Improved antibodies - oligonucleotide conjugates

Abstract

To provide improved biologically active compounds or compositions comprising such improved biologically active compounds, in certain embodiments of the present invention.SOLUTION: The present invention relates to a ligand-effector moiety provided with at least one saponin and an antibody-effector moiety provided with at least one saponin. One embodiment of the invention is a composition comprising a ligand-effector moiety provided with at least one saponin or an antibody-effector moiety provided with at least one saponin of the invention. The present invention also relates to antibody-drug conjugates comprising a covalently linked saponin and antibody-oligonucleotide conjugates comprising a covalently linked saponin. One aspect of the present invention relates to a pharmaceutical composition comprising at least one saponin-provided ligand-effector moiety or at least one saponin-provided antibody-effector moiety of the present invention, and optionally further comprising a pharmaceutically acceptable excipient. The present invention also relates to a ligand-effector moiety provided with at least one saponin or an antibody-effector moiety provided with at least one saponin for use as a medicament. The present invention also relates to a ligand-effector moiety provided with at least one saponin or an antibody-effector moiety provided with at least one saponin of the present invention for use in the treatment or prevention of cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to ligand-effector moieties provided with at least one saponin and antibody-effector moieties provided with at least one saponin, such as antibody-drug conjugates and antibody-oligonucleotide conjugates. One embodiment of the present invention relates to a composition comprising a ligand-effector moiety provided with at least one saponin of the present invention or an antibody-effector moiety provided with at least one saponin of the present invention. The present invention also relates to antibody-drug conjugates comprising a covalently linked saponin and antibody-oligonucleotide conjugates comprising a covalently linked saponin. One embodiment of the present invention relates to a pharmaceutical composition comprising a ligand-effector moiety provided with at least one saponin of the present invention or an antibody-effector moiety provided with at least one saponin, optionally further comprising a pharmaceutically acceptable excipient. The present invention also relates to a ligand-effector moiety provided with at least one saponin or an antibody-effector moiety provided with at least one saponin for use as a pharmaceutical. The present invention also relates to a ligand-effector moiety provided with at least one saponin of the invention or an antibody-effector moiety provided with at least one saponin for use in the treatment or prevention of cancer. [Background technology]

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

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

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

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

[0006] Examples of ADCs with clinical potential include those being evaluated as treatment options for lymphoid malignancies and multiple myeloma, such as brentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin. Polatuzumab vedotin, which binds to CD79b on (malignant) B cells, and pinatuzumab vedotin, which binds to CD22, are being tested in clinical trials. Each ADC has been combined with rituximab, a monoclonal antibody that binds to CD20, and no payload has been delivered (B. Yu and D. Liu, Antibody-drug conjugates in clinical trials for lymphoid malignancies and multiple myeloma; Journal of Hematology & Oncology (2019) 12:94). Combinations of monoclonal antibodies, such as these examples, are yet another approach, attempting to arrive at a "magic bullet" that combines many or even all of the previously mentioned desirable characteristics of ADCs.

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

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

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

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

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

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

[0013] In some embodiments of the present invention, it is a primary goal to provide improved bioactive compounds or compositions containing such improved bioactive compounds.

[0014] It is one of several objects of embodiments of the present invention to provide a solution to the problem of non-specificity faced when administering small molecule therapeutically active compounds to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of drugs having suboptimal specificity for disease-driving biological factors or biological processes. It is one of several objects of embodiments of the present invention to provide a solution to the problem of inadequate safety characteristics of current drugs when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of current drugs being less effective than desired when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem of current drugs not being adequately targeted to diseased cells with little or no off-target activity against non-diseased cells when administered to human patients in need thereof. It is one of several objects of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently timely mechanism of action (e.g., the administered drug molecule should reach the target site in the human patient within a certain time frame and should remain at the target site for a certain time frame).It is one of several objects of embodiments of the present invention to provide a solution to the problem that current drugs, when administered to a human patient in need thereof, do not have a sufficiently long-lasting therapeutic activity in the patient's body.

[0015] At least one of the above objects of embodiments of the present invention is achieved by providing an antibody-drug conjugate (ADC) or antibody-oligonucleotide (AOC), e.g., antibody-BNA, covalent complex of the present invention, comprising a cell-targeting moiety according to the present invention and at least one saponin and at least one effector moiety, e.g., a proteinaceous toxin and / or oligonucleotide, e.g., a BNA. The ADC and / or AOC are also suitable for use as a medicament according to the present invention.

[0016] While the present invention will be described with reference to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

[0017] One aspect of the invention relates to a conjugate comprising a cell surface targeting molecule and at least one effector moiety, and further comprising at least one covalently attached saponin.

[0018] One embodiment is a conjugate of the invention, wherein at least one saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, and / or a saponin isolated from any one or more of Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, e.g., Quillaja saponaria.

[0019] In one embodiment is a conjugate of the invention, wherein at least one saponin is a single particular saponin or a mixture of two or more different saponins.

[0020] One embodiment is a conjugate of the invention, wherein at least one saponin has a molecular mass of 3,000 daltons or less, preferably 2,500 daltons or less, more preferably 2,300 daltons or less, most preferably 2,000 daltons or less, for example 1,500 to 1,900 daltons.

[0021] In one embodiment is a conjugate of the invention, wherein the at least one saponin is a saponin of Table A1 or Scheme I, 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 albumenum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereoisomers and / or any combination thereof. Preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21, and / or a saponin having a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group, and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group. and / or 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilayic acid, 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside. More preferably, the at least one saponin is SO1861 and / or QS-21.

[0022] In one embodiment is a conjugate of the invention, wherein at least one saponin is a bisdesmosidic saponin, has a molecular mass of at least 1,500 daltons, comprises an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, and has a first branched carbohydrate side chain at the C-3 position, which optionally contains glucuronic acid. The saponin contains an ester group with a second branched carbohydrate side chain at the C-28 position, this second branched carbohydrate chain preferably containing at least four carbohydrate units, optionally containing at least one acetyl residue, for example two acetyl residues, and / or optionally containing a deoxy carbohydrate, and / or optionally containing quinovose, and / or optionally containing glucose, and / or optionally containing 4-methoxycinnamic acid, and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally containing 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, linked to the carbohydrate via an ester bond. Alternatively, the at least one saponin is QS-21 or any one or more of 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, QS-18, QS-1861, protonated QS1861 (QS1862), and Quil-A.

[0023] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule comprises or consists of a ligand or proteinaceous ligand or proteinaceous binding molecule for binding to a cell surface molecule.

[0024] One embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule comprises or consists of a non-proteinaceous and / or proteinaceous ligand for binding to a cell surface molecule, such as EGF or a cytokine, and / or comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin, and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, which is capable of binding to a cell surface molecule.

[0025] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule is a tumor cell surface molecule, preferably a tumor cell receptor, e.g., 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, PSMA, CanAg, integrin - capable of binding to a receptor selected from alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, and EGFR.

[0026] An embodiment is a conjugate of the invention, wherein the tumor cell receptor is internalized by the tumor cell after binding to the cell surface molecule targeting molecule of the invention, or the tumor cell receptor is internalized by the tumor cell after binding to the conjugate of the invention, preferably, binding of the cell surface molecule targeting molecule to the tumor cell receptor and binding of the conjugate is followed by internalization of the complex of the conjugate and tumor cell receptor mediated by the tumor cell receptor, e.g., via endocytosis.

[0027] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment or domain thereof, preferably comprises or consists of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, any one of the antibodies of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof.

[0028] In one embodiment is a conjugate of the invention, wherein at least one effector moiety comprises or consists of any one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid, preferably a vector, a gene, a transgene that induces cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), a DNA aptamer, an RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoroa The nucleic acid may be selected from any one or more of midate morpholino oligomers (PMOs), locked nucleic acids (LNAs), bridged nucleic acids (BNAs), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNAs), plasmids, glycol nucleic acids (GNAs), and threose nucleic acids (TNAs), or derivatives thereof, more preferably BNAs, for example BNAs for silencing HSP27 protein expression.

[0029] In one embodiment is a conjugate of the invention, wherein at least one effector moiety comprises or consists of any one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid, preferably a vector, a gene, a transgene that induces cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), a DNA aptamer, an RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoroa The oligonucleotides may be selected from one or more of midate morpholino oligomers (PMOs), locked nucleic acids (LNAs), bridged nucleic acids (BNAs), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNAs), plasmids, glycol nucleic acids (GNAs), and threose nucleic acids (TNAs), or derivatives thereof, more preferably BNAs, for example, BNAs for silencing HSP27 protein expression. At least one effector moiety may be, for example, two different (antisense) oligonucleotides for silencing two different genes.

[0030] In one embodiment is a conjugate of the invention, wherein the at least one effector moiety comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of a peptide, a protein, an enzyme such as urease and Cre recombinase, a ribosome-inactivating protein, a proteinaceous toxin selected from Table A5, more preferably a viral toxin, such as apoptin, a bacterial toxin, such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins, such as alpha-sarcin; plant toxins including ribosome-inactivating proteins and the A chain of type 2 ribosome-inactivating proteins, such as giantin, e.g., giantin-30 or giantin-32, saporin, e.g., saporin-S3 or saporin-S6, bouganin, the deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog RNase, or granzyme B or angiogenin from humans, or any fragment or derivative thereof; preferably, the protein toxin is giantin and / or saporin.

[0031] In one embodiment is a conjugate according to the invention, wherein the at least one effector molecule comprises or consists of at least one payload, preferably one or more of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, pasudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N -acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepine, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, Pseudomonas The steroid inhibitor is selected from one or more of a truncated form of P. aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or a derivative thereof.

[0032] One embodiment is a conjugate of the invention, wherein at least one effector moiety is either covalently attached to the cell surface molecule targeting molecule via at least one linker or is directly attached to the cell surface molecule targeting molecule.

[0033] In one embodiment is a conjugate of the invention, wherein at least one effector moiety is covalently attached to a cell surface targeting molecule, thereby forming gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin antibody-drug conjugates, and any one of the antibody-drug conjugates of Table A2 and Table A3.

[0034] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecular targeting molecule, preferably an amino acid residue of the cell surface molecular targeting molecule, via an aldehyde functional group on the saponin, and / or to at least one effector moiety, preferably via an amino acid residue on at least one effector moiety, via an aldehyde functional group on the saponin, preferably an aldehyde functional group at position C-23 of a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type.

[0035] One embodiment is a conjugate of the invention, in which the aldehyde functional group of at least one saponin, preferably the aldehyde functional group at position C-23 of at least one saponin, is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled via a thio-ether bond to a sulfhydryl group, e.g., a sulfhydryl group of a cysteine, on a cell surface targeting molecule and / or on at least one effector moiety.

[0036] One embodiment is a conjugate of the invention, wherein at least one saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type, has an aldehyde functional group at position C-23, and comprises a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, and the saponin is covalently attached to an amino acid residue of a cell surface molecular targeting molecule and / or to at least one effector moiety via said glucuronic acid functional group, preferably via an amino acid residue on the at least one effector moiety.

[0037] One embodiment is a conjugate of the invention, in which a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of at least one saponin is covalently coupled to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled via an amide bond to an amine group on the cell surface molecule targeting molecule and / or at least one effector moiety, for example, a lysine or N-terminal amine group of the cell surface molecule targeting molecule and / or at least one effector moiety.

[0038] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface targeting molecule and / or to at least one effector moiety, either directly or via at least one linker, such as a bifunctional linker based on N-ε-maleimidocaproic acid hydrazide and / or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, or a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B.

[0039] In one embodiment is a conjugate of the invention, wherein the trifunctional linker comprises a second chemical group to which at least one saponin is covalently attached, a third chemical group for covalently attaching to a cell surface targeting molecule, and a first chemical group for covalently attaching to at least one effector moiety, preferably the trifunctional linker is of Scheme II and Structure B.

[0040] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and to at least one effector moiety via at least one linker comprising a trifunctional linker to which both the cell surface molecule targeting molecule and the at least one effector moiety are attached, preferably the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0041] An embodiment is a conjugate of the invention, wherein at least one linker comprises at least one cleavable linker, optionally said cleavable linker undergoes cleavage under acidic, reductive, enzymatic, or photoinduced conditions, preferably the cleavable linker comprises a cleavable bond selected from a hydrazone or hydrazide bond that undergoes cleavage under acidic conditions, and / or a bond that is susceptible to proteolysis, e.g., proteolysis by cathepsin B, and / or a bond that is susceptible to cleavage under reductive conditions, e.g., a disulfide bond.

[0042] In one embodiment is a conjugate of the invention, wherein at least one linker comprises at least one cleavable linker, said cleavable linker undergoing cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦5.5.

[0043] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a lysine side chain forming an amide bond and / or to a cysteine ​​side chain forming a thio-ether bond or a disulfide bond, wherein the lysine and / or cysteine ​​is included in a cell surface molecule targeting molecule and / or is included in at least one effector moiety, and wherein the at least one saponin is either directly attached to the lysine and / or cysteine ​​or is attached via at least one linker, optionally including a cleavable linker and / or a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B.

[0044] In one embodiment is a conjugate of the invention, wherein the linker is based on N-ε-maleimidocaproic acid hydrazide, and / or based on 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B, a cleavable linker, and / or involves any one or more of a disulfide bond, a thio-ether bond, an amide bond, or a hydrazide bond.

[0045] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and / or to at least one effector moiety via at least one linker, the linker being or comprising a scaffold comprising a polymeric or oligomeric structure and further comprising at least one fourth chemical group for covalently attaching the scaffold to the cell surface molecule targeting molecule and / or to at least one effector moiety.

[0046] In one embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via a cleavable bond and / or via a non-cleavable bond.

[0047] One embodiment is a conjugate of the invention, wherein the cleavable bond undergoes cleavage under any of acidic, reductive, enzymatic, and photoinduced conditions, preferably the cleavable bond comprises a hydrazone or hydrazide bond that undergoes cleavage under acidic conditions, and / or a bond that is susceptible to proteolysis, e.g., proteolysis by cathepsin B, and / or a bond that undergoes cleavage under reductive conditions, e.g., a disulfide bond.

[0048] In one embodiment is a conjugate of the invention, wherein the cleavable bond undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦5.5.

[0049] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via any one or more of an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thio-ether bond, an amide bond, a peptide bond, or an ester bond, preferably via at least one linker.

[0050] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via any one or more of an imine bond, a hydrazone bond, and a hydrazide bond, which bond is preferably cleavable according to the invention, and preferably the at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via an aldehyde functionality at position C-23 of the at least one saponin.

[0051] One embodiment is a conjugate of the invention, in which the aldehyde functionality at position C-23 of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled via a thio-ether bond to a sulfhydryl group, e.g., a sulfhydryl group of cysteine, on a backbone polymeric or oligomeric structure.

[0052] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via an amide bond, and preferably at least one saponin, when present, is covalently attached to the backbone polymeric or oligomeric structure via a glucuronic acid functionality as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin.

[0053] One embodiment is a conjugate of the invention, in which a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin is covalently coupled to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled via an amide bond to an amine group on a backbone polymeric or oligomeric structure, for example, a lysine or N-terminal amine group of the backbone polymeric or oligomeric structure.

[0054] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure, the covalent attachment involving an aldehyde functionality at position C-23 of at least one saponin, when present, and / or the covalent attachment involving a glucuronic acid functionality as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin, when present.

[0055] In one embodiment is a conjugate of the invention, wherein at least one fourth chemical group of the scaffold for covalently coupling the scaffold to a cell surface targeting molecule and / or to at least one effector moiety is a click chemistry group, preferably selected from any one or more of a tetrazine, an azide, an alkene, or an alkyne, or cyclic derivatives of these groups, preferably an azide group.

[0056] In one embodiment is a conjugate of the invention, wherein the backbone polymeric or oligomeric structure comprises a linear, branched, and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures, which assembly is preferably assembled by covalent crosslinking.

[0057] In one embodiment is a conjugate of the invention, wherein the at least one saponin is a defined number or range of saponins, preferably 1 to 128 saponins, or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 saponins, or any number therebetween, for example, 7, 9, 12 saponins.

[0058] One embodiment is a conjugate of the invention, wherein the conjugate comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1-100 saponins, or any number of saponins therebetween, e.g., 7, 9, 12 saponins, covalently attached directly to an amino acid residue of a cell surface targeting molecule and / or to at least one effector moiety, preferably via an amino acid residue of at least one effector moiety, preferably to a cysteine ​​and / or a lysine, and and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1 to 8 such backbones or 2 to 4 such backbones, of any one of claims 28 to 40, and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1 to 8 such backbones or 2 to 4 such backbones, and 1 to 32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins, or any number of saponins in between, for example 7, 9, 12 saponins, are covalently attached to the at least one backbone.

[0059] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and to at least one effector moiety via a trifunctional linker, the trifunctional linker comprising a second chemical group to which the at least one saponin is covalently attached either directly or via a linker such as a cleavable linker and / or via a backbone comprising a polymeric or oligomeric structure, and a fourth chemical group in accordance with the invention for covalent coupling of the backbone to the trifunctional linker, the trifunctional linker further comprising a third chemical group for covalent attachment to the cell surface molecule targeting molecule and a first chemical group for covalent attachment to at least one effector moiety, the cell surface molecule targeting molecule being attached to the third chemical group and / or the at least one effector moiety being attached to the first chemical group, preferably the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0060] One 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.

[0061] An aspect of the invention relates to a conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0062] Certain aspects of the invention relate to a conjugate of the invention or a pharmaceutical composition of the invention for use in the treatment or prevention of cancer or autoimmune disease.

