, such as human cells, including human tumor cells.

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

[0042] The embodiments of the invention described herein can work in combination and cooperation, unless otherwise specified.

[0043] Furthermore, references to various embodiments as "preferably" or "eg" or "for example" or "particularly" are not intended to limit the scope of the invention but rather to be construed as exemplary modes in which the invention may be practiced.

[0044] The term "comprising" as used in the claims should not be construed as being limited to, for example, the subsequently recited composition elements or method steps or components; 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 feature, integer, (method) step, or component as referenced, but not as excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the phrase "a method comprising steps A and B" should not be limited to a method consisting solely of steps A and B; rather, for the present invention, the recited steps of the method are only A and B, and the claims should be construed as including equivalents of those method steps. Thus, the scope of the phrase "a composition comprising components A and B" should not be limited to a composition consisting solely of components A and B; rather, for the present invention, the recited components of the composition are only A and B, and the claims should be construed as including equivalents of those components.

[0045] In addition, reference to an element or component by the indefinite article "a" or "an" does not exclude the possibility that more than one element or component is present, unless the context clearly requires that there is only one element or component. Thus, the indefinite article "a" or "an" normally means "at least one."

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

[0047] The term "Quillaja saponin" has its ordinary scientific meaning and, as used herein, refers to the saponin fraction of Quillaja saponaria, and therefore the source for all other QS saponins, containing primarily QS-18 and QS-21.

[0048] "QS-21" or "QS21" has its ordinary scientific meaning and, as used herein, refers to a mixture of QS-21 A-apio (about 63%), QS-21 A-xylo (about 32%), QS-21 B-apio (about 3.3%), and QS-21 B-xylo (about 1.7%).

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

[0050] Similarly, "QS-21B" has its ordinary scientific meaning and refers herein 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 from Quillaja saponaria, containing variable amounts of over 50 different saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3β-OH group, found in QS-7, QS-17, QS18, and QS-21. The saponins found in Quil-A are listed in Table 2 of van Setten (1995) [Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer, and Gideon FAKersten, Glycosyl Compositions and Structural Characteristics of the Potential Immuno-adjuvant Active Saponins in the Quillaja saponaria Molina Extract Quil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 660-666 (1995)]. Quil-A and Quillaja saponins are also saponin fractions derived from Quillaja saponaria, and both contain a wide variety of different saponins with largely overlapping contents. The two fractions differ in their 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 28 [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171]. The structure described is that of the api-variant of QS-7, QS1862. The molecular mass is 1862 daltons, since this is the formal mass of glucuronic acid, including the proton. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in the negative ion mode, the observed mass is 1861 daltons. [Brief explanation of the drawings]

[0053] [Figure 1a](Figure 1A): Targeted two-component approach (single target). SO1861 and a toxin (e.g., a ribosome-inactivating protein) are each separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) The mAb-toxin and VHH-SO1861 bind to cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, allowing endolysosomal escape, 4) release of the toxin into the cytoplasm, and 5) the toxin induces cell death. (Figure 1B): Targeted two-component approach (two targets). SO1861 and a toxin (a ribosome-inactivating protein) are each separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) mAb-toxin and VHH-SO1861 bind to their corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 1b](Figure 1C): Targeted two-component approach (two targets). SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH for delivery and internalization into target cells. 1) VHH-toxin and VHH-SO1861 bind to their corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 becomes active, allowing endolysosomal escape, 4) release of the toxin into the cytoplasm, and 5) the toxin induces cell death. (Figure 1D): Targeted two-component approach (two targets). SO1861 and a toxin (ribosome-inactivating protein) are each separately conjugated to a VHH or mAb for delivery and internalization into target cells. 1) VHH-toxin and mAb-SO1861 bind to their corresponding cell surface receptors, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active allowing endolysosomal escape, 4) release of the toxin into the cytoplasm occurs, and 5) the toxin induces cell death. [Figure 1c](Figure 1E): Targeted two-component approach (one target). SO1861 and a toxin (ribosome-inactivating protein) are separately conjugated to a VHH or antibody (mAb) for delivery and internalization into target cells. 1) The VHH-toxin and mAb-SO1861 bind to cell surface receptors; 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex); 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, allowing endolysosomal escape; 4) release of the toxin into the cytoplasm occurs; and 5) the toxin induces cell death. (Figure 1F): Schematic diagram showing exemplary molecules and conjugates of the present invention. An IgG antibody is shown covalently conjugated to four saponin molecules "S" attached to the antibody's light chain and with an effector molecule "E" covalently attached to the constant domain of the antibody's heavy chain. [Figure 1d] (Figure 1G): Schematic diagram showing exemplary molecules and conjugates of the invention. An IgG antibody covalently conjugated with four trivalent linkers (each linker is covalently bound to a saponin and covalently bound to an effector molecule) is shown. The trivalent linkers are covalently bound to the antibody light chain. (Figure 1H): Schematic diagram showing exemplary molecules and conjugates of the invention. A single domain antibody covalently conjugated with two trivalent linkers (each linker is covalently bound to a saponin and covalently bound to an effector molecule) is shown. (Figure 1I): Schematic diagram showing exemplary molecules and conjugates of the invention. A single domain antibody covalently conjugated with a trivalent linker (the trivalent linkers are covalently bound to a saponin and covalently bound to an effector molecule) is shown. [Figure 1e](Figure 1J): (S)n-(L)(E) concept: mAb-(SO1861)n(protein toxin). Both SO1861 covalently linked at a cysteine ​​residue (Cys) and a protein toxin (ribosome-inactivating protein) at a lysine residue are conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-L-SO1861)4 (Lys-protein toxin)2 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of the conjugate occurs (binding of the conjugate to the receptor is followed by internalization of the conjugate / receptor complex), 3) at low endolysosomal pH and appropriate concentration, SO1861 becomes activated, allowing endolysosomal escape, 4) release of the toxin into the cytoplasm, and 5) the toxin induces cell death. [Figure 1f] (Figure 1K): (S)n-(L)(E) concept: mAb-(SO1861)n(antisense BNA oligo)m. Both SO1861 linked to a cysteine ​​residue (Cys) and an antisense BNA oligonucleotide linked to a lysine residue are conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-SO1861)4(Lys-BNA oligo)2 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor is followed by internalization of the conjugate / receptor complex), 3) at low endosomal, lysosomal, and endolysosomal pH and at appropriate concentrations, SO1861 becomes active, allowing endosomal, lysosomal, and endolysosomal escape, 4) release of the BNA oligo into the cytoplasm, and 5) induction of target gene silencing. [Figure 1g](Figure 1L): (S)n-(L)(E) concept: mAb-(SO1861-scaffold-antisense BNA oligo)n. (SO1861-trifunctional linker-BNA oligo)n is conjugated to an antibody (mAb) for delivery and internalization into target cells. The antibody can be, for example, an IgG or an sdAb such as a VHH. 1) mAb-(SO1861-trifunctional linker-BNA oligo)4 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex), 3) at low endosomal pH and appropriate concentration, SO1861 becomes active, allowing endosomal escape, 4) release of the BNA oligo into the cytoplasm occurs, and 5) target gene silencing is induced. The term "scaffold" in relation to the conjugates of the present invention is to be understood as an oligomeric or polymeric molecule having one or more chemical groups for covalently binding to one or more further molecules, such as saponin molecules and / or effector molecules, such as protein toxins or oligonucleotides, and having at least one chemical group for covalently coupling to a protein, such as an antibody, such as an IgG or sdAb. [Figure 2] (Figure 2): Cell killing (MTS) assay of combination treatment according to the invention of VHH(HER2)-SO1861(DAR1) + 50 pM trastuzumab-saporin (DAR4) or 10 pM CD71mab-saporin (DAR4) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 3] (Figure 3): Cell killing (MTS) assay of combination treatment according to the invention of trastuzumab-saporin (DAR4) or CD71-saporin (DAR4) + 900 nM HER2VHH-SO1861 (DAR1) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 4](Figure 4): Cell killing (MTS) assay of the combination treatment according to the invention of VHH(HER2)-SO1861(DAR1) + 50 pM CD71VHH-Gianthin(DAR1) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 5] (Figure 5): Cell killing (MTS) assay of the combination treatment according to the invention of CD71VHH-Gianthin (DAR1) + 900 nM HER2VHH-SO1861 (DAR1) on SK-BR-3 cells (HER2++ / CD71+) (A) and MD-MB-468 cells (HER2- / CD71+) (B). [Figure 6] (Figure 6): Cell killing (MTS) assay of combination treatments according to the invention of CD71VHH-Gianthin (DAR1) + cetuximab-SO1861 (DAR4) or HER2VHH-Gianthin (DAR1) + cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + cetuximab-SO1861 (DAR4) against A431 cells (EGFR+= / HER+ / - / CD71+) (A) and A2058 cells (EGFR- / HER+ / - / CD71+) (B). [Figure 7] (Figure 7): Cell killing (MTS) assay of combination treatments according to the invention of CD71VHH-Gianthin (DAR1) + 77nM cetuximab-SO1861 (DAR4) or HER2VHH-Gianthin (DAR1) + 77nM cetuximab-SO1861 (DAR4) or EGF VHH-Gianthin (DAR1) + 77nM cetuximab-SO1861 (DAR4) on SK-BR-3 cells (HER2++ / EGFR+ / CD71+) (A) and MDA-MB-468 cells (HER2- / EGFR++ / CD71+) (B). [Figure 8a](Figure 8): Tumor-targeted protein toxin delivery results in reduced tumor volume and inhibited tumor growth in tumor-bearing mice in vivo. A) Dose-escalating (intraperitoneal, i.p.) doses of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice demonstrate reduced tumor volume at day 26 compared to controls. B, C) Dose-escalating (intraperitoneal, i.p. (B) or intravenous, i.v. (C)) doses of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice demonstrate reduced tumor growth compared to controls. The term "Cys-L-" in reference to the covalent conjugates of the present invention is to be understood as a covalent bond to a cysteine ​​side chain, the bond being labile and cleavable under acidic conditions such as those present in the endosomes and lysosomes of mammalian cells, such as human cells, including human tumor cells. The term "Lys-S-" in relation to the covalent conjugates of the present invention will be understood as a covalent bond to a lysine side chain, which bond is stable and non-cleavable under acidic conditions such as those present in the endosomes and lysosomes of mammalian cells, such as human cells, including human tumor cells. [Figure 8b](Figure 8): Tumor-targeted protein toxin delivery results in reduced tumor volume and inhibited tumor growth in tumor-bearing mice in vivo. A) Dose-escalating (intraperitoneal, i.p.) doses of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice demonstrate reduced tumor volume at day 26 compared to controls. B, C) Dose-escalating (intraperitoneal, i.p. (B) or intravenous, i.v. (C)) doses of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice demonstrate reduced tumor growth compared to controls. The term "Cys-L-" in reference to the covalent conjugates of the present invention is to be understood as a covalent bond to a cysteine ​​side chain, the bond being labile and cleavable under acidic conditions such as those present in the endosomes and lysosomes of mammalian cells, such as human cells, including human tumor cells. The term "Lys-S-" in relation to the covalent conjugates of the present invention will be understood as a covalent bond to a lysine side chain, which bond is stable and non-cleavable under acidic conditions such as those present in the endosomes and lysosomes of mammalian cells, such as human cells, including human tumor cells. [Figure 9] (Figure 9): Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg cetuximab-(Cys-L-SO1861)3,9(Lys-L-HSP27BNA)1,8 in A431 tumor-bearing mice induced efficient tumor-targeted gene silencing compared to controls. [Figure 10] (Figure 10): Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice. 30 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 in A431 tumor-bearing mice induced efficient tumor-targeted gene silencing compared to controls. [Figure 11a](Figure 11): HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells (tumor cells). A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-Gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-Gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). Note: The legend for Figure 11B also refers to the graph in Figure 11A. C, D) Cetuximab-(Cys-L-SO1861)3,9(Lys-L-dianthine)2,0 or cetuximab-(Cys-L-SO1861)3,9(Lys-S-dianthine)2,0 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 11D also refers to the graph in Figure 11C. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 11b] (Figure 11): HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells (tumor cells). A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-Gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-Gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). Note: The legend for Figure 11B also refers to the graph in Figure 11A. C, D) Cetuximab-(Cys-L-SO1861)3,9(Lys-L-dianthine)2,0 or cetuximab-(Cys-L-SO1861)3,9(Lys-S-dianthine)2,0 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 11D also refers to the graph in Figure 11C. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 11c](Figure 11): HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells (tumor cells). A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-Gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-Gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). Note: The legend for Figure 11B also refers to the graph in Figure 11A. C, D) Cetuximab-(Cys-L-SO1861)3,9(Lys-L-dianthine)2,0 or cetuximab-(Cys-L-SO1861)3,9(Lys-S-dianthine)2,0 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 11D also refers to the graph in Figure 11C. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 11d] (Figure 11): HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells (tumor cells). A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-Gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-Gianthine)1,7 treatment and control in SK-BR-3 cells (HER2++) and MDA-MB-468 cells (HER2-). Note: The legend for Figure 11B also refers to the graph in Figure 11A. C, D) Cetuximab-(Cys-L-SO1861)3,9(Lys-L-dianthine)2,0 or cetuximab-(Cys-L-SO1861)3,9(Lys-S-dianthine)2,0 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 11D also refers to the graph in Figure 11C. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 12a](Figure 12): EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)3,8 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 12B also refers to the graph in Figure 12A. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 12b] (Figure 12): EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)3,8 treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: The legend for Figure 12B also refers to the graph in Figure 12A. Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 13] (Figure 13): HER2-targeted antisense BNA oligo delivery and gene silencing in cancer cells. Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)3,5 treatment and control on SK-BR-3 cells (HER2++). Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 14] (Figure 14): EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells. A, B) Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) treatment and control for A431 cells (EGFR++) and A2058 cells (EGFR-). Note: See Table A2 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 15a](Figure 15): 1-Target 2-Component (EGFR-High Expression). EGFR-targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) cells by therapeutic combination. A, B) Cetuximab-S01861 titration combined with a fixed concentration of 10 pM cetuximab-saporin demonstrates that a 100-400-fold reduced concentration of conjugated SO1861 is required relative to unconjugated SO1861 to induce cell killing by cetuximab-saporin. C, D) Cetuximab-saporin titration combined with 278 nM cetuximab-S01861 kills cells in contrast to 300 nM unconjugated SO1861 + cetuximab-saporin. 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination because both cetuximab conjugates compete for the same EGFR receptor. Only simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 15B also refers to the graph in Figure 15A. Note: The legend shown in Figure 15D also refers to the graph in Figure 15C. [Figure 15b](Figure 15): 1-Target 2-Component (EGFR-High Expression). EGFR-targeted cell killing in A431 (EGFR+++) and CaSKi (EGFR++) cells by therapeutic combination. A, B) Cetuximab-S01861 titration combined with a fixed concentration of 10 pM cetuximab-saporin demonstrates that a 100-400-fold reduced concentration of conjugated SO1861 is required relative to unconjugated SO1861 to induce cell killing by cetuximab-saporin. C, D) Cetuximab-saporin titration combined with 278 nM cetuximab-S01861 kills cells in contrast to 300 nM unconjugated SO1861 + cetuximab-saporin. 1500 nM unconjugated SO1861 + cetuximab-saporin is more efficient compared to the therapeutic combination because both cetuximab conjugates compete for the same EGFR receptor. Only simultaneous targeted delivery of both cetuximab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. Note: The legend shown in Figure 15B also refers to the graph in Figure 15A. Note: The legend shown in Figure 15D also refers to the graph in Figure 15C. [Figure 16a] (Figure 16): 1-Target 2-Component (No / Low EGFR Expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Cetuximab-S01861 titration combined with a fixed concentration of 10 pM cetuximab-saporin does not induce cell killing by cetuximab-saporin. C, D) Cetuximab-saporin titration combined with 278 nM cetuximab-S01861 fails to induce cell killing. Low EGFR receptor expression is prohibitive for sufficient S01861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated S01861 induces cell killing. Note: The legend shown in Figure 16B also refers to the graph in Figure 16A. Note: The legend shown in Figure 16D also refers to the graph in Figure 16C. [Figure 16b](Figure 16): 1-Target 2-Component (No / Low EGFR Expression). EGFR-targeted cell killing in HeLa (EGFR+) and A2058 (EGFR-) cells by therapeutic combination. A, B) Cetuximab-S01861 titration combined with a fixed concentration of 10 pM cetuximab-saporin does not induce cell killing by cetuximab-saporin. C, D) Cetuximab-saporin titration combined with 278 nM cetuximab-S01861 fails to induce cell killing. Low EGFR receptor expression is prohibitive for sufficient S01861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated S01861 induces cell killing. Note: The legend shown in Figure 16B also refers to the graph in Figure 16A. Note: The legend shown in Figure 16D also refers to the graph in Figure 16C. [Figure 17] (Figure 17): 1-Target 2-Component (High HER2 Expression). HER2-targeted cell killing in SKBR3 (HER2+++) cells by therapeutic combination. A) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 673 pM trastuzumab-saporin shows that a 1000-fold reduced concentration of conjugated SO1861 is required relative to unconjugated SO1861 to induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 9.4 nM trastuzumab-SO1861 kills cells in contrast to 10 nM unconjugated SO1861 + trastuzumab-saporin. 1075nM unconjugated SO1861 + trastuzumab-saporin is more effective than therapeutic combinations because both trastuzumab conjugates compete for the same HER2 receptor. Only the simultaneous targeted delivery of both trastuzumab conjugates results in efficient cell killing, in contrast to monotherapy with either conjugate alone. SPT001 is SO1861. [Figure 18a](Figure 18): 1-Target 2-Component (No / Low HER2 Expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Trastuzumab-S01861 titration combined with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Trastuzumab-saporin titration combined with 10 nM trastuzumab-S01861 fails to induce cell killing. Low HER2 receptor expression is prohibitive for sufficient SO1861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 18B also refers to the graph in Figure 18A. Note: The legend shown in Figure 18D also refers to the graph in Figure 18C. SPT001 is SO1861. [Figure 18b] (Figure 18): 1-Target 2-Component (No / Low HER2 Expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by therapeutic combination. A, B) Trastuzumab-S01861 titration combined with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Trastuzumab-saporin titration combined with 10 nM trastuzumab-S01861 fails to induce cell killing. Low HER2 receptor expression is prohibitive for sufficient SO1861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated SO1861 induces efficient cell killing. Note: The legend shown in Figure 18B also refers to the graph in Figure 18A. Note: The legend shown in Figure 18D also refers to the graph in Figure 18C. SPT001 is SO1861. [Figure 19a](Figure 19): Two-target, two-component (EGFR high expression and HER2 low expression). EGFR / HER2-targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Cetuximab-SO1861 titration combined with a fixed concentration of 50 pM trastuzumab-saporin shows that a 100-fold reduced concentration of conjugated SO1861 is required relative to unconjugated SO1861 to induce cell killing by trastuzumab-saporin. C, D) Trastuzumab-saporin titration combined with 278 nM cetuximab-SO1861 kills cells in contrast to 300 nM unconjugated SO1861 + trastuzumab-saporin. 1500 nM unconjugated SO1861 + trastuzumab-saporin has equivalent cell killing efficiency compared to the therapeutic combination, 278 nM cetuximab-SO1861 + trastuzumab-saporin, because both conjugates do not compete for the same receptor. Only simultaneous targeted delivery of both conjugates results in efficient cell killing, as opposed to monotherapy with either conjugate alone. Note: The legend shown in Figure 19B also refers to the graph in Figure 19A. Note: The legend shown in Figure 19D also refers to the graph in Figure 19C. SPT001 is SO1861. [Figure 19b](Figure 19): Two-target, two-component (EGFR high expression and HER2 low expression). EGFR / HER2-targeted cell killing in A431 (EGFR+++ / HER2+ / -) and Caski (EGFR++ / HER2+ / -) cells by therapeutic combination. A, B) Cetuximab-SO1861 titration combined with a fixed concentration of 50 pM trastuzumab-saporin shows that a 100-fold reduced concentration of conjugated SO1861 is required relative to unconjugated SO1861 to induce cell killing by trastuzumab-saporin. C, D) Trastuzumab-saporin titration combined with 278 nM cetuximab-SO1861 kills cells in contrast to 300 nM unconjugated SO1861 + trastuzumab-saporin. 1500 nM unconjugated SO1861 + trastuzumab-saporin has equivalent cell killing efficiency compared to the therapeutic combination, 278 nM cetuximab-SO1861 + trastuzumab-saporin, because both conjugates do not compete for the same receptor. Only simultaneous targeted delivery of both conjugates results in efficient cell killing, as opposed to monotherapy with either conjugate alone. Note: The legend shown in Figure 19B also refers to the graph in Figure 19A. Note: The legend shown in Figure 19D also refers to the graph in Figure 19C. SPT001 is SO1861. [Figure 20a](Figure 20): Two-target, two-component (low EGFR expression and no / low HER2 expression). EGFR / HER2-targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-S01861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-S01861 does not enhance cell killing, while 1500 nM unconjugated S01861 induces efficient cell killing. Low EGFR receptor expression is prohibitive for sufficient S01861 to be delivered via antibody-mediated delivery. Note: The legend shown in Figure 20B also refers to the graph in Figure 20A. Note: The legend shown in Figure 20D also refers to the graph in Figure 20C. SPT001 is SO1861. [Figure 20b] (Figure 20): Two-target, two-component (low EGFR expression and no / low HER2 expression). EGFR / HER2-targeted cell killing in HeLa (EGFR+ / HER2+ / -) and A2058 (EGFR- / HER2+ / -) cells by therapeutic combination. A, B) Titration of cetuximab-S01861 in combination with a fixed concentration of 50 pM trastuzumab-saporin does not induce cell killing by trastuzumab-saporin. C, D) Titration of trastuzumab-saporin in combination with 278 nM cetuximab-S01861 does not enhance cell killing, while 1500 nM unconjugated S01861 induces efficient cell killing. Low EGFR receptor expression is prohibitive for sufficient S01861 to be delivered via antibody-mediated delivery. Note: The legend shown in Figure 20B also refers to the graph in Figure 20A. Note: The legend shown in Figure 20D also refers to the graph in Figure 20C. SPT001 is SO1861. [Figure 21](Figure 21): 2-target 2-component (HER2 high expression and EGFR low expression). HER2-targeted cell killing in SKBR3 (HER2+++ / EGFR+ / -) cells by therapeutic combination. A) Trastuzumab-SO1861 titration combined with a fixed concentration of 1.5 pM EGF dianthine shows that a 400-fold lower concentration of conjugated SO1861 is required to induce cell killing by EGF dianthine relative to unconjugated SO1861. B) EGF dianthine titration combined with 9.4 nM trastuzumab-SO1861 can kill cells in contrast to 10 nM unconjugated SO1861 + EGF dianthine. 1075nM unconjugated SO1861 + EGF dianthine has equivalent cell killing efficiency compared to the therapeutic combination, 9.4nM trastuzumab-SO1861 + EGF dianthine, because both conjugates do not compete for the same receptor. Only the simultaneous targeted delivery of both conjugates results in efficient cell killing, as opposed to monotherapy with either conjugate alone. SPT001 is SO1861. [Figure 22a](Figure 22): 2-target, 2-component (low HER2 expression and low or high EGFR expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by a therapeutic combination according to the present invention. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 5 pM cetuximab-saporin does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression is prohibitive for sufficient SO1861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels (D) for delivery of cetuximab-saporin did not alter its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck for cell killing activity was that HER2 expression levels were too low, resulting in insufficient SO1861 inside target cells to switch to endosomal escape. Note: The legend shown in Figure 22B also refers to the graph in Figure 22A. Note: The legend shown in Figure 22D also refers to the graph in Figure 22C. SPT001 is SO1861. [Figure 22b](Figure 22): 2-target, 2-component (low HER2 expression and low or high EGFR expression). HER2-targeted cell killing in JIMT1 (HER2+) and A431 (HER2+ / -) cells by a therapeutic combination according to the present invention. A, B) Titration of trastuzumab-SO1861 in combination with a fixed concentration of 5 pM cetuximab-saporin does not induce cell killing by cetuximab-saporin. C, D) Titration of cetuximab-saporin in combination with 10 nM trastuzumab-SO1861 fails to induce cell killing. Low HER2 receptor expression is prohibitive for sufficient SO1861 to be delivered via antibody-mediated delivery, yet 1500 nM unconjugated SO1861 induces efficient cell killing. Even high EGFR receptor expression levels (D) for delivery of cetuximab-saporin did not alter its efficacy in the presence of trastuzumab-SO1861, indicating that the bottleneck for cell killing activity was that HER2 expression levels were too low, resulting in insufficient SO1861 inside target cells to switch to endosomal escape. Note: The legend shown in Figure 22B also refers to the graph in Figure 22A. Note: The legend shown in Figure 22D also refers to the graph in Figure 22C. SPT001 is SO1861. [Figure 23] (Figure 23): Two-target, two-component versus T-DM1. Cells with high EGFR expression and low HER2 expression (A431) are efficiently killed by the therapeutic combination, but T-DM1 is not effective at such low-toxic concentrations. T-DM1 is trastuzumab-emtansine (Kadcyla®), which carries 3.5 DM1 toxin molecules per antibody. [Figure 24a](Figures 24A-E): Relative cell viability when trastuzumab (Figure 24A), cetuximab (Figure 24B), or T-DM1 (Figure 24C), free toxins saporin (Figure 24D) and dianthin (Figure 24D), saporin coupled to non-cell-binding IgG (Figure 24D), and saporin coupled to non-cell-binding IgG combined with free saponin SO1861 (Figure 24E) are contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, and BT-474. Note: The legend shown in Figure 24C also refers to the curves in Figures 24A and 24B. [Figure 24b] (Figures 24A-E): Relative cell viability when trastuzumab (Figure 24A), cetuximab (Figure 24B), or T-DM1 (Figure 24C), free toxins saporin (Figure 24D) and dianthin (Figure 24D), saporin coupled to non-cell-binding IgG (Figure 24D), and saporin coupled to non-cell-binding IgG combined with free saponin SO1861 (Figure 24E) are contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, and BT-474. Note: The legend shown in Figure 24C also refers to the curves in Figures 24A and 24B. [Figure 24c] (Figures 24A-E): Relative cell viability when trastuzumab (Figure 24A), cetuximab (Figure 24B), or T-DM1 (Figure 24C), free toxins saporin (Figure 24D) and dianthin (Figure 24D), saporin coupled to non-cell-binding IgG (Figure 24D), and saporin coupled to non-cell-binding IgG combined with free saponin SO1861 (Figure 24E) are contacted with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, and BT-474. Note: The legend shown in Figure 24C also refers to the curves in Figures 24A and 24B. [Figure 25](Figure 25): 1T2C in vivo activity. The 1T2C combination of 50 mg / kg cetuximab-(Cys-L-SO1861)4 + 25 mg / kg cetuximab-(-L-HSP27BNA)4 in A431 tumor-bearing mice demonstrates potent tumor-targeted gene silencing compared to controls. [Figure 26] (Figure 26): 1T2C in vivo activity. The 1T2C combination of 40 mg / kg trastuzumab-(Cys-L-SO1861)4 + 0.02 / 0.03 mg / kg trastuzumab-saporin in a PDX tumor mouse model (high HER2 expression) shows effective tumor growth inhibition. [Figure 27] (FIG. 27): The 2T2 component system tested in an A431 tumor-bearing mouse model demonstrates tumor regression. [Figure 28] (FIG. 28): The 2T2 component system tested in the A431 tumor-bearing mouse model demonstrates tumor regression and eradication. [Figure 29a] (Figure 29): Two-target, two-component. EGFR / HER2-targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by a therapeutic combination according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control on A431 cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control on CaSKi cells. Legend and / or axes are the same for A, B, and legend is the same for figures C and D. [Figure 29b](Figure 29): Two-target, two-component. EGFR / HER2-targeted cell killing in A431 cells (EGFR++ / HER2+ / -) (A, C) and CaSKi cells (EGFR++ / HER2+ / -) (B, D) by a therapeutic combination according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration + fixed concentration of 50 pM trastuzumab-saporin and control on A431 cells. C, D) Trastuzumab-saporin titration + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control on CaSKi cells. Legend and / or axes are the same for A, B, and legend is the same for figures C and D. [Figure 30a] (Figure 30): Two-target, two-component. EGFR / HER2-targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by a therapeutic combination according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration on HeLa cells + fixed concentration of 50 pM trastuzumab-saporin and control. C, D) Trastuzumab-saporin titration on A2058 cells + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control. Legend and / or axes are the same for C and D. [Figure 30b] (Figure 30): Two-target, two-component. EGFR / HER2-targeted cell killing in HeLa cells (EGFR+ / - / HER2+ / -) (A, C) and A2058 cells (EGFR- / HER2+ / -) (B, D) by a therapeutic combination according to the invention. A, B) Cetuximab-(Cys-L-SO1861)3,7 titration on HeLa cells + fixed concentration of 50 pM trastuzumab-saporin and control. C, D) Trastuzumab-saporin titration on A2058 cells + fixed concentration of 75 nM cetuximab-(Cys-L-SO1861)3,7 and control. Legend and / or axes are the same for C and D. [Figure 31](Figure 31): Two targets, two components. HER2 / EGFR-targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by a therapeutic combination according to the invention. A) Trastuzumab-(Cys-L-SO1861)4 titration on SKBR3 cells + a fixed concentration of 1.5 pM EGF-giantine and control. B) EGF-giantine titration on SKBR3 cells + a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control. [Figure 32a] (Figure 32): Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / - / EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by a therapeutic combination according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 1.5 pM EGF-giantine and control on JIMT-1 cells. C, D) EGF-giantine titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on MDA-MB-468 cells. Legend and / or axes are the same for C and D. [Figure 32b] (Figure 32): Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / - / EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by a therapeutic combination according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 1.5 pM EGF-giantine and control on JIMT-1 cells. C, D) EGF-giantine titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on MDA-MB-468 cells. Legend and / or axes are the same for C and D. [Figure 33](Figure 33): Two-target, two-component. HER2 / EGFR-targeted cell killing in SKBR3 cells (HER2++ / EGFR+ / -) (A, B) by a therapeutic combination according to the invention. A) Trastuzumab-(Cys-L-SO1861)4 titration on SKBR3 cells + a fixed concentration of 10 pM cetuximab-saporin and a control. B) Cetuximab-saporin titration on SKBR3 cells + a fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and a control. [Figure 34a] (Figure 34): Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / - / EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by a therapeutic combination according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 10 pM cetuximab-saporin and control on JIMT-1 cells. C, D) Cetuximab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on MDA-MB-468 cells. Legend and / or axes are the same for A, B, and legend is the same for C and D. [Figure 34b] (Figure 34): Two targets, two components. HER2 / EGFR-targeted cell killing in JIMT-1 cells (HER2+ / - / EGFR+ / -) (A, C) and MDA-MB-468 cells (HER2- / EGFR++) (B, D) by a therapeutic combination according to the invention. A, B) Trastuzumab-(Cys-L-SO1861)4 titration + fixed concentration of 10 pM cetuximab-saporin and control on JIMT-1 cells. C, D) Cetuximab-saporin titration + fixed concentration of 2.5 nM trastuzumab-(Cys-L-SO1861)4 and control on MDA-MB-468 cells. Legend and / or axes are the same for A, B, and legend is the same for C and D. DETAILED DESCRIPTION OF THE INVENTION