[0063] One embodiment of the present invention relates to any of the following ADCs and AOCs and semi-finished conjugates thereof, which comprise a cell surface molecule targeting molecule of the present invention and either comprise at least one effector moiety of the present invention or comprise at least one saponin of the present invention, or both:

[0064] Anti-EGFR antibody-saponin; Anti-EGFR antibody-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin; Anti-EGFR antibody-SO1861; Anti-EGFR antibody-GE1741; Anti-EGFR antibody-SA1641; Anti-EGFR antibody-Quil-A; Anti-EGFR antibody-QS-21; Anti-EGFR antibody-saponin in the water-soluble saponin fraction of Quillaja saponaria;

[0065] Cetuximab-saponin; Cetuximab-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 and optionally containing a glucuronic acid function as carbohydrate substituent on the C-3 beta-OH group of the saponin; Cetuximab-SO1861; Cetuximab-GE1741; Cetuximab-SA1641; Cetuximab-Quil-A; Cetuximab-QS-21; Cetuximab-saponin in the water-soluble saponin fraction of Quillaja saponaria;

[0066] Anti-HER2 antibody-saponin; Anti-HER2 antibody-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin; Anti-HER2 antibody-SO1861; Anti-HER2 antibody-GE1741; Anti-HER2 antibody-SA1641; Anti-HER2 antibody-Quil-A; Anti-HER2 antibody-QS-21; Anti-HER2 antibody-saponin in the water-soluble saponin fraction of Quillaja saponaria;

[0067] Trastuzumab-saponin; Trastuzumab-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 and optionally containing a glucuronic acid function as carbohydrate substituent on the C-3 beta-OH group of the saponin; Trastuzumab-SO1861; Trastuzumab-GE1741; Trastuzumab-SA1641; Trastuzumab-Quil-A; Trastuzumab-QS-21; Trastuzumab-saponin in the water-soluble saponin fraction of Quillaja saponaria;

[0068] anti-CD71 antibody-saponin; anti-CD71 antibody-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin; 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;

[0069] OKT-9-saponin; OKT-9-triterpenoid saponin and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde function at position C-23 and optionally containing a glucuronic acid function as carbohydrate substituent on the C-3 beta-OH group of the saponin; OKT-9-SO1861; OKT-9-GE1741; OKT-9-SA1641; OKT-9-Quil-A; OKT-9-QS-21; OKT-9-saponin in the water-soluble saponin fraction of Quillaja saponaria;

[0070] Anti-EGFR antibody-oligonucleotide; Anti-EGFR antibody-antisense oligonucleotide; Anti-EGFR antibody-siRNA; Anti-EGFR antibody-antisense BNA; Anti-EGFR antibody-antisense BNA (HSP27); Anti-EGFR antibody-proteinaceous toxin; Anti-EGFR antibody-ribosome-inactivating protein; Anti-EGFR antibody-giantin; Anti-EGFR antibody-saporin;

[0071] Cetuximab-oligonucleotide; Cetuximab-antisense oligonucleotide; Cetuximab-siRNA; Cetuximab-antisense BNA; Cetuximab-antisense BNA (HSP27); Cetuximab-proteineous toxin; Cetuximab-ribosome-inactivating protein; Cetuximab-giantin; Cetuximab-saporin;

[0072] Anti-HER2 antibody-oligonucleotide; Anti-HER2 antibody-antisense oligonucleotide; Anti-HER2 antibody-siRNA; Anti-HER2 antibody-antisense BNA; Anti-HER2 antibody-antisense BNA (HSP27); Anti-HER2 antibody-proteinaceous toxin; Anti-HER2 antibody-ribosome-inactivating protein; Anti-HER2 antibody-giantin; Anti-HER2 antibody-saporin;

[0073] Trastuzumab-oligonucleotide; Trastuzumab-antisense oligonucleotide; Trastuzumab-siRNA; Trastuzumab-antisense BNA; Trastuzumab-antisense BNA (HSP27); Trastuzumab-proteinaceous toxin; Trastuzumab-ribosome-inactivating protein; Trastuzumab-gianthin; Trastuzumab-saporin; Anti-CD71 antibody-oligonucleotide; Anti-CD71 antibody-antisense oligonucleotide; Anti-CD71 antibody-siRNA; Anti-CD71 antibody-antisense BNA; Anti-CD71 antibody-antisense BNA (HSP27); Anti-CD71 antibody-proteinaceous toxin; Anti-CD71 antibody-ribosome-inactivating protein; Anti-CD71 antibody-gianthin; Anti-CD71 antibody-saporin;

[0074] OKT-9-oligonucleotide; OKT-9-antisense oligonucleotide; OKT-9-siRNA; OKT-9-antisense BNA; OKT-9-antisense BNA (HSP27); OKT-9-protein toxin; OKT-9-ribosome-inactivating protein; OKT-9-gianthin; OKT-9-saporin;

[0075] anti-EGFR antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, an siRNA, an antisense BNA, and an antisense BNA (HSP27), the saponin is any one or more of a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and a saponin in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab;

[0076] anti-EGFR antibody (-proteinaceous toxin) (-saponin), wherein 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 bisdesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-EGFR antibody is preferably cetuximab;

[0077] anti-HER2 antibody (-oligonucleotide) (-saponin), wherein the oligonucleotide is any one or more of an antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), the saponin is any one or more of triterpenoid saponins and / or bisdesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab;

[0078] anti-HER2 antibody (-proteinaceous toxin) (-saponin), wherein 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 bisdesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-HER2 antibody is preferably trastuzumab;

[0079] anti-CD71 antibody (-oligonucleotide) (-saponin), where the oligonucleotide is any one or more of an antisense oligonucleotide, siRNA, antisense BNA, and antisense BNA (HSP27), the saponin is any one or more of triterpenoid saponins and / or bisdesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9;

[0080] Anti-CD71 antibody (-proteinaceous toxin) (-saponin). The proteinaceous toxin is any one or more of a ribosome-inactivating protein, dianthin, and saporin, and the saponin is any one or more of triterpenoid saponins and / or bisdesmosidic triterpene saponins belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in the water-soluble saponin fraction of Quillaja saponaria, and the anti-CD71 antibody is preferably OKT-9.

[0081] One embodiment is a semi-finished conjugate of the invention or a conjugate of the invention, wherein the cell surface molecule targeting molecule is selected from cetuximab, trastuzumab, OKT-9, 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 in the water-soluble saponin fraction of Quillaja saponaria.

[0082] In one embodiment is a conjugate according to the invention, wherein the cell surface molecule targeting molecule is selected from cetuximab, trastuzumab, OKT-9, 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 a saponin in the water-soluble saponin fraction of Quillaja saponaria.

[0083] One embodiment of the present invention relates to an ADC, AOC, or semi-finished ADC conjugate or semi-finished AOC conjugate of structure C, comprising a cell surface molecule targeting molecule 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 molecule; S is a saponin; E is an effector moiety; b=0-64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64, or any integer or fraction therebetween; and c=0-8, preferably 0, 1, 2, 3, 4, 6, 8, or any integer or fraction therebetween, and 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.

[0084] An embodiment is structure C of the invention, wherein A is an anti-EGFR antibody, e.g., cetuximab, an anti-HER2 antibody, e.g., trastuzumab, an anti-CD71 antibody, e.g., OKT-9, and / or wherein S is a saponin, triterpenoid saponin, and / or bisdesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane having an aldehyde functionality at position C-23 and optionally containing a glucuronic acid functionality as the carbohydrate substituent of the C-3 beta-OH group of the saponin, such as SO1861, GE1741, SA1641, Quil-A, QS-21, and Quillaja. and / or wherein E is any one or more of the saponins in the water-soluble saponin fraction of S. saponaria, and / or wherein 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.

[0085]

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

[0086] One aspect of the present invention relates to the use of any of the previously mentioned conjugates, ADCs, AOCs, semi-finished ADCs, and semi-finished AOCs as a medicament.

[0087] One aspect of the present invention relates to the use of any of the previously mentioned conjugates, ADCs, AOCs, semi-completed ADCs, semi-completed AOCs for use in the treatment or prevention of cancer or autoimmune disease.

[0088] definition The term "linker" has its usual scientific meaning and herein refers to a linear stretch or chemical moiety of amino acid residues conjugated via a peptide bond, which attaches a molecule or atom to another molecule, such as a ligand or an effector molecule or a scaffold. Typically, a linker comprises a chain of atoms linked by a chemical bond. Any linker molecule or linker technology known in the art can be used in the present disclosure. Where indicated, a linker is a linker for covalently attaching molecules via a chemical group on such molecule suitable for forming a covalent bond or covalent linkage with the linker. The linker can be a non-cleavable linker. For example, the linker is stable under physiological conditions. The linker can be a cleavable linker, for example, a linker that is cleavable in the presence of an enzyme, at a specific pH range or value, or under physiological conditions, such as the intracellular conditions of endosomes, such as lysosomes and late endosomes, of mammalian cells, such as human cells. Exemplary linkers that may be used in the context of the present disclosure include, but are not limited to, N-ε-maleimidocaproic acid hydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate, or 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

[0089] The term "trifunctional linker" has its usual scientific meaning and refers herein to a linker that attaches three molecules via their respective chemical groups. Those skilled in the art can design such trifunctional linkers based on this disclosure and common general knowledge. Such trifunctional linkers may exhibit, for example, maleimide groups that can be used for conjugation to targeting ligands that exhibit thiol groups for thiol-ene reactions. Additionally, trifunctional linkers may exhibit dibenzocyclooctyne (DBCO) groups for so-called strain-promoted alkyne-azide cycloaddition (SPAAC, click chemistry) with azide-bearing saponins. Finally, trifunctional linkers may acquire a third functional group, such as a trans-cyclooctyne (TCO) group, to undergo so-called inverse electron-demand Diels-Alder (IEDDA) reactions with tetrazine (Tz)-bearing effector molecules. Those skilled in the art will understand that the three chemical groups of a trifunctional linker can all be the same or different, or the linker can contain two of the same chemical groups for linking molecules to the trifunctional linker. The bond formed between the trifunctional linkers can be covalent or non-covalent, with covalent bonds being preferred. The bond formed between the trifunctional linker and one, two, or three binding molecules via each chemical group can be a cleavable (labile) bond, cleavable under acidic conditions in cells, such as the endosomes and lysosomes of mammalian cells, such as human cells, or a non-cleavable bond. It should be understood that a trifunctional linker can contain one or two chemical groups for forming a covalent bond, and two or one additional chemical group(s) for forming a non-covalent bond, respectively. It will be appreciated that a trifunctional linker may include one or two chemical groups for forming a cleavable bond, and an additional two or one chemical group(s), respectively, for forming a non-cleavable bond.

[0090] The term "cleavable," as used, for example, in the term "cleavable linker" or "cleavable bond," has its usual scientific meaning, and herein refers to cleavage under acidic, reductive, enzymatic, or photoinduced conditions. For example, a cleavable linker may be cleaved under acidic conditions. Preferably, the cleavable linker is cleaved in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH of 4.0 to 6.5, more preferably at a pH of 5.5 or less. As another example, the cleavable linker may be cleaved by an enzyme, for example, a cathepsin. Furthermore, an example of a covalent bond cleavable under reductive conditions is a disulfide bond.

[0091] The terms "oligomer" and "polymer" in the context of oligomeric or polymeric backbones have their customary scientific meanings. Here, a polymer refers to a substance having a molecular structure built up primarily or entirely from a large number of equal or similar units linked together; here, an oligomer refers to a polymer whose molecules consist of a relatively small number of repeating units. For example, a structure containing 5-10 or fewer equal or similar units may be referred to as an oligomeric structure, a structure containing 10-50 or more monomeric units may be referred to as a polymeric structure, and a structure of 10 monomeric units may be referred to as either oligomeric or polymeric.

[0092] The term "binding site" has its usual scientific meaning and refers here to a region or epitope on one molecule, such as a protein, DNA, or RNA, to which another molecule can bind.

[0093] The term "scaffold" has its usual scientific meaning, and herein refers to an oligomeric or polymeric template or carrier or base (base molecule or base structure) to which one or more molecules, such as ligand molecules, saponins of the invention, glycosides, effector molecules, can be covalently attached, either directly or via a linker, such as a cleavable linker. The scaffold may comprise a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or may comprise an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The backbone may comprise polymeric or oligomeric structures, such as poly- or oligo(amines), e.g., polyethyleneimine and poly(amidoamine); or polyethylene glycol, poly- or oligo(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers; or poly(dextrins), poly- or oligosaccharides, e.g., cyclodextrin or polydextrose; or natural and / or artificial poly- or oligoamino acids, e.g., polylysine, or peptides or proteins, DNA oligo- or polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not exhibit substantial acute or chronic toxicity in organisms and can either be excreted intact or can be completely degraded by the body's metabolism into excretable and / or physiological compounds.

[0094] The term "ligand" has its usual scientific meaning and refers herein to any molecule or molecules that can selectively bind to a target cell surface molecule or target cell surface receptor expressed on a target cell, e.g., a target cancer cell or a target autoimmune cell. A ligand can bind to an epitope contained by a receptor or other antigen on a target cell. Preferably, the cell-binding ligand is an antibody.

[0095] As used herein, the term "antibody" is used in the broadest sense and can refer to an immunoglobulin (Ig), defined as a protein belonging to the classes IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), or to a functional binding fragment or binding domain of an immunoglobulin. In the context of the present invention, a "binding fragment" or "binding domain" of an immunoglobulin is defined as an antigen-binding fragment or domain of a parent immunoglobulin or other derivative, which essentially retains the antigen-binding activity of such parent immunoglobulin. Functional fragments and functional domains are antibodies in the sense of the present invention, even if their affinity for antigen is lower than that of the parent immunoglobulin. "Functional fragments and domains" according to the present invention include, but are not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments, scFv, dsFv, single-domain antibodies (sdAbs), monovalent IgG, scFv-Fc, reduced IgG (rIgG), minibodies, diabodies, triabodies, tetrabodies, Fc fusion proteins, nanobodies, variable V domains such as VHH and Vh, and other types of antigen-recognizing immunoglobulin fragments and domains. Fragments and domains can be engineered to minimize or completely eliminate intermolecular disulfide interactions that occur between CH1 and CL domains. Due to their smaller size, functional fragments and domains offer the advantage of greater tumor penetration. In addition, functional fragments or domains may be more uniformly distributed in tumor masses compared to whole immunoglobulins.

[0096] The antibodies (immunoglobulins) of the present invention may be bifunctional or multifunctional. For example, a bifunctional antibody has one arm with specificity for one receptor or antigen, and the other arm recognizes a different receptor or antigen. Alternatively, each arm of a bifunctional antibody may have specificity for a different epitope of the same receptor or antigen on a target cell.

[0097] The antibodies (immunoglobulins) of the present invention can be, but are not limited to, polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, resurfaced antibodies, anti-idiotypic antibodies, mouse antibodies, rat antibodies, rat / mouse hybrid antibodies, llama antibodies, heavy chain-only llama antibodies, heavy chain-only antibodies, and veterinary antibodies. Preferably, the antibodies (immunoglobulins) of the present invention are monoclonal antibodies. Resurfaced, chimeric, humanized, and fully human antibodies are also more preferred because they are less likely to cause immunogenicity in humans. The antibodies of the ADCs of the present invention preferably specifically bind to antigens expressed on the surface of cancer cells, autoimmune cells, diseased cells, and abnormal cells while leaving any healthy cells essentially unchanged (e.g., by not binding to such normal cells or by binding to such healthy cells with a lesser degree of number and / or affinity).

[0098] Specific antibodies that can be used in the ADCs of the invention include anti-HER2 monoclonal antibodies, such as trastuzumab and pertuzumab, anti-CD20 monoclonal antibodies, such as rituximab, ofatumumab, tositumomab, and ibritumomab, anti-CA125 monoclonal antibodies, such as oregovomab, anti-EpCAM (17-1A) monoclonal antibodies, such as edrecolomab, anti-EGFR monoclonal antibodies, such as cetuximab, panitumumab, and nimotuzumab, anti-CD30 monoclonal antibodies, such as brentuximab, anti-CD33 monoclonal antibodies, such as gemtuzumab and huMy9-6, anti-vascular integrin alpha-v beta-3 monoclonal antibodies, such as etaracizumab, and anti-CD52 monoclonal antibodies, such as alemtuzumab. anti-CD22 monoclonal antibodies, such as epratuzumab, anti-CEA monoclonal antibodies, such as labetuzumab, anti-CD44v6 monoclonal antibodies, such as bivatuzumab, anti-FAP monoclonal antibodies, such as sibrotuzumab, anti-CD19 monoclonal antibodies, such as huB4, anti-CanAg monoclonal antibodies, such as huC242, anti-CD56 monoclonal antibodies, such as huN901, anti-CD38 monoclonal antibodies, such as daratumumab, anti-CA6 monoclonal antibodies, such as DS6, anti-IGF-IR monoclonal antibodies, such as cixutumumab and 3B7, anti-integrin monoclonal antibodies, such as CNTO95, and anti-syndecan-1 monoclonal antibodies, such as B-B4. One embodiment is Stemline's protein-toxin conjugate: ELZONRIS™ (tagraxofusp, SL-401), a novel targeted therapy directed against interleukin-3 (IL-3) receptor alpha (CD123), a target present on a wide range of malignancies.

[0099] Any molecule other than an antibody that binds to a cell receptor or antigen on a target cell can also be used as the cell-binding ligand of the ligand-drug conjugate of the present invention. The ligand is provided in accordance with the present invention as a covalently attached saponin. These ligands include, but are not limited to, proteins, polypeptides, peptides, and small molecules. Examples of these non-antibody ligands include interferons (e.g., IFN-α, IFN-β, and IFN-γ), transferrin, lectins, epidermal growth factor (EGF) and EGF-like domains, gastrin-releasing peptide (GRP), platelet-derived growth factor (TGF), transforming growth factor (TGF), vaccinia growth factor (VGF), insulin and insulin-like growth factors (IGFs, e.g., IGF-1 and IGF-2), other suitable hormones, such as thyrotropin-releasing hormone (TRH), melanocyte-stimulating hormone (MSH), and steroids. Hormones (e.g., estrogen and androgen), somatostatin, lymphokines (e.g., IL-2, IL-3, IL-4, and IL-6), colony-stimulating factors (CSFs, e.g., G-CSF, M-CSF, and GM-CSF), bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g., AS-1411, GBI-10, RNA aptamers against HIV glycoproteins), small molecules (e.g., folic acid, anisamidophenylboronic acid), vitamins (e.g., vitamin D), carbohydrates (e.g., hyaluronic acid, galactose).

[0100] An "effector molecule" or "effector moiety" or "payload" has its usual scientific meaning and, in the context of the present invention, is any substance that affects cellular metabolism by interacting with an effector molecule target within the cell. This effector molecule target is any molecule or structure within the cell that excludes the lumen of compartments and vesicles of the endocytic and recycling pathways, but includes the membranes of these compartments and vesicles. Said structures within the cell therefore include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol.

[0101] The effector molecule or moiety is a drug substance, such as a toxin, e.g., a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide. A drug substance in the present invention is an effector molecule or moiety used to achieve a beneficial result in an organism, preferably a vertebrate, more preferably a mammal, such as a non-human subject or a human / human subject. Benefits include diagnosis, prognosis, treatment, cure, and prevention (prophylaxis) of a disease and / or condition and / or health problem. A drug substance may also lead to unwanted and possibly even harmful side effects (e.g., adverse events observed during clinical trials). In this case, the pros and cons must be weighed to determine whether the drug substance is suitable in a particular case. If the effect of a drug substance in a cell is overwhelmingly beneficial to the organism as a whole, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the organism as a whole, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, target and off-target cells depend on the purpose and are defined by the user. Examples of effector molecules and moieties are drugs, toxins, polypeptides (eg, enzymes), polynucleotides (including polypeptides and polynucleotides that contain unnatural amino acids or nucleic acids), and any combination thereof.