[0054] For a bioactive molecule (e.g., an effector molecule) to function, it must be able to bind to its target, e.g., in serum, on the outside of a cell surface or inside a cell or organelle. The active moiety of almost all protein-based targeted toxins must, for example, enter the cytosol of the target cell or mediate its target-modulating effect. In many cases, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound to the outside of the cell, (2) it is recycled back to the cell surface after internalization, or (3) it is transported to the endolysosome where it is degraded. These fundamental problems have been known for decades, and although over 500 targeted toxins have been investigated in the past few decades, the problems have not yet been solved; only paired antibody-targeted protein toxins have been approved for the market, albeit 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 and Ontak.

[0055] To overcome these problems, many strategies have been described, including approaches that target toxins to intracellular membrane transport complexes of biosynthetic pathways in the endoplasmic reticulum and techniques that disrupt or weaken the membrane integrity of endosomes, i.e., compartments of the endocytic pathway within cells, thereby promoting endosomal escape. These include the use of lysosomotropic amines, carboxyionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human, and synthetic origin, other organic molecules, and light-induced approaches. The efficacy of targeted toxins is usually increased by 100 or 1,000 times in cell culture, and in exceptional cases, by more than a million times. However, the requirement to co-administer endosomal escape enhancers with other substances introduces new problems, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell-type-dependent variability.

[0056] All strategies involving physicochemical methods require enhancer molecules that interact more or less directly with membranes and essentially include small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not target cell specific and distribute with different kinetics than targeted toxins. This is one major drawback of existing approaches.

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

[0058] It is an object of the present invention to provide conjugates that combine an effector molecule activity-enhancing molecule and an ADC or AOC for use in therapy, such as anti-cancer therapy. By providing such conjugates of the present invention, the therapeutic window of the effector molecule that is part of the conjugate, such as an ADC or AOC, is effectively broadened.

[0059] At least one of the above objects is achieved by providing improved ADCs and improved AOCs that are conjugates that further comprise 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, but rather only by the claims. While the present invention has been described in terms of several embodiments, alternatives, modifications, permutations and equivalents thereof will be apparent to those skilled in the art upon reading the specification and examining the drawings and graphs. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0061] The present inventors have established that the therapeutic window of a conjugate, such as an antibody drug conjugate or an antibody-oligonucleotide conjugate, is increased when the conjugate contains at least one covalently attached saponin when administered to a tumor-bearing mammal (mouse) to which the conjugate is administered (see, e.g., Figures 8-14 in the Examples section for examples of the effect of saponins covalently attached to effector molecules and antibodies on a series of in vitro tumor cells and in vivo tumor models). Saponins are conjugated to effector molecules such as antibodies and oligonucleotides, such as protein toxins and BNAs. The present inventors have demonstrated that antibodies, such as full-length intact IgG, or VNAs, can enhance the therapeutic window of a conjugate. HH We have first established and determined that conjugating the saponins of the present invention with a ligand for binding to a cell surface molecule, such as an sdAb, provides a conjugate for cell-specific delivery of the saponin at the cell surface of a target cell that exposes the cell surface molecule on its cell surface, followed by delivery of the saponin to the interior of the cell, such as the cellular endosomes, endolysosomes, lysosomes, and ultimately into the cytosol of the cell. Examples of such cell-targeted saponin conjugates include, for example, saponin-V, as outlined in the Examples section herein below. HH Conjugates are provided in Figures 2-5 and Figures 5-7, 15-23, and 25-34. Saponins are used to target EGF, Her2, and V HH or conjugated to a ligand such as IgG, EGFR-targeted IgG, etc.

[0062] Conjugates of the present invention, including sdAbs or full-length antibodies or other immunoglobulin (Ig) formats as cell surface molecule-binding molecules in the conjugates of the present invention, preferably, and in all illustrative examples, have at least one glycoside, such as a saponin of the present invention, covalently attached thereto, more preferably via a (cleavable) linker. Without wishing to be bound by any theory, saponins enhance the therapeutic efficacy of effector moieties attached to cell surface molecule-targeting molecules (antibodies, sdAbs), presumably by enhancing endosomal escape to the cytosol, where the effector moiety's activity is desired. In this way, even at doses of effector molecules lower than conventional doses of ADCs or AOCs, i.e., at low doses of the conjugates of the present invention, the therapeutic effect is established under the influence of the presence of the saponin-containing conjugate, whereby the saponin is delivered to the vicinity of, the target cells, and / or the interior of the target cells. The target cells are, for example, diseased cells, such as tumor cells, autoimmune cells, or B cells associated with a B-cell disorder. 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 the outside of a cell into said 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, covalently linked to each other either directly or via at least one linker, wherein the at least one saponin is a mono-desmosidic triterpene glycoside or a bi-desmosidic triterpene glycoside, and wherein the sdAb is capable of binding to a cell surface molecule of said cell.

[0064] The present inventors disclose herein that covalent coupling of saponins, such as the saponins in the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, to cell surface targeting molecules, e.g., antibodies, sdAbs, via, for example, a trifunctional linker, e.g., a trifunctional linker of Structure A (shown herein below), or via a scaffold oligomeric or polymeric structure comprising the covalently bound saponin, results in enhanced cytotoxicity exerted by an effector moiety, such as a toxin, comprised by the conjugate of the invention under the influence of the covalently coupled saponin in the conjugate.

[0065] Thus, one aspect of the present invention relates to an endosomal escape-enhancing molecule, i.e., a conjugate comprising a saponin, an effector moiety, and a binding molecule, e.g., an sdAb, whereby a glycoside molecule and an effector molecule are attached to one and the same binding molecule, e.g., in the endosomal escape-enhancing conjugate, and the endosomal escape-enhancing conjugate is capable of specifically binding to a target cell-specific surface molecule or structure, thereby inducing receptor-mediated endocytosis of the complex of the conjugate and the target cell-specific surface molecule (binding of the conjugate to the receptor followed by internalization of the conjugate / receptor complex). Preferably, the combination of the saponin and the effector moiety in the endosomal escape-enhancing conjugate allows the saponin to enhance endosomal escape of the effector moiety. By doing so, the conjugate preferably improves the effect of the effector molecule 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 cellular uptake of substances and the terminology used herein will first be described. The uptake of extracellular substances into cells via vesicle budding is called endocytosis. 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) nonspecific liquid uptake (pinocytosis), or (4) nonspecific particle uptake (phagocytosis). All types of endocytosis result in the following cellular processes of vesicle trafficking and material sorting, called endocytic pathways. Endocytic pathways are complex and incompletely understood. Previously, organelles were thought to form de novo and then mature into the next organelle via the endocytic pathway. It is now hypothesized that endocytic pathways involve stable compartments connected by vesicle trafficking. Compartments are complex, multifunctional membrane-based organelles specialized for specific sets of essential functions for the cell. Vesicles are considered simpler transient organelles in composition and are defined as membrane-enclosed containers that newly form by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early and late endosomes are stable compartments in the endocytic pathway, but early endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and even lysosomes can also become vesicles. Endocytic vesicles most notably arise from clathrin-coated pits at the plasma membrane, first fuse with early endosomes, and are the major sorting compartment at approximately pH 6.5. Most internalized cargo and membranes are recycled back to the plasma membrane via recycling vesicles (the recycling pathway). Components to be degraded are transported via multivesicular bodies to the acidic late endosomes (pH below 6). Lysosomes are vesicles that can assemble mature lysosomal enzymes and deliver them to late endosomal compartments when needed. The resulting organelles are called hybrid organelles or endolysosomes.Lysosomes bud off hybrid organelles in a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are highly dynamic organelles, and it is often difficult to distinguish between them. Degradation of molecules encapsulated in the plasma membrane occurs within endolysosomes. Endosomal escape is the active or passive release of substances into the cytoplasm from the lumen of any type of compartment or vesicle derived from the endocytic pathway, preferably clathrin-mediated endocytosis, or the recycling pathway. 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 substances may be transported to other cellular units, such as the nucleus. In the context of the present invention, glycoside molecules (saponins) are compounds that can enhance the effects of effector molecules, particularly by promoting endosomal escape. Glycoside molecules interact with the membranes of compartments and vesicles of the endocytic and recycling pathways, making them leaky for the effector molecules and resulting in enhanced endosomal escape.

[0067] The term "improving the effectiveness of an effector molecule" means that the saponin increases the functional effectiveness of the effector molecule (e.g. the therapeutic index of a toxin or drug; the metabolic effectiveness of a modifier in a biotechnological process; the effectiveness of gene transfection in cell culture research experiments), preferably by enabling or improving 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 stronger therapeutic effect with lower administration and / or fewer side effects. "Improving the effectiveness of an effector molecule" may also mean that an effector molecule that could not be used due to lack of effectiveness (e.g., was not known to be an effector molecule) becomes effective when used in combination with the present invention. Any other effect that is beneficial or desirable as provided by the present invention and that can be attributed to the combination of an effector moiety and a saponin in one conjugate is considered to be an "improved effect". In the context of the present invention, the saponins of the present invention are "enhancers" of the functional effectiveness of the effector molecules in the conjugates of the invention.

[0068] One major drawback of the enhancement of targeted toxins with glycosides, such as saponins, up until the present invention is that targeted toxins are internalized by receptor-mediated endocytosis (binding of the conjugate to a receptor followed by internalization of the conjugate / receptor complex), while glycosides passively diffuse through the cell membrane and reach the endosomal membrane, likely via interaction with cholesterol. In principle, glycosides can enter any cell, even non-target cells (off-target cells), causing inefficient use of enhancers in target cells for effective release of the targeted toxin and potentially side effects in non-target cells. One major problem is that the translocation of targeted toxins and glycosides proceeds with different kinetics, and these kinetics vary from cell line to cell line and tissue to tissue. As a result, the exact time difference for application of the two substances (ADC, free saponin) can vary widely from tumor to tumor (cell line). Furthermore, the release, absorption, distribution, metabolism, and excretion of these substances also vary in living organisms. Furthermore, non-specific uptake of glycosides by non-targeted cells can induce undesired effects in these cells. This can be, for example, delivery of compounds that should be delivered to the lysosome to the cytosol, interference with antigen presentation, etc. Non-targeted administration of glycosides and targeted drugs can also be problematic in drug development and can prevent or at least delay marketing approval by relevant authorities (e.g., FDA or EMA). In the context of the present invention, a targeted toxin or targeted drug refers to a toxin or drug that is specifically targeted to a membrane-bound molecule on a target cell, for example, bound to a ligand of a membrane receptor or bound to an antibody that specifically recognizes a structure on the cell membrane of the target cell.

[0069] It would therefore be very useful to direct the glycoside via the same pathway as the effector molecule, for example via targeting a ligand to the target cell so that the enhancer becomes available at effective concentrations within the acidic compartments of the endocytic pathway of the target cell and acts synergistically with the toxin. The present invention therefore provides a novel approach to redirecting both the effector molecule and the endosomal escape enhancer (i.e. the saponin of the present invention) via targeting a ligand (binding molecule) to the acidic compartments of the endocytic pathway of the target cell.

[0070] The present inventors have established that the effector molecule that is part of a conjugate containing an sdAb is delivered to the interior of cells with high efficiency under the influence of saponin that is also contained in the conjugate, when the effect of the effector molecule inside the cells is taken into account. HH Despite the relatively small size of sdAbs such as V, binding of conjugates containing such sdAbs to cell surface receptors still occurs when both the effector molecule and saponin are V HH This occurs when the conjugate contains an sdAb such as V. HH The binding of saponin to an sdAb such as V and the binding of an effector molecule can also be combined to produce a V that binds to a cell surface molecule, e.g. HH This does not cause steric hindrance when the ability of the conjugate to bind to the effector molecule is taken into account. That is, contacting tumor cells with a suboptimal dose of, for example, an ADC, does not result in intracellular effector molecule activity in the absence of a saponin covalently coupled to the ADC (the target cells are not efficiently killed when exposed to the biological activity of the effector molecule). However, when target tumor cells are contacted with a conjugate of the invention comprising an effector molecule, comprising a saponin, and further comprising a target cell-binding sdAb, efficient tumor cell killing is achieved.

[0071] By targeting a single cell surface molecule with a conjugate of the invention, delivery of the saponin and effector moiety attached to a cell surface molecule-targeting antibody such as an sdAb in a conjugate of the invention to the cytosol and interior of a targeted cell that exposes the cell surface molecule on its cell surface will be improved and more specific compared to, for example, contacting the cell with only a conventional ADC lacking the saponin of the invention and therefore without the presence of a cell-targeting saponin (a conjugate of the invention). The diseased cells selected for targeting by the cell surface molecule-targeting sdAb of the conjugate ideally have a high level of epitopes on the cell surface molecule to which the cell surface molecule-targeting molecule can bind (i.e., expression of the targeted cell surface molecule on targeted cells, e.g., tumor cells or autoimmune cells, is relatively higher than expression on non-targeted cells, e.g., healthy cells), and / or expose epitopes in the targeted cell surface molecule for binding by the cell surface molecule-targeting sdAb of the conjugate, particularly when (adjacent) healthy 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 relatively highly and / or specifically expressed on the targeted (disease, tumor) cells compared to healthy cells. In one embodiment, the conjugate of the present invention is a cell surface molecule targeted by the cell surface molecule-targeting sdAb of the conjugate, such as a tumor cell receptor, which is specifically expressed or expressed at a relatively high level compared to the expression of the cell surface molecule on the surface of healthy (adjacent) cells. Therefore, the epitope on the targeted cell surface molecule is ideally unique to the targeted diseased cells and is at least specifically present and exposed on the surface of the targeted cells. Binding of the conjugate of the present invention to the epitope on the cell surface molecule on the targeted cell is followed by endocytosis of the complex of the conjugate and the cell surface molecule (binding of the conjugate to the receptor is followed by internalization of the conjugate / receptor complex).Because the conjugate can only enter target cells via binding interactions with cell surface molecules that are specifically or uniquely expressed to a sufficient degree on the targeted cells compared to healthy cells that should not be targeted, accumulation of therapeutically effective amounts of the effector moiety and saponin contained by the conjugate inside the target cells is only possible and occurs if the expression level of the targeted cell surface molecule exceeds a certain minimum expression threshold. At the same time, the fact that the effector moiety attached to the cell surface molecule-targeting sdAb of the conjugate can only exert its intracellular (e.g., cytotoxic or gene silencing) activity in the presence of the very same conjugate with a covalently attached saponin also provides protection against negative, unwanted side effects of the effector moiety on, for example, healthy cells and tissues that would not be targeted or affected by the effector moiety, compared to exposure of cells to an ADC without a covalently attached saponin. That is, the sufficiently low expression or absence of exposed cell surface molecules to which the conjugate may bind ideally prevents the conjugate from entering (non-targeted) healthy cells to an extent that would result in endosomal escape of the effector moiety under the influence of the saponin contained by the conjugate. Because an ADC having a coupled saponin or an AOC having a covalently coupled saponin according to the present invention can be used at a lower dose than when an ADC or AOC without a coupled saponin is applied in a treatment regimen, the low degree of entry of an ADC having a coupled saponin or entry of an AOC having a coupled saponin in healthy cells already carries a lower risk of causing unwanted side effects, for example, when targeting and killing target disease cells such as tumor cells and autoimmune cells is considered.

[0072] Thus, the inclusion of sdAbs in conjugates has numerous advantages over the inclusion of antibodies such as IgG, or binding fragments or binding domains thereof. Importantly, because sdAbs do 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. Therefore, the risk profile of the conjugates of the present invention is improved compared to ADCs and AOCs based on IgG, or compared to ADCs or AOCs that contain an Fc tail. In addition, because the conjugates of the present invention cannot bind via Fc receptors, the conjugates are already effective at lower doses than those required to achieve the same effector molecule activity as ADCs and AOCs based on full-length antibodies, due to reduced or no unwanted capture of the conjugate by cell surface receptors different from the intended target cell surface molecule. Furthermore, the relatively small size of sdAbs, compared to, for example, Fabs, scFvs, and IgGs, improves tissue delivery, advantageously allowing the conjugate to reach target cells when administered to a patient in need of therapy. All these advantages of the application of sdAbs in the conjugates of the present invention, when compared to the application of larger antibodies or fragments thereof, such as IgGs containing an Fc tail, result in an improved therapeutic window for the effector molecule when included by the conjugates of the present invention, as with ADCs or AOCs. For example, an ADC based on an sdAb may achieve improved killing of target cells in the case of targeted tumor cells when the effector molecule is, for example, a toxin, whereas at the same dose, an ADC based on IgG and containing the same effector molecule is ineffective or only suboptimally effective. Due to this embodiment of the present invention, it is now possible to treat patients with a lower dose of the effector molecule as part of a conjugate comprising an sdAb, i.e., a conjugate of the present invention, and thereby achieve the same or improved effector molecule-mediated effect in the target cells, compared to the higher doses required when an ADC or AOC based on an antibody containing the same effector molecule is used.The administration of such conjugate of the present invention at a lower dose reduces the risk of patient's occurrence of side effects, for example, due to non-targeting and non-specific entry into healthy cells.This is important, for example, when the cell surface molecule targeted by the sdAb contained in the conjugate is more highly expressed on target (tumor) cells, but is not inherently expressed on such target cells.The lower dose of conjugate reduces the risk of conjugate binding to such low expression bodies, such as non-tumor healthy cells.

[0073] The inventors have also found that the therapeutic range of the conjugates of the invention is broadened due to the incorporation of a covalently bound saponin in the conjugates of the invention. That is, when an ADC or AOC provided with a saponin (i.e., a conjugate of the invention) is contacted with a target cell upon binding of the sdAb to its binding partner on the surface of the target cell, the saponin contained by the conjugate of the invention is also brought into close proximity, i.e., to the surface of the target cell, along with the effector molecule of the conjugate. When a target cell bearing a cell surface molecule, i.e., a target for the sdAb contained by the conjugate, is contacted with a conjugate of the invention, both the effective amount of the effector molecule and the effective amount of saponin are lower than when the target cell is contacted with an ADC or AOC in the absence of saponin or in the presence of free (non-targeting) saponin. The presence of a targeting saponin as part of a conjugate of the invention enhances the activity of the effector molecule in the target cell, thereby broadening the therapeutic range of the conjugate, and thereby the therapeutic range of the effector molecule. Sufficient effector molecule efficiency is achieved at lower doses when target cells are contacted with the conjugates of the present invention. A similar effect, when the effector molecule-enhancing activity of saponin is taken into account, is observed by the inventors when an ADC or AOC is contacted with target cells in the presence of saponin or a functional derivative thereof, but at concentrations of free saponin (derivative) that are 100- to 1000-fold higher than the established effective dose when the conjugates of the present invention comprising both the effector molecule and the effector molecule activity-enhancing saponin described herein are applied together with an sdAb for targeted binding of the conjugate to target (tumor) cells. Thus, providing a saponin or derivative thereof with a binding molecule (i.e. an sdAb comprised by a conjugate of the invention) and also providing an effector molecule (i.e. an effector molecule comprised by a conjugate of the invention) in the very same conjugate results in an improved effector molecule activity enhancement effect when the conjugate of the invention is contacted with a target cell expressing a cell surface molecule on its surface, i.e. a binding target for the sdAb.Although targeted saponins are already effective at lower doses than free saponin in delivering effector molecules into target cells and from endosomes or lysosomes to the cytosol of said cells, and the effector molecule should bind to its target binding partner and exert its biological activity (e.g. cell killing where the target cell is a tumour cell and the effector molecule is, for example, a toxin), the inventors have found that the combination of saponin, targeting moiety (sdAb) and effector molecule (e.g. a toxin, AON) in a single molecule is even more effective.

[0074] The present inventors have therefore provided a pharmaceutical composition comprising a conjugate comprising a saponin (derivative), an effector molecule and an sdAb for targeted delivery of the conjugate at a target cell, which, when compared to existing ADCs based on Fc comprising a full-length antibody or construct thereof that is not provided with a covalently linked saponin, has an improved therapeutic window, a reduced risk of inducing side effects when an effective dose of the effector molecule comprised by the conjugate is administered to a patient in need of effector molecule-based therapy, and improved effector molecule activity due to improved delivery of the conjugate to the interior of target cells, more particularly to the interior of the cytosol of such target cells, under the influence of the targeting saponin as part of the conjugate of the invention. While it is part of the invention that such conjugates of the invention are administered to a patient in need of effector molecule-based therapy together with a dose of free saponin (derivative), application of the conjugate alone is preferred.

[0075] One embodiment is an antibody, preferably a V derived from a heavy chain of immunoglobulin G origin, preferably of human origin. H domain; V derived from the light chain of an antibody, preferably of immunoglobulin G origin, preferably of human origin L Domain: A novel antigen receptor of camelid origin or variable heavy chain (V NAR V such as those derived from heavy chain-only antibodies (HCAbs) such as Ig-NAR origin, such as the ) domainHH domain (preferably the HCAb is from Camelidae origin); preferably the at least one sdAb is from Camelidae origin (Camelidae V), such as from HCAb from camel, llama, alpaca, dromedary, vicuna, guanaco and Bactrian camel. H ) V derived from HCAb HH A conjugate of the present invention is a domain.

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

[0077] One embodiment is a conjugate of the invention comprising a single first sdAb covalently bound to one of the at least one effector molecule and / or to one of the at least one saponin, or two or more sdAbs, where 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, with all of the 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 wherein the 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 is a conjugate of the invention comprising 1 to 8 sdAbs capable of binding to the same binding site on a cell surface molecule, wherein at least one effector molecule and / or at least one saponin is attached 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 attached to two or more of the sdAbs, and wherein the at least one effector molecule and the at least one saponin are attached to the same sdAb or to different sdAbs, preferably wherein each of the at least one effector molecule is attached to a separate sdAb and / or each of the at least one saponin is attached to a separate sdAb, and the effector molecule and saponin are attached to the same sdAb or to separate sdAbs.

[0080] By providing a conjugate of the invention comprising a (linear) string of multiple sdAbs covalently linked to each other, one can provide the advantage of the ability of the conjugate to bind to target cells with higher avidity, which can result in improved uptake (endocytosis) of the conjugate by the target cell (binding of the conjugate to a receptor followed by internalization of the conjugate / receptor complex).

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

[0082] Without wishing to be bound by any theory, in view of the failures observed when treating tumor-bearing animals with ADCs in combination with free saponins, it is preferable to synchronize the presence of both at least one saponin and an effector moiety, preferably a toxin or oligonucleotide, in compartments or vesicles of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. With ADCs and free saponins, synchronizing the presence of the molecules in late endosomes to obtain a synergistic effect in vivo has not been advantageously achieved according to the inventors' attempts. In one aspect, the present invention preferably solves at least the following problems with combining an effector moiety and a saponin in a single conjugate molecule: without wishing to be bound by any theory, for example, only one suitable chemical group within the saponin that can be used for (covalent), in particular a single cleavable, retainable coupling, is required for endosomal escape activity. For example, although the significant endosomal escape enhancer effect of the saponins of the invention exemplified herein has been known for over a decade, it is most likely due to known limitations that saponins have not been used in clinical trials in combination with pharmaceutically active substances other than in vaccination regimes, where the use of immune-enhancing adjuvant substances has been implied. For example, providing the conjugates of the invention with covalently bound saponins, for example in the context of scaffolds bearing several saponins, at least partially overcomes these difficulties. Surprisingly, saponins previously applied for their immune-enhancing activity in the context of vaccinations involving saponins as adjuvant components are now also suitable for (covalently) coupling to cell surface molecule-targeting antibodies, such as sdAbs, comprised by the conjugates of the invention for in vitro or in vivo anti-tumor activity.

[0083] In an embodiment, the at least one sdAb is a single sdAb or is at least two, preferably two sdAbs, and the sdAb is capable of binding to a cell surface molecule of a cell, such as HIV gp41, or the sdAb binds to 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, The conjugate of the present invention is capable of binding to a receptor selected from any one or more of Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC-1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5, CD16, CXCR7 (ACKR3), and glucocorticoid-inducible TNFR-related protein (GITR), most preferably selected from HER2, c-Met, VEGFR2, CXCR7, CD71, and EGFR1.

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

[0085] In one embodiment, the at least one sdAb is a single sdAb or at least two, preferably two, and the sdAbs are selected from the group consisting of anti-CD71 sdAb, anti-HER2 sdAb, anti-CD20 sdAb, anti-CA125 sdAb, anti-EpCAM (17-1A) sdAb, anti-EGFR sdAb, anti-CD30 sdAb, anti-CD33 sdAb, anti-vascular integrin 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-HIV gp41, and the sdAb is preferably selected from V HH , more preferably camelid V H The conjugate of the present invention is

[0086] In one embodiment, at least one sdAb comprises an sdAb capable of binding to HER2, CD71, HIV gp41 and / or EGFR, said sdAb preferably comprising HH , more preferably camelid V H The conjugate of the present invention is

[0087] In certain embodiments, the at least one sdAb comprises an sdAb for binding to HER2 selected from sdAbs generated by clone 11A4, clone 18C3, clone 22G12, clone Q17 or clone Q17-C-tag; or an sdAb for binding to EGFR and generated by clone anti-EGFR Q86-C-tag; or an sdAb for binding to CD71 and generated by clone anti-CD71 Q52-C-tag; or an sdAb for binding to HIV gp41 and generated by clone anti-HIV gp41 or an sdAb encoded by the cDNA of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31; or any one of the sdAbs having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36-72, optionally the conjugate further comprises a further sdAb different from the at least one sdAb, the further sdAb for binding to albumin being any one or more of the further sdAbs having the amino acid sequence of SEQ ID NOs: 33, 34 and 35, preferably the further sdAb is HH , more preferably camelid V H The conjugate of the present invention is

[0088] Suitable V for incorporation in the conjugates of the present invention HH can be found, for example, in the Single Domain Antibody Database (Wilton, EE et al. (2018)), patent applications US Patent Application Publication No. 20160251440 (anti-CD123, anti-CEACAM), US Patent Application Publication No. 9683045 (anti-c-Met), US Patent Application Publication No. 20090252681 (anti-EGFR, anti-IGF-1R), US Patent Application Publication No. 9969805 (anti-HER2), US Patent Application Publication No. 20190023796A1 (anti-HER3), and Kijanka et al. (2013) for anti-HER2, and Mercier et al. (2019) for anti-HER2.HH In view of their ability to bind to tumor cell-specific receptors, amino acid sequences and / or cDNA sequences of 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 are also provided herein as SEQ ID NOs: 1-32 and 36-72. In particular, Vs that can bind to the binding site of any of the tumor cell-specific receptors HER2, VEGFR and CD71 are also provided herein. HH are suitable for incorporation in the conjugates of the present invention. We have identified V that target any one of such receptors. HH have been shown to be effective in delivering effector molecules attached to sdAbs. See, e.g., the Examples section and Figures 4-7.

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

[0090] In one embodiment, the conjugate according to the present invention is one in which the effector molecule is a pharmaceutically active substance, such as a toxin, e.g., a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide. A pharmaceutically active substance in this invention is an effector molecule used to achieve a beneficial outcome in an organism, preferably a vertebrate, more preferably a human. Benefits include diagnosis, prognosis, treatment, cure, and prevention of diseases and / or conditions. Pharmaceutically active substances may also cause unwanted and harmful side effects. In this case, the advantages and disadvantages must be weighed to determine whether a pharmaceutically active substance is suitable for a particular case. If the effect of a pharmaceutically active substance inside a cell is predominantly beneficial to the entire organism, e.g., a human patient, the cell is called a target cell. If the effect inside the cell is predominantly harmful to the entire organism, the cell is called an off-target cell. In artificial systems, such as cell cultures and bioreactors, target and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules are drugs, toxins, polypeptides (such as enzymes), and polynucleotides, including polypeptides and polynucleotides that contain 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)boganin, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, pseudomonas exotoxin), metabolic enzymes (argininosuccinate lyase, argininosuccinate synthase), 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).Toxins, as used herein, are defined as pharmaceutically active substances that can kill or inactivate cells. Preferably, targeted toxins are toxins that are only, or at least predominantly, toxic to target cells but not to off-target cells. The net effect of targeted toxins is preferably beneficial to the entire organism.

[0091] In one embodiment is a conjugate of the invention, wherein at least one effector molecule is selected from any one or more of a vector, a gene, a cell suicide-inducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotides (ASO, AON), short interfering RNA (siRNA), anti-microRNA (anti-miRNA), DNA aptamer, RNA aptamer, mRNA, small circular DNA, peptide nucleic acid (PNA), phosphoramidite morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 3'-fluorohexitol nucleic acid (FHNA), plasmid, glycol nucleic acid (GNA) and threose nucleic acid (TNA), or derivatives thereof, more preferably a BNA, for example a BNA for silencing HSP27 protein expression or a BNA for silencing apolipoprotein B expression.

[0092] In certain embodiments, the at least one effector molecule is selected from the group consisting of short interfering RNA (siRNA), short hairpin RNA (shRNA), anti-hairpin microRNA (miRNA), single-stranded RNA, aptamer RNA, double-stranded RNA (dsRNA), anti-microRNA (anti-miRNA, anti-miR), antisense oligonucleotide (ASO), mRNA, DNA, antisense DNA, locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-O,4'-aminoethylene bridged nucleic acid (BNA), and the like. NC ), BNA-based siRNA, and BNA-based antisense oligonucleotides (BNA-AONs).

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

[0094] In one embodiment, at least one effector molecule is an oligonucleotide capable of silencing a gene when present in a cell containing such gene, wherein the gene is any one of the following genes: apolipoprotein B (apoB), transthyretin (TTR), proprotein convertase subtilisin / kexin type 9 (PCSK9), delta-aminolevulinic acid synthase 1 (ALAS1), antithrombin 3 (AT3), glycolate oxidase (GO), complement component C5 (CC5), hepatitis B virus (HBV) X gene, HBV S gene, alpha-1 antitrypsin (AAT), and lactate dehydrogenase (LDH); and / or a conjugate of the invention capable of targeting an aberrant miRNA when present in a cell containing such aberrant miRNA.

[0095] In one embodiment, the effector molecule is an oligonucleotide capable of targeting an mRNA when present in a cell containing such mRNA, wherein the mRNA is involved in the expression of any one of the following proteins: apoB, TTR, PCSK9, ALAS1, AT3, GO, CC5, an expression product of the HBV X gene, an expression product of the HBV S gene, AAT, and LDH, or a conjugate of the invention capable of attenuating or restoring miRNA function, such as inhibition of oncogenic miRNA (onco-miR) or suppression of onco-miR expression, when present in a cell containing such miRNA.

[0096] The present inventors demonstrate that a tumor cell-targeting monoclonal antibody provided with a covalently coupled antisense BNA, such as a BNA (HSP27), and a covalently coupled saponin of the present invention (both the BNA and saponin are coupled to the antibody (e.g., cetuximab) via a cleavable bond), contacted with tumor cells, can silence HSP27 in vivo in tumors compared to a control and compared to an AOC having only the BNA but no saponin (SO1861, Quil-A). Thus, administration of 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, confers anti-tumor cell activity to the ADC-saponin or AOC-saponin that is not seen with an ADC alone or an AOC alone without a saponin covalently coupled to the monoclonal antibody at the same dose. Notably, when AOC and a separate monoclonal antibody having a covalently coupled saponin as a combination of two separate conjugates were administered separately to tumor-bearing mice in separate groups of mice, they increased HSP27 expression in tumor cells compared to the control group (administered only solvent). Only the administration of the AOC-saponin conjugate of the present invention containing an effector moiety of the present invention showed a decrease in HSP27 expression compared to the control. The antisense BNA (HSP27) was a BNA having an oligonucleotide sequence according to Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liao, P Kraft, R Bandaru, Y Wu, LM Greenberger, and ID Horak, "Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotides without transfection," Gene Therapy (2011) 18, 326-333]. Notably, to the best of our knowledge, BNAs are designed for application as free nucleic acids.The inventors have now demonstrated for the first time that antisense BNAs can be covalently coupled to ligands or antibodies via (non-)cleavable linkers in such a way that gene silencing activity is retained in vitro and, more importantly, in vivo in tumor cells in tumor-bearing animals. This approach to providing BNA-based AOCs opens up a novel method for administering targeted BNAs to human (cancer) patients in need.