[0102] Drug effector molecules or effector moieties may include, but are not limited to, anti-cancer agents, anti-inflammatory agents, and anti-infective (e.g., anti-fungal, anti-bacterial, anti-parasitic, anti-viral) agents. Preferably, the drug molecules of the present invention are anti-cancer or anti-autoimmune agents. Suitable anti-cancer agents include, but are not limited to, alkylating agents, metabolic inhibitors, spindle poison plant alkaloids, cytotoxic / anti-tumor antibiotics, topoisomerase inhibitors, photosensitizers, and kinase inhibitors. The definition of "anti-cancer agent" also includes, for example, (i) antihormonal agents that act to control or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands; (iii) antiandrogens; (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation; (vii) ribozymes, such as VEGF expression inhibitors and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines; topoisomerase 1 inhibitors; (ix) angiogenesis inhibitors; and pharmaceutically acceptable salts, acids, solvates, and derivatives of any of the above.

[0103] Toxin effector molecules or moieties can include, but are not limited to, proteinaceous toxins (e.g., bacterial and plant-derived toxins), toxins that target tubulin filaments, toxins that target DNA, and toxins that target RNA. Examples of proteinaceous toxins include saporin, dianthin, ricin, modeccin, abrin, volkensin, viscumin, Shiga toxin, Shiga-like toxins, Pseudomonas exotoxin (PE, also known as exotoxin A), diphtheria toxin (DT), and cholera toxin. Examples of tubulin filament-targeting toxins include maytansinoids (e.g., DM1 and DM4), auristatins (e.g., monomethylauristatin E (MMAE) and monomethylauristatin F (MMAF)), toxoids, tubulysins, cryptophycins, and rhizoxins. Examples of DNA-targeted toxins are calicheamicin: N-acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycin, doxorubicin, methotrexate, benzodiazepines, camptothecin analogs, and anthracyclines. Examples of DNA-targeted toxins are amanitin, splicehostatin, and Thailanstatin. The toxins used in the present invention are defined as drug substances that can kill or inactivate cells. Preferably, targeted toxins are toxins that are only, or at least predominantly, toxic to target cells, but not to off-target cells. The net effect of the targeted toxin is preferably beneficial to the organism as a whole.

[0104] An effector molecule or moiety that is a polypeptide can be, for example, a polypeptide that restores a lost function, such as, for example, an enzyme replacement or a gene regulatory function, or a toxin. Examples of polypeptides as effector molecules include, for example, Cas9; toxins (e.g., saporin, dianthin, gelonin, (de)bouganin, agrostin, ricin (toxin A chain); pokeweed antiviral protein, apoptin, diphtheria toxin, Pseudomonas exotoxin), metabolic enzymes (e.g., argininosuccinate lyase, argininosuccinate synthetase), enzymes of the coagulation cascade, repair enzymes; enzymes of cell signaling; cell cycle regulators; gene regulators (transcription factors, e.g., NF-κB, or gene repressors, e.g., methionine repressor).

[0105] An effector molecule or effector moiety that is a polynucleotide can be, for example, a polynucleotide containing coding information, such as a gene or open reading frame encoding a protein. It can also contain regulatory information, such as a promoter or regulatory element binding region, or a sequence encoding a microRNA. Such polynucleotides can include natural and artificial nucleic acids. Artificial nucleic acids include, for example, peptide nucleic acids (PNAs), morpholinos and locked nucleic acids (LNAs), as well as glycol nucleic acids (GNAs) and threose nucleic acids (TNAs). Each of these is distinguished from naturally occurring DNA or RNA by modifications to the backbone of the molecule. Examples of nucleotides as effector molecules include, but are not limited to, DNA: single-stranded DNA (e.g., adenine phosphoribosyltransferase DNA); linear double-stranded DNA (e.g., coagulation factor IX gene); circular double-stranded DNA (e.g., plasmid); RNA: mRNA (e.g., TAL effector molecule nuclease), tRNA, rRNA, siRNA, miRNA, antisense RNA; antisense oligonucleotides (ASO, AON, e.g., PNA, PMO, LNA, and BNA).

[0106] For example, the term "proteinaceous," as used in "proteinaceous molecule" and "proteinaceous toxin," refers to molecules and toxins comprising at least one string of amino acid residues that can be obtained as an expression product from a single mRNA. Such molecules or toxins can further comprise any post-translational modifications, carbohydrates, e.g., N- or O-linked carbohydrates, disulfide bonds, phosphorylation, sulfation, etc., resulting from any post-translational modification, and / or can further comprise any other modifications, e.g., resulting from chemical modifications (e.g., resulting from the attachment of an effector moiety, saponin, scaffold, ligand, etc., either directly, e.g., to an amino acid side chain, or via at least one linker that is (covalently) bound to the molecule and chemically bound to the proteinaceous molecule to chemically modify the proteinaceous molecule). The term "proteinaceous" also encompasses and includes aggregates of such molecules, e.g., homodimers, heterotrimers, heterohexamers, or complex assemblies, e.g., ribosomes.

[0107] For example, in the context of "specific binding" and "a receptor or molecular target specifically present or expressed on the surface of tumor cells," and the like, the terms "specific" and "specifically" have their ordinary scientific meaning known in the art. Here, for example, it refers to a binding interaction of a first molecule with a second molecule that occurs with a relatively higher affinity relative to any putative binding of the first molecule to an additional molecule different from the second molecule, or, when considering a number of receptors or molecular targets, the expression or higher degree of expression of a cell surface receptor or molecular target on the surface of a first type of cell, such as a tumor cell, autoimmune cell, diseased cell, or abnormal cell, relative to the degree of expression of the same receptor or molecular target on a second type of cell, such as a healthy cell. Expression in a second type of cell may be completely absent or very low relative to any degree of expression on tumor cells. Furthermore, for example, in "specific binding," the term "specific" has its ordinary scientific meaning known in the art, and here refers to a molecule that can interact with another molecule with a higher binding affinity than background interactions between molecules. Similarly, the term "specificity" refers to an interaction, e.g., between two molecules or between a cell and a molecule, that has a higher binding affinity than background interactions between molecules. Binding molecules, such as immunoglobulins, bind via their binding site, e.g., the immunoglobulin variable region of an immunoglobulin, to a binding site on a molecule, e.g., an epitope, cell surface receptor, etc., with a higher binding affinity than background interactions between molecules. In the context of the present invention, background interactions typically have a K of 10E-4 M or less. D Similarly, a "specific binding domain" is a domain that preferentially binds to a binding site on a molecule, e.g., an epitope, cell surface receptor, etc., with a binding affinity higher than background interactions between molecules. In the context of the present invention, a "background interaction" is typically a K of 10E-4 M or less. D Preferably, the specific binding domain has a K of about 10E-5 M. D It binds with higher affinity than

[0108] The term "binding" is defined as an interaction between molecules that can be distinguished from background interactions.

[0109] As used herein, the term "fragment" refers to an amino acid sequence that is part of a protein domain or that makes up an intact protein domain. Binding fragments according to the invention must have binding specificity for their respective targets, e.g., cell surface receptors on the surface of diseased cells, e.g., tumor cells.

[0110] The term "ADC" or "antibody-drug conjugate" has its usual scientific meaning known to those of skill in the art and, herein, refers to a class of biopharmaceutical drugs designed as targeted therapies, for example, to treat cancer. Unlike chemotherapy, ADCs are intended to target and kill tumor cells while sparing healthy cells. ADCs consist of an antibody linked to a bioactive cytotoxic (anticancer) payload or drug. ADCs combine the targeting capabilities of monoclonal antibodies with the cancer-killing capabilities of cytotoxic drugs. They are designed with the intent of discriminating between healthy cells and diseased tissue, such as tumor cells in tumors.

[0111] The term "Saponinum album" has its ordinary meaning and herein refers to a mixture of saponins produced by Merck KGaA (Darmstadt, Germany) containing saponins from Gypsophila paniculata and Gypsophila arostii, including SA1657 and primarily SA1641.

[0112] The term "Quillaja saponin" has its ordinary meaning and refers herein to the saponin fraction of Quillaja saponaria, and therefore the source of all other QS saponins, containing primarily QS-18 and QS-21.

[0113] "QS-21" or "QS21" has its customary scientific meaning and herein refers to a mixture of QS-21 A-apio (~63%), QS-21 A-xylo (~32%), QS-21 B-apio (~3.3%), and QS-21 B-xylo (~1.7%).

[0114] Similarly, "QS-21A" has its customary scientific meaning and refers herein to a mixture of QS-21 A-apio (-65%) and QS-21 A-xylo (-35%).

[0115] Similarly, "QS-21B" has its customary scientific meaning and refers herein to a mixture of QS-21 B-apio (-65%) and QS-21 B-xylo (-35%).

[0116] The term "Quill-A" refers to a commercially available semi-purified extract from Quillaja saponaria, which contains variable quantities of more than 50 distinct saponins, many of which incorporate the triterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at the C-3 beta-OH group, found in QS-7, QS-17, QS18, and QS-21. The saponins found in Quil-A are listed in Table 2 of van Setten (1995) (Dirk C. van Setten, Gerrit van de Werken, Gijsbert Zomer, and Gideon 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 saponin are also fractions of saponins from Quillaja saponaria, and both contain a large variety of different saponins with largely overlapping contents. Because the two fractions are obtained by different purification procedures, the two fractions differ in their specific composition.

[0117] The terms "QS1861" and "QS1862" refer to QS-7 and QS-7 api. QS1861 has a molecular mass of 1861 daltons, and QS1862 has a molecular mass of 1862 daltons. QS1862 is listed in line 28 of Table 1 in Fleck et al. (2019) (Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereira da Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira and Simone Gasparin Verza, Saponins from Quillaja saponaria and Quillaja brasiliensis: Particular Chemical Characteristics and Biological Activities, Molecules 2019, 24, 171; doi:10.3390 / molecules24010171). The structure shown is that of QS1862, an API variant of QS-7. The molecular mass is 1862 daltons, because this mass is the nominal mass including the proton in the glucuronic acid. At neutral pH, the molecule is deprotonated. When measured by mass spectrometry in the negative ion mode, the measured mass is 1861 daltons.

[0118] The terms first, second, third, and the like in the specification and claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. The terms are interchangeable in appropriate circumstances. Embodiments of the invention may operate in sequences other than those described or illustrated herein.

[0119] Furthermore, although various embodiments may be referred to as "preferably" or "eg" or "for example" or "specifically," these should not be construed as limiting the scope of the invention, but rather as exemplary ways in which the invention may be implemented.

[0120] The term "comprising" used in the claims should not be construed as being limited to the subsequently listed elements or steps; it does not exclude other elements or steps. It needs to be construed as specifying the presence of a stated feature, integer, step, or component, but does not exclude the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the scope of the phrase "a pharmaceutical composition comprising A and B" should not be limited to a pharmaceutical composition consisting solely of components A and B; for the purposes of the present invention, the recited components of the pharmaceutical composition are only A and B, and the claim should be construed to encompass equivalents of those components. Similarly, the scope of the phrase "a method comprising steps A and B" should not be limited to a method consisting solely of steps A and B; rather, for the purposes of the present invention, the recited steps of the method are only A and B, and the claim should be construed to encompass equivalents of those steps.

[0121] Additionally, unless the context clearly requires that there be only one of the feature, reference to a feature by the indefinite article "a" or "an" does not exclude the possibility that more than one of the feature may be present, e.g., a component, excipient, saponin, etc. Thus, the indefinite article "a" or "an" normally means "at least one." [Brief explanation of the drawings]

[0122] [Figure 1-1] Tumor-targeted protein toxin delivery results in tumor volume reduction and tumor growth inhibition in tumor-bearing mice. A) Dose-escalating administration (intraperitoneal i.p.) of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice reveals tumor volume reduction compared to controls. B, C) Dose-escalating administration (intraperitoneal i.p. (B) or intravenous i.v. (C)) of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice reveals tumor growth reduction compared to controls. [Figure 1-2]Tumor-targeted protein toxin delivery results in tumor volume reduction and tumor growth inhibition in tumor-bearing mice. A) Dose-escalating administration (intraperitoneal i.p.) of cetuximab-(Cys-L-SO1861)3,9(Lys-S-gianthine)2 in A431 tumor-bearing mice reveals tumor volume reduction compared to controls. B, C) Dose-escalating administration (intraperitoneal i.p. (B) or intravenous i.v. (C)) of cetuximab-(Cys-L-SO1861)3,9(Lys-L-gianthine)2 in A431 tumor-bearing mice reveals tumor growth reduction compared to controls. [Figure 2] Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice compared to controls. 30 mg / kg cetuximab-(Cys-L-SO1861)3,9(Lys-L-HSP27BNA)1,8 in A431 tumor-bearing mice demonstrates efficient tumor-targeted gene silencing compared to controls. [Figure 3] Tumor-targeted antisense BNA oligonucleotide delivery and gene silencing in tumor-bearing mice compared to controls. 30 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 in A431 tumor-bearing mice reveals induced efficient tumor-targeted gene silencing compared to controls. [Figure 4-1]HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 4-2] HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 4-3]HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 4-4] HER2- or EGFR-targeted protein toxin delivery and cell killing in cancer cells according to the present invention. A, B) Trastuzumab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or trastuzumab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in SK-BR-3 cells (HER2) and MDA-MB-468 cells (HER2-). C, D) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-gianthine)1,7 or cetuximab-(Cys-L-SO1861)3,8(Lys-S-gianthine)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 5-1] EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 5-2]EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)1,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 6] HER2-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. SK-BR-3 cells (HER2) treated with trastuzumab-(Cys-L-SO1861)3,8(Lys-L-HSP27BNA)3,5 and control. Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 7] EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B) Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 treatment and control in A431 cells (EGFR) and A2058 cells (EGFR-). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 8-1] Control treatments for all cell lines. AD) Cell viability when the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, and BT-474 are treated with trastuzumab (A), cetuximab (B), T-DM1, (C), free toxins: saporin and dianthin (D), or saporin coupled to non-cell-binding IgG (D). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 8-2]Control treatments for all cell lines. AD) Cell viability when the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058, and BT-474 are treated with trastuzumab (A), cetuximab (B), T-DM1, (C), free toxins: saporin and dianthin (D), or saporin coupled to non-cell-binding IgG (D). Note: See Table 23 for target receptor expression data (determined by FACS analysis) for each cell line. [Figure 9] (S)n-(L)(E) Concept: mAb-(SO1861)n (protein toxin). For delivery and internalization into target cells, SO1861 is conjugated to a cysteine ​​residue (Cys) and a protein toxin (ribosome-inactivating protein) is conjugated to a lysine residue on the same antibody (mAb). 1) mAb-(Cys-L-SO1861)4 (Lys-protein toxin)2 binds to its corresponding cell surface receptor, 2) endocytosis mediated by the conjugated receptor occurs, 3) at a low endolysosomal pH and appropriate concentration, SO1861 becomes active, enabling endolysosomal escape, 4) release of the toxin into the cytoplasm, and 5) the toxin induces cell death. [Figure 10] (S)n-(L)(E) Concept: mAb-(SO1861)n (antisense BNA oligo)n. Both SO1861 at a cysteine ​​residue (Cys) and an antisense BNA oligonucleotide at a lysine residue are conjugated to the same antibody (mAb) for delivery and internalization into target cells. 1) mAb-(Cys-SO1861)4 (Lys-BNA oligo)2 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active, enabling endolysosomal escape, 4) release of the BNA oligo into the cytoplasm, and 5) induction of target gene silencing. [Figure 11](S)n-(L)(E) Concept: mAb-(SO1861-scaffold-antisense BNA oligo)n. (SO1861-trifunctional linker-BNA oligo)n is conjugated to an antibody (mAb) for delivery and internalization into target cells. 1) mAb-(SO1861-trifunctional linker-BNA oligo)4 binds to its corresponding cell surface receptor, 2) receptor-mediated endocytosis of both conjugates occurs, 3) at low endolysosomal pH and appropriate concentrations, SO1861 becomes active and enables endolysosomal escape, 4) release of the BNA oligo into the cytoplasm occurs, and 5) targeted gene silencing is induced. [Figure 12] Antibody-SO1861 Conjugation Procedure. The coupling reaction of four moieties of the plant-derived saponin SO1861 to four cysteines on the light chain of an antibody is shown. First, the disulfide bonds on the IgG are blocked under the influence of exposure to TCEP (tris(2-carboxyethyl)phosphine). Second, the saponin SO1861 containing a chemical linker attached to it is added with trifluoroacetic acid, linking the four saponin moieties to the IgG. To produce a cleavable "ready-to-conjugate" saponin, the aldehyde group of SO1861 was reacted with an EMCH (ε-maleimidocaproic acid hydrazide) linker. The hydrazide group of EMCH forms an acid-cleavable hydrazone bond with the aldehyde of SO1861. At the same time, the EMCH linker presents a maleimide group that is thiol (sulfhydryl) reactive and can therefore be conjugated to a thiol of an IgG, i.e., a ligand moiety, thereby providing an endosomal escape-enhancing conjugate of the invention and / or providing a first binding molecule of the invention. [Figure 13] SO1861-EMCH synthesis [Figure 14-1] Synthesis of dendron-(-L-SO1861)4. [Figure 14-2] Synthesis of dendron-(-L-SO1861)4. [Figure 14-3] Synthesis of dendron-(-L-SO1861)4. [Figure 14-4]Synthesis of dendron-(-L-SO1861)4. [Figure 14-5] Synthesis of dendron-(-L-SO1861)4. [Figure 14-6] Synthesis of dendron-(-L-SO1861)4. [Figure 15-1] Synthesis of dendron (-L-SO1861)8. [Figure 15-2] Synthesis of dendron (-L-SO1861)8. [Figure 15-3] Synthesis of dendron (-L-SO1861)8. [Figure 15-4] Synthesis of dendron (-L-SO1861)8. [Figure 15-5] Synthesis of dendron (-L-SO1861)8. [Figure 16-1] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 16-2] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 16-3] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 16-4] SO181-L-trifunctional linker-L-HSP27BNA synthesis. [Figure 17] Synthesis of HSP27BNA-dendron-(-L-SO1861)4. [Figure 18] Synthesis of dendron (NEM)4. [Figure 19] A scaffold precursor with four amino groups for saponin linkages and an azide group for click chemistry. [Figure 20] Evidence of saponin coupling to the model scaffold. The inset shows the theoretically expected peak and intensity distribution for the bound saponin. Experimental data obtained by LC-MS / ESI-MS showed almost exactly the same peaks at m / z 758-760 Da, demonstrating successful saponin coupling. [Figure 21]Cytotoxicity assay using the targeted toxin dianthine-epidermal growth factor (dianthine-EGF). Untreated cells were normalized to 1. The polymeric structure (pentrimer) had no effect on cell viability in the presence or absence of dianthine-EGF and saponin (SA1641), indicating the lack of intrinsic cytotoxicity of the polymeric structure. The clickable targeted toxin (dianthine-EGF-alkyne) had significantly reduced activity. This is the result of toxin modification but not any relationship to the scaffold. The functionalized polymeric structure had the same activity as the unclicked targeted toxin, indicating that scaffold functionalization does not impair effector molecule activity. The effect of saponin was identical in the presence and absence of the polymeric structure, indicating that the polymeric structure does not impair the efficacy of saponin in the two-component system. [Figure 22-1] H-NMR spectra of (A) SO1861 and (B) SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide). (A) The peak at 9.43 ppm (Ha) corresponds to the aldehyde proton of SO1861. (B) The peak at 6.79 ppm (Hc) corresponds to the maleimide proton of SO1861-EMCH, and the peak at 7.68 ppm (Hb) corresponds to the hydrazone proton. The absence of the signal at 9.43 ppm indicates quantitative conversion of the aldehyde group. [Figure 22-2] H-NMR spectra of (A) SO1861 and (B) SO1861-EMCH (EMCH = N-ε-maleimidocaproic acid hydrazide). (A) The peak at 9.43 ppm (Ha) corresponds to the aldehyde proton of SO1861. (B) The peak at 6.79 ppm (Hc) corresponds to the maleimide proton of SO1861-EMCH, and the peak at 7.68 ppm (Hb) corresponds to the hydrazone proton. The absence of the signal at 9.43 ppm indicates quantitative conversion of the aldehyde group. [Figure 23]MALDI-TOF-MS spectra of (A) SO1861-EMCH and (B) SO1861-EMCH-mercaptoethanol. (A) RP mode: m / z 2124 Da ([MK], saponin-EMCH), m / z 2109 Da ([MK], SO1861-EMCH), m / z 2094 Da ([MNa], SO1861-EMCH). (B) RP mode: m / z 2193 Da ([MK], saponin-EMCH-mercaptoethanol), m / z 2185 Da ([MK], SO1861-EMCH-mercaptoethanol), m / z 2170 Da ([MNa], SO1861-EMCH-mercaptoethanol). [Figure 24] SO1861 structure with highlighted chemical groups for conjugation of saponin to the polymeric structure to enhance endosomal escape. The highlighted groups are aldehyde (black circle), carboxylic acid (dashed circle), alkene (dashed pentagon), and alcohol (dashed box). The aldehyde group (arrow) is the most suitable group for chemoselective and reversible conjugation reactions. [Figure 25] Strategy for producing (A) stable and (B) cleavable "ready-to-conjugate" endosomal escape-enhancing factor saponins. [Figure 26] Hydrolysis of the hydrazone bond of SO1861-EMCH under acidic conditions. [Figure 27] SO1861-EMCH structure. (A) Standard molecular structure and (B) 3D model. Maleimide groups are marked with circles. [Figure 28-1] (A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 28-2](A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 28-3] (A) Synthesis scheme of SO1861-EMCH. (B) MALDI-TOF-MS spectra of SO1861 (m / z 1861 Da) and (C) SO1861-EMCH (m / z 2068 Da) in negative reflector mode. TFA: trifluoroacetic acid, rt: room temperature, h: time, and MW: molecular weight. [Figure 29-1] MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl solution at pH 3. [Figure 29-2] MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl solution at pH 3. [Figure 30] Reaction scheme for conjugation of SO1861-EMCH to either amine-bearing polymer structure. [Figure 31] MALDI-TOF-MS spectra of (A) BSA-SO1861 (m / z 70.0 kDa, 72.1 kDa, 74.2 kDa) and (B) BSA (m / z 66.6 kDa). [Figure 32] (B) Reaction scheme for the conjugation of (A) SO1861-EMCH and (B) SO1861-HATU (HATU = 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) to cyanine 3 dye-labeled polyamidoamine (PAMAM) G5 dendrimer. [Figure 33-1]MALDI-TOF-MS spectra of (A) Cy3-PAMAM and (B-D) Cy3-PAMAM-SO1861 with increasing SO1861-EMCH loading equivalents from (B) to (D). (B) corresponds to Cy3-PAMAM-SO1861 with 5 SO1861 atoms attached per PAMAM, (C) corresponds to Cy3-PAMAM-SO1861 with 13 SO1861 atoms attached per PAMAM, and (D) corresponds to Cy3-PAMAM-SO1861 with 51 SO1861 atoms attached per PAMAM. [Figure 33-2] MALDI-TOF-MS spectra of (A) Cy3-PAMAM and (B-D) Cy3-PAMAM-SO1861 with increasing SO1861-EMCH loading equivalents from (B) to (D). (B) corresponds to Cy3-PAMAM-SO1861 with 5 SO1861 atoms attached per PAMAM, (C) corresponds to Cy3-PAMAM-SO1861 with 13 SO1861 atoms attached per PAMAM, and (D) corresponds to Cy3-PAMAM-SO1861 with 51 SO1861 atoms attached per PAMAM. [Figure 34] MALDI-TOF-MS spectra of (A) Cy3-PAMAM-SO1861 prepared by SO1861-EMCH at a loading of 5 equivalents and (B) Cy3-PAMAM-SO1861 prepared by SO1861-EMCH at a loading of 30 equivalents. [Figure 35] MALDI-TOF-MS spectrum of Cy3-PAMAM-NC-SO1861 (NC = stable bond (“non-cleavable”)). [Figure 36-1] (A) Reaction scheme and MALDI-TOF-MS spectra of (B) Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO), (C) Cy3-PAMAM-(SO1861)5-DBCO, and (D) Cy3-PAMAM-(SO1861)27-DBCO. [Figure 36-2](A) Reaction scheme and MALDI-TOF-MS spectra of (B) Cy3-PAMAM-NC-SO1861-dibenzocyclooctyne (DBCO), (C) Cy3-PAMAM-(SO1861)5-DBCO, and (D) Cy3-PAMAM-(SO1861)27-DBCO. [Figure 37-1] Reaction schemes of (A) dianthine-EGF-Alexa488 and (B) dianthine-EGF-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine-EGF, (D) dianthine-EGF-Alexa488, and (E) dianthine-EGF-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 37-2] Reaction schemes of (A) dianthine-EGF-Alexa488 and (B) dianthine-EGF-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine-EGF, (D) dianthine-EGF-Alexa488, and (E) dianthine-EGF-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 38-1] Reaction scheme of (A) dianthine-Alexa488 and (B) dianthine-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine, (D) dianthine-Alexa488, and (E) dianthine-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 38-2] Reaction scheme of (A) dianthine-Alexa488 and (B) dianthine-Alexa488-SS-PEG-N3. MALDI-TOF-MS spectra of (C) dianthine, (D) dianthine-Alexa488, and (E) dianthine-Alexa488-SS-PEG-N3; Alexa488: Alexa Fluor 488 dye. [Figure 39]Fluorescent image of SDS-PAGE gel performed by VersaDoc imaging system. M = marker, P = Cy3-PAMAM-(SO1861)27-DBCO, D = dianthine-EGF-Alexa488-SS-PEG-N3, C1 = Cy3-PAMAM-(SO1861)5-dianthine-EGF-Alexa488, C2 = Cy3-PAMAM-NC-SO1861-dianthine-EGF-Alexa488, and C3 = Cy3-PAMAM-(SO1861)27-dianthine-EGF-Alexa488. [Figure 40] (A) Synthesis scheme of Cy3-PAMAM-NC-SO1861 by reductive amination. (B and C) Respective MALDI-TOF-MS spectra. [Figure 41] Reaction scheme for the production of poly(SO1861) using SO1861-EMCH as the monomer, the APS / TMEDA system as the polymerization initiator, and aminopropanethiol as the radical quenching agent. [Figure 42] MALDI-TOF-MS spectra of poly(SO1861) reaction batches: (A) SO1861-EMCH at 60 °C, (B) SO1861-EMCH 11-3 equivalents APS at 60 °C, and (C) SO1861-EMCH 11-3 equivalents APS / TMEDA at 60 °C. [Figure 43] DNA approach. Use of the principles of DNA origami to generate DNA-based scaffolds capable of conjugating and releasing glycoside molecules. In addition, one of the DNA strands yields a click chemistry moiety that can be used for conjugation to targeted toxins to form a functionalized scaffold. bp: base pair. [Figure 44] Polypeptide (peptide-SO1861) approach: Use of peptide sequences that can conjugate and release glycoside molecules and react with them to form poly(peptide-SO1861) constructs. The terminus of the poly(peptide) chain can be further modified with click chemistry moieties (e.g., BCN-NHS linkers) that can be used for conjugation to toxins. [Figure 45]MALDI-TOF-MS spectra of (A) the native peptide and (B) the peptide-SO1861 conjugate. [Figure 46] Molecular structure of a G4-dendron with a protected amino group. [Figure 47] Synthetic schemes for the generation of dendron-based and functional scaffolds. [Figure 48] (A) Reaction scheme for partial dye-labeling and deprotection of G4-dendron. (B) MALDI-TOF-MS spectrum of deprotected and partially dye-labeled G4-dendron. [Figure 49] MALDI-TOF-MS spectra of G4-dendron-SO1861 scaffolds with (A) 22 equivalents of SO1861-EMCH, (B) 10 equivalents of SO1861-EMCH, and (C) 3 equivalents of SO1861-EMCH. [Figure 50-1](A) Cell viability curves of treated HeLa cells according to EGFR cell surface expression determined by FACS analysis of HeLa cells. (B, see Table 19) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF (Dia-EGF), SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, and SO1861 Gianthine-EGF 667 nM dendron. (C) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF, SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, SO1861 Gianthine-EGF 500 nM PAMAM-(SH)16, SO1861 Gianthine-EGF 500 nM PAMAM-(SH)65, and SO1861 Gianthine-EGF 500 nM PAMAM-(SH)108. (D) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF, SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)3, SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)8, and SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)18. [Figure 50-2](A) Cell viability curves of treated HeLa cells according to EGFR cell surface expression determined by FACS analysis of HeLa cells. (B, see Table 19) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF (Dia-EGF), SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, and SO1861 Gianthine-EGF 667 nM dendron. (C) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF, SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, SO1861 Gianthine-EGF 500 nM PAMAM-(SH)16, SO1861 Gianthine-EGF 500 nM PAMAM-(SH)65, and SO1861 Gianthine-EGF 500 nM PAMAM-(SH)108. (D) Cell viability of HeLa cells treated with SO1861 Gianthine-EGF, SO1861 Gianthine-EGF 500 nM chloroquine, SO1861 Gianthine-EGF 500 nM PAMAM, SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)3, SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)8, and SO1861 Gianthine-EGF 500 nM PAMAM-(mPEG)18. [Figure 51-1] (A) Reaction scheme for thiolation of PAMAM using the thiolation reagent 2-iminothiolane. MALDI-TOF-MS spectra of (B) native PAMAM, (C) thiolated PAMAM-(SH)16, (D) thiolated PAMAM-(SH)65, and (E) thiolated PAMAM-(SH)108. [Figure 51-2] (A) Reaction scheme for thiolation of PAMAM using the thiolation reagent 2-iminothiolane. MALDI-TOF-MS spectra of (B) native PAMAM, (C) thiolated PAMAM-(SH)16, (D) thiolated PAMAM-(SH)65, and (E) thiolated PAMAM-(SH)108. [Figure 52-1](A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 52-2] (A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 52-3] (A) Reaction scheme for PEGylation of PAMAM using the PEGylation reagent mPEG2k-NHS. MALDI-TOF-MS spectra of (B) native PAMAM, (C) PEGylated PAMAM-(mPEG2k)3, (D) PEGylated PAMAM-(mPEG2k)8, and (E) PEGylated PAMAM-(mPEG2k)18. [Figure 53] A platform with click chemistry functional groups for linking any desired effector molecule. The user determines the location of the click chemistry position on the effector molecule, as well as any further properties of the effector molecule, such as the choice and location of any optional ligands. [Figure 54] A functionalized scaffold with pre-bound effector molecules and click chemistry functional groups for linking any desired ligand. Optionally, a pH-sensitive linker can be provided for release of the effector molecule from the scaffold after reaching the endosome. DETAILED DESCRIPTION OF THE INVENTION

[0123] For a bioactive molecule to work, it must be able to engage its target, for example, in serum, on the cell surface, or within a cell or organelle. The active moiety of nearly all protein-based targeted toxins, for example, must enter the cytosol of the target cell to mediate its target-modulating effect. In many constellations, the toxin remains ineffective because (1) the targeting moiety is poorly internalized and remains bound to the outside of the cell, (2) it is recycled to the cell surface after internalization, or (3) it is transported to the endolysosome, where it is degraded. These fundamental issues have been known for decades, and more than 500 targeted toxins have been investigated over the past several decades. However, the problem has not yet been solved, and only a few antibody-targeted protein toxins have been approved for sale to date, albeit with warning labels about 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.

[0124] Many strategies to overcome these problems have been described, including approaches to target toxins to endogenous membrane transport complexes of the biosynthetic pathway in the endoplasmic reticulum and techniques to block or weaken the membrane integrity of endosomes, i.e., compartments of the endocytic pathway in cells, thereby facilitating endosomal escape. These include the use of lysosomotropic amines, carboxylic acid ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human, and synthetic origin, other organic molecules, and light-inducible techniques. While the efficacy of targeted toxins has typically been increased by 100- or 1,000-fold in cell culture, and in exceptional cases by more than 1,000,000-fold, the requirement to coadminister endosomal escape-enhancing agents with other substances introduces new challenges, including additional side effects, loss of target specificity, difficulty in determining the therapeutic window, and cell-type-dependent variability.

[0125] All strategies involving physicochemical techniques require enhancer molecules that interact more or less directly with membranes and essentially include small chemical molecules, secondary metabolites, peptides, and proteins. A common feature of all these substances is that they are not target cell specific themselves and distribute with kinetics other than targeted toxins. This is one major drawback of current approaches.

[0126] While the present invention will be described with reference to specific embodiments, the invention is not limited thereto but only by the claims. The embodiments of the invention described herein can work in combination and in concert, unless otherwise specified.

[0127] While the present invention has been described in terms of several embodiments, it is contemplated that alternatives, modifications, permutations, and equivalents thereof will become apparent to those skilled in the art upon reading and understanding the specification and upon studying the drawings and graphs. The present invention is in no way limited to the illustrated embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0128] One aspect of the invention relates to a conjugate comprising a cell surface targeting molecule and at least one effector moiety, and further comprising at least one covalently attached saponin.

[0129] 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 is administered to a tumor-bearing mammal (mouse). The conjugate comprises at least one covalently attached saponin. The conjugates of the present invention have at least one glycoside, such as a saponin, attached thereto, preferably covalently, more preferably via a cleavable linker. The saponin increases the therapeutic efficacy of an effector moiety attached to a cell surface targeting molecule, presumably by improving endosomal escape of the effector moiety to the cytosol, where the effector moiety's activity is desired. In this manner, a therapeutic effect is established under the influence of the presence of a conjugate containing saponin(s) at a dose already lower than conventional doses of ADCs or AOCs, i.e., the conjugates of the present invention. This brings the saponin(s) to, near, and / or within the target cells. The target cell may be, for example, a diseased cell, such as a tumor cell or an autoimmune cell or a B cell associated with a B cell disorder, etc. According to the present invention, the effector moiety is, for example, an oligonucleotide such as a toxin as part of an ADC or an antisense BNA as part of an AOC.

[0130] One embodiment is a conjugate of the invention, wherein at least one saponin is a triterpenoid saponin and / or a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position C-23 and optionally containing a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, and / or a saponin isolated from any one or more of Gypsophila species and / or Saponaria species and / or Agrostemma species and / or Quillaja species, e.g., Quillaja saponaria.

[0131] In one embodiment is a conjugate of the invention, wherein at least one saponin is a single particular saponin or a mixture of two or more different saponins.

[0132] One embodiment is a conjugate of the invention, wherein at least one saponin has a molecular mass of 3,000 daltons or less, preferably 2,500 daltons or less, more preferably 2,300 daltons or less, most preferably 2,000 daltons or less, for example 1,500 to 1,900 daltons.

[0133] In one embodiment is a conjugate of the invention, wherein the at least one saponin is a saponin of Table A1 or Scheme I, 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 albumenum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereoisomers and / or any combination thereof. Preferably, the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21, and / or a saponin having a quillaric acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3 beta-OH group, and a Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent at the C-28-OH group. and / or 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquilayic acid, 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside. More preferably, the at least one saponin is SO1861 and / or QS-21.

[0134] The inventors herein disclose that covalently coupling a saponin, such as the water-soluble fraction of Quillaja saponaria, QS-21, SA1641, SO1861, saponins of Table A1, Scheme I, to a cell surface targeting molecule, such as a proteinaceous molecule, for example via a trifunctional linker, such as the trifunctional linker of Scheme II, or via a backbone oligomeric or polymeric structure, and including a covalently bound saponin, results in improved cytotoxicity exerted by an effector moiety, such as a toxin, included in the conjugate under the influence of the covalently coupled saponin on the conjugate.

[0135] In one embodiment is a conjugate of the invention, comprising a saponin comprising one or some or all of the indicated structural features of the saponin of Structure A in Scheme I (a saponin of Structure A is said to have an "ideal" structure when it has endosomal escape-enhancing activity for an effector moiety in the endosomes of a cell contacted with a conjugate of the invention), and / or a saponin selected from any one or more of the additional saponins of Scheme I:

[0136] [ka]

[0137] [ka]

[0138] [ka]

[0139] [ka]

[0140] [ka]

[0141] According to the present invention, glycosides such as saponins according to the present invention attached to cell surface molecule targeting molecules included in the conjugates of the present invention, which have an "ideal" structure for the purpose of enhancing endosomal escape of effector molecules included in the conjugates of the present invention, are bisdesmosidic saponins according to Structure A of Scheme I, which have a molecular mass of at least 1,500 daltons and comprise an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, and which have a first branched carbohydrate side chain at the C-3 position, which optionally contains glucuronic acid. The saponin contains an ester group with a second branched carbohydrate side chain at the C-28 position, this second branched carbohydrate chain preferably containing at least four carbohydrate units, optionally containing at least one acetyl residue, for example two acetyl residues, and / or optionally containing a deoxy carbohydrate, and / or optionally containing quinovose, and / or optionally containing glucose, and / or optionally containing 4-methoxycinnamic acid, and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally containing 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, linked to the carbohydrate via an ester bond.

[0142] SO1861 differs from the "ideal structure" represented by Structure A in Scheme I only by having only one acetyl residue on the quinovose and by having an additional xylose. The "ideal structure" of a saponin for enhancing endosomal escape of an effector molecule or effector moiety is preferably a saponin having Structure A in Scheme I, and saponins that exhibit endosomal escape-enhancing activity have one or more structural features represented by Structure A in Scheme I. Without wishing to be bound by any theory, the inventors believe that Structure A in Scheme I represents an "ideal saponin" (and not a minimum requirement saponin) for endosomal escape-enhancing activity. This means that not all of the structures (chemical groups) can or must be present on every saponin that has at least sufficient endosomal escape-enhancing activity to promote accumulation of the effector moiety in the cytosol. This also means that some saponins may have other structural elements, such as acyl chains, and / or that for still other saponins that exhibit endosomal escape-enhancing activity, the sugars may differ from those represented by Scheme I. For example, when considering the idealized structure A in Scheme I, QS-21 saponin and some of the saponins in the water-soluble fraction of Quillaja saponaria (Quillaja saponin; Quil-A) differ in carbohydrate modifications at C-28, such as the presence of an acyl chain in QS-21. Saponins in the water-soluble fraction of Quillaja saponaria, such as QS-7 and QS1862, are similar to the idealized structure A and similar to QS1861.