[0097] In one embodiment, the conjugate of the present invention comprises at least one effector molecule, preferably comprising or consisting of at least one proteinaceous molecule selected from any one or more of a peptide, a protein, an enzyme, and a protein toxin. The inventors have found that highly effective tumor cell killing is achieved when an sdAb that binds to HER2, VEGFR, or 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 an sdAb and an effector molecule are provided in the Examples section and shown in Figures 4-7.

[0098] In certain embodiments, the 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 toxins, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins such as alpha-sarcin; ribosome-inactivating proteins and giantins, e.g., giantin-30 or giantin-32, saporins, e.g., saporin-S3 or saporin-S6, boganin or deimmunized derivatives of boganin, deboganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, model toxins, In the conjugate of the present invention, the protein toxin is selected from any one or more of: a plant toxin comprising the A chain of a type 2 ribosome-inactivating protein, such as cusin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or an animal or human toxin, such as frog RNase, or granzyme B or human angiogenin, or any toxic fragment or toxic derivative thereof; preferably, the protein toxin is dianthin and / or saporin.

[0099] One embodiment is a conjugate of the invention wherein at least one effector molecule comprises or consists of at least one payload.

[0100] In certain embodiments, the at least one effector molecule is at least one of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, passudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N-acetyl-γ-calicheamicin, pyrrolobenzodiamine, ... Azepine (PBD) dimers, benzodiazepines, CC-1065 analogs, duocarmycins, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxanthone, tubulysin, indolinobenzodiazepines, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracyclines, camptothecin analogs, SN-38, DX-8951f, exatecan mesylate, Pseudomonas aeruginosa aeruginosa exotoxin (PE38), duocarmycin derivatives, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or derivatives thereof.

[0101] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effect. For example, some effector molecules, such as Pseudomonas exotoxin, are rerouted to other organelles before the "late endosomal stage" and therefore would not normally benefit from incorporation in a conjugate 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 rerouting. In particular, toxins that are highly toxic and require only one molecule to escape the endosome and kill a cell can be modified to be less potent. It is preferred to use toxins that kill cells when at least two, more preferably at least five, 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). It is further preferred that the conjugates of the present invention comprise a covalently conjugated functionalized scaffold, i.e., a scaffold such as an oligomeric or polymeric scaffold or a trifunctional linker, comprising one or more covalently attached effector moieties for targeting the scaffold comprising one or more bound effector moieties at target cells, such as tumor cells or autoimmune cells. Furthermore, to reduce off-target toxicity, cell membrane-impermeant small molecule toxins are preferred effector molecules over cell membrane-permeant toxins.

[0102] Preferably, the effector moiety comprised by the conjugate of the invention, whose effect is enhanced by the saponin comprised by the conjugate, is detached from the conjugate, for example from an antibody, such as an sdAb, present in the conjugate as the cell surface molecule targeting moiety of the conjugate upon plasma membrane invagination. This can be achieved, for example, by a cleavable bond that cleaves under acidic, reductive, enzymatic or light-induced conditions.

[0103] In one embodiment is a conjugate of the invention, wherein the conjugate comprises an antibody-drug conjugate (ADC), such as an ADC comprising 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 of such a human antibody may be H The domains have several improvements in domain stability known in the art (human V H It will be appreciated that this may require "camelization" of the domain.

[0104] In some embodiments, the at least one saponin is selected from the group consisting of (Group C): 2alpha-hydroxyoleanolic acid; 16 alpha-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16alpha,23-dihydroxyoleanolic acid; Gypsogenin; Quila acid; Protoesigenin-21(2-methylbut-2-enoate)-22-acetate; 23-Oxo-vallingtogenol C-21,22-bis(2-methylbut-2-enoate); 23-Oxo-vallingtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; digitogenin; 3,16,28-trihydroxyoleanan-12-ene; gypsogenic acid; or Its derivatives and comprising an aglycone core structure selected from Preferably, at least one saponin comprises an aglycone core structure selected from quillic acid and gypsogenin, more preferably a conjugate of the present invention wherein at least one saponin comprises the aglycone core structure quillic acid.

[0105] Without wishing to be bound by any theory, the presence of an aldehyde group (or a derivative thereof) in the aglycone core structure of a saponin (also referred to herein as "aglycone") is beneficial to the ability of the saponin to stimulate and / or enhance the endosomal escape of effector molecules comprised by the conjugates of the present invention, when such saponin coexists with these effector molecules in a cell, in the endosomes of said cell as part of the conjugates of the present invention, or when in free form in the endosome (e.g., dissociates from the conjugate once the conjugate is delivered inside a target cell endosome or lysosome). Thus, conjugates of the present invention comprising saponins having an aglycone with an aldehyde group are preferred. In quillic acid and gypsogenin, the aldehyde group is located at the C of the aglycone. 23 It's in the atom.

[0106] In some embodiments, at least one saponin is at least one C3 atom or C4 atom of the aglycone core structure of the at least one saponin. 28 a first sugar chain attached to the C atom, preferably attached to the C3 atom of the aglycone core structure of at least one saponin; 28 The conjugates of the present invention comprise one or both of a first sugar chain and a second sugar chain attached to the atom, and preferably at least one saponin comprises a first sugar chain and a second sugar chain. Thus, when the saponin comprised by the conjugates 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 C4 position of the aglycone core structure of the saponin. 28 are joined at the

[0107] In some embodiments, At least one saponin (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-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and any derivative thereof and a first sugar chain selected from and / or At least one saponin (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 comprises a second sugar chain selected from:

[0108] Thus, when the saponin comprised 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 of the saponin, and the second sugar chain is attached at the C4 position of the aglycone core structure of the saponin. 28 Preferably, the saponin has an aldehyde group in the aglycone.

[0109] In one embodiment, at least one saponin comprises a first sugar chain and a second sugar chain according to Group A and Group B, respectively, the first sugar chain comprising two or more sugar moieties and the second sugar chain comprising two or more sugar moieties, and the aglycone core structure is preferably querric acid or gypsogenin, more preferably querric acid; i. the aldehyde group in the aglycone core structure is derivatized; ii. the carboxyl group of the glucuronic acid moiety in the first glycan is derivatized; and iii. At least one acetoxy (Me(CO)O-) group in the second glycan is derivatized. The conjugate of the present invention is one, two or three, preferably one or two of the above.

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

[0111] In some embodiments, the at least one saponin is selected from the group consisting of Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macranthoidine A, esculentoside A, phytolaccagenin, esinate, 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-017 775, 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 The conjugate of the present invention is any one or more of B-xylo, beta-escin, escin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitonin, primulic acid 1 and AS64R, or derivatives thereof, or stereoisomers thereof, and / or 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 saponin derivatives according to the present invention.

[0112] In some embodiments, the at least one saponin is 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, Quillaja saponin, Saponinum album album), QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, or derivatives thereof, or stereoisomers thereof, and / or any combination thereof, and preferably the saponin derivative is a SO1861 derivative and / or a GE1741 derivative and / or a SA1641 derivative and / or a QS-21 derivative, more preferably the saponin derivative is a SO1861 derivative or a QS21 derivative, and most preferably the saponin derivative is a SO1861 derivative according to the present invention.

[0113] Such triterpene glycoside-type saponins can enhance endosomal escape of effector molecules contained in a conjugate and present in a cellular endosome (or lysosome) when the saponin, either as part of a 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 greater than approximately 100-1000 times stronger when the saponin is contacted with cells as part of a conjugate of the present invention. When such cells are contacted with an effector molecule and saponin as part of a certain cell-targeting conjugate, such as an ADC or AOC, the free saponin can stimulate delivery of an effector molecule into the cytosol of a cell at a saponin concentration that is 100-1000 times higher than the concentration of the same saponin contained in a conjugate of the present invention required to achieve the same degree of delivery of the effector molecule from the outside of the target cell to the inside of the endosome and ultimately to the cytosol of the cell. Saponins that exhibit such endosomal escape-enhancing activity, and saponins that share a high structural similarity with saponins that have an established ability to enhance cytosolic delivery of an effector molecule comprised by a conjugate, are listed in Table A1. Thus, when a saponin is part of a conjugate of the invention, upon binding of the conjugate's sdAb, binding to a targeted cell surface binding site on a target cell, binding to the cell, and following endocytosis, targeted delivery of the saponin into the endosome of the cell is about 100-1000 times more effective than contacting the same cells with a free, untargeted saponin (derivative) that is not provided with a binding molecule such as an antibody or sdAb for binding to a cell surface molecule of the target cell.

[0114] For example, the small size of the sdAb of the conjugates of the present invention compared to IgG-type antibodies or fragments thereof, such as Fab and scFv, contributes to efficient uptake, e.g., endocytosis, by target cells that expose a binding site for the sdAb contained in the conjugate. Typically, the sdAb in the conjugates of the present invention can bind to a cell surface receptor on the target cell, such as a tumor cell-specific cell surface receptor. In this way, the conjugates of the present invention are particularly suitable for endocytosis into, for example, tumor cells expressing the cell surface receptor.

[0115] An embodiment is a conjugate of the invention wherein at least one sdAb comprises an sdAb for binding to a cell surface molecule of a cell, and the cell is a tumor cell, an autoimmune cell, an infected cell such as a virally infected cell, or an abnormal cell such as a cell containing a genetic or enzymatic defect.

[0116] In one embodiment, at least one sdAb is an sdAb for binding to a cell surface molecule of a cell, and is selected from the group consisting of immunoglobulins: anti-CD71 antibodies such as IgG OKT-9, anti-HER2 antibodies such as trastuzumab (Herceptin), pertuzumab, anti-CD20 antibodies such as rituximab, ofatumumab, tositumomab, obinutuzumab ibritumomab, anti-CA125 antibodies such as oregovomab, anti-EpCAM (17-1A) antibodies such as edrecolomab, anti-EGFR antibodies such as cetuximab, matuzumab, panitumumab, nimotuzumab, anti-CD30 antibodies such as brentuximab, anti-CD33 antibodies such as gemtuzumab, huMy9-6, etaracizumab, etc. anti-vascular integrin alpha-v beta-3 antibodies, anti-CD52 antibodies such as alemtuzumab, anti-CD22 antibodies such as epratuzumab and pinatuzumab, binding fragments (Fv) of the anti-CD22 antibody moxetumomab, humanized monoclonal antibodies inotuzumab, anti-CEA antibodies such as labetuzumab, anti-CD44v6 antibodies such as bivatuzumab, anti-FAP antibodies such as sibrotuzumab, anti-CD19 antibodies such as huB4, anti-CanAg antibodies such as huC242, anti-CD56 antibodies such as huN901, anti-CD38 antibodies such as daratumumab, OKT-10 anti-CD38 monoclonal antibody, anti-CA6 antibodies such as DS6, anti-IGF-1R antibodies such as cizutumumab, 3B7, CNTO 95, an anti-syndecan-1 antibody such as B-B4, an anti-CD79b such as polatuzumab, an anti-HIV gp41 antibody, preferably any one of an anti-HIV gp41 antibody, an anti-CD71 antibody, an anti-HER2 antibody and an anti-EGFR antibody, more preferably trastuzumab, pertuzumab, cetuximab, matuzumab, an anti-CD71 antibody, OKT-9, and most preferably trastuzumab, cetuximab, or the anti-CD71 antibody OKT-9.

[0117] These cell surface molecules are usually present on tumor cells, with at least a certain degree of tumor cell specificity. Tumor cell specificity makes these receptors suitable targets for the conjugates of the present invention, and therefore the sdAb in the conjugates can bind to such cell surface receptors. The saponin contained in the conjugates of the present invention can stimulate the release and delivery of the effector molecule contained in the conjugates of the present invention into the cytosol of cells, such as (tumor) cells targeted by the sdAb contained in the conjugates of the present invention, and is therefore particularly suitable for selection as the target (tumor) cell surface molecule for the sdAb, which is a cell surface receptor known for its suitability to function as a target for ADCs and AOCs. In addition, the conjugates of the present invention are suitable for simultaneous delivery of the effector molecule that is part of the conjugate together with the saponin contained in the very same conjugate of the present invention, which conjugate is an improved ADC or improved AOC containing both the sdAb and the saponin. Targeting tumor cell-specific receptors with the conjugates of the present invention promotes endocytosis and delivery of the saponin as part of the conjugate to the target cell endosomes and / or lysosomes. When tumor cells are contacted with the conjugates of the present invention, the effector molecule contained by the conjugates of the present invention is co-delivered to the endosomes or lysosomes, and under the influence of the co-localized saponin, the effector molecule is subsequently translocated into the cytosol of the target cell. As explained earlier in this specification, application of the targeted saponin as part of the conjugates of the present invention results in an approximately 100- to 1000-fold improvement in the enhancing effect of the saponin compared to application of free saponin lacking a cell-targeting binding molecule such as a receptor ligand, antibody, or sdAb, when the biological activity of the effector molecule contained by the conjugates of the present invention is taken into account.

[0118] Camelid V in the conjugates of the present invention HThe application of small sdAbs such as these prevents or slows the clearance of the conjugates of the present invention from the circulation and from the body of a human subject to which the conjugate is administered, compared to the clearance rates typically observed for antibody-based ADCs. In addition, due to the relatively small size of sdAbs, the risk of limiting or interfering with saponin activity inside target cells due to the presence of linked protein domains is limited when such antibodies are conjugated to saponins, compared to larger sizes, such as antibodies. Generally, the smaller the size of the molecule linked to the saponin, the greater the chance of the conjugated molecule, e.g., V HH The presence of sdAbs such as sdAbs reduces the risk of interference with intracellular saponin activity. Furthermore, the relatively small size of sdAbs allows for rapid distribution in tissues, such as tumor tissue, allowing the conjugates of the present invention to improve target cell delivery and thereby improve the degree of binding to target cells compared to the relatively large size of IgGs commonly used in, for example, ADCs and OACs. One of the many advantages of using sdAbs in the conjugates of the present invention is the absence of an Fc tail, which is common in conventional antibodies, for example, IgG type. The absence of an Fc tail in the sdAb in 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 conjugates are administered to patients in need thereof. The absence of an Fc tail eliminates the risk of side effects generated by Fc binding to cells in patients to whom the antibody-based ADC is administered. Conjugates containing the sdAbs of the present invention do not have this risk of Fc-mediated unwanted side effects.

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

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

[0121] Coupling of the saponin to the sdAb and / or effector molecule via a linker allows flexibility when considering attachment sites for coupling of the saponin to the sdAb and / or effector molecule. Furthermore, such linkers may act as spacers between the sdAb, saponin and effector molecule, such that the sdAb maintains its ability to bind to binding sites on cell surface molecules, 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 towards its intracellular binding partner.

[0122] In one embodiment, at least one saponin is covalently attached via a thioether bond to a sulfhydryl group on one of the at least one sdAb and / or one of the at least one effector molecule, the covalent attachment preferably occurring between the C and C of the aglycone core structure of the saponin. 23 The conjugates of the invention are those via the linker N-ε-maleimidocaproic acid hydrazide (EMCH) which is covalently attached to an aldehyde group at position 1 of the sdAb and to sulfhydryl groups on the sdAb and / or effector molecule, such as sulfhydryl groups of cysteine.

[0123] In one embodiment, the at least one saponin is C 23 a bi-desmosidic triterpene saponin or derivative thereof belonging to the 12,13-dehydrooleanane type optionally bearing an aldehyde functional group at position 1 and comprising a glucuronic acid unit in a first sugar chain linked at the C3 beta-OH group of the aglycone core structure of the saponin, wherein the saponin is covalently linked to an amino acid residue of at least one sdAb and / or at least one effector molecule via the carboxyl group of the glucuronic acid unit in the first sugar chain, preferably via a linker, wherein the amino acid residue is preferably selected from cysteine ​​and lysine.

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

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

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

[0127] In some embodiments, the molecular structure (II): (Saponin-Linker-) a Immunoglobulin-effector portion (Structure (II)) (wherein a=1 to 4, preferably 1, 2, or 4, and the immunoglobulin is preferably an sdAb) Or molecular structure (III): (Saponin-dendron (-saponin) x ) b -immunoglobulin-effector moiety (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, or 4, and the immunoglobulin is preferably an sdAb) Or molecular structure (IV): (Saponin-trifunctional linker (-effector moiety)) c -immunoglobulin (Structure (IV)) (wherein c=1 to 4, preferably 1, 2, or 4, and the immunoglobulin is preferably an sdAb) Or molecular structure (V): ((Saponin-dendron(-saponin) y )-trifunctional linker (-effector moiety) d -Ig (Structure (V)) wherein 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, or 4; and the immunoglobulin ("Ig") is preferably an sdAb.

[0128] Preferably, x or y is 3, 7, or 15. Preferably, b or d is 1, 2, or 4, although in some embodiments, b or d is 3 and the immunoglobulin is V HH When the saponin is an 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 attached 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, and lysosomes). Preferably, the linker is EMCH. Preferably, the trifunctional linker is a linker having structure A as shown herein below. With regard to structures II and III, preferably, the effector moiety is attached to the Ig via a linker such as a cleavable linker.

[0129] In one embodiment is a conjugate of the invention wherein the two or more covalently attached saponin moieties are part of a covalent saponin conjugate comprising at least one oligomeric or polymeric molecule and two or more saponins covalently attached thereto, and the covalent saponin conjugate is covalently attached to at least one of at least one sdAb and / or at least one of at least one effector molecule.

[0130] Such covalent saponin conjugates function as carriers for multiple saponin moieties, which can be attached to the sdAb comprised by the conjugate via a single bond, preferably via a (cleavable) linker. The application of such covalent saponin conjugates offers flexibility when considering the number of saponin moieties in the conjugates of the invention, as the covalent saponin conjugates can have any selected number of covalently attached saponin moieties, such as 1 to 200 saponin moieties associated with a selected type of oligomeric or polymeric structure containing attachment sites for covalently linking these saponins. For example, with respect to cytosolic delivery of effector molecules comprised by the conjugates of the invention, the number of saponins present in the conjugates of the invention can be tailored by providing a covalent saponin conjugate having a number of saponin moieties that is sufficient and sufficient to stimulate cytosolic delivery of the effector molecule when it is part of a conjugate of the invention and when the effector molecule co-exists with the saponin as an integral part of one and the same conjugate in the endosome or lysosome of the target cell where the effector molecule is to exert its biological activity.

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

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

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

[0134] An embodiment is a conjugate of the invention wherein the cleavable linker is susceptible to cleavage under acidic, reductive, enzymatic and / or photoinduced conditions, preferably wherein the cleavable linker comprises a cleavable bond selected from hydrazone and hydrazide bonds, which are susceptible to cleavage under acidic conditions, and / or bonds susceptible to proteolysis, e.g., proteolysis by cathepsin B, and / or bonds susceptible to cleavage under reductive conditions, such as disulfide bonds.

[0135] One embodiment is a conjugate of the invention wherein the cleavable linker is subject to cleavage in vivo under acidic conditions, such as those present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0-6.5, more preferably at a pH of ≦5.5.

[0136] Such cleavable linkers, which are cleavable under conditions such as those found in endosomes and lysosomes, facilitate delivery of the free saponin inside the endosome or lysosome upon cleavage (separation) of the saponin from the remainder of the conjugate of the invention. In this way, the conjugates of the invention combine the advantages of cell-targeted delivery of the saponin upon specific binding of the sdAb to a cell surface molecule on the target cell, and the presence of the free saponin intracellularly, i.e., inside the endosome (or lysosome), which contributes to the ability of the free saponin to stimulate and / or facilitate delivery of the effector molecule comprised by the conjugate of the invention from the endosome (or lysosome) to the cytosol of the target cell.

[0137] One embodiment is a conjugate of the invention wherein an oligomeric or polymeric molecule of the covalent saponin conjugate is covalently attached to at least one of the at least one sdAb and / or at least one of the 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 wherein at least one saponin is covalently attached to an oligomeric or polymeric molecule of the covalent saponin conjugate via a cleavable linker according to the invention.

[0139] One embodiment is a conjugate of the invention wherein at least one saponin is covalently bonded to the oligomeric or polymeric molecule of the covalent saponin conjugate via any one or more of an imine bond, hydrazone bond, hydrazide bond, oxime bond, 1,3-dioxolane bond, disulfide bond, thioether bond, amide bond, peptide bond or ester bond, preferably via a linker.