[0143] In one embodiment is a conjugate of the invention, wherein at least one saponin is a bisdesmosidic saponin, has a molecular mass of at least 1,500 daltons, comprises an oleanane-type triterpene containing an aldehyde group at the C-23 position and optionally a hydroxyl group at the C-16 position, and has a first branched carbohydrate side chain at the C-3 position, which optionally contains glucuronic acid. The saponin contains an ester group with a second branched carbohydrate side chain at the C-28 position, this second branched carbohydrate chain preferably containing at least four carbohydrate units, optionally containing at least one acetyl residue, for example two acetyl residues, and / or optionally containing a deoxy carbohydrate, and / or optionally containing quinovose, and / or optionally containing glucose, and / or optionally containing 4-methoxycinnamic acid, and / or optionally containing 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, and / or optionally containing 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, linked to the carbohydrate via an ester bond. Alternatively, the at least one saponin is QS-21 or any one or more of 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, QS-18, QS-1861, protonated QS1861 (QS1862), and Quil-A.

[0144] Table A1 and Scheme I and the above embodiments summarize a series of saponins that have been identified for their endosomal escape-enhancing activity when contacted in free form with a second molecule (e.g., an effector moiety or molecule, e.g., a toxin, an oligonucleotide) to mammalian cells, particularly human tumor cells. Reference is also made to saponins with such endosomal escape-enhancing activity listed in Tables 9 and 10 of Böttger (2013) (Stefan Böttger, "Understanding synergistic effects of saponins and ribosomal inactive proteins, Type I, Dissertation on Erlangung"). des akademischen grades des doktors der (Naturwissenschaften (Dr. rer. nat.), eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universitat Berlin, Table 9, Table 10, pages 67-74). Indeed, in cell-based bioassays using human tumor cells, it has been established that for the saponins tabulated in Table A1 and those of Scheme I and various embodiments of the invention described herein, when part of a conjugate of the invention, under the influence of these saponins, effector moieties, e.g., nucleic acids and / or toxins, e.g., protein toxins (e.g., one or more of the protein toxins listed in Table A5), included in the conjugate are delivered to the cytosol with increased efficiency and / or effectiveness, presumably by intracellular release from (late) endosomes and lysosomes. That is, endosomal and / or lysosomal escape of such effector moieties, e.g., nucleic acids and / or toxins, attached by the conjugates of the invention is less efficient in the absence of saponin.

[0145] Surprisingly, the inventors have now discovered that QS-21 and its family members QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 Water-soluble saponin fractions from Quillaja saponaria, including B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18, and Quil-A, also demonstrate the ability to enhance the in vitro biological effects of, for example, nucleic acids conjugated to monoclonal antibodies or protein toxins conjugated to monoclonal antibodies (examples of conjugates of the invention comprising payloads such as covalently attached oligonucleotides or (protein) toxins). In this case, tumor cells of a mammalian species (human) are administered a monoclonal antibody (a cell surface molecule targeting molecule of the present invention) in the form of a covalent conjugate comprising an attached effector moiety and at least one glycoside, such as QS-21 and its family member saponin (e.g., the water-soluble fraction of Quillaja saponaria) contained in the conjugate as a covalent conjugate. The effector molecule and glycoside, such as the saponin fraction of Quillaja saponaria, QS-21, SO1861, SA1641, GE1741, are covalently attached to, for example, the cell surface molecule targeting molecule, either directly or via a linker, or via a polymeric or oligomeric structure, either directly or via at least one linker. Without wishing to be bound by any theory, the observed stimulation or enhancement of, for example, antisense BNA-mediated reduction of tumor cell HSP27 expression (HSP27 gene silencing) in the presence of saponin from Quillaja saponaria in vitro may (also) be related to activation of inflammasomes in tumor cells by saponin, leading to, for example, tumor cell pyroptosis.The present inventors have established that when the conjugate comprises a saponin, such that the saponin(s) are targeted to the same (tumor) cells, the cell surface molecule-targeting molecules of the present invention conjugated to, for example, antisense BNAs, dianthin, or saporin, exert any or improved antitumor cell activity in vitro when contacted with cells in a bio-based cell assay. On the other hand, in the absence of saponin(s), i.e., in conventional ADC formats without linked saponin(s), no such activity against tumor cells was observed. The presence of a covalently coupled saponin on the conjugates of the present invention has been found to be essential for the efficient exertion of the activity of the effector moiety on the conjugate.

[0146] QS-21 and water-soluble saponin fractions containing QS-21 from Quillaja saponaria have also been known for a long time and have been actively applied, for example, due to their immune-enhancing ability as adjuvants for subunit vaccines. For example, QS-21 has been applied in two phase III clinical trials in human patients. They were vaccinated with a subunit vaccine mixed with an adjuvant containing QS-21 (Glaxo-Smith-Kline, MAGRIT trial, DERMA study). The subunit was the MAGE-A3 protein, which is specifically expressed and presented by tumor cells. QS-21-enhanced antitumor vaccination aimed to extend disease-free survival in cancer patients (melanoma; non-small cell lung cancer). Additionally, QS-21 has been tested as an adjuvant in clinical trials for the development of anti-cancer vaccine treatments, vaccines for HIV-1 infection, vaccines for hepatitis B, and anti-malaria vaccines using QS-21 with Glaxo-Smith-Kline's adjuvants AS01 and AS02. Previous studies have demonstrated that under the influence of QS-21 saponin containing adjuvants, an immune response is elicited against MAGE-A3 peptides displayed on the surface of cancer cells (AS15; GSK). To the inventor's surprise, the saponin fraction of Quillaja saponaria, and therefore likely QS-21 (as part of the water-soluble saponin fraction of Quillaja saponaria), enhances the anti-tumor cell activity of a payload, such as a protein toxin (dianthin), attached to a cell surface targeting molecule (e.g., EGF ligand) on the conjugate of the present invention (e.g., EGF ligand).

[0147] For example, targeting a single cell surface molecule with a conjugate of the present invention results in improved and more specific delivery of the effector moiety and saponin attached to the cell surface molecule targeting molecule on the conjugate of the present invention in and within the cytosol of target cells that expose the cell surface molecule on the cell surface, compared to contacting cells with a conventional ADC alone that lacks the saponin of the present invention, and therefore without the cell-targeting saponin (conjugate of the present invention). The diseased cells selected for targeting with the cell surface molecule targeting molecule of the conjugate ideally have a high degree (i.e., relatively high expression of the target cell surface molecule on target cells, e.g., tumor cells or autoimmune cells, compared to expression on non-target cells, e.g., healthy cells) of epitopes on the cell surface molecule to which the cell surface molecule targeting molecule can bind, and / or, when considered in conjunction with (adjacent) healthy cells of the patient, specifically expose epitopes on the target cell surface molecule for binding of the cell surface molecule targeting molecule of the conjugate. Preferably, the cell surface molecule targeted by the cell surface molecule targeting molecule of the conjugate of the present invention is relatively highly and / or specifically expressed in target (disease, tumor) cells compared to healthy cells. One embodiment is a conjugate of the present invention, wherein the cell surface molecule targeted by the cell surface molecule targeting molecule of the conjugate, e.g., a tumor cell receptor, is specifically or relatively highly expressed when compared to the expression of the cell surface molecule on the surface of healthy (adjacent) cells. Therefore, the epitope on the target cell surface molecule is ideally unique to the target diseased cells, or at least specifically present and exposed on the surface of the target cells. Binding of the conjugate of the present invention to the epitope on the cell surface molecule on the target cell is followed by endocytosis of the complex of the conjugate and the target cell surface molecule.Because the conjugate can only enter target cells via binding interactions with cell surface molecules that are specifically or inherently expressed to a sufficient degree on the target cells, compared to healthy cells that should not be targeted, accumulation of a therapeutically effective amount of the effector moiety and saponin contained in the conjugate within the target cells is possible and will only occur if the expression level of the target cell surface molecule is above a certain minimum expression threshold. At the same time, the fact that the effector moiety attached to the cell surface molecule-targeting molecule 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 a safeguard against negative, unwanted side effects of the effector moiety on, for example, healthy cells and tissues not intended to be targeted and affected by the effector moiety, compared to exposure of cells to an ADC without the covalently attached saponin(s). That is, sufficiently low expression or absence of exposed cell surface molecules to which the conjugate can bind ideally does not allow the conjugate to enter (non-target) healthy cells to an extent that would result in endosomal escape of the effector moiety under the influence of the saponin contained in 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 compared to when an ADC or AOC without coupled saponin is applied in a therapeutic regimen, entry of the AOC having a coupled saponin in healthy cells or entry of the AOC having a coupled saponin to a low extent already carries a lower risk of causing unwanted side effects when targeting and killing target diseased cells, such as tumor cells and autoimmune cells, is considered.

[0148] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule comprises or consists of a ligand or proteinaceous ligand or proteinaceous binding molecule for binding to a cell surface molecule.

[0149] One embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule comprises or consists of a non-proteinaceous and / or proteinaceous ligand for binding to a cell surface molecule, such as EGF or a cytokine, and / or comprises or consists of an immunoglobulin, at least one binding domain of an immunoglobulin, and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising or consisting of a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment, which is capable of binding to a cell surface molecule.

[0150] The present inventors have established that such immunoglobulins, their domains, ligands, etc., are particularly suitable for application as cell surface molecule targeting molecules in conjugates containing a binding site for binding to a target cell surface molecule. For example, antibodies and antibody binding domains are suitable for targeting epitopes on selected cell surface molecules exposed on the cell surface, resulting in the conjugate, and thus the effector moiety and saponin, being targeted together to target cells expressing the cell surface molecule targeted by the cell surface molecule targeting molecule of the conjugate. Similarly, a ligand such as EGF that targets EGFR on target cells is suitable for application as a cell surface molecule targeting molecule in the conjugate. Cell surface molecule targeting molecules that bind to epitopes on target cell surface molecules that are specific for binding of the conjugate to the cell surface molecule are preferred. Cell surface molecule targeting molecules of the present invention based on antibodies or their domains or binding fragments enable such desired specificity for selected epitopes on selected cell surface molecules, such as selected cells for targeting, e.g., diseased cells, tumor cells, autoimmune cells, etc. Thus, cell surface molecule targeting molecules based on antibodies, such as (human, humanized, chimeric) monoclonal antibodies or binding molecules (fragments, domains) are preferred for the conjugates of the present invention.

[0151] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule is a tumor cell surface molecule, preferably a tumor cell receptor, e.g., 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, PSMA, CanAg, integrin The conjugate of the present invention may bind to a receptor selected from alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, and VEGFR2, preferably CD71, HER2, and EGFR. These receptors are preferred examples of molecules bearing epitopes to which the cell surface molecule targeting molecule of the conjugate of the present invention can bind. These molecules are sufficiently specific, or even inherently expressed, on the surface of the target cells selected for conjugate binding. As a result, the conjugate binds uniquely and / or specifically, or at least preferentially and at least to a greater extent, to said target cells, e.g., abnormal cells, tumor cells, malignant cells, diseased cells, B cells associated with malignancies, autoimmune cells, etc., compared to binding of the conjugate to cells that express and expose the receptor to a lesser extent or that do not expose the receptor on their cell surface.

[0152] An embodiment is a conjugate of the invention, wherein the tumor cell receptor is internalized by the tumor cell after binding to the cell surface molecule targeting molecule of the invention, or the tumor cell receptor is internalized by the tumor cell after binding to the conjugate of the invention, preferably, binding of the cell surface molecule targeting molecule to the tumor cell receptor and binding of the conjugate is followed by internalization of the complex of the conjugate and tumor cell receptor mediated by the tumor cell receptor, e.g., via endocytosis.

[0153] In one embodiment, the tumor cell receptor is internalized by the tumor cell upon binding to the conjugate of the present invention. And / or, internalization is induced by binding to the cell surface molecule targeting molecule of the present invention. Alternatively, the tumor cell receptor is internalized by the tumor cell after binding to the conjugate of the present invention, or is internalized by and / or due to binding of the conjugate to the tumor cell receptor. Preferably, binding of the cell surface molecule targeting molecule to the tumor cell receptor and binding of the conjugate induces and / or results in tumor cell receptor-mediated internalization of the complex of the conjugate and tumor cell receptor, e.g., via endocytosis.

[0154] Synchronization is the missing link between a successful delivery strategy in mice and its application in humans. Indeed, the inventors have established in a series of in vivo mouse tumor models that separately administering one dose of free saponin and one dose of ADC without coupled saporin to mice does not result in any desired antitumor activity, such as delayed tumor growth, tumor regression, diminished, or slower tumor growth, compared to control animals not treated with the ADC in the presence of free saponin. Free saponin was administered using various routes of administration and at various times (before, during, and after ADC administration) compared to the moment of ADC administration. The ADCs tested in the in vivo tumor models were cetuximab-giantin (with free SO1861) or trastuzumab-saporin (with free SO1861). Varying the dose of free saponin did not result in effective antitumor activity. The referenced ADC was administered at a dose that did not by itself produce any beneficial antitumor effect in tumor-bearing animals. Surprisingly, the inventors have now established that beneficial antitumor activity in various in vitro mammalian cell-based bioassays using human tumor cells and / or in 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). 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 linkage, and / or an effector moiety (e.g., dianthin, gene silencing antisense BNA (HSP27)) covalently attached via a non-cleavable or non-cleavable bond. The scaffold is covalently linked to the cell surface targeting molecule of the conjugate, such as a monoclonal antibody, such as cetuximab, trastuzumab, or OKT-9. Alternatively, the scaffold can be a dendron, for example, a dendron to which four moieties, such as, for example, four saponin molecules, can be attached, for example, a G4-dendron. Alternatively, for example, a dendron for binding two saponin molecules and two effector molecules.The dendrons contain chemical groups for (covalent) coupling of the conjugate to cell surface targeting molecules, such as ligands or antibodies or fragments or domains thereof. Reference is made to the Further Embodiments and Examples section, which illustrates various of these scaffolds according to the invention. They have shown in vivo and / or in vitro antitumor cell activity, for example, when considering cytotoxicity exerted by proteinaceous toxins or gene silencing in tumor cells.

[0155] Without wishing to be bound by any theory, in light of the failures observed when treating tumor-bearing animals with ADCs together with free saponins, it is preferable to synchronize the presence of both at least one saponin and an effector molecule, preferably a toxin or oligonucleotide, in a compartment or vesicle of the endocytic pathway of target cells, such as tumor cells or autoimmune cells. With ADCs and free saponins, synchronizing the presence of the molecules in late endosomes to achieve a synergistic effect in vivo has not been beneficially achieved according to the inventors' attempts. In certain embodiments, with respect to combining an effector moiety and saponin(s) on a single conjugate molecule, the present invention preferably solves at least the following problem: without wishing to be bound by any theory, for example, only one rational chemical group on the saponin that can be used for (covalent), particularly single and cleavable, retainable coupling is required for endosomal escape activity. Most likely, known limitations are the reason why, although the significant endosomal escape-enhancing effects of saponins, e.g., those listed in Table A1 and Scheme I, have been known for more than a decade, saponins have not been used in combination with active pharmaceutical ingredients in clinical studies other than the application of saponins in vaccination regimens involving the use of immune-enhancing adjuvant substances. For example, providing a covalently bound saponin to the conjugates of the present invention at least partially overcomes these difficulties, e.g., in the context of several saponin-bearing scaffolds. Surprisingly, saponins previously applied for their immune-enhancing activity in vaccination contexts involving saponins as adjuvant components are also suitable for (covalent) coupling to cell surface targeting molecules included in the conjugates of the present invention for in vitro and in vivo anti-tumor activity.

[0156] An embodiment is a conjugate of the invention, wherein the cell surface molecule targeting molecule is or comprises a monoclonal antibody or at least one cell surface molecule binding fragment or domain thereof, preferably comprises or consists of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, any one of the antibodies of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one cell surface molecule binding fragment or domain thereof.

[0157] As stated, targeting cell surface molecules with the cell surface molecule targeting molecules of the conjugates of the present invention results in improved and more specific delivery of saponin and effector moieties in and within the cytosol of the very same target cells. The diseased cells selected for targeting with the cell surface molecule targeting molecules of the conjugates ideally have cell surface molecules to a greater extent and / or specifically than the patient's (adjacent) healthy cells are considered. Therefore, the epitope on the target cell surface molecule is ideally unique to the target diseased cells, and is at least specifically present and exposed on the surface of the target cells. Binding of the conjugate is followed by endocytosis of the complex between the conjugate and the target cell surface molecule.

[0158] Tables A2, A3, and A4 list preferred examples of cell surface molecules that comprise the epitope of the cell surface molecule targeting molecule of the conjugates of the present invention. When the cell surface molecule is specifically expressed on the target cell, binding of the conjugate to the cell surface molecule preferably results in specific targeting of the conjugate to the desired target cell, such as a tumor cell that exposes the tumor cell surface molecule. Meanwhile, other cells, such as healthy cells that do not express the cell surface molecule or express the cell surface molecule to a lower extent compared to the expression of the cell surface molecule(s) on the target (abnormal) cell, are not targeted or are targeted only to a lower extent by the conjugate.

[0159] A drug substance in the present invention is an effector moiety that is used to achieve a beneficial result in an organism, preferably a vertebrate, more preferably a human, such as a cancer or autoimmune patient. Benefits include diagnosis, prognosis, treatment, cure, and / or prevention of a disease and / or condition. A drug substance may also lead to unwanted and harmful side effects. In this case, the pros and cons must be weighed to determine whether the drug substance is suitable in a particular case. If the effect of the drug substance in a cell is overwhelmingly beneficial to the whole organism, the cell is called a target cell. If the effect in a cell is overwhelmingly harmful to the whole organism, the cell is called an off-target cell. In artificial systems such as cell culture and bioreactors, target and off-target cells depend on the purpose and are defined by the user.

[0160] A polypeptide effector moiety can be, for example, a polypeptide that restores a lost function, such as an enzyme replacement, a gene regulatory function, or a toxin.

[0161] In one embodiment is a conjugate of the invention, wherein at least one effector moiety comprises or consists of any one or more of an oligonucleotide, a nucleic acid, and a xenonucleic acid, preferably a vector, a gene, a transgene that induces cell suicide, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense oligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA (miRNA), a DNA aptamer, an RNA aptamer, mRNA, minicircle DNA, peptide nucleic acid (PNA), phosphoroa The nucleic acid may be selected from any one or more of midate morpholino oligomers (PMOs), locked nucleic acids (LNAs), bridged nucleic acids (BNAs), 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 2'-O,4'-aminoethylene-bridged nucleic acids, 3'-fluorohexitol nucleic acids (FHNAs), plasmids, glycol nucleic acids (GNAs), and threose nucleic acids (TNAs), or derivatives thereof, more preferably BNAs, for example BNAs for silencing HSP27 protein expression.

[0162] The present inventors demonstrate that a tumor cell-targeting monoclonal antibody provided with a covalently coupled antisense BNA, e.g., a BNA(HSP27), and provided with a covalently coupled saponin of the invention (which is contacted with tumor cells, where both the BNA and saponin are coupled to the antibody (e.g., cetuximab) via a cleavable bond) can silence HSP27 in tumors in vivo compared to a control and compared to an AOC bearing only the BNA but not the saponin (SO1861, Quil-A). Thus, administering an ADC-saponin conjugate of the invention or an antibody-oligonucleotide conjugate-saponin conjugate (AOC-saponin) of the 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 the ADC alone or the AOC alone, at the same dose, without the saponin covalently attached to the monoclonal antibody. Notably, AOC and another monoclonal antibody with a covalently coupled saponin, as a combination of two separate conjugates, when administered separately to tumor-bearing mice in separate groups of mice, increased HSP27 expression in tumor cells compared to a control group (administered vehicle only). Only administration of the AOC-saponin conjugate of the present invention containing an effector moiety of the present invention resulted in reduced HSP27 expression compared to the control. The antisense BNA (HSP27) was a BNA with the oligonucleotide sequence 5'-GGCacagccagtgGCG-3' according to Zhang et al. (2011) (Y Zhang, Z Qu, S Kim, V Shi, B Liao, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid(LNA)-based antisense oligonucleotides without transfection, Gene Therapy (2011) 18, 326-333). Notably, to the best of the inventor's knowledge, BNAs have been designed for application as free nucleic acids. Here, the inventors demonstrate 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 of tumor-bearing animals. This approach to providing BNA-based AOCs opens a new avenue for administering targeted BNAs to human (cancer) patients in need thereof.

[0163] In one embodiment is a conjugate of the invention, wherein the at least one effector moiety comprises or consists of at least one proteinaceous molecule, preferably selected from any one or more of a peptide, a protein, an enzyme such as urease and Cre recombinase, a ribosome-inactivating protein, a proteinaceous toxin selected from Table A5, more preferably a viral toxin, such as apoptin, a bacterial toxin, such as Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncated diphtheria toxin (DT), cholera toxin; fungal toxins, such as alpha-sarcin; plant toxins including ribosome-inactivating proteins and the A chain of type 2 ribosome-inactivating proteins, such as giantin, e.g., giantin-30 or giantin-32, saporin, e.g., saporin-S3 or saporin-S6, bouganin, the deimmunized derivative of bouganin, debouganin, Shiga-like toxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin, viscumin A chain; or animal or human toxins, such as frog RNase, or granzyme B or angiogenin from humans, or any fragment or derivative thereof; preferably, the protein toxin is giantin and / or saporin.

[0164] In one embodiment is a conjugate according to the invention, wherein the at least one effector molecule comprises or consists of at least one payload, preferably one or more of a ribosome-targeting toxin, an elongation factor-targeting toxin, a tubulin-targeting toxin, a DNA-targeting toxin, and an RNA-targeting toxin, more preferably emtansine, pasudotox, maytansinoid derivative DM1, maytansinoid derivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin), calicheamicin, N -acetyl-γ-calicheamicin, pyrrolobenzodiazepine (PBD) dimer, benzodiazepine, CC-1065 analog, duocarmycin, doxorubicin, paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide, docetaxel, 5-fluorouracil (5-FU), mitoxantrone, tubulysin, indolinobenzodiazepine, AZ13599185, cryptophycin, rhizoxin, methotrexate, anthracycline, camptothecin analog, SN-38, DX-8951f, exatecan mesylate, Pseudomonas The steroid inhibitor is selected from one or more of a truncated form of P. aeruginosa exotoxin (PE38), a duocarmycin derivative, amanitin, α-amanitin, splicostatin, tylanstatin, ozogamicin, tesirin, amberstatin 269, and soravtansine, or a derivative thereof.

[0165] Effector moieties useful in the present invention preferably rely on late endosomal escape to exert their effect. Some effectors, such as Pseudomonas exotoxin, are rerouted to other organelles before the "late endosomal stage" and therefore would not normally benefit from coupling to a second proteinaceous molecule according to the present invention. However, such toxins can be adapted for use in the present invention, for example, by deleting the rerouting carried out by the signal peptide. Specifically, toxins that are highly toxic and would require only one molecule to escape the endosome and kill a cell can be modified to be less potent. It is preferred to use toxins that kill cells when at least 2, more preferably at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 50, and most preferably at least 100 toxin molecules escape the endosome. It is further preferred that the conjugates of the present invention comprise a covalently conjugated functionalized scaffold. That is, scaffolds such as oligomeric or polymeric scaffolds or trifunctional linkers containing covalently attached effector moieties for targeting the scaffold containing the attached effector moieties to target cells such as tumor cells or autoimmune cells. Furthermore, to reduce off-target toxicity, cell membrane-impermeable small molecule toxins are preferred effector molecules over cell membrane-permeable toxins.

[0166] The term "ligand" as used herein has its usual meaning, preferably a molecule or structure capable of binding to another molecule or structure on the cell surface of a target cell. The molecule or structure on the cell surface can be endocytosed, preferably absent or less pronounced on off-target cells. Preferably, the molecule or structure on the cell surface is constitutively endocytosed. More preferably, the conjugate, or more specifically, the cell surface molecule targeting molecule, in the present invention, induces endocytosis of the molecule or structure on the cell surface of the target cell after binding to the molecule or structure. This applies, for example, to epidermal growth factor receptor (EGFR), HER2, and CD71, which are present on the surface of various cancer cells. Examples of molecules or structures on the cell surface of target cells that are constitutively endocytosed include claudin-1 or major histocompatibility complex class II glycoprotein. The cell surface molecule targeting molecule can be, for example, an antibody, growth factor, or cytokine. In the conjugates of the present invention, combining a toxin with a cell surface molecule targeting molecule and at least one saponin of the present invention is one possibility for creating a targeted toxin. A toxin that is toxic only in target cells because it interferes with processes occurring only in the target cells can also be considered a targeted toxin (it cannot exert its toxic effect in off-target cells, such as apoptin). Preferably, the targeted toxin is a toxin that is combined with a cell surface molecule targeting molecule, such as a monoclonal antibody, so that it is active in target cells (because it is only bound to and endocytosed by the target cells) and not in off-target cells. For example, in conjugates of the present invention that include a functionalized scaffold containing an effector moiety and / or saponin(s), the cell surface molecule targeting molecule, such as a monoclonal antibody, guides the effector moiety and / or saponin(s) through the scaffold to the target cell. After internalization, at least one glycoside, preferably a saponin, contained in the conjugates of the present invention mediates endosomal escape of the effector moiety.The saponin is typically a saponin listed in Table A1 and Scheme I, preferably the saponin is SO1861 and / or QS-21 and / or SA1641 and / or GE1741.

[0167] Preferably, the effector moiety included in the conjugate of the invention, whose effect is enhanced by the saponin included in the conjugate, is detached from the conjugate upon endocytosis, e.g., from an antibody present on the conjugate as a cell surface molecule targeting molecule. This can be achieved, for example, by a cleavable bond that breaks under acidic, reductive, enzymatic, or light-induced conditions.

[0168] One embodiment is a conjugate of the invention, wherein at least one effector moiety is either covalently attached to the cell surface molecule targeting molecule via at least one linker or is directly attached to the cell surface molecule targeting molecule.

[0169] In one embodiment is a conjugate of the invention, wherein at least one effector moiety is covalently attached to a cell surface targeting molecule, thereby forming gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin antibody-drug conjugates, and any one of the antibody-drug conjugates of Table A2 and Table A3.

[0170] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecular targeting molecule, preferably an amino acid residue of the cell surface molecular targeting molecule, via an aldehyde functional group on the saponin, and / or to at least one effector moiety, preferably via an amino acid residue on at least one effector moiety, via an aldehyde functional group on the saponin, preferably an aldehyde functional group at position C-23 of a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type.

[0171] One embodiment is a conjugate of the invention, in which the aldehyde functional group of at least one saponin, preferably the aldehyde functional group at position C-23 of at least one saponin, is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled via a thio-ether bond to a sulfhydryl group, e.g., a sulfhydryl group of a cysteine, on a cell surface targeting molecule and / or on at least one effector moiety.

[0172] One embodiment is a conjugate of the invention, wherein at least one saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type, has an aldehyde functional group at position C-23, and comprises a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of the saponin, and the saponin is covalently attached to an amino acid residue of a cell surface molecular targeting molecule and / or to at least one effector moiety via said glucuronic acid functional group, preferably via an amino acid residue on the at least one effector moiety.

[0173] One embodiment is a conjugate of the invention, in which a glucuronic acid functional group as a carbohydrate substituent of the C-3 beta-OH group of at least one saponin is covalently coupled to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled via an amide bond to an amine group on the cell surface molecule targeting molecule and / or at least one effector moiety, for example, a lysine or N-terminal amine group of the cell surface molecule targeting molecule and / or at least one effector moiety.

[0174] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface targeting molecule and / or to at least one effector moiety, either directly or via at least one linker, such as a bifunctional linker based on N-ε-maleimidocaproic acid hydrazide and / or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, or a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B.

[0175] In one embodiment is a conjugate of the invention, wherein the trifunctional linker comprises a second chemical group to which at least one saponin is covalently attached, a third chemical group for covalently attaching to a cell surface targeting molecule, and a first chemical group for covalently attaching to at least one effector moiety, preferably the trifunctional linker is of Scheme II and Structure B.

[0176] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and to at least one effector moiety via at least one linker comprising a trifunctional linker to which both the cell surface molecule targeting molecule and the at least one effector moiety are attached, preferably the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0177] One embodiment is a trifunctional linker of the invention, for example, a trifunctional linker of Scheme II and Structure B and / or FIG. 16 of the invention.

[0178] One embodiment is the use of a trifunctional linker in accordance with the invention, such as the trifunctional linker of Scheme II and structure B, for the preparation of a conjugate, such as an ADC or AOC, preferably a conjugate comprising at least one saponin of the invention, an ADC-saponin conjugate in accordance with the invention, and / or an AOC-saponin conjugate.

[0179] One embodiment is a semi-finished product comprising a trifunctional linker of the present invention. At least one saponin according to the present invention is covalently bound to the trifunctional linker. And / or at least one effector moiety according to the present invention is covalently bound to the trifunctional linker, either directly or via at least one linker and / or via at least one oligomeric or polymeric backbone of the present invention. The linker is preferably a cleavable linker according to the present invention, the effector moiety of the present invention is preferably a toxin, such as a protein toxin selected from dianthin and saporin, and / or the saponin of the present invention is preferably any one or more of Quil-A, QS-21, QS-7, QS1861, SO1861, SA1641, GE1741, and the water-soluble saponin fraction of Quillaja saponaria.

[0180] An embodiment is a conjugate of the invention, wherein at least one linker comprises at least one cleavable linker, optionally said cleavable linker undergoes cleavage under acidic, reductive, enzymatic, or photoinduced conditions, preferably the cleavable linker comprises a cleavable bond selected from a hydrazone or hydrazide bond that undergoes cleavage under acidic conditions, and / or a bond that is susceptible to proteolysis, e.g., proteolysis by cathepsin B, and / or a bond that is susceptible to cleavage under reductive conditions, e.g., a disulfide bond.

[0181] In one embodiment is a conjugate of the invention, wherein at least one linker comprises at least one cleavable linker, said cleavable linker undergoing cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦5.5.

[0182] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a lysine side chain forming an amide bond and / or to a cysteine ​​side chain forming a thio-ether bond or a disulfide bond, wherein the lysine and / or cysteine ​​is included in a cell surface molecule targeting molecule and / or is included in at least one effector moiety, and wherein the at least one saponin is either directly attached to the lysine and / or cysteine ​​or is attached via at least one linker, optionally including a cleavable linker and / or a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B.

[0183] In one embodiment is a conjugate of the invention, wherein the linker is based on N-ε-maleimidocaproic acid hydrazide, and / or based on 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, a trifunctional linker, such as the trifunctional linker of Scheme II and Structure B, a cleavable linker, and / or involves any one or more of a disulfide bond, a thio-ether bond, an amide bond, or a hydrazide bond.

[0184] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and / or to at least one effector moiety via at least one linker, the linker being or comprising a scaffold comprising a polymeric or oligomeric structure and further comprising at least one fourth chemical group for covalently attaching the scaffold to the cell surface molecule targeting molecule and / or to at least one effector moiety.

[0185] In one embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via a cleavable bond and / or via a non-cleavable bond.

[0186] One embodiment is a conjugate of the invention, wherein the cleavable bond undergoes cleavage under any of acidic, reductive, enzymatic, and photoinduced conditions, preferably the cleavable bond comprises a hydrazone or hydrazide bond that undergoes cleavage under acidic conditions, and / or a bond that is susceptible to proteolysis, e.g., proteolysis by cathepsin B, and / or a bond that undergoes cleavage under reductive conditions, e.g., a disulfide bond.

[0187] In one embodiment is a conjugate of the invention, wherein the cleavable bond undergoes cleavage in vivo under acidic conditions present in the endosomes and / or lysosomes of mammalian cells, preferably human cells, preferably at a pH between 4.0 and 6.5, more preferably at a pH ≦5.5.

[0188] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via any one or more of an imine bond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulfide bond, a thio-ether bond, an amide bond, a peptide bond, or an ester bond, preferably via at least one linker.

[0189] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure via any one or more of an imine bond, a hydrazone bond, and a hydrazide bond, which bond is preferably cleavable according to the invention, and preferably the at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via an aldehyde functionality at position C-23 of the at least one saponin.

[0190] One embodiment is a conjugate of the invention, in which the aldehyde functionality at position C-23 of at least one saponin is covalently coupled to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently coupled via a thio-ether bond to a sulfhydryl group, e.g., a sulfhydryl group of cysteine, on a backbone polymeric or oligomeric structure.

[0191] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via an amide bond, and preferably at least one saponin, when present, is covalently attached to the backbone polymeric or oligomeric structure via a glucuronic acid functionality as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin.

[0192] One embodiment is a conjugate of the invention, in which a glucuronic acid functional group as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin is covalently coupled to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is covalently coupled via an amide bond to an amine group on a backbone polymeric or oligomeric structure, for example, a lysine or N-terminal amine group of the backbone polymeric or oligomeric structure.

[0193] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a backbone polymeric or oligomeric structure, the covalent attachment involving an aldehyde functionality at position C-23 of at least one saponin, when present, and / or the covalent attachment involving a glucuronic acid functionality as a carbohydrate substituent on the C-3 beta-OH group of at least one saponin, when present.

[0194] In one embodiment is a conjugate of the invention, wherein at least one fourth chemical group of the scaffold for covalently coupling the scaffold to a cell surface targeting molecule and / or to at least one effector moiety is a click chemistry group, preferably selected from any one or more of a tetrazine, an azide, an alkene, or an alkyne, or cyclic derivatives of these groups, preferably an azide group.

[0195] In one embodiment is a conjugate of the invention, wherein the backbone polymeric or oligomeric structure comprises a linear, branched, and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures, which assembly is preferably assembled by covalent crosslinking.

[0196] In one embodiment is a conjugate of the invention, wherein the at least one saponin is a defined number or range of saponins, preferably 1 to 128 saponins, or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 128 saponins, or any number therebetween, for example, 7, 9, 12 saponins.

[0197] One embodiment is a conjugate of the invention, wherein the conjugate comprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, or 1-100 saponins, or any number of saponins therebetween, e.g., 7, 9, 12 saponins, covalently attached directly to an amino acid residue of a cell surface targeting molecule and / or to at least one effector moiety, preferably via an amino acid residue of at least one effector moiety, preferably to a cysteine ​​and / or a lysine, and and / or via at least one linker and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1 to 8 such backbones or 2 to 4 such backbones, of any one of claims 28 to 40, and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, preferably 1 to 8 such backbones or 2 to 4 such backbones, and 1 to 32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins, or any number of saponins in between, for example 7, 9, 12 saponins, are covalently attached to the at least one backbone.

[0198] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule and to at least one effector moiety via a trifunctional linker, the trifunctional linker comprising a second chemical group to which the at least one saponin is covalently attached either directly or via a linker such as a cleavable linker and / or via a backbone comprising a polymeric or oligomeric structure, and a fourth chemical group in accordance with the invention for covalent coupling of the backbone to the trifunctional linker, the trifunctional linker further comprising a third chemical group for covalent attachment to the cell surface molecule targeting molecule and a first chemical group for covalent attachment to at least one effector moiety, the cell surface molecule targeting molecule being attached to the third chemical group and / or the at least one effector moiety being attached to the first chemical group, preferably the trifunctional linker is the trifunctional linker of Scheme II and Structure B.

[0199] In accordance with the present invention, the saponin is typically one of those listed in Table A1, Scheme I. When considering intracellular entry and cytosolic accumulation of the effector moiety covalently coupled to the cell surface molecule targeting molecule of the conjugate of the present invention, the activity of the saponin, e.g., intracellular endosomal escape-enhancing activity, has proven beneficial when the saponin is covalently coupled to the cell surface molecule targeting molecule (either directly or indirectly via a first bond to the effector moiety, which is directly coupled to the cell surface molecule targeting molecule) and involves a hydrazone bond and / or hydrazide bond and / or disulfide bond. Such bond types are readily cleaved under acidic and / or reductive conditions within the (late) endosomes and lysosomes of mammalian cells, e.g., human cells. Alternatively, the inventors have also demonstrated that covalent coupling of saponins to cell surface targeting molecules via bonds that are not readily cleavable under physiological conditions within cells, e.g., (late) endosomes, lysosomes, cytosol, is also beneficial for enhancing the activity of saponins on the biological effects of effector moieties such as nucleic acids (e.g., BNAs that silence HSP27) and proteinaceous toxins such as saporin. An example of such a bond is an amide bond to couple saponins to lysine side chains, either directly or via a (cleavable) linker and / or via an oligomeric or polymeric backbone. In this application, including the claims, when referring to a conjugate of the present invention, e.g., a conjugate optionally comprising a backbone having a saponin coupled to a cell surface molecule targeting molecule via a linker and / or backbone via a hydrazone bond or a disulfide bond, the terms "cleavable linker," "cleavable bond," etc., are also referred to as "labile linker" ("L") and "labile bond," e.g., in the context of cleavage of such bond or linker in (late) endosomes and / or lysosomes. For example, Figures 2 and 3 show in vivo HSP27 gene silencing in human tumors in mice. Tumor-bearing mice were treated with a conjugate of the present invention.According to the present invention, a monoclonal antibody is provided having a saponin bonded thereto via a labile linker (hydrazone bond) and an antisense BNA for silencing the HSP27 gene in tumor cells, covalently coupled to the monoclonal antibody via a disulfide bond. Without being bound by any theory, once the conjugate of the present invention is internalized, for example by endocytosis, the hydrazone bond and the disulfide bond are cleaved in the (late) endosomes and / or lysosomes of target tumor cells expressing an epitope on the cell surface of a target cell surface molecule, in this case, EGFR. Considering the entry of the BNA from endosomes and / or lysosomes into the cytosol, cleavage of the bond likely contributes to the endosomal escape-enhancing activity of the saponin, but such cleavage is not essential for observing the gene silencing effect of the cetuximab-SO1861-BNA conjugate of the present invention.