[0140] In one embodiment, the at least one saponin is C 23and at least one saponin optionally comprises a glucuronic acid functional group in the first sugar chain at the C3 beta-OH group of the aglycone core structure of the saponin, wherein the aldehyde functional group is involved in covalent attachment to the oligomeric or polymeric molecule of the covalent saponin conjugate, and / or when present, the glucuronic acid functional group is involved in covalent attachment of the saponin to the oligomeric or polymeric molecule of the covalent saponin conjugate via a direct covalent bond or via a linker (the linker being a cleavable linker or a stable linker). As used herein, stable refers to the bond between the saponin and the sdAb or effector molecule, or the bond between the saponin and the oligomeric or polymeric structure, which bond remains intact (is not cleaved) under acidic conditions within a cell, particularly the acidic conditions within the endosomes or lysosomes of such cells. In addition, such stable bonds remain intact (i.e., are not cleaved) in the circulation and organs of a human subject to which a conjugate of the invention, including, for example, a covalent saponin conjugate, is administered. In contrast, a cleavable linker, or oligomeric or polymeric structure in connection with the attachment of a saponin to an sdAb or effector molecule comprised by the conjugate, refers to a bond that is cleaved under acidic conditions such as those found inside the endosomes and lysosomes of human cells, e.g., mammalian cells such as tumor cells, but such a cleavable linker remains intact (is not cleaved) when a conjugate comprising such a cleavable bond is present in the circulation or organs, i.e., outside the cells of, for example, a human subject to which a conjugate of the invention is administered.

[0141] In some embodiments, the C of the aglycone core structure of at least one saponin is 23The conjugates of the present invention comprise a conjugate in which an aldehyde functional group at position 1 is covalently attached to a linker EMCH, and EMCH is covalently attached via a thioether bond to a sulfhydryl group in the oligomeric or polymeric molecule of the covalent saponin conjugate, such as a sulfhydryl group of cysteine. Attachment of the EMCH linker to the aldehyde group of the aglycone of the saponin results in the formation of a hydrazone bond. Such a hydrazone bond is a typical example of a bond that is cleavable under acidic conditions in endosomes and lysosomes. The saponin coupled to the sdAb contained in the conjugate of the present invention, or the effector molecule contained in the conjugate of the present invention, or the oligomeric or polymeric structure of the covalent saponin conjugate, is releasable from the conjugate of the present invention upon delivery to the endosome or lysosome of a target cell that is coupled to the sdAb or effector molecule of the conjugate and exposes a cell surface molecule to which the sdAb of the conjugate can bind. In this way, the saponin coupled to the sdAb or effector molecule in the conjugates of the present invention is transported from the outside of the cell to the endosome (or lysosome), where it is released from the remainder of the conjugate upon pH-driven cleavage of the hydrazone bond. In the endosome (or lysosome), the free saponin can exert its stimulatory activity when delivery of the effector molecule comprised by the conjugates of the present invention to the cytosol is considered. Surprisingly, the present inventors have established that, with respect to saponin, the presence of the saponin in free form in the endosome or lysosome is not essential for the endosomal escape-enhancing activity of the saponin. Furthermore, for example, the saponin comprised by certain conjugates enhances 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 certain conjugate are contacted with the same target cell.

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

[0143] One embodiment is a conjugate of the invention wherein the covalent saponin conjugate polymeric or oligomeric molecule is attached 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 an amino acid residue of the sdAb and / or an amino acid residue of the effector molecule, and comprises a click chemistry group on the covalent saponin conjugate polymeric or oligomeric molecule, wherein the click chemistry group is preferably selected from a tetrazine, an azide, an alkene or alkyne, or cyclic derivatives of these groups, more preferably the click chemistry group is an azide.

[0144] In one embodiment, the polymeric or oligomeric molecule of the covalent saponin conjugate comprises a polymeric and / or 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 glycols, oligo-ethylene glycols (OEGs) such as OEG3, OEG4, and OEG5, or assemblies of these polymeric and / or oligomeric structures, where the assemblies are preferably constructed by covalent crosslinking, preferably wherein the polymeric or oligomeric molecule of the covalent saponin conjugate is a dendron such as a poly-amidoamine (PAMAM) dendrimer. The type and size or length of the oligomeric or polymeric structure is selected to be driven by the number of selected saponins to be incorporated in the conjugates of the invention. That is, the number of saponins to be coupled to the sdAb or effector molecule comprised by the conjugate for formation of the conjugate of the invention can determine the selection of a suitable oligomeric or polymeric structure having a sufficient number of attachment sites for coupling the desired number of saponins, thereby providing a covalent saponin conjugate having a selected number of saponin moieties to be coupled to the sdAb or effector molecule for providing a conjugate of the invention. For example, the length of the OEG or the size of the dendron or poly-lysine molecule will determine the maximum number of saponins that can be covalently linked to such an oligomeric or polymeric structure.

[0145] Thus, the conjugates according to the invention comprise at least one saponin. "At least one" in this context means that the conjugate comprises one saponin molecule, but may also comprise a pair (e.g., 2, 3, or 4) of saponins or a number (e.g., 10, 20, or 100) of saponins. Depending on the application, the conjugate may comprise a covalently attached scaffold with a covalently attached saponin (covalent saponin conjugate), and the scaffold may be designed so that it comprises a defined number of saponins. Preferably, the conjugates according to the invention comprise a defined number or range of saponins, rather than a random number. This is particularly advantageous for drug development in connection with marketing authorization. A defined number in this context means that the conjugate preferably comprises a previously defined number of saponins. This can be achieved, for example, by designing a scaffold comprising a polymer structure with a specific number of possible moieties for saponin attachment. Under ideal circumstances, all of these moieties are coupled to saponins, and the scaffold comprises a previously defined number of saponins. It is envisioned that a standard set of scaffolds containing, for example, 2, 4, 8, 16, 32, 64, etc. saponins may be provided, allowing the user to easily test the optimal number based on their needs. One embodiment is a conjugate of the present invention (covalent saponin conjugate of the present invention) comprising a scaffold of the present invention, wherein the saponin is present in a defined range, for example, because, under non-ideal circumstances, not all moieties present in the polymer structure will be bound to the saponin. Such a range may be, for example, 2-4 saponin molecules per scaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules per scaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules per scaffold, etc. Thus, in such cases, a conjugate comprising a scaffold according to the present invention will contain 2, 3, or 4 saponins when the range is defined as 2-4.

[0146] The scaffold is essentially independent of the type of saponin covalently bound to it, and the scaffold is subsequently (sequentially) covalently coupled to the conjugate. Thus, the conjugates of the present invention comprising a scaffold (the covalent saponin conjugates of the present invention) are the foundational products for platform technology. Because at least one covalently bound saponin mediates the intracellular delivery of an effector molecule bound to a cell surface molecule-targeting sdAb contained by the conjugate of the present invention, the scaffold technology according to the present invention is a system that mediates the controlled intracellular delivery of an effector moiety by saponin. The scaffold provides an optimized, functionally active unit that can be linked to the saponin and cell surface molecule-targeting sdAb contained by the conjugate at a single, defined position on the sdAb.

[0147] One embodiment is a conjugate of the invention (covalent saponin conjugate of the invention) comprising a scaffold according to the invention in which the number of monomers in the polymeric or oligomeric structure is a precisely defined number or range. Preferably, the polymeric or oligomeric structure comprises structures such as poly(amines), e.g., polyethyleneimines and poly(amidoamines), or poly(esters), such as polyethylene glycols, poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins), or peptides or proteins, or natural and / or artificial polyamino acids, e.g., poly-lysine, DNA polymers, stabilized RNA polymers or PNA (peptide nucleic acid) polymers, appearing as linear, branched or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers or aggregates of these structures, pure or mixed. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not exhibit substantial acute or chronic toxicity in living organisms and can be excreted intact or completely degraded into compounds and / or physiological compounds that can be excreted by the body's metabolism. The assemblies can be constructed by covalent crosslinking or non-covalent bonds and / or attractive forces. They can therefore also form nanogels, microgels, or hydrogels, or can be bound to carriers such as inorganic nanoparticles, colloids, liposomes, micelles, or particle-like structures containing cholesterol and / or phospholipids. The polymeric or oligomeric structures preferably have a precisely defined number or range of coupling moieties (chemical groups) for coupling glycoside molecules (and / or carrier molecules such as effector molecules and / or their fragments, e.g., ligands, monoclonal antibodies, or sdAbs).Preferably at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably (approximately) 100% of the precisely defined number or range of coupling moieties (chemical groups) in the polymeric or oligomeric structure are occupied by glycoside molecules (saponin of the invention) in a scaffold according to the invention (covalent saponin conjugate of the invention).

[0148] Preferably, dendrons are branched, well-defined, dendritic polymers with a single chemically addressable group at the origin of the tree, called the focal point. Dendrimers are two or more dendrons joined at their focal points. Dendronized polymers are polymers in which the focal points of one or more dendrons are joined. In a preferred embodiment, a scaffold according to the present invention is provided, wherein the polymeric or oligomeric structure comprises a linear, branched, or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or pure or mixed aggregates of these structures, which can be assembled by covalent crosslinking or non-covalent attraction and can form nanogels, microgels, or hydrogels, and preferably the polymer is a structure such as a derivative of poly(amine), e.g., polyethyleneimine and poly(amidoamine), and poly(esters), such as polyethylene glycol, poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), as well as a structure such as poly-lysine, or a natural and / or artificial polyamino acid, such as a peptide or protein, or a DNA polymer, stabilized RNA polymer, or PNA (peptide nucleic acid) polymer. Preferably, the polymeric or oligomeric structure is biocompatible.

[0149] In one embodiment, at least one saponin is linked to a trifunctional linker, preferably structure A:

[0150] [ka]

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

[0152] [ka]

[0153] and having a molecular structure represented by A conjugate of the invention 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 a trifunctional linker and a linkage between the saponin or covalent saponin conjugate, the effector molecule and the sdAb, respectively, or L1, L2 and L3 are linkers (L1, L2 and L3 are the same or different).

[0154] Unless otherwise indicated, whenever the term "endosome" or "endosomal escape" is used herein, it also includes endolysosomes and lysosomes, and escape from endolysosomes and lysosomes, respectively, particularly when relating to the endosomal escape mechanism of the saponins of the present invention. After entering the cytosol, the material may travel to other cellular units, such as the nucleus.

[0155] In formal terms, a glycoside is any molecule in which a sugar group is attached to another group via its anomeric carbon by a glycosidic bond. Without wishing to be bound by any theory, glycoside molecules such as saponins in the context of the present invention are particularly those 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 as exemplified herein and in the claims) interact with the membranes of compartments and vesicles in the endocytic and recycling pathways, rendering them leaky to the effector moiety, resulting in enhanced endosomal escape. The term "the scaffold is capable of enhancing endosomal escape of the effector moiety" means that at least one saponin (glycoside molecule) that is coupled to a cell surface molecule-targeting antibody such as an sdAb via a linker or directly, or coupled via the polymeric or oligomeric structure of the scaffold (covalent saponin conjugate of the invention), when both molecules are within an endosome, e.g., a late endosome, optionally and preferably after the at least one saponin has been released from the conjugate, such as from a linker or polymeric or oligomeric structure comprised by said conjugate, e.g., by cleavage of a cleavable bond between the at least one glycoside (saponin) and the conjugate (e.g., via the polymeric or oligomeric structure of the scaffold and / or via the linker). Even if the bond between at least one saponin according to the invention and a cell surface molecule targeting sdAb of a conjugate of the invention, optionally via a linker or scaffold, is a "stable bond", this does not mean that such a bond cannot be cleaved, for example, by an enzyme in an endosome. For example, a saponin, optionally combined with part of the oligomeric or polymeric structure of the linker or scaffold, may be cleaved from the remaining linker fragment or oligomeric or polymeric structure.For example, a protease may cleave the (proteinaceous) linker or proteinaceous polymer structure, such as albumin, thereby liberating at least one saponin. However, it is preferred that the glycoside molecule (preferably saponin) is released in an active form, preferably in the original form it had before being coupled (prepared to be) to the cell surface molecule-targeting sdAb of the conjugate of the invention, optionally via a linker and / or oligomeric or polymeric scaffold (covalent saponin conjugate of the invention), so that while the glycoside (saponin) has its native structure after such cleavage or the glycoside (saponin) has a chemical group or (part of) a linker attached to it after such cleavage, the glycoside biological activity (saponin biological activity), e.g. endosomal / lysosomal escape enhancing activity towards an effector moiety present in the same endosome or lysosome, is maintained or restored upon said cleavage of the bond between the glycoside (saponin) and the cell surface molecule-targeting antibody, such as an sdAb, optionally comprising a linker and / or scaffold of the invention. The term "stable", in relation to the present invention, for example, with respect to the bond between a saponin and an amino acid residue of a cell surface molecule-targeting sdAb in a conjugate, linker, polymer or oligomeric structure (of a scaffold also known as a covalent saponin conjugate of the present invention), ligand, (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or effector (effector moiety, effector molecule), means that the bond is not easily cleaved, or at least is not designed to be easily cleaved, for example, by pH differences, salt concentrations, or UV light, or reductive conditions. The term "cleavable", in relation to the present invention, for example, with respect to the bond between a saponin and a cell surface molecule-targeting sdAb, linker, amino acid residue, polymer or oligomeric structure of a covalent saponin conjugate, ligand, antibody and / or effector molecule, means that the bond is designed to be easily cleaved, for example, by pH differences, salt concentrations, under reductive conditions, etc. Those skilled in the art are familiar with such cleavable bond types and how to prepare them.

[0156] Prior to the present invention, one of the major hurdles to bringing ADCs and AOCs to market was their narrow therapeutic window: therapeutically effective doses of ADCs or AOCs were accompanied by (unacceptable) side effects that hindered their development and value in treating patients with ADCs. By applying the conjugates of the present invention, such as ADC-saponin conjugates and AOC-saponin conjugates, it is now possible to target one or more glycoside molecules (saponins) to (target) cells together with an ADC bearing a payload or together with a (monoclonal) antibody (sdAb) conjugated to an oligonucleotide, such as a BNA, according to the present invention. In particular, it was not previously possible to specifically target the effector moiety of an ADC or AOC, or any other conjugate of a payload and a (proteinaceous) cell surface molecule targeting molecule, and a (predetermined, controllable) specific number or range of glycoside molecules (saponins) per effector moiety simultaneously to the cytosol of a cell, such as via the endocytic pathway of the cell.

[0157] The solution provided by the present invention involves the covalent attachment of at least one saponin to the cell surface molecule targeting molecule, i.e., sdAb, of the conjugate of the present invention. A further solution provided by the present invention involves (initial) polymerizing a glycoside molecule (saponin) using an oligomeric or polymeric scaffold and providing the cell surface molecule targeting molecule comprised by the conjugate of the present invention with a group of covalently attached saponins, allowing for remonomerization of one or more saponins at the intracellular site where the saponin's mechanism of action is desired, for example, after endocytosis. "Polymerize" in this context means the reversible and / or irreversible multiple conjugation of saponin molecules to an sdAb via a linker, or directly or via a polymeric or oligomeric structure to form a scaffold (covalent saponin conjugate of the invention) or the reversible and / or irreversible multiple conjugation of (modified) saponins to form a polymeric or oligomeric structure to form a scaffold (covalent saponin conjugate of the invention). In this context, "remonomerization" means, for example, cleaving the saponin from the conjugate, from the linker connecting the saponin to the cell surface molecule-targeting sdAb of the conjugate, or from the scaffold, after endocytosis, to restore the (native) chemical state of the unconjugated saponin, which may or may not include additional chemical groups, such as chemical groups for connecting the saponin to the linker, amino acid residues or scaffold of the conjugate, and / or (chemical) linkers attached to chemical groups of the saponin, such as aldehyde or carboxylic acid groups. Due to the complex chemistry of saponins, for example, "polymerization" of saponins with a scaffold or other connecting linker and their "remonomerization" at the desired location, e.g., within a cell after endocytosis, has been a difficult challenge. In particular, the chemical reactions used to provide linkers and conjugates, e.g., scaffolds comprising covalently linked glycosides for covalently binding to triterpenoid saponins (polymerization of glycosides), typically occur in organic solvents that do not contain water, whereas the saponins and, e.g., biocompatible polymers applied as scaffolds to have the conjugated saponins, are water-soluble molecules.The chemical properties of unmodified saponin further prohibit its own polymerization. Another possible solution, directly conjugating multiple saponins to effector molecules, was presumed to be less promising because effector molecules (drugs, toxins, polypeptides, or polynucleotides) usually do not provide sufficient conjugation sites, the coupling product would be highly heterogeneous, and / or the coupling of a biologically active molecule such as a saponin and, for example, a peptide, toxin, or nucleic acid together would carry the risk of affecting and interfering with the activity of one or both of the molecules conjugated together to a conjugate comprising such a saponin. Furthermore, when a saponin is coupled to, for example, an ADC or antibody-oligonucleotide conjugate (AOC), there is a significant risk that the effector moiety contained by the conjugates of the present invention will 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 a conjugate of the present invention and optionally a pharmaceutically acceptable excipient and / or a pharmaceutically acceptable diluent.

[0159] Whether a saponin-containing conjugate of the present invention, with or without further comprising one or more (cleavable) linkers and / or optionally a scaffold (covalent saponin conjugate of the present invention), can interfere with the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be readily determined as known in the art by assays such as those described in the Examples section. Inhibition is described as the "fold increase in the amount of glycoside (saponin of the present invention) required to induce 50% cell death." Preferably, the scaffold does not result in an increase that is at least the increase in glycoside molecules (saponin) required to obtain 50% cell death observed when using chloroquine as a positive control. Alternatively, and preferably, a saponin-containing conjugate, with or without further comprising one or more (cleavable) linkers and / or optionally a scaffold, does not result in at least a four-fold increase, more preferably at least a two-fold increase, of glycoside molecules to induce 50% cell death. The fold increase will be measured in an assay where chloroquine as a positive control induces a two-fold increase in the amount of glycoside, preferably the amount of saponin, where the saponin is any one or more of the saponins of the present invention (previous embodiments) where 50% cell killing is observed.

[0160] As mentioned above, the at least one saponin comprised by the conjugate according to the invention increases the efficacy of at least existing and novel effector moieties as defined in the present invention. Potential side effects are reduced without a decrease in efficacy due to the reduced administration of the effector moiety comprised by the conjugate. Thus, the present invention provides a conjugate according to the invention for use in medicine or as a medicament. A third aspect of the present invention relates to a pharmaceutical composition of the invention for use as a medicament.

[0161] Several preferred characteristics may be formulated for the endosomal escape enhancers comprised by the conjugates of the present invention, i.e., the saponins of the present invention: (1) they are preferably not toxic and do not provoke an immune response, (2) they preferably do not mediate the cytosolic uptake of the effector moiety into off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector moiety, (4) they are preferably biodegradable or excretable, and (5) they preferably do not substantially interfere with biological processes of the organism that are unrelated to the biological activity of the effector molecule with which the endosomal escape enhancer is combined, e.g., do not interact with hormones. Examples of saponins of the present invention that meet, at least to some extent, the foregoing criteria are bidesmosidic triterpenes, preferably bidesmosidic triterpene saponins, such as SO1861, SA1641, QS-21, GE1741, and the additional saponins listed throughout this specification.

[0162] Also provided is the use of a conjugate according to the present invention for the manufacture of a medicament. In particular, cancer medicaments, and in particular conventional chemotherapy medicaments, are notorious for their side effects. Due to the targeting and synchronization of the timing and location of both the pharmaceutically active substance contained by the conjugate and the saponin contained by the very same conjugate molecule, the therapeutic conjugate according to the present invention is particularly useful for use as a medicament, in particular for use in a method for treating cancer. Thus, the present invention provides a therapeutic conjugate according to the present invention for use in a method for treating acquired or hereditary disorders, in particular monogenic deficiency disorders. Thus, the therapeutic conjugate comprises at least one saponin and at least one effector moiety, as well as an sdAb for targeting the conjugate to abnormal target cells, such as tumor cells or autoimmune cells. Thus, one aspect of the present invention relates to a therapeutic conjugate according to the present invention, wherein the conjugate comprises a covalently attached effector moiety and comprises a covalently attached saponin and a cell surface molecule-binding antibody such as an sdAb, for use in a method for the treatment of cancer or an autoimmune disease.

[0163] A further application of the conjugates of the present invention in medicine is the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting disease can be hereditary or acquired. In many cases, only symptomatic treatment is possible, and for some rare diseases, insufficient treatment options lead to a shortened lifespan of the patient. An example of such a disease is phenylketonuria, an inborn error of metabolism that causes impaired metabolism of the amino acid phenylalanine. The disease is characterized by mutations in the gene for the liver enzyme phenylalanine hydroxylase. Phenyletonuria is not curable to date. The incidence is approximately 1:10,000, with the highest known incidence being 1:2,600 in Turkey. Cell surface molecule-targeting antibodies, preferably V, contained by the conjugates of the present invention with conjugated phenylalanine hydroxylase or conjugated polynucleotides encoding phenylalanine hydroxylase, are also useful. HH An sdAb such as can be used to target liver cells and replace the defective enzyme in the liver cells by use of a suitable specific antibody or sdAb. This is one example of the use of a therapeutic conjugate of the invention comprising a conjugated saponin according to the invention and a conjugated enzyme or oligonucleotide for replacement or gene therapy. In a preferred embodiment, there is provided a therapeutic conjugate according to the invention for use in methods of gene therapy or replacement therapy.