[0200] Those skilled in the art will understand that trifunctional linkers are suitable scaffolds of the present invention for covalently coupling one, two, or three saponin moieties. In trifunctional linkers, covalent coupling of one or two saponin moieties is preferred. The second and / or third binding site is suitable for covalently coupling, for example, a proteinaceous ligand, such as a cell surface molecule targeting molecule. Typical proteinaceous ligands are EGF for targeting (tumor) cells expressing EGFR on their cell surface, and cytokines for targeting tumor cells or autoimmune cells. Furthermore, the second or third binding site of a trifunctional linker is suitable for covalently coupling an immunoglobulin, such as a monoclonal antibody, i.e., a cell surface molecule targeting molecule for binding to a cell surface molecule, such as a tumor cell surface molecule, preferably a tumor cell-specific molecule, more preferably a tumor cell receptor specifically (over)expressed on the surface of tumor cells. Similarly, immunoglobulins, or any fragment(s) and / or domain(s) thereof encompassing the binding specificity of an immunoglobulin, are suitable for binding to cell surface molecules, such as receptors expressed on the surface of autoimmune cells. Thus, in one embodiment, the conjugates of the invention comprise a trifunctional linker, which comprises or consists of a covalently attached saponin, e.g., QS-21, SO1861, and a covalently attached cell surface molecule targeting molecule, e.g., a cell targeting moiety, e.g., a ligand or antibody for (specific) binding to tumor cells, autoimmune cells, diseased cells, abnormal cells, non-healthy cells, B-cell disorders.

[0201] One embodiment is a conjugate of the invention, which comprises an oligomeric trifunctional linker as a backbone core structure according to Scheme II:

[0202] [ka]

[0203] The saponin and effector moieties are covalently attached to the trifunctional linker scaffold via a labile cleavable hydrazone linker (acid sensitive) and / or via a maleimide-containing bond, while attachment of the scaffold to a cell surface targeting molecule such as an antibody is established via a maleimide-containing bond to cysteines of the binding site, such as one, two, three, or four cysteines, and / or via a labile cleavable hydrazone linker (acid sensitive), thereby forming structure B:

[0204] [ka]

[0205] As a result, one to four scaffolds are covalently attached to a single cell surface targeting molecule, eg, an antibody such as a monoclonal antibody.

[0206] In one embodiment, the conjugate of the present invention is one in which the linkage between the saponin and the cell surface targeting molecule is preferably via an acid-labile bond that is stable at pH 7.4 and releases the saponin below pH 6.5, more preferably between pH 6.5 and 5.0. This is achieved, for example, by an imine formed by an aldehyde group on the saponin and an amino group on the linker connecting the saponin and the cell surface targeting molecule. Other chemical bonds that satisfy the pH conditions can also be used for aldehyde coupling. For example, certain hydrazones or acetals require hydrazide and hydroxyl groups as functional groups on the linker, respectively. When the bond is cleavable, the saponin is preferably attached to the backbone polymer or oligomer structure via one of the carboxyl groups or an aldehyde functional group on the saponin, more preferably via an aldehyde functional group, preferably an aldehyde functional group at position 23. Alternatively, the saponin is preferably attached to the cell surface molecule targeting molecule either via the backbone polymeric or oligomeric structure, via a linker connecting the backbone polymeric or oligomeric structure, via a carboxylic acid functional group or via an aldehyde functional group on the saponin.

[0207] In one embodiment, the conjugate of the present invention is one in which at least one saponin is bound to a cell surface molecule targeting molecule via a stable bond. In a more preferred embodiment, the stable bond between the saponin and the cell surface molecule targeting molecule preferably occurs via amide coupling or amine formation. This is achieved, for example, by carbodiimide-mediated amide bond formation between an amino group of the polymer or oligomer backbone structure linking the saponin and the cell surface molecule targeting molecule and an activated glucuronic acid group of the saponin. Chemical bonds that meet the definition of a stable bond can also be used for aldehyde coupling. For example, specific amines are derivatized after reductive amination, requiring a primary amino group as a functional group on the polymer or oligomer structure of the linker or backbone. When the bond is a stable bond, the saponin is preferably attached to the linker or backbone via one of the carboxyl groups of the saponin, and the linker or backbone is further linked to the cell surface molecule targeting molecule.

[0208] An embodiment is a conjugate of the invention, wherein the saponin is coupled to a cell surface targeting molecule via a scaffold according to the invention, and the chemical group for covalent coupling of the scaffold to the binding site is a click chemistry group.

[0209] One embodiment is a conjugate of the present invention, in which a saponin is coupled to a cell surface targeting molecule via a scaffold according to the present invention, and the click chemistry group is a tetrazine, azide, alkene, or alkyne, or a cyclic derivative of any of these groups, preferably azide. The click chemistry group is a chemical functional group suitable for click chemistry, which is defined as a reaction that is modular, broad in scope, gives very high yields, produces only benign by-products, offers high selectivity and high tolerance for different functional groups, and is stereospecific. Required process features include simple reaction conditions, readily available starting materials and reagents, no solvent or the use of a mild (e.g., water) or easily removed solvent, and simple product isolation. The click chemistry group for coupling the saponin, optionally via a backbone or linker, to the cell surface targeting molecule on the conjugate of the invention is preferably a tetrazine, azide, alkene, or alkyne, or a reactive derivative thereof, such as methyl-tetrazine or maleimide (alkene), more preferably an alkyne, or a cyclic derivative of these groups, such as a cyclooctyne (e.g., aza-dibenzocyclooctyne, difluorocyclooctyne, bicyclo[6.1.0]non-4-yne, dibenzocyclooctyne).

[0210] Thus, the conjugates according to the present invention comprise at least one saponin. By "at least one" in this context, it is meant that the conjugate comprises one saponin molecule, but may also comprise several (e.g., 2, 3, or 4) saponins or a large number (e.g., 10, 20, or 100) saponins. Depending on the application, the conjugate may comprise a covalently attached scaffold with a covalently attached saponin. The scaffold may be designed so that it comprises a defined number of saponins. Preferably, the conjugates according to the present invention comprise a defined number or range of saponins rather than a random number. This is particularly advantageous for drug development and with respect to marketing approval. In this regard, a defined number means that the conjugate preferably comprises a predetermined number of saponins. This is achieved, for example, by designing a scaffold comprising a polymer structure with a certain number of possible moieties for attachment of the saponin(s). In an ideal situation, all of these moieties are coupled to saponins, and the scaffold then contains a predetermined number of saponins. It is envisioned to provide a standard set of scaffolds containing, for example, 2, 4, 8, 16, 32, 64, etc. saponins, so that the optimal number can be easily tested by the user according to their needs. One embodiment is a conjugate of the present invention comprising a scaffold of the present invention. In a non-ideal situation, not all moieties present on the polymer structure bind saponins, but rather the saponins are present in a predetermined range. Such a range can be, for example, 2 to 4 saponin molecules per scaffold, 3 to 6 saponin molecules per scaffold, 4 to 8 saponin molecules per scaffold, 6 to 8 saponin molecules per scaffold, 6 to 12 saponin molecules per scaffold, etc. Thus, in such cases, if the range is defined as 2 to 4, a conjugate comprising a scaffold according to the present invention will contain 2, 3, or 4 saponins.

[0211] The scaffold is essentially independent of the type of saponin covalently attached to it, and the scaffold is subsequently (sequentially) covalently coupled to the conjugate. Therefore, conjugates comprising the scaffold are the foundational products of a new platform technology. Because at least one covalently attached saponin mediates the intracellular delivery of an effector moiety attached to a cell surface molecule targeting molecule included in the conjugate of the invention, the scaffold technology according to the present invention is the first known system for mediating controlled intracellular effector moiety delivery by saponins. The scaffold provides an optimized and functionally active unit that can be linked to the saponin(s) included in the conjugate and to the cell surface molecule targeting molecule, e.g., a ligand, antibody, etc., at a single, defined position.

[0212] In one embodiment, a conjugate comprises a scaffold according to the present invention, wherein the number of monomers in the polymeric or oligomeric structure is a precisely defined number or range. Preferably, the polymeric or oligomeric structure comprises structures such as poly(amines), e.g., polyethyleneimines and poly(amidoamines), or polyethylene glycols; structures such as poly(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, poly(dextrins); peptides or proteins; or structures such as natural and / or artificial polyamino acids, e.g., polylysine, DNA polymers, stabilized RNA polymers, or PNA (peptide nucleic acid) polymers. They may appear as either pure or mixed linear, branched, or cyclic polymers, oligomers, dendrimers, dendrons, dendronized polymers, dendronized oligomers, or aggregates of these structures. Preferably, the polymeric or oligomeric structures are biocompatible, meaning that they do not exhibit substantial acute or chronic toxicity in living organisms and can either be excreted intact or completely degraded by the body's metabolism into excretable and / or physiological compounds. The aggregates can be assembled by covalent crosslinking or non-covalent bonds and / or attractive forces. Thus, they can also form nanogels, microgels, or hydrogels. Alternatively, they can be attached to carriers, such as inorganic nanoparticles, colloids, liposomes, micelles, or particle-like structures containing cholesterol and / or phospholipids. The polymeric or oligomeric structures preferably have a precisely defined number or range of coupling moieties for coupling glycoside molecules (and / or effector molecules and / or carrier molecules, such as ligands, monoclonal antibodies, or fragments thereof). Preferably, at least 50%, more preferably at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% of a precisely defined number or range of coupling moieties on a polymeric or oligomeric structure are occupied by glycoside molecules in the backbone according to the present invention.

[0213] Preferably, a dendron is a branched, well-defined dendritic polymer with a single chemically addressable group at the origin of the tree, called the focal point. A dendrimer is the connection of two or more dendrons at their focal points. A dendronized polymer is the connection of the focal points of one or more dendrons to a polymer. In a preferred embodiment, a scaffold according to the invention is provided, wherein the polymeric or oligomeric structure comprises a linear, branched, or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, or aggregates of these structures, either pure or mixed. The aggregates can be assembled by covalent crosslinking or non-covalent attractive forces to form nanogels, microgels, or hydrogels. Preferably, the polymer is a derivative of poly(amine), such as polyethyleneimine and poly(amidoamine), and polyethylene glycol, a poly(ester) structure, such as poly(lactide), poly(lactam), polylactide-co-glycolide copolymer, and poly(dextrin), and a structure such as natural and / or artificial polyamino acids, such as polylysine, or a peptide or protein or DNA polymer, stabilized RNA polymer, or PNA (peptide nucleic acid) polymer. Preferably, the polymer or oligomeric structure is biocompatible.

[0214] An embodiment is a conjugate of the invention for use in accordance with the invention, wherein the conjugate comprises more than one covalently attached saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128, or 1-100 saponins, or any number therebetween, for example 7, 9, 12 saponins, or any.

[0215] One embodiment is a conjugate of the invention, wherein at least one saponin is covalently attached to the polymeric or oligomeric structure of the oligomeric or polymeric backbone via at least one cleavable linker according to the invention.

[0216] In one embodiment is a conjugate of the invention, wherein the chemical group of the oligomer or polymer backbone for covalent coupling of the oligomer or polymer backbone to an amino acid residue of a cell surface targeting molecule on the conjugate is a click chemistry group, preferably selected from tetrazine, azide, alkene, or alkyne, or cyclic derivatives of these groups. More preferably, the chemical group is azide.

[0217] In one embodiment is a conjugate of the invention, wherein the polymeric or oligomeric structure of the polymeric or oligomeric backbone comprises a linear, branched, and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures, which assembly is preferably assembled by covalent crosslinking.

[0218] The inventors have established that covalent coupling of a saponin, preferably via a cleavable bond or linker, to a cell surface molecule targeting molecule on a conjugate of the invention according to any of the above embodiments herein provides efficient and cell-targeted enhancement of the activity of the effector moiety included in said conjugate. Coupling a saponin, either directly or via a linker, to a cysteine ​​or lysine side chain of a cell surface molecule targeting molecule on a conjugate, such as a monoclonal antibody, has been found to be a valuable way of specific and efficient delivery of the effector moiety-enhanced activity within target cells. In this case, the effector moiety is also delivered to the same target cells by using the very same cell surface molecule targeting molecule to which a saponin(s) is also covalently attached.

[0219] To explain the present invention in more detail, the process of cellular uptake of substances (although the inventors do not wish to be bound by any theory) and the terminology used herein will be described. The uptake of extracellular substances into cells by vesicle budding is called endocytosis. Such vesicle budding can be characterized by (1) receptor-dependent ligand uptake mediated by the cytosolic protein clathrin, (2) lipid raft uptake mediated by the cholesterol-binding protein caveolin, (3) nonspecific fluid uptake (pinocytosis), or (4) nonspecific particle uptake (phagocytosis). All types of endocytosis enter into subsequent cellular processes of vesicle trafficking and material sorting, referred to as endocytic pathways. Endocytic pathways are complex and incompletely understood. Without wishing to be bound by any theory, organelles can form de novo and mature into subsequent organelles along the endocytic pathway. However, it is hypothesized here that endocytic pathways involve stable compartments connected by vesicle trafficking. Compartments are complex, multifunctional membrane organelles specialized for specific sets of essential cellular functions. Vesicles are considered transient organelles, simpler in composition and defined as membrane-enclosed containers formed de novo by budding from pre-existing compartments. In contrast to compartments, vesicles can undergo maturation, a series of physiologically irreversible biochemical changes. Early and late endosomes represent stable compartments in the endocytic pathway, while primary endocytic vesicles, phagosomes, multivesicular bodies (also called endosomal carrier vesicles), secretory granules, and even lysosomes represent vesicles. Endocytic vesicles, most notably those originating from clathrin-coated pits at the plasma membrane, first fuse with early endosomes, the major sorting compartment with a pH of approximately 6.5. The majority of internalized cargo and membranes are recycled to the plasma membrane via recycling vesicles (the recycling pathway). Components to be degraded are transported via multivesicular bodies to acidic late endosomes (pH lower than 6). Lysosomes are vesicles that can store mature lysosomal enzymes and deliver them to late endosomal compartments when needed.The resulting organelles are called hybrid organelles or endolysosomes. Lysosomes bud from hybrid organelles through a process called lysosomal remodeling. Late endosomes, lysosomes, and hybrid organelles are extremely dynamic organelles, and it is often difficult to distinguish between them. Degradation of endocytosed molecules occurs within endolysosomes or lysosomes. Endosomal escape is the active or passive release of materials from the lumen of any type of compartment or vesicle from the endocytic pathway, preferably from clathrin-mediated endocytosis or recycling pathways to the cytosol. Therefore, endosomal escape includes, but is not limited to, release from endosomes, endolysosomes, or lysosomes, including their intermediates and hybrid organelles.

[0220] Unless specifically indicated otherwise, and particularly when relating to the endosomal escape mechanisms of glycoside molecules such as the saponins of the present invention, whenever the words "endosome" or "endosomal escape" are used herein, it also encompasses escape from endolysosomes and lysosomes and from endolysosomes and lysosomes, respectively. After entering the cytosol, the material may translocate to other cellular units, such as the nucleus.

[0221] In formal terms, a glycoside is any molecule in which a sugar group is linked to another group via its anomeric carbon by a glycosidic bond. Without wishing to be bound by any theory, glycoside molecules such as saponins in the context of the present invention are such molecules that can further improve the efficacy of an effector moiety, particularly by facilitating endosomal escape of the effector moiety. Without wishing to be bound by any theory, glycoside molecules (saponins, such as those listed in Table A1) interact with membranes of compartments and vesicles of the endocytic and recycling pathways, making them leaky for the effector moiety, resulting in increased endosomal escape. By the term "scaffold capable of increasing endosomal escape of the effector moiety," it is meant that at least one saponin (glycoside molecule) coupled via a linker or directly to a cell surface molecule targeting molecule or via the polymeric or oligomeric structure of the scaffold can improve endosomal escape of the effector moiety. At this time, both molecules are in an endosome, e.g., a late endosome, and optionally and preferably after at least one glycoside, such as a saponin, has been released from the conjugate, e.g., from a linker or polymeric or oligomeric structure contained in the conjugate, e.g., by cleavage of a cleavable bond between at least one glycoside (saponin) and the conjugate (e.g., via the backbone polymeric or oligomeric structure and / or via the linker). Although the bond between at least one glycoside, e.g., a saponin according to the present invention, and a cell surface molecule targeting molecule of a conjugate of the present invention, optionally via a linker or backbone, may be a "stable bond," this does not mean that such a bond cannot be cleaved in the endosome, e.g., by an enzyme. For example, the glycoside or saponin, optionally together with a portion of the linker or backbone oligomeric or polymeric structure, may be cleaved from the remaining linker fragment or oligomeric or polymeric structure.For example, a protease may cleave a (proteinaceous) linker or a proteinaceous polymer structure, such as albumin, thereby releasing at least one glycoside, such as a saponin. However, it is preferred that the glycoside molecule (preferably a saponin) is released in an active form, preferably in the original form it had before it was (prepared to be) coupled to the cell surface molecule targeting molecule of the conjugate, optionally via a linker and / or oligomeric or polymeric backbone; therefore, the glycoside (saponin) retains its native structure after such cleavage, or the glycoside (saponin) retains (part of) a chemical group or linker attached thereto after such cleavage. On the other hand, the glycoside biological activity (saponin biological activity), such as endosomal / lysosomal escape-enhancing activity against an effector moiety present in the same endosome or lysosome, is maintained or restored by said cleavage of the bond between the glycoside (saponin) and a cell surface molecule targeting molecule, such as an antibody, optionally comprising a linker and / or backbone of the present invention. In the present invention, the term "stable", for example, between a saponin and an amino acid residue of a cell surface molecule targeting molecule on a conjugate, a linker, a polymeric or oligomeric structure (backbone), a ligand, a (monoclonal) immunoglobulin or binding domain or fragment thereof, and / or a further effector (effector moiety, effector molecule), means that the bond is not easily broken, or at least is not designed to be easily broken, for example, by pH differences, salt concentrations, UV light, or reducing conditions. In the present invention, the term "cleavable", for example, between a saponin and a cell surface molecule targeting molecule, a linker, an amino acid residue, a polymeric or oligomeric structure (backbone), a ligand, an antibody, and / or an effector, means that the bond is designed to be easily cleaved, for example, by pH differences, salt concentrations, under reducing conditions, etc. Those skilled in the art are familiar with such cleavable bonds and how to prepare them.