[0164] The conjugates of the present invention now make it possible to design and manufacture one-component, non-viral, clinically applicable gene delivery technologies. For example, the conjugates of the present invention enable the development of non-viral gene delivery technologies that enhance therapeutic efficacy with lower therapeutic doses, thereby improving patient health. The conjugates of the present invention, particularly when containing covalently attached cell surface molecule-targeting antibodies, such as monoclonal antibodies or sdAbs, for binding to (tumor, autoimmune) cell surface-specific molecules, and when coupled to effector moieties such as oligonucleotides, e.g., BNAs, overcome a long-standing major bottleneck in the field of gene delivery: enabling efficient, safe, and cost-effective cytosolic / nucleosolic translocation of gene therapy products across the endosomal membrane. Indeed, gene therapy is one of the most promising therapeutic options for future advanced therapies in a wide range of diseases. Successful gene delivery requires target cell recognition and cytosolic and nucleosolic uptake of the gene. One of the major problems in the field of non-viral gene therapy is the inefficient and unsafe delivery of genetic material for therapeutic use in patients.

[0165] Thus, by applying the conjugates of the present invention, which comprise a ligand or, preferably, a cell-targeting cell surface molecule targeting molecule such as an antibody (fragment, domain thereof, preferably sdAb) and an oligonucleotide such as an antisense BNA, the inventors have now made it possible to overcome a long-standing major bottleneck in the field of gene delivery: the safe passage of gene therapy products across the endosomal membrane into the cytosol / nucleosol. The conjugates of the present invention allow the targeting of any addressable cell type by all known genetic factors, thereby making it a technology designed to ensure successful patient therapy, not only for genetic disorders but also for cancer therapy, and therefore important for a large patient population. The conjugate-based technology of the present invention allows the targeting of targeting ligands or (monoclonal) (tumor cell-specific) antibodies, or fragments thereof, or preferably V, for endosomal escape enhancers (EEEs), such as saponins as exemplified herein and saponins of the embodiments according to the present invention. HHThe conjugates of the present invention may comprise polymeric or oligomeric scaffolds (covalent saponin conjugates of the present invention) that function as carriers for cell surface molecule targeting molecules, such as sdAbs, and effector moieties, such as LNAs or BNAs, as described herein. For example, the use of conjugates of the present invention comprising cell-targeting antibodies (fragments) or oligonucleotides, such as sdAbs and BNAs, has the potential to deliver all kinds of biomacromolecules to the cytosol and nucleus. The development of novel targeting ligands, sdAbs and monoclonal (human, humanized) antibodies, is continuously being explored by numerous research groups and companies worldwide. The same is true for oligonucleotides intended for delivery in the cytosol of diseased cells, such as cancer cells. Thus, the conjugates of the present invention also act as molecular interfaces to which current and future targeting sdAbs and antibodies, as well as current and future therapeutic oligonucleotides (and payloads such as protein toxins), can be attached or linked, for example, via click chemistry, to the oligomeric or polymeric scaffold modules of the present invention (the covalent saponin conjugates 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 include antibodies and ligands as cell surface molecular targeting molecules, although sdAbs are preferred. The global market for gene therapy drugs is growing rapidly and encompasses potential treatments for a wide range of disease areas, including cancer, cardiovascular disease, Parkinson's disease, Alzheimer's disease, HIV, and many rare (monotopic) diseases. Existing viral vector-based gene therapy technologies have significant challenges, including safety, manufacturing logistics, and the associated high costs. The conjugates of the present invention enable their use in technology platforms that offer an alternative to existing viral gene delivery technologies. The conjugates of the present invention are therefore suitable for implementation in approaches to developing non-viral gene therapies for diseases such as cancer, cardiovascular disease, Parkinson's disease, Alzheimer's disease, HIV infection and many rare (monotopic) diseases.The conjugates of the present invention are suitable for developing novel therapies to revolutionize the fields of antibody-drug conjugates (ADCs) and oligonucleotide-based therapeutics by generating gene therapy drugs based on non-viral vectors, such as those based on targeted antisense BNAs. In particular, the application of the conjugates of the present invention in covalent conjugates comprising an antibody, such as an sdAb, an oligonucleotide, such as a BNA, and at least one saponin is one of many advantageous approaches made possible by the present invention. For example, the use of the conjugates of the present invention now makes it possible to exploit the endocytic pathway of mammalian cells. Endocytosis is exploited for the delivery of therapeutic agents, and the conjugates of the present invention contribute to improved uptake and endosomal escape of, for example, siRNA contained by the conjugate. The conjugates of the present invention are suitable for use with, for example, small molecules that act as delivery enhancers for the payload, oligonucleotides. In addition, conjugates of the present invention can be used with covalently coupled oligonucleotides, such as BNAs, to bind to a ligand and preferably an antibody (domain or fragment, preferably a V). HHConjugates of the present invention having a covalently coupled cell-targeting moiety such as a saponin of the present invention provide a solution to existing problems found in existing endosomal escape enhancers and gene therapy products related to their application as two components, thereby complicating therapeutic approval and clinical applicability, since such conjugates of the present invention are single-conjugated therapeutic molecules encompassing a saponin, a gene product such as a 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, in which endosomal escape enhancers (e.g., glycosides according to embodiments of the present invention and examples provided), gene therapy products (oligonucleotides according to the present invention, such as BNAs), and targeting ligands or antibodies (e.g., sdAbs according to embodiments of the present invention and exemplified herein below in the Examples section) are all encompassed by the conjugates of the present invention. Thus, such conjugates of the present invention offer therapeutic opportunities for existing and future macromolecular drugs for a wide range of diseases and large patient populations. The application of such conjugates of the present invention, which comprise at least one saponin, at least one oligonucleotide, and at least one specific cell-targeting moiety, such as an immunoglobulin or sdAb, addresses an obvious problem with existing methods of separately applying an endosomal escape enhancer and a gene therapy product, which do not guarantee that both compounds are simultaneously present at the interaction site. 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 technique with increased synchronization (time and location) of both compounds, i.e., the saponin and the gene product, such as a BNA.

[0166] Gene therapy has the potential to help treat inherited, previously incurable diseases such as cystic fibrosis, cholera, Huntington's disease, or hemophilia. However, several current challenges remain: for example, therapeutic genes must be precisely delivered to specific target cells in the body. On the other hand, therapeutic genes must be absorbed by the targeted cells, but must not be destroyed. Existing gene therapy approaches use viruses as a vehicle for genes. However, these procedures involve significant risks and cannot be adapted for the introduction of other biomolecules. One embodiment is a conjugate of the present invention containing a (plant-derived) glycoside (e.g., any one of the saponins of the present invention) for use in platform technology, which, when incorporated by the conjugate as a carrier molecule, enables not only the delivery of genes but also the delivery of different therapeutic biomolecules to be introduced into target cells. Thus, the conjugate of the present invention can be used to develop nucleic acid-based therapies for cystic fibrosis, cholera, Huntington's disease, or hemophilia. In addition, the conjugates of the present invention enable novel gene therapy strategies to improve the health of patients with genetic diseases, including those 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 endosomal escape enhancer (glycoside; i.e., the saponin of the present invention), a gene therapy product, and a targeting ligand (i.e., an sdAb), all contained in a single conjugate. The resulting non-viral gene therapy based on the conjugates of the present invention exhibits an approximately 40-fold increase in delivery efficiency at lower dosages than currently available strategies. Additionally, the conjugates of the present invention are suitable for use in clinical applications, such as repairing or replacing defective genes, such as in cystic fibrosis patients, and for targeted delivery of specific genes to destroy cancer cells. Indeed, the conjugates of the present invention are suitable for application in treatment regimens for any disease caused by a genetic defect that is currently incurable, such as cystic fibrosis, Huntington's disease, and hemophilia.Gene therapy utilizing the conjugates of the present invention is useful in overcoming two existing problems: first, the conjugates of the present invention allow for the delivery of therapeutic genes to specific target cells in the body; second, the therapeutic gene enters these cells but is not destroyed due to the presence of a targeting moiety such as an antibody or sdAb for binding to the target cells, an oligonucleotide product and a saponin, all covalently linked together in the conjugates of the present invention by using, for example, an oligomeric or polymeric scaffold (covalent saponin conjugates of the present invention).

[0167] The present invention also provides a method of treating cancer, comprising 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 for administration are known in the art and include toxicity, solubility, route of administration, and maintenance of activity. For example, a pharmacological composition injected into the bloodstream should be soluble.

[0169] Suitable dosage forms depend, in part, on the use or route of entry, e.g., transdermal or by injection. Such dosage forms must allow the compound to reach the target cells, whether or not the target cells are present in a multicellular host. Other factors are known in the art and include considerations such as toxicity and dosage forms that delay the compound or composition from exerting its effect.

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

[0171] Surprisingly, the inventors have found that certain doses of ADC that do not result in the killing of any tumor cells are not sufficient to treat tumors in which such ADCs are targeted by tumor cell-targeting antibodies or VDCs. HH It has been found that when contacted with tumor cells in the presence of saponin coupled to ADC anti-CD71 V, it is sufficient and effective for tumor cell killing. Examples are provided in Figures 2-7. For example, a dose of ADC anti-CD71 V HH -Toxin, anti-HER2 V HH -toxin or anti-EGFR V HH ADCs, such as toxins, do not exert toxic effects on tumor cells when contacted with them. Typical toxins are protein toxins, such as dianthin and saporin. However, when such ADCs are co-administered with a conjugate containing a saponin, efficient tumor cell killing is achieved. The conjugate containing a saponin can be used to treat, for example, anti-HER2 V HH -SO1861, anti-CD71 V HH -SO1861, anti-EGFR V HH ADCs include, for example, V-type agonists capable of binding to HER2, CD71, or EGFR. HH For example, the ADC comprises a protein toxin such as dianthin or saporin. HHBy applying a conjugate containing the saponin, the dose of saponin required to achieve efficient biological activity of the effector molecule contained by the ADC, i.e., ADC or AOC, inside target cells is approximately 100-1000 times lower when the saponin-containing conjugate is co-administered with the ADC, i.e., ADC or AOC, to target cells compared to the dose of free saponin required when the ADC is co-administered with free saponin to target cells. In addition, the saponin-containing conjugate enhances the ADC, i.e., sdAb, containing the ADC or AOC, at doses of the ADC or AOC that would otherwise be ineffective on tumor cells when administered to a patient in need thereof in the absence of the targeting saponin. In addition, the saponin-containing conjugate is already sufficiently effective at relatively low doses when enhancement of the effector molecule of the ADC or AOC is considered, i.e., doses at which free saponin not provided with a tumor cell-targeting binding molecule such as an sdAb would not be sufficiently effective in enhancing the effector molecule. In combination, the inventors believe that ADCs or AOCs may already be capable of targeting tumor cell-targeting antibodies or VDCs. HHThis provides an improved method for treating human patients in need of treatment with an ADC or AOC comprising a therapeutically effective amount of a tumor cell-targeting sdAb, because the ADC or AOC is effective at a lower dose when co-administered to a patient in the presence of a conjugate comprising a saponin. One of the many advantages of this combination is the absence of an Fc tail in the ADC, and preferably the sdAb portion of the conjugate comprising a saponin. The absence of an Fc tail prevents unwanted binding of the conjugate to off-target patient cells that have Fc receptors, which could otherwise result in side effects when such an Fc tail is present, such as those seen with many conventional ADCs and AOCs. ADCs and AOCs based on antibodies that contain an Fc tail suffer from reduced efficacy due to unwanted binding of such IgG-based ADCs and AOCs to Fc receptors. As a result of Fc receptor binding, the effective dose of such IgG-based ADCs and AOCs is reduced. Additionally, the absence of an Fc tail provides several advantages in this regard. Off-target and undesired binding of ADCs, AOCs, and improved ADCs and improved AOCs of the present invention (i.e., conjugates of the present invention) to Fc receptors is not possible. In addition, the ADCs, AOCs, and conjugates of the present invention have lower effective doses when target receptor-mediated endocytosis and delivery of the ADCs, AOCs, and conjugates of the present invention to the inside of endosomes are considered because the conjugates are not "lost" due to Fc receptor binding, a defect seen in IgG-based conjugates. As a result, the therapeutic window of the ADCs, AOCs, and conjugates of the present invention, all of which comprise a cell-targeting sdAb instead of an Fc-containing antibody, is wider than the therapeutic window that would be achieved if the sdAb were replaced with a conventional IgG containing an Fc tail. Similarly, as a result, the therapeutic window of a conjugate containing a saponin is wider than the therapeutic window that would be achieved if the tumor cell-targeting sdAb and the sdAb in such a conjugate of saponin were replaced with a binding molecule for binding to a cell surface molecule on the target cell, such as a conventional IgG containing an Fc tail.

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

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

[0174] A fifth aspect of the present invention provides an in vitro or ex vivo method for translocating an effector molecule of the present invention from the exterior of a cell to the interior of said cell, preferably to the cytosol of said cell, comprising the steps of: a) providing a cell which expresses on its cell surface a binding site for at least one sdAb comprised by a conjugate of the invention, 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 comprising a genetic defect, cells comprising an enzyme defect and tumor cells; b) providing a conjugate of the invention, said conjugate comprising an effector molecule to be transferred to the cells provided in step a); and c) contacting the cells of step a) in vitro or ex vivo with the conjugate of step b) Including, Thus, the present invention relates to a method comprising the steps of translocating the conjugate comprising an effector molecule from the outside of a cell to the inside of the cell, and translocating the effector molecule from the outside of a cell to the inside of the cell, preferably to the cytosol of the cell, by translocation of the conjugate.

[0175] A sixth aspect of the present invention provides an in vitro or ex vivo method for transferring a conjugate of the present invention from the exterior of a cell to the interior of said cell, comprising: a) providing a cell which expresses on its cell surface a binding site for at least one sdAb comprised by a conjugate of the invention, 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 comprising a genetic defect, cells comprising an enzyme defect and tumor cells; b) providing a conjugate of the invention; and c) contacting the cells of step a) in vitro or ex vivo with the conjugate of step b), thereby translocating the conjugate from the outside of the cells to the inside of said cells.

[0176] [Table 1]

[0177] [Table 2]

[0178] [Table 3]

[0179] [Table 4]

[0180] An aspect of the invention relates to a kit comprising a container containing an endosomal escape-enhancing conjugate according to the invention, the kit further comprising instructions for using the conjugate.

[0181] Certain aspects of the present invention relate to any of the following ADCs and AOCs provided with at least one covalently linked saponin, which comprise a cell surface molecule targeting molecule (i.e., an sdAb) of the present invention and which comprise at least one effector moiety of the present invention or at least one saponin of the present invention resulting in an ADC or AOC, and semi-completed conjugates thereof, wherein the following antibody is an sdAb: Anti-EGFR antibody-saponin; anti-EGFR antibodies—triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleananes 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; 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 derived from cetuximab - triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde function at the C-23 position and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3 beta-OH group of the saponin; V of cetuximab H or V L sdAb-SO1861 derived from; V of cetuximabH or V L sdAb-GE1741 derived from; V of cetuximab H or V L sdAb-SA1641 derived from; V of cetuximab H or V L sdAb-Quil-A derived from; V of cetuximab H or V L sdAb-QS-21 derived from; V of cetuximab H or V L sdAb derived from -Saponin in the water-soluble saponin fraction of Quillaja saponaria; Anti-HER2 antibody-saponin; anti-HER2 antibodies—triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the 12,13-dehydrooleanan type 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; 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 sdAb-saponin derived from; V of trastuzumab H or V L sdAb derived from - triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde function at the C-23 position and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3 beta-OH group of the saponin; V of trastuzumab H or V L sdAb-SO1861 derived from; V of trastuzumab H or V L sdAb-GE1741 derived from; V of trastuzumab H or V L sdAb-SA1641 derived from; V of trastuzumab H or V L sdAb-Quil-A derived from; V of trastuzumab H or V L sdAb-QS-21 derived from; V of trastuzumab H or V L sdAb derived from -Saponin in the water-soluble saponin fraction of Quillaja saponaria; anti-CD71 antibody-saponin; anti-CD71 antibodies—triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the 12,13-dehydrooleanan type 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; Anti-CD71 antibody-SO1861; Anti-CD71 antibody-GE1741; Anti-CD71 antibody-SA1641; Anti-CD71 antibody-Quil-A; Anti-CD71 antibody-QS-21; Anti-CD71 antibody - Saponin in the water-soluble saponin fraction of Quillaja saponaria; OKT-9 V H or V L sdAb-saponin derived from; OKT-9 V H or V LsdAb derived from - triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde function at the C-23 position and optionally containing a glucuronic acid function in the carbohydrate substituent at the C-3 beta-OH group of the saponin; OKT-9 V H or V L sdAb-SO1861 derived from; OKT-9 V H or V L sdAb-GE1741 derived from; OKT-9 V H or V L sdAb-SA1641 derived from; OKT-9 V H or V L sdAb-Quil-A derived from; OKT-9 V H or V L sdAb-QS-21 derived from; OKT-9 V H or V L sdAb derived from -Saponin in the water-soluble saponin fraction of Quillaja saponaria; anti-EGFR antibody-oligonucleotide; anti-EGFR antibody-antisense oligonucleotide; Anti-EGFR antibody-siRNA; anti-EGFR antibody-antisense BNA; anti-EGFR antibody-antisense BNA (HSP27); Anti-EGFR antibodies-proteinaceous toxins; anti-EGFR antibody-ribosome-inactivating protein; Anti-EGFR antibody-Giantin; Anti-EGFR antibody-Saporin; V of cetuximab H or V L sdAb-oligonucleotides derived from; V of cetuximab H or V L sdAb-antisense oligonucleotides derived from; V of cetuximab H or V L sdAb-siRNA derived from; V of cetuximab H or V L sdAb-antisense BNA derived from; V of cetuximab H or V L sdAb-antisense BNA derived from (HSP27); V of cetuximab H or V L sdAb-proteinaceous toxins derived from; V of cetuximab H or V L sdAb-ribosome-inactivating protein derived from; V of cetuximab H or V L sdAb-gianthin 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-protein toxin; anti-HER2 antibody-ribosome-inactivating protein; Anti-HER2 antibody-Giantin; 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 L sdAb-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 derived from (HSP27); V of trastuzumab H or V L sdAb-proteinaceous toxins derived from; V of trastuzumab H or V L sdAb-ribosome-inactivating protein derived from; V of trastuzumab H or V L sdAb-gianthin 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-Giantin; anti-CD71 antibody-Saporin; OKT-9 V H or V L sdAb-oligonucleotides derived from; OKT-9 V H or V L sdAb-antisense oligonucleotides derived from; OKT-9 V H or V L sdAb-siRNA derived from; OKT-9 V H or V L sdAb-antisense BNA derived from; OKT-9 VH or V L sdAb-antisense BNA derived from (HSP27); OKT-9 V H or V L sdAb-proteinaceous toxins derived from; OKT-9 V H or V L sdAb-ribosome-inactivating protein derived from; OKT-9 V H or V L sdAb-gianthin derived from; OKT-9 V H or V L sdAb-saporin derived from; anti-EGFR antibody (-oligonucleotide) (-saponin) (wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), and the saponin is any one or more of a triterpenoid saponin and / or a bidesmosidic triterpene saponin belonging to the 12,13-dehydrooleanan type 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, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab); anti-EGFR antibody (-proteinaceous toxin) (-saponin) (the proteinaceous toxin is any one or more of ribosome-inactivating proteins, dianthins, and saporins, and the saponins are any one or more of triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the 12,13-dehydrooleanan type 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, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab); anti-HER2 antibody (-oligonucleotide) (-saponin) (wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), and the saponin is any one or more of a triterpenoid saponin and / or a bidesmosidic triterpene saponin belonging to the 12,13-dehydrooleanan type 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, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab); anti-HER2 antibody (-proteinaceous toxin) (-saponin) (the proteinaceous toxin is any one or more of ribosome-inactivating proteins, dianthins, and saporins, and the saponins are any one or more of triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the 12,13-dehydrooleanan type 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, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab); anti-CD71 antibody (-oligonucleotide) (-saponin) (wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), and the saponin is any one or more of a triterpenoid saponin and / or a bidesmosidic triterpene saponin belonging to the 12,13-dehydrooleanan type 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, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9); and Anti-CD71 antibody (-proteinaceous toxin) (-saponin) (The proteinaceous toxin is any one or more of ribosome-inactivating proteins, dianthins, and saporins, and the saponins are any one or more of triterpenoid saponins and / or bidesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type 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, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9).

[0182] In one embodiment, the cell surface molecule targeting molecule is selected from the group consisting of cetuximab, trastuzumab, OKT-9, and V. H or V L sdAb derived from (i.e., sdAb is the V of such monoclonal antibody H or V L 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 Quillaja saponaria, SO1861, GE1741, SA1641, Quil-A, QS-21 and Quillaja saponaria.

[0183] In one embodiment, the cell surface molecule targeting molecule is selected from the group consisting of cetuximab, trastuzumab, OKT-9, and V. H or V L sdAb derived from (i.e., sdAb is the V of such monoclonal antibody H or V Land 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 SO1861, GE1741, SA1641, Quil-A, QS-21 and saponin or a derivative thereof in the water-soluble saponin fraction of Quillaja saponaria.

[0184] One embodiment of the invention provides an ADC or AOC, or semi-completed ADC conjugate or semi-completed AOC conjugate of structure C, comprising a cell surface molecule targeting molecule sdAb of the invention and comprising at least one effector moiety of the invention and / or comprising at least one saponin of the invention: A(-S) b (-E) c (Structure C) (wherein A is a cell surface molecule-targeting 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 decimal point therebetween; c=0 to 8, preferably 0, 1, 2, 3, 4, 6, 8 or any integer or decimal point 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] In certain embodiments, 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, a triterpenoid saponin and / or bidesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functionality at the C-23 position and optionally containing a glucuronic acid functionality in the carbohydrate substituent at the C-3 beta-OH group of the saponin, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and Quillaja saponaria (Quillaja saponaria). and / or structure C of the present invention, wherein E is any one or more of the saponins in the water-soluble saponin fraction of Saccharomyces cerevisiae (Saccharomyces cerevisiae), and / or E is any one or more of an oligonucleotide, an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), and / or any one or more of a proteinaceous toxin, a ribosome-inactivating protein, a dianthin, and a saporin.

[0186] Certain embodiments include those in which the saponin (if present), and / or effector moiety (if present) are covalently coupled via at least one linker, such as a cleavable linker, and / or via a covalent saponin conjugate (i.e., at least one oligomeric or polymeric scaffold), such as linkers based on N-ε-maleimidocaproic acid hydrazide (EMCH), 3-(2-pyridyldithio)propanoic acid succinimidyl or 3-(2-pyridyldithio)propanoic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and covalent saponin conjugates (scaffolds) based on dendrons, such as G4-dendrons, or trifunctional linkers, such as the trifunctional linkers of structure A, and / or a cell surface molecule-targeting antibody, preferably a V HHand at least a lysine side chain and / or a cysteine ​​side chain of an sdAb such as is involved in a covalent bond to a saponin and / or effector moiety and / or linker and / or cleavable linker and / or covalent saponin conjugate, preferably wherein the saponin and / or effector moiety is covalently linked to a cell surface molecule targeting molecule, preferably an antibody such as an sdAb, and the covalent linkage comprises or consists of an amide bond, hydrazone bond, disulfide bond, Structure C of the invention, a conjugate of the invention or a semi-finished conjugate of the invention.

[0187] Certain aspects of the present invention relate to the use of any of the aforementioned conjugates, ADCs comprising a covalently linked saponin, AOCs comprising a covalently linked saponin, semi-completed ADCs, and semi-completed AOCs as a medicament.

[0188] One aspect of the present invention relates to the use of any of the above-described conjugates, ADCs comprising a covalently linked saponin, AOCs comprising a covalently linked saponin, semi-completed ADCs, and semi-completed AOCs for use in treating or preventing cancer or autoimmune diseases. [Example]

[0189] Examples and exemplary embodiments Example 1.V HH -SO1861 + mAb-saporin (1T2C and 2T2C) One-target, two-component systems (1T2C) are HH -SO1861 and mAb-protein toxin combination therapy, V HH The two-target, two-component system (2T2C) is a system that targets V HH -SO1861 and mAb-protein toxin combination therapy, V HH SO1861-EMCH recognizes and binds to a different cell surface receptor than mAbs (Figure 1B). SO1861-EMCH binds to the anti-HER2V receptor DAR1 via the (labile) terminal cysteine ​​residue (Cys). HHconjugated to (HER2V HH -SO1861). HER2V HH -SO1861 was titrated against a fixed concentration of 10 pM CD71mab-saporin (CD71 monoclonal antibody conjugated to a protein toxin, saporin, with a DAR of 4) or 50 pM trastuzumab-saporin (trastuzumab conjugated to a protein toxin, saporin, with a DAR of 4). ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + Targeted protein toxin-mediated cell killing against HER2V was determined in SK-BR-3 cells for both combinations with 10 pM CD71mab-saporin or 50 pM trastuzumab-saporin. HH The results showed that SO1861 enhanced cell death (IC50 = 300 nM; Figure 2A). HH -SO1861 alone induced cell death at high concentrations (IC50=4,000nM), while the same concentration of HER2V HH , HER2V HH +CD71mab-saporin or HER2V HH +Trastuzumab-saporin failed to induce cell killing activity (IC50>5000nM; Figure 2A). MDA-MB-468 (HER2 - / CD71 + ) in HER2V HH While the combination of SO1861 + 10 pM CD71mab-saporin revealed cell-killing activity at high concentrations (IC50 = 2,000 nM; Figure 2B), HER2V HH The combination of SO1861 and 50 pM trastuzumab-saporin demonstrated cell-killing activity at much higher concentrations (IC50 > 5,000 nM; Figure 2B). HH , HER2V HH +CD71mab-saporin or HER2V HH+Trastuzumab-saporin failed to induce cell killing activity in MDA-MB-468 cells (IC50>5,000 nM; Figure 2B).

[0190] All of this is due to the HER2-targeted V HH have shown that conjugation to enhances endosomal escape and cytoplasmic delivery of targeted protein toxins (targeting the same or different cell surface receptors), resulting in cell killing of HER2-expressing cells.

[0191] Next, trastuzumab-saporin or CD71mab-saporin was administered at 900 nM HER2V HH -Titered against a fixed concentration of SO1861 and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + We determined targeted protein toxin-mediated cell killing against HER2V 2 cells treated with 900 nM trastuzumab-saporin or CD71mab-saporin in combination with low concentrations of HER2V 2 cells. HH While SO1861 already induced efficient cell killing of SK-BR-3 (IC50 = 0.0001 pM; Figure 3A), CD71mab-saporin + 900 nM HER2V HH or trastuzumab-saporin + 900nM HER2V HH However, they were only able to induce cell death at high concentrations (IC50 = 50 pM; IC50 = 400 pM, respectively; Figure 3). - / CD71 + ) in CD71mab-saporin + 900nM HER2V HH -SO1861 demonstrated cell killing with IC50=0.01pM, while trastuzumab-saporin + 900nM HER2V HH -SO1861 showed activity with an IC50 of 2,000 pM. Trastuzumab-saporin + 900 nM HER2V HH or CD71mab-saporin + 900nM HER2V HHshowed cell killing only at IC50 > 10,000 pM and IC50 = 20 pM, respectively (Fig. 3B). This indicates that relatively low concentrations of trastuzumab-saporin or CD71mab-saporin inhibit the HER2 ++ / CD71 + Relatively low HER2V expression in expressing cells HH -SO1861(DAR1) in combination with other 5-HT-10 agonists is effective and can induce cell death.

[0192] Example 2.V HH -SO1861+V HH -Jianqing (2T2C) Two-target, two-component systems (2T2C) are HH 1-SO1861 and V HH It is a combination treatment of two protein toxins, each V HH recognizes a different cell surface receptor (Figure 1C). HH conjugated to the terminal cysteine ​​residue of HER2V HH -SO1861(DAR1) was generated. HER2V HH -SO1861, 50 nM CD71V HH -Titered against a fixed concentration of dianthine and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + ) was determined, which was shown to be effective against relatively low concentrations of V HH -HER2-L-SO1861 enhanced cell death (SK-BR-3; IC50 = 300 nM; Figure 4A). HH -SO1861 alone induced cell death at high concentrations (IC50=4,000nM), while the same concentration of HER2V HH , HER2V HH +50 pM CD71VHH-dianthin failed to induce cell death (IC50>5,000 nM; Figure 4A). - / CD71 +) in HER2V HH -SO1861+50pM CD71V HH -dianthine combination revealed cell-killing activity at higher concentrations (IC50 = 600 nM; Figure 4B), whereas HER2V HH , HER2V HH -SO1861 or HER2V HH +50pM CD71V HH -dianthin failed to induce cell killing activity (IC50>5,000 nM; Fig. 4B).

[0193] Next, CD71V HH -Dianthin, 900nM HER2V HH -Titered against a fixed concentration of SO1861 and SK-BR-3 (HER2 ++ / CD71 + ) and MDA-MB-468 (HER2 - / CD71 + We determined cell killing mediated by a targeted protein toxin against CD71V. HH - 900nM HER2V in combination with Gianthin HH -SO1861 induced efficient cell killing of SK-BR-3 cells (IC50 = 0.05 pM; Figure 5A), while CD71V HH Giantin or CD71V HH -Jianthin + 900nM HER2V HH CD71V could induce cell death only at high concentrations (IC50>10,000 pM; Fig. 5A). HH -Gianthin was also titrated against a fixed concentration of 77 nM trastuzumab-SO1861 (DAR4), which also inhibited SK-BR-3 (HER2 ++ / CD71 + ) cells (IC50<0.0001pM). - / CD71 + ) in CD71V HH -Jianthin + 900nM HER2V HH-SO1861 only showed cell killing at much higher concentrations (IC50 = 10 pM, Figure 5B), whereas CD71V HH -Jianqing, CD71V HH -Jianthin + 900nM HER2V HH or CD71V HH -Giantine + trastuzumab-SO1861 (DAR4) showed cell killing only with an IC50 of 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 the combination of HER2-SO1861 conjugate concentrations is effective and can induce cell death.

[0195] MDA-MB-468 cells (HER2 - / CD71 + The combination according to the present invention in (1) did not demonstrate any cell-killing activity, indicating that in the absence of sufficient receptor expression, the effective intracellular delivery concentration of SO1861 does not reach the threshold for inducing endosomal escape and cytoplasmic 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 same cell surface receptors (Figure 1E). HH - a combination therapy of a protein toxin, a mAb and a V HH recognizes a different cell surface receptor (Fig. 1D).

[0197] Gianthin-C (giantin with a terminal cysteine) was used to target HER2. HH , V targeting CD71HH or V targeting EGFR HH conjugated to the terminal cysteine ​​residue of HER2V HH -Jiantin (DAR1), CD71V HH -Giantin (DAR1) and EGFRV HH -Gianthin (DAR1) was produced.

[0198] CD71V HH -Jianqing, HRE2V HH -Dianthin or EGFRV HH -Gianthin was titrated against a fixed concentration of cetuximab-SO1861 (DAR4) and A431 (EGFR ++ / HER2 + / - / CD71 + ) and A2058 (EGFR - / HER2 + / - / CD71 + We determined targeted protein toxin-mediated cell killing against CD71V in combination with 77 nM cetuximab-SO1861. HH -gianthin induced efficient cell killing of A431 cells (IC50<0.0001 pM; Figure 6A), while CD71V HH -Dianthin alone showed activity with IC50=2000pM. The other two combinations showed activity against EGF-1. HH -Giantine + 77nM Cetuximab -SO1861 and HER2V HH -Giantine + 77nM cetuximab -SO1861 showed efficient cell killing with IC50=20pM and IC50=50pM, respectively, while EGFRV HH -Giantin or HER2V HH -Dianthine alone failed to induce efficient cell killing in A431 cells (IC50>10,000 pM; Figure 6A). - / HER2 + / - / CD71 + ) in CD71V HH -Giantin and CD71V HH- Gianthin + 77 nM cetuximab - SO1861 showed cell killing activity with IC50 = 3,000 pM and IC50 = 1,000 pM, respectively, while all other treatments or combinations showed cell killing activity with IC50 = 10,000 pM V in A2058 cells. HH -toxin did not cause cell death (Figure 6B).

[0199] This indicates that cetuximab-SO1861 (DAR4) is a potent anti-cancer drug with three different V HH This shows that efficient induction of endosomal escape of the -dianthine conjugate can induce enhanced cell death in A431 cells.

[0200] Next, CD71V HH -Jianqing, HRE2V HH -Dianthin or EGFRV HH - Gianthin was titrated against a fixed concentration of trastuzumab-SO1861 (DAR4) and SK-BR-3 (HER2 ++ / EGFR = / CD71 + ) and MDA-MB-468 cells (HER2 - / EGFR ++ / CD71 + We determined targeted protein toxin-mediated cell killing against CD71V at very low concentrations in combination with 77 nM trastuzumab-SO1861. HH -gianthin induced efficient cell killing of SK-BR-3 cells (IC50<0.0001 pM; Figure 7A), while CD71V HH -Dianthin alone showed activity with an IC50 of 10,000 pM. The other two combinations showed activity against EGF-1. HH -Giantine + 77nM trastuzumab -SO1861 and HER2V HH -Giantine + 77nM trastuzumab -SO1861 showed efficient cell killing with IC50=400pM and IC50=6pM, respectively, while EGFRV HH -Giantin or HER2V HH-Dianthine alone failed to induce efficient cell killing in SK-BR-3 cells (IC50>10,000 pM; Figure 7A). - / EGFR ++ / CD71 + ) in CD71V HH -Giantin and CD71V HH - Gianthin + 77 nM cetuximab - SO1861 showed cell killing activity with IC50 = 3000 pM and IC50 = 2000 pM, respectively, while all other treatments or combinations showed cell killing activity with IC50 = 10,000 pM V in MDA-MB-468 cells. HH -giantin showed no cell killing until trastuzumab-SO1861 (DAR4) was administered to three different V HH This shows that efficient induction of endosomal escape of the -dianthine conjugate can induce enhanced cell death in SK-BR-3 cells.

[0201] Materials and Methods material SO1861 was isolated and purified by Analyticon Discovery GmbH from a crude plant extract obtained from Saponaria officinalis. HH QVQ, Utrecht, The Netherlands (HER2V HH :Clone name: Q17c;CD71V HH :Clone name:Q52c EGFRV HHAnti-CD71mab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA). Dianthine-Cys (Dia-Cys, a dianthine variant with a single C-terminal cysteine) was manufactured by Proteogenix, France. Trastuzumab (Tras, Herceptin®, Roche) and cetuximab (Cet, Erbitux®, Merck KGaA) were purchased from a pharmacy (Charite, Berlin). CD71 monoclonal antibody was purchased from BioCell (Okt9, #BE0023). Custom trastuzumab-saporin and anti-CD71mab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA). Dianthine-Cys (Dia-Cys, a dianthine variant 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 columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standard solution (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), 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), polyethylene glycol sorbitan monolaurate (TWEEN® 20, Sigma-Aldrich), Dulbecco's phosphate-buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-N a2, 99%, Sigma-Aldrich), sterile filters 0.2 μm and 0.45 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP, Thermo-Fisher), HSP27 BNA disulfide 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), Ultrapure Lab Water Deionized water (DI) was obtained freshly from Systems (MilliQ, Merck), nickel-nitrilotriacetic acid agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5-dithiobis(2-nitrobenzoic acid) (Ellman's reagent, DTNB, 98%, Sigma-Aldrich), S-acetylmercaptosuccinic anhydride fluorescein (SAMSA reagent, Invitrogen), sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), a PD MiniTrap desalting column (GE Healthcare) with Sephadex G-25 resin, and a PD10 G25 desalting column (GE Healthcare).Zeba spin desalting columns (Thermo-Fisher) in 5, 2, 5, and 10 mL volumes, Vivaspin centrifugal filters T4 10 kDa MWCO, T4 100 kDa MWCO, and T15 (Sartorius), Biosep s3000 aSEC columns (Phenomenex), Vivacell ultrafiltration units 10 and 30 kDa MWCO (Sartorius), and 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) was added methanol (extra-dried, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was subjected to preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS: 96%. LRMS(m / z):2069[M-1] 1- LC-MS rt (min): 1.08 4

[0204] Cell viability assay After treatment, cells were incubated at 37°C for 72 hours, after which cell viability was determined by an MTS assay performed according to the manufacturer's instructions (CellTiter 96® AQueous One-Solution Cell Proliferation Assay, Promega). Briefly, the MTS solution was diluted 20-fold in DMEM without phenol red (PAN-Biotech GmbH) supplemented with 10% FBS. After washing the cells once with 200 μL / well of PBS, 100 μL of the diluted MTS solution was added per well. The plate was incubated at 37°C for approximately 20-30 minutes. The OD at 492 nm was then measured on a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" well was subtracted from all other wells, and the percentage of cell viability of treated / untreated cells was calculated by dividing the background-corrected signal of the treated wells by the background-corrected signal of the untreated wells (x100).

[0205] FACS analysis Cells were seeded at 500,000 cells / plate in 10 cm dishes 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 they reached 90% confluency. Cells were then trypsinized (TryplE Express, Gibco Thermo Scientific) to single cells. 0.75x10 6 The cells were transferred to a 15 mL Falcon tube and centrifuged (1,400 rpm, 3 min). The supernatant was discarded, leaving the cell pellet submerged. The pellet was dissociated by gently tapping the Falcon tube on a vortex shaker, and the cells were resuspended in 4 mL of cold PBS (with Mg 2+ and Ca 2+ After washing, the cells were resuspended in 3 mL of cold PBS (Mg-free, 2% FBS). 2+and Ca 2+ The cells were resuspended in 2% FBS (free, 2% FBS) and divided equally into three round-bottom FACS tubes (1 mL / tube). The cells were centrifuged again and resuspended in 200 μL of cold PBS (Mg 2+ and Ca 2+ free, 2% FBS) or 195 μL of cold PBS (Mg 2+ and Ca 2+ The cells were resuspended in 200 μL antibody solution containing 5 μL of antibody in 2% FBS (free). EGFR receptors were stained using APC mouse IgG1, kappa APC anti-human EGFR (#352906, Biolegend). HER2 receptors were stained using PE anti-human HER2 APC anti-human CD340 (erbB2 / HER-2) (#324408, Biolegend), with PE mouse IgG2a, kappa isotype Ctrl FC (#400212, Biolegend) used as a matching isotype control. CD71 receptors were stained using PE anti-human CD71 (#334106, Biolegend), with PE mouse IgG2a, kappa isotype Ctrl FC (#400212, Biolegend) used as a matching isotype control. Samples were incubated on a tube roller mixer at 4°C for 30 minutes. Cells were then resuspended in cold PBS (Mg 2+ and Ca 2+ The cells were washed three times with PBS (free, 2% FBS) and fixed for 20 minutes at room temperature using a 2% PFA solution in PBS. The cells were washed twice with cold PBS and resuspended in 250-350 μL of cold PBS for FACS analysis. Samples were analyzed using a BD FACSCanto II flow cytometry system (BD Biosciences) and FlowJo software. The results of the analysis of cell surface expression of EGFR, HER2, and CD71 on various cells are summarized in Table A2.

[0206] [Table 5]

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

[0208] After incubation, an aliquot of V HH The -SO1861 mixture was removed and characterized by Ellman's assay to confirm incorporation of SO1861. The conjugate was purified on a 1.6 x 35 cm Superdex 200PG column eluted with DPBS pH 7.5 to give purified V HH An aliquot was filtered to 0.2 μm, concentrated, and standardized to 1.0 mg / ml to give V HH -SO1861 was obtained.

[0209] V HH -Procedure for conjugation of dianthine Gianthin-Cys was concentrated by ultrafiltration using a Vivaspin T15 10 KDa MWCO centrifugal filter and buffer-exchanged into TBS pH 7.5. An aliquot of freshly prepared TCEP solution (10.0 mg / ml) was added to the concentrated Gianthin-Cys, and the mixture was vortexed briefly, followed by incubation at 20°C for 60 minutes with roller mixing. After incubation, the resulting Gianthin-SH was purified by gel filtration using a Zeba spin desalting column, followed by repeated centrifugal wash cycles using a Vivaspin T15 10 KDa MWCO centrifugal filter to TBS pH 7.5. The resulting Gianthin-SH was reacted with freshly prepared DTME solution (10 mg / ml) in DMSO, and the mixture was vortexed briefly, followed by incubation at 20°C for 60 minutes. Gianthin-DTME was then obtained after purification by gel filtration using a Zeba spin desalting column to TBS pH 7.5. Dianthine-DTME was stored at 20°C until conjugation. HH was concentrated by ultrafiltration using a Vivaspin T15 10 KDa MWCO centrifugal filter and buffer exchanged into TBS pH 7.5. HH An aliquot of freshly prepared TCEP solution (10.0 mg / ml) was added to the mixture, which was vortexed briefly and then incubated for 60 minutes at 37°C with roller mixing. After incubation, the resulting V HH was purified by gel filtration using a Zeba spin desalting column, followed by repeated centrifugal wash cycles in TBS pH 7.5 using Vivaspin T4 5KDa MWCO centrifugal filters. HH -SH was reacted with dianthine-DTME, and the mixture was vortexed briefly followed by overnight incubation at 20° C. The reaction mixture was then concentrated using a Vivaspin T4 10 KDa MWCO centrifuge tube and purified by gel filtration using a 1.6 × 35 cm Superdex 200PG column eluted with DPBS pH 7.5.

[0210] Antibody-(L-SO1861)4 Trastuzumab and cetuximab are subsequently referred to as "Ab." The Ab was conjugated to the saponin SO18161-EMCH via a Michael-type thiol-ene conjugation reaction at DARs of 1, 2, 3, 4, 5, and 6. The SO1861-EMCH molecule obtains a labile (L) hydrazone bond between its structure and its maleimide functional group, creating a labile bond between the saponin and Ab. The procedure is exemplarily described for trastuzumab-(L-SO1861)4.

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

[0212] To four portions of cetuximab (9.73 mg, 4.864 mg / mL, 65 nmol, respectively), aliquots of freshly prepared TCEP solution (0.5–2.0 mg / mL, 1.15–7.02 molar equivalents, 75–455 nmol) were added. The mixtures were briefly vortexed and then incubated at 20°C for 300 min with roller mixing. After incubation (before the addition of SO1861-EMCH), approximately 1 mg (0.210 mL) aliquots of Ab-SH were removed from each mixture and purified by gel filtration using Zeba spin desalting columns in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-Ab ratios = 2.0, 4.2, 5.9, and 6.8, respectively). To each bulk Ab-SH, an aliquot of freshly prepared SO1861-EMCH solution (2 mg / ml, 1.3 molar equivalents per "thiol," 0.15–0.61 μmol, 0.16–0.63 ml) was added, and the mixture was vortexed briefly and then incubated for 120 min at 20 °C. In addition to each conjugation reaction, two aliquots of desalted Ab-SH (0.25 mg, 1.67 nmol) were reacted with NEM (1.3 molar equivalents per "thiol," 4.3–17.4 nmol, 2.2–8.7 μl of a 0.25 mg / ml solution) or TBS pH 7.5 buffer (2.2–8.7 μl) as positive and negative controls, respectively, for 120 min at 20 °C. After incubation (before the addition of NEM), a 0.200 ml aliquot of the Ab-SO1861-EMCH mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of SO1861-EMCH. To the bulk Ab-SO1861-EMCH mixture, an aliquot of freshly prepared NEM solution (2.5 mg / ml, 2.5–10 molar equivalents, 0.15–0.58 μmol) was added, and the mixture was purified using a Zeba spin desalting column eluted with DPBS pH 7.5 to obtain the purified cetuximab-(L-SO1861) conjugate. The product was standardized to 2.5 mg / ml, filtered to 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 Table A3 and Table A4.

[0213] [Table 6]

[0214] [Table 7]

[0215] material Throughout the specification, claims and drawings, "VHH", "Vhh", "V hh " and "V HH " should be understood as referring to single domain antibodies of the same type. h " and "V H The same is true for single domain antibodies of the type referred to as either "antibodies" or "antibodies with a specific structure."

[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 columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standard solution (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), 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), polyethylene glycol sorbitan monolaurate (TWEEN® 20, Sigma-Aldrich), Dulbecco's phosphate-buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA-N a2, 99%, Sigma-Aldrich), sterile filters 0.2 μm and 0.45 μm (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), Ultrapure Lab Water Freshly obtained deionized water (DI) from 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), sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), PD MiniTrap desalting columns with Sephadex G-25 resin (GE Healthcare), PD10 G25 desalting columns (GE Healthcare), Zeba spin desalting columns (Thermo-Fisher) in 0.5, 2, 5, and 10 mL volumes, 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), acrylamide (99.The reagents used were sodium dodecyl sulfate (98%, Sigma-Aldrich), ammonium persulfate (APS, 98%, Sigma-Aldrich), glycerol (99%, Sigma-Aldrich), bromophenol blue (Sigma-Aldrich), and 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 These HSP27-Mal derivatives were: 1) Mal-trifunctional linker-(L-SO1861)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was performed to identify HER2-VHH -[S-trifunctional linker-(L-SO1861)-(L-HSP27 BNA)] is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then 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 These HSP27-Mal derivatives were: 1) Mal-trifunctional linker-(S-SO1861)-(L-HSP27 BNA), 2) Mal-trifunctional linker-(blocking)-(L-HSP27 BNA). The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-SO1861)-(L-HSP27 BNA)] is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then 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 HHare subsequently referred to as "Ab." The Ab was conjugated to two different maleimides (Mal) bearing HSP27 BNA derivatives, subsequently referred to as "HSP27-Mal," via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were: 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: 4, 8, or more than 8. The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-dendron-(L-SO1861)4)-(L-HSP27 BNA)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then aliquoted for biological evaluation.

[0223] 4.V HH -[S-trifunctional linker-(L-QS21)-(L-HSP27 BNA)] 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 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 performed to identify HER2-V HH -[S-trifunctional linker-(L-QS21)-(L-HSP27 BNA)] is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then aliquoted for biological evaluation.

[0225] 5.V HH -[S-trifunctional linker-(S-QS21)-(L-HSP27 BNA)] 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 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 performed to identify HER2-V HH -[S-trifunctional linker-(S-QS21)-(L-HSP27 BNA)] is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then 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 HHThese are hereafter referred to as "Ab." The Ab was conjugated to two different maleimides (Mal) bearing HSP27 BNA derivatives, hereafter referred to as "HSP27-Mal," via a Michael-type thiol-ene reaction. These HSP27-Mal derivatives were: 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: 4, 8, or more than 8. The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-dendron-(L-QS21)4)-(L-HSP27 BNA)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of HSP27 BNA-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per "thiol," 40 nmol and 43 nmol, respectively). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-HSP27 BNA derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and Ellman's assay alongside positive and negative controls to obtain 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 a 1.6 x 30 cm Sephadex G50M gel eluted 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. The product was filtered to 0.2 μm and then aliquoted for biological evaluation.

[0229] 7.V HH -[S-trifunctional linker-(L-SO1861)-(L-dianthine)]4 HER2-V HH -[S-tri-(L-SO1861)-(L-dianthin)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(L-SO1861)-(L-dianthin)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-V HH -[S-tri-(L-SO1861)-(L-dianthin)]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) bearing dianthine derivatives, later referred to as "dianthine-Mal," via a Michael-type thiol-ene reaction. These dianthine-Mal derivatives were: 1) Mal-trifunctional linker-(L-SO1861)-(L-dianthine), 2) Mal-trifunctional linker-(blocking)-(L-dianthine). The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(L-SO1861)-(L-dianthine)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of the dianthine-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per thiol, 40 nmol and 43 nmol). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-dianthine derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of dianthine 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 a 1.6 x 30 cm Sephadex G50M column eluted 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.The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0231] 8.V HH -[S-trifunctional linker-(S-SO1861)-(L-dianthine)]4 HER2-V HH -[S-tri-(S-SO1861)-(L-dianthin)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(S-SO1861)-(L-dianthine)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-V HH -[S-tri-(S-SO1861)-(L-dianthin)]4, CD71-V HH -[S-tri-(blocking)-(L-dianthine)]4, HER2-V HH , EGFR-V HH , and CD71-V HH (hereinafter referred to as "Ab"). Ab was conjugated to two different maleimides (Mal) bearing dianthine derivatives, later referred to as "dianthine-Mal", via a Michael-type thiol-ene reaction. These dianthine-Mal derivatives were: 1) Mal-trifunctional linker-(S-SO1861)-(L-dianthine), 2) Mal-trifunctional linker-(blocking)-(L-dianthine). The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-SO1861)-(L-dianthine)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of the dianthine-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per thiol, 40 nmol and 43 nmol). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-dianthine derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of dianthine 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 a 1.6 x 30 cm Sephadex G50M column eluted 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.The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0233] 9.V HH -[S-trifunctional linker-(S-dendron-(L-SO1861) n )-(L-dianthin)]4 HER2-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-dianthin)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-dianthine)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-V HH -[S-tri-(S-dendron-(L-SO1861)n)-(L-dianthin)]4, CD71-V HH -[S-tri-(blocking)-(L-dianthine)]4, HER2-V HH , EGFR-V HH , and CD71-V HH are subsequently referred to as "Ab." Ab was conjugated to two different maleimides (Mal) bearing dianthine derivatives, subsequently referred to as "dianthine-Mal," via a Michael-type thiol-ene reaction. These dianthine-Mal derivatives were: 1) Mal-trifunctional linker-(S-dendron-(L-SO1861)n)-(L-dianthine), 2) Mal-trifunctional linker-(blocked)-(L-dianthine). "n" refers to the number of SO1861 molecules: 4, 8, or more than 8. The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-dendron-(L-SO1861)4)-(L-dianthine)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of the dianthine-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per thiol, 40 nmol and 43 nmol). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-dianthine derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of dianthine 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 a 1.6 x 30 cm Sephadex G50M column eluted 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.The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0235] 10.V HH -[S-trifunctional linker-(L-QS21)-(L-dianthine)]4 HER2-V HH -[S-tri-(L-QS21)-(L-dianthin)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(L-QS21)-(L-dianthine)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-V HH -[S-tri-(L-QS21)-(L-dianthin)]4, CD71-V HH -[S-tri-(blocking)-(L-dianthine)]4, HER2-V HH , EGFR-V HH , and CD71-V HH (L-QS21)-(L-Gianthine), 2) Mal-trifunctional linker-(blocking)-(L-Gianthine). The procedure was performed to conjugate the Ab to two different maleimides (Mal) bearing dianthine derivatives, later referred to as "Dianthine-Mal", via a Michael-type thiol-ene reaction. These dianthine-Mal derivatives were: 1) Mal-trifunctional linker-(L-QS21)-(L-Gianthine), 2) Mal-trifunctional linker-(blocking)-(L-Gianthine). The procedure was performed to conjugate the Ab to two different maleimides (Mal) bearing dianthine derivatives, later referred to as "Dianthine-Mal", via a Michael-type thiol-ene reaction. HH -[S-trifunctional linker-(L-QS21)-(L-dianthine)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was 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), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of the dianthine-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per thiol, 40 nmol and 43 nmol). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-dianthine derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of dianthine 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 a 1.6 x 30 cm Sephadex G50M column eluted 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.The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0237] 11.V HH -[S-trifunctional linker-(S-QS21)-(L-dianthine)] HER2-V HH -[S-tri-(S-QS21)-(L-dianthine)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(S-QS21)-(L-dianthine)]4, EGFR-V HH -[S-tri-(blocking)-(L-dianthine)]4, CD71-VHH-[S-tri-(S-QS21)-(L-gianthine)]4, CD71-VHH-[S-tri-(blocking)-(L-gianthine)]4, HER2-V HH , EGFR-V HH , and CD71-V HH These Abs were conjugated to two different maleimides (Mal) bearing dianthine derivatives, later referred to as "dianthine-Mal," via a Michael-type thiol-ene reaction. These dianthine-Mal derivatives were: 1) Mal-trifunctional linker-(S-QS21)-(L-dianthine), 2) Mal-trifunctional linker-(blocking)-(L-dianthine). The procedure was performed to identify HER2-V HH -[S-trifunctional linker-(S-QS21)-(L-dianthine)]4 is illustratively described: The Ab was reconstituted to 21 mg / ml in deionized (DI) water 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) was added to obtain 50 mM TBS, 2.5 mM EDTA buffer, pH 7.5.

[0238] To Ab (2.1 mg, 0.5 mg / ml, 0.14 μmol), an aliquot 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 with roller mixing. After incubation (before addition of the construct), an 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 in TBS pH 7.5. These aliquots were characterized by UV-vis analysis and Ellman's assay (thiol-to-ab ratio = 4.0). Bulk Ab-SH was divided into two aliquots (0.11 mg, 7.6 nmol and 0.12 mg, 8.3 nmol). To each aliquot was added an aliquot of each of the dianthine-Mal derivatives 1 and 2 (freshly prepared in TBS pH 7.5 at 2 mg / ml, 1.3 molar equivalents per thiol, 40 nmol and 43 nmol). The mixture was vortexed briefly and then incubated for 120 min at 20°C. In addition to the conjugation reaction of Ab-dianthine derivative 2, two 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 a 0.25 mg / ml solution) or TBS pH 7.5 buffer (6.7 μl) as positive and negative controls, respectively, for 120 min at 20°C. After incubation (before the addition of NEM), a 0.100 ml aliquot of the Ab-Construct 2 mixture was removed and purified by gel filtration using a Zeba spin desalting column in TBS pH 7.5. This aliquot was characterized by UV-vis and by Ellman's assay alongside positive and negative controls to determine the incorporation of dianthine 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 a 1.6 x 30 cm Sephadex G50M column eluted 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.The product was filtered to 0.2 μm before being aliquoted for biological evaluation.

[0239] 12.V HH -[S-trifunctional linker-(S-dendron-(L-QS21) n )-(L-dianthin)]4 HER2-V HH -[S-tri-(S-dendron-(L-QS21)n)-(L-dianthin)]4, HER2-V HH -[S-tri-(blocking)-(L-dianthine)]4, EGFR-V HH -[S-tri-(S-dendron-(L-QS21)n)-(L-dianthine)]4, EGFR-V HH -[S-tri-(bl...

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 the cell; Single domain antibodies, further referred to as sdAbs; saponin, said effector molecule, said sdAb, and said saponin are covalently linked; said effector molecule being an oligonucleotide; The saponin is a bi-desmoside triterpene glycoside containing an aglycone core structure selected from quillaric acid or gypsogenin; the saponin comprises a first glycan attached to the C3 atom of the aglycone core structure and a second glycan attached to the C28 atom of the aglycone core structure; the first glycan is branched and comprises a glucuronic acid unit; the second glycan is branched and comprises one or more carbohydrate units selected from fucose (Fuc), rhamnose (Rha) and quinovose (Qui); A conjugate, wherein said sdAb is capable of binding to a cell surface molecule of said cell.

2. V derived from the heavy chain of an antibody Ｈ domain, V derived from the light chain of an antibody Ｌ Domain, and V derived from heavy chain-only antibodies (HCAbs) of Camelidae origin or Ig-NAR origin ＨＨ domain 2. The conjugate of claim 1, comprising at least one sdAb selected from any one or more of:

3. 3. The conjugate of claim 1 or 2, comprising at least two sdAbs, at least one of said at least two sdAbs being covalently bound to said effector molecule and / or said saponin.

4. The conjugate of claim 3, wherein the at least two sdAbs are the same sdAb.

5. A conjugate according to any one of claims 1 to 4, comprising 1 to 8 sdAbs capable of binding to the same binding site on a cell surface molecule.

6. A conjugate described in any one of claims 1 to 5, wherein at least one of the sdAbs is capable of binding to CD71.

7. A conjugate according to any one of claims 1 to 6, wherein the sdAb is capable of binding to a tumour cell surface receptor which is a tumour cell specific receptor.

8. The tumor cell specific receptor is CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, prostate specific membrane antigen (PSMA), CanAg, integrin alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, D 8. The conjugate of claim 7, selected from any one or more of LL3, 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).

9. The conjugate of claim 7 or 8, wherein the tumor cell-specific receptor is selected from HER2, c-Met, VEGFR2, CXCR7, CD71 and EGFR1.

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

11. 11. The conjugate of claim 10, wherein the conjugate further comprises an additional, different sdAb that binds albumin.

12. 12. The conjugate of claim 11 , wherein the further different sdAb comprises any one or more of the amino acid sequences of SEQ ID NOs: 33, 34 and 35.

13. The oligonucleotide may be a short interfering RNA (siRNA), a short hairpin RNA (shRNA), an anti-hairpin microRNA (miRNA), a single stranded RNA, an aptamer RNA, a double stranded RNA (dsRNA), an anti-microRNA (anti-miRNA, anti-miR), an antisense oligonucleotide (ASO), an mRNA, a DNA, an antisense DNA, a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a 2'-O,4'-aminoethylene bridged nucleic acid (BNA), or a combination thereof. ＮＣ ), BNA-based siRNA, and BNA-based antisense oligonucleotide (BNA-AON).

14. The conjugate of claim 13 , wherein the oligonucleotide is selected from a chemically modified siRNA, a metabolically stable siRNA, and a chemically modified and metabolically stable siRNA.

15. 15. The conjugate of any one of claims 1 to 14, wherein the oligonucleotide is capable of silencing 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).

16. The conjugate according to any one of claims 1 to 15, wherein the 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.

17. 17. The conjugate of any one of claims 1 to 16, wherein the saponin comprises an aglycone core structure that is quinoline.

18. 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-; 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 18. The conjugate of any one of claims 1 to 17, selected from 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.

19. The saponin is i. the aldehyde groups in the aglycone core structure are derivatized; ii. A carboxyl group of the glucuronic acid moiety in the first glycan is derivatized; and iii. At least one acetoxy (Me(CO)O-) group in the second glycan is derivatized; The conjugate according to any one of claims 1 to 18, wherein

20. The at least one saponin is i. - reduction to alcohol; - conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH); - conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH); or - an aglycone core structure containing an aldehyde group that has been derivatized by conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid] hydrazide (KMUH); ii. a first glycan comprising a carboxyl group that has been derivatized by conversion to an amide bond via reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); iii. a second glycan containing an acetoxy group (Me(CO)O-) that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. Any combination of two or three derivatizations i., ii., and / or iii. The conjugate according to any one of claims 1 to 19, comprising:

21. The saponin is NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP -017888, NP-017889, NP-018108, SA1641, NP-017674, NP-017810, AG1, NP-003881, NP -017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, S O1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, QS-7, QS1861, QS-7 21. The conjugate of any one of claims 1 to 20, which is any one or more of: api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, QS-21, QS-21A, QS-21B, or stereoisomers thereof, and / or any combination thereof.

22. The conjugate according to any one of claims 1 to 21, wherein the saponin is any one or more of QS-21, SO1861, SA1641 and GE1741.

23. The conjugate according to any one of claims 1 to 22, wherein the saponin is SO1861.

24. The sdAb is an anti-CD71 antibody OKT-9, trastuzumab, pertuzumab, rituximab, ofatumumab, tositumomab, obinutuzumab ibritumomab, oregovomab, edrecolomab, cetuximab, matuzumab, panitumumab, nimotuzumab, brentuximab, gemtuzumab, huMy9-6, etaracizumab, alemtuzumab, 24. The conjugate of any one of claims 1 to 23, derived from or based on any one or more of the immunoglobulins: platuzumab, pinatuzumab, moxetumomab, inotuzumab, labetuzumab, bivatuzumab, sibrotuzumab, anti-CD19 antibody huB4, anti-CanAg antibody huC242, anti-CD56 antibody huN901, anti-CD38 antibody OKT-10, daratumumab, anti-CA6 antibody DS6, anti-IGF-1R antibody 3B7, cizutumumab, anti-integrin antibody CNTO 95, anti-syndecan-1 antibody B-B4, and polatuzumab.

25. 25. The conjugate of any one of claims 1 to 24, wherein the sdAb is derived from or based on any one or more of the immunoglobulins of trastuzumab, pertuzumab, cetuximab, matuzumab, and the anti-CD71 antibody OKT-9.

26. 26. The conjugate of any one of claims 1 to 25, wherein the effector molecule is covalently attached to the sdAb directly or via a linker.

27. 27. The conjugate of any one of claims 1 to 26, wherein the saponin is covalently attached to the sdAb and / or the effector molecule directly or via a linker.

28. 28. The conjugate of any one of claims 1 to 27, wherein the conjugate comprises a trifunctional linker, and the sdAb, the saponin and the effector molecule are separately covalently attached to the trifunctional linker, either directly or via a linker.

29. The saponin is a C-type saponin comprising the aglycone core structure of the saponin. ２３ 29. The conjugate of any one of claims 1 to 28, 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 N-ε-maleimidocaproic acid hydrazide (EMCH).

30. The saponin is a C-type saponin comprising the aglycone core structure of the saponin. ３ 30. The conjugate of any one of claims 1 to 29, which is a bi-desmosidic saponin comprising a glucuronic acid unit in a first glycan linked at a beta-OH group, said saponin being covalently attached to an amino acid residue of said at least one sdAb and / or said at least one effector molecule via a carboxyl group of said glucuronic acid unit in said first glycan.

31. The at least one saponin has a C ３ 31. The conjugate of claim 30, comprising a glucuronic acid unit in the first saccharide chain at a beta-OH group, the glucuronic acid unit being covalently attached to a linker.

32. 32. The conjugate of any one of claims 1 to 31, further comprising an oligomeric or polymeric molecule attached to said sdAb or said effector molecule, said conjugate comprising one or more saponins.

33. 33. The conjugate of claim 32, wherein the oligomeric or polymeric molecule is dendron-based.

34. The conjugate of any one of claims 1 to 33, wherein the saponin is covalently attached via a cleavable linker.

35. The conjugate of claim 34, wherein the cleavable linker is subject to cleavage under acidic conditions.

36. 36. The conjugate of claim 34 or 35, wherein the cleavable linker is subject to cleavage at a pH of ≦5.

5.

37. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 36 as an active medical ingredient.

38. 38. The pharmaceutical composition of claim 37 for treating any one or more human diseases selected from 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.

39. The pharmaceutical composition of claim 37 or 38, wherein the saponin is SO1861 or QS-21.

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