[0222] 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, hindering their development and value in treating patients with ADCs. The application of the conjugates of the present invention, such as ADC-saponin conjugates and AOC-saponin conjugates, now makes it possible to guide one or more glycoside molecules (saponin(s)) together with a payload-bearing ADC or together with a (monoclonal) antibody conjugated to an oligonucleotide, such as a BNA, to a (target) cell. In particular, it was not previously possible to specifically guide the effector moiety of either an ADC or AOC or other conjugate of a payload, a (proteinaceous) cell surface molecule targeting molecule, and a (predetermined and controllable) specific number or range of glycoside molecules (saponins) per effector moiety simultaneously to the cytosol of a cell, e.g., via the endocytic pathway of the cell.

[0223] The solution provided by the present invention involves the covalent attachment of at least one saponin to a cell surface molecule targeting molecule of a conjugate of the present invention. A further solution provided by the present invention involves (first) polymerizing a glycoside molecule (saponin) using an oligomeric or polymeric backbone and providing a cluster of covalently attached saponins to a cell surface molecule targeting molecule comprised in a conjugate of the present invention, thereby enabling remonomerization of one or more saponins at the intracellular site where the saponin's mechanism of action is desired, for example after endocytosis. In this context, "polymerizing" refers to the reversible and / or irreversible multiple conjugation of a saponin molecule to a first proteinaceous molecule, either via a linker or directly, or via a polymeric or oligomeric structure to form the backbone, or the reversible and / or irreversible multiple conjugation of a (modified) saponin thereby forming a polymeric or oligomeric structure to form the backbone. In this context, "remonomerization" refers to the cleavage of the saponin(s) from the conjugate, from the linker linking the saponin(s) to the cell surface targeting molecule of the conjugate, or from the backbone, e.g., after endocytosis, and regaining the (native) chemical state of the unconjugated saponin. These unconjugated saponins may or may not contain additional chemical groups, e.g., chemical groups for linking the saponin to the linker, amino acid residue, or backbone of the 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 in the backbone or other connecting linker and their "remonomerization" at a desired location, e.g., within a cell, e.g., after endocytosis, has been a difficult task. In particular, the chemical reactions (glycoside polymerization) used to provide the scaffold and linker comprising the covalently linked glycoside, e.g., triterpenoid saponin, for covalent attachment to the conjugate typically occur in an organic solvent that is free of water, while the saponin and, e.g., the biocompatible polymer applied as the scaffold to carry the attached saponin, are water-soluble molecules.The chemical properties of unmodified saponin further prevented its polymerization by itself. Another solution for (directly) conjugating multiple saponins to effector molecules was deemed less promising because the effector molecules (drugs, toxins, polypeptides, or polynucleotides) typically did not provide sufficient conjugation sites. The coupling products were highly heterogeneous, and / or coupling a biologically active molecule such as a saponin to, for example, a peptide, toxin, or nucleic acid carried the risk of affecting and interfering with the activity of one or even both molecules conjugated together in a conjugate containing such a saponin. Furthermore, when a saponin was coupled to, for example, an ADC or antibody-oligonucleotide conjugate (AOC), there was a significant risk that the effector moiety included in the conjugate of the present invention would lose its function. Embodiments of the present invention address at least one of these drawbacks.

[0224] In the context of the present invention, an effector molecule or effector moiety is any substance that affects cellular metabolism by interacting with an effector molecule target within the cell, which may be any molecule or structure within the cell, excluding the compartments of the endocytic and recycling pathways and the lumen of vesicles, but including the membranes of these compartments and vesicles. Therefore, such structures within the cell include the nucleus, mitochondria, chloroplasts, the endoplasmic reticulum, the Golgi apparatus, other transport vesicles, the interior of the plasma membrane, and the cytosol. Cytosolic delivery of an effector moiety in the context of the present invention preferably means that the effector moiety is capable of escaping endosomes (and / or lysosomes) (which also includes escaping endolysosomes and lysosomes, as defined above) and preferably is capable of reaching the effector moiety target described herein. The present invention also encompasses a new type of molecule called a scaffold. This serves to simultaneously bring both the effector moiety and at least one glycoside molecule, such as a saponin of the present invention, into endosomes in a predetermined ratio when the effector moiety and the saponin are both included in the conjugate of the present invention. In the context of the present invention, the backbone polymeric or oligomeric structure is a structurally ordered formation, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or it is an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stabilized polymeric micelle, or liposome, but excludes structures consisting of non-covalent aggregates of monomers, such as cholesterol / phospholipid mixtures. The terms "polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer" have their usual meaning. Specifically, a polymer is a substance having a molecular structure assembled primarily or entirely from a large number of equal or similar units linked together, while an oligomer is a polymer whose molecules are composed of a relatively small number of repeating units. There is no consensus on one particular cutoff for "many" and "few" used in the above definitions of polymer and oligomer, respectively.However, because the backbone can comprise a polymeric or oligomeric structure, or both, the full range of numbers of similar units linked together applies to such structures, i.e., from 2 monomeric units to 100 monomeric units, 1000 monomeric units, and more. For example, a structure containing 5 or fewer monomeric units may be referred to as an oligomeric structure, and a structure containing 50 monomeric units may be referred to as a polymeric structure. A structure of 10 monomeric units may be referred to as either an oligomer or a polymer. The backbone as defined herein further comprises at least one glycoside molecule, such as a saponin of the present invention. The backbone preferably includes polymeric or oligomeric structures, such as poly- or oligo(amines), e.g., polyethyleneimine and poly(amidoamines), and biocompatible structures, e.g., polyethylene glycol, poly- or oligo(esters), e.g., poly(lactides), poly(lactams), polylactide-co-glycolide copolymers, and poly(dextrins), poly- or oligosaccharides, e.g., cyclodextrins or polydextrose, and poly- or oligoamino acids, e.g., polylysine or peptides or proteins, or DNA oligo- or polymers. The assembled polymeric structure defined herein comprises at least one backbone and, optionally, other individual polymeric or oligomeric structures. The other individual polymeric or oligomeric structures of the assembly can be (a) a backbone (thus comprising at least one glycoside molecule, e.g., a saponin of the present invention), (b) a functionalized backbone (thus comprising at least one glycoside molecule, e.g., a saponin, and at least one effector moiety), or (c) a polymeric or oligomeric structure without glycoside molecules but having at least one effector moiety, such as a saponin of the present invention (see, e.g., Table A1). A functionalized assembled polymeric structure is an assembled polymeric structure containing at least one backbone and at least one polymeric structure comprising, e.g., (a) at least one functionalized backbone or (b) at least one effector moiety, which is attached to the cell surface molecule targeting molecule of the conjugate of the present invention.The polymeric or oligomeric structure on the assembled polymeric structure, which does not contain any of the molecules mentioned above (i.e., no glycosides such as saponin, no effector moieties), is specifically added as a structural component of the assembled structure. This helps to assemble or stabilize the assembled structure ("glue-like"). Without wishing to be bound by any theory, an acidic environment appears to be a prerequisite for the synergy between the glycoside (saponin) and the effector moiety.

[0225] Whether a conjugate of the present invention comprising a saponin, either with or without one or more (cleavable) linkers and / or optionally a backbone, can disrupt the acidic environment and inhibit the endosomal escape function of at least one glycoside (saponin) can be readily determined by assays as described in Example 12 and as known in the art. Inhibition is described as a "-fold increase in the amount of glycoside required to induce 50% cell kill." Preferably, the backbone does not result in an increase that is at least the increase in glycoside molecules (saponin) required to obtain 50% cell kill observed when chloroquine is used as a positive control. Alternatively and preferably, a conjugate comprising a saponin, either with or without one or more (cleavable) linkers and / or optionally a backbone, does not result in at least a four-fold increase, more preferably at least a two-fold increase, of glycoside molecules to induce 50% cell kill. The fold increase would be measured in an assay essentially as described in Example 12, with chloroquine inducing a 2-fold increase in the amount of glycoside, preferably saponin, as a positive control, where the saponin is any one or more of the saponins of the invention to observe 50% cell killing (see Table A1, Scheme I, previous embodiments).

[0226]

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

[0227] A number of preferred characteristics can be formulated for the endosomal escape-enhancing factors, i.e., glycosides or saponins, preferably saponins according to the invention, comprised in the conjugates of the invention: (1) they preferably are not toxic and do not provoke an immune response, (2) they preferably do not mediate cytosolic uptake of the effector moiety into off-target cells, (3) their presence at the site of action is preferably synchronized with the presence of the effector moiety, (4) they preferably are biodegradable or excretable, and (5) they preferably do not substantially interfere with, e.g., interact with hormones, biological processes of an organism unrelated to the biological activity of the effector molecule with which the endosomal escape-enhancing factor is combined. Examples of glycoside molecules, such as saponins, of the invention that meet, at least to some extent, the previously mentioned criteria are bisdesmosidic triterpenes, preferably bisdesmosidic triterpene saponins, such as SO1861, SA1641, QS-21, GE1741, and the saponins of Table A1, Scheme I.

[0228] One embodiment is a conjugate of the invention, e.g., an antibody-drug conjugate or antibody-oligonucleotide conjugate or ligand-drug conjugate of the invention, wherein the antibody is selected from the group consisting of CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanA g, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, or EGFR. and / or is or comprises any one of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab, herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or at least one tumor cell receptor-binding fragment and / or at least one tumor cell receptor-binding domain thereof. and / or the antibody-drug conjugate comprises gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and any one of the antibody-drug conjugates of Tables A2 and A3. Alternatively, the ligand-drug conjugate comprises at least one ligand for binding to a cell surface molecule, such as EGF or a cytokine.

[0229] As previously stated, at least one saponin contained in a conjugate according to the present invention increases the efficacy of at least the existing and new effector moieties defined in the present invention. Potential side effects will be reduced without reducing efficacy due to the reduced dosage of the effector moiety contained in the conjugate. Thus, the present invention provides a conjugate according to the present invention for use in medicine or as a medicament. Therefore, one aspect of the present invention relates to a conjugate according to the present invention for use as a medicament, the conjugate comprising at least a saponin and at least one effector moiety. The use of a conjugate according to the present invention for the manufacture of a medicament is also provided. In particular, cancer medicine, especially classical chemotherapy drugs, is notorious for their side effects. Due to the temporal and spatial synchronization and targeting of both the drug substance contained in the conjugate and the saponin contained in the very same conjugate molecule, the therapeutic drug conjugate according to the present invention is particularly advantageous for use as a medicament, in particular for use in a method for treating cancer. Therefore, the present invention provides a therapeutic drug conjugate according to the present invention for use in a method for treating cancer. The present invention also provides a therapeutic drug conjugate according to the invention for use in a method for treating an acquired or inherited disorder, in particular a monogenic deficiency. The therapeutic drug conjugate therefore comprises at least one saponin and at least one effector moiety. Thus, one aspect of the invention relates to a therapeutic drug conjugate according to the invention for use in a method for the treatment of cancer or an autoimmune disease, wherein the conjugate comprises a covalently attached effector moiety and comprises a covalently attached saponin.

[0230] A further medical application of the conjugates of the present invention is the replacement of intracellular enzymes in target cells that produce these enzymes in insufficient amounts or with insufficient functionality. The resulting disease can be hereditary or acquired. In most cases, only symptomatic treatment is possible, and in several rare diseases, inadequate treatment options lead to a shortened lifespan for patients. An example of such a disease is phenylketonuria, an inborn error of metabolism resulting in impaired metabolism of the amino acid phenylalanine. The disease is characterized by mutations in the gene encoding the hepatic enzyme phenylalanine hydroxylase. To date, phenylketonuria is incurable. The incidence is approximately 1:10,000, with the highest known incidence being 1:2,600 in Turkey. The cell surface molecule targeting molecule, preferably an antibody, contained in the conjugates of the present invention, having attached phenylalanine hydroxylase or an attached polynucleotide encoding phenylalanine hydroxylase, can be used to target liver cells and replace the defective enzyme in liver cells by using a suitable specific antibody. This is one example of the use of a therapeutic drug conjugate of the invention comprising a saponin in accordance with the invention attached thereto and an enzyme or oligonucleotide attached thereto, for replacement or gene therapy. In a preferred embodiment there is provided a therapeutic drug conjugate in accordance with the invention for use in methods of gene therapy or replacement therapy.

[0231] 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 allow the development of non-viral gene delivery technologies, which improve therapeutic efficacy with lower therapeutic doses, thereby improving patient health. Particularly when containing a cell surface molecule targeting molecule, such as a covalently attached monoclonal antibody for binding to a (tumor, autoimmune) cell surface-specific molecule, and when coupled to an effector moiety, such as an oligonucleotide, e.g., BNA, the conjugates of the present invention overcome a long-standing and major bottleneck in the field of gene delivery: the efficient, safe, and cost-effective transfer of gene therapy products from the endosomal membrane to the cytosol / nucleosol. Indeed, gene therapy is one of the most promising treatment 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 insufficiently safe delivery of genetic material for therapeutic use to patients.

[0232] Thus, by applying the conjugates of the present invention, which comprise a cell surface molecule targeting molecule, such as a ligand or preferably an antibody (fragment, domain thereof) and an oligonucleotide such as an antisense BNA, to target cells, the inventors have now made it possible to overcome a long-standing and major bottleneck in the field of gene delivery: the safe transfer of gene therapy products from the endosomal membrane to the cytosol / nucleosol. The conjugates of the present invention represent a technology designed to allow targeting of any addressable cell type by all known gene drugs, thereby ensuring better patient treatment, not only for genetic diseases but also for cancer treatment, and thus important for a large patient population. The conjugate-based technology of the present invention may comprise a polymeric or oligomeric backbone, which serves as a carrier for an endosomal escape enhancer (EEE), e.g., the saponin of any of the embodiments according to Table A1 and Scheme I, for a cell surface molecule targeting molecule such as a targeting ligand or (monoclonal) (tumor cell-specific) antibody, and for an effector moiety, here an effector gene, e.g., an LNA or BNA. The use of conjugates of the present invention, including oligonucleotides such as cell-targeting antibodies (fragments) and BNAs, has the potential to deliver any type of biological macromolecule to the cytosol and nucleus. The development of new targeting ligands and monoclonal (human, humanized) antibodies is under continuous investigation by numerous research groups and companies worldwide. The same is true for oligonucleotides targeted for delivery in the cytosol of diseased cells, such as cancer cells. Therefore, the conjugates of the present invention also present a molecular interface, whereby current and future targeting ligands and antibodies, as well as current and future therapeutic oligonucleotides (and payloads, such as protein toxins) can be linked or attached to the oligomeric or polymeric backbone modules of the present invention, for example, via click chemistry, allowing for custom drug applications and future developments in the field of tissue and cell targeting technologies. The conjugates of the present invention may include antibodies and ligands as cell surface molecular targeting molecules.The global market for gene therapy is growing rapidly, covering a wide range of disease areas, including potential treatments for cancer, cardiovascular disease, Parkinson's disease, Alzheimer's disease, HIV, and many rare (monogenic) diseases. Current viral vector-based gene therapy technologies have significant challenges, such as safety, manufacturing logistics, and associated high costs. The conjugates of the present invention allow for the use of technology platforms that represent an alternative to current viral gene delivery technologies. Thus, the conjugates of the present invention are 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 (monogenic) diseases. The conjugates of the present invention are suitable for developing novel treatments to transform the fields of antibody-drug conjugates (ADCs) and oligonucleotide-based therapeutics, for example, by creating non-viral vector-based gene therapy drugs based on targeted antisense BNAs. The application of the conjugates of the present invention, particularly as covalent conjugates of oligonucleotides such as antibodies and BNAs with at least one saponin, is one of many beneficial approaches made possible by the present invention. For example, the use of the conjugates of the present invention allows for the exploitation of 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, the siRNA contained in the conjugate. The conjugates of the present invention are suitable for use with, for example, small molecules that act as payloads, or delivery enhancers for oligonucleotides. Thus, the conjugates of the present invention, which have a covalently coupled oligonucleotide, e.g., a BNA, and a covalently coupled cell targeting moiety, e.g., a ligand and preferably an antibody (domain or fragment), and a saponin of the present invention, offer a solution to current problems with current endosomal escape enhancers and gene therapy products that complicate their application as two components and therefore the approval and clinical applicability of therapeutic agents.This is because such conjugates of the present invention are therapeutic drug molecules that incorporate a gene product, such as a saponin or BNA, and a (tumor) cell-targeting moiety, such as a (monoclonal) antibody, in a single conjugate. Therefore, the present invention provides a non-viral gene delivery technology, in which an endosomal escape-enhancing agent (e.g., glycosides according to embodiments of the present invention, Table A1, Scheme I), a gene therapy product (an oligonucleotide according to the present invention, e.g., a BNA), and a targeting ligand or antibody (e.g., Tables A2, A3, A4, according to embodiments of the present invention) are all included in the conjugates of the present invention. Therefore, such conjugates of the present invention offer current and future therapeutic opportunities for macromolecular drugs for a wide range of diseases and large patient populations. The application of such conjugates of the present invention, comprising at least one saponin, at least one oligonucleotide, and at least one specific cell-targeting moiety, e.g., an immunoglobulin, addresses the problems evident in current methods of separately applying an endosomal escape-enhancing agent and a gene therapy product. These current methods do not guarantee that both compounds will be present at the interaction site simultaneously. This problem is now overcome by using the conjugates of the present invention, which provide a non-viral gene delivery technology with increased synchronization (in time and space) of gene products of both compounds, i.e., saponins and BNAs.

[0233] Gene therapy can be useful for hereditary and previously incurable diseases, such as cystic fibrosis, collagen, Huntington's disease, or hemophilia. However, several problems remain to be overcome: 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 target cells but not destroyed. Current gene therapy approaches use viruses as gene ferries. However, these procedures involve significant risks and are not transferable to the introduction of other biomolecules. One embodiment is a conjugate of the present invention containing (plant-derived) glycosides for use in platform technology, which not only allows the delivery of genes when included in the conjugate as carrier molecules, but also allows 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 treatments for cystic fibrosis, collagen, Huntington's disease, or hemophilia. The conjugates of the present invention thus enable new 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 this invention, a non-viral gene delivery technology has been developed that combines a plant-derived endosomal escape enhancer (glycoside), a gene therapy product, and a targeting ligand, all contained in a single conjugate. The resulting non-viral gene therapy based on the conjugates of the present invention exhibits approximately 40-fold increased delivery efficiency at lower doses compared to currently available strategies. This makes the conjugates of the present invention suitable for clinical applications, such as repairing or replacing defective genes, as in cystic fibrosis patients, and for targeted delivery of specific genes to destroy cancer cells, for example. Indeed, the conjugates of the present invention are suitable for application in treatment regimens for any disease caused by genetic defects and currently incurable, such as cystic fibrosis, Huntington's disease, and hemophilia.Gene therapy utilizing the conjugates of the invention helps overcome two current problems: first, the conjugates of the invention allow therapeutic genes to be delivered to specific target cells in the body; and second, the therapeutic genes enter these cells but are not destroyed due to the presence of targeting moieties such as saponin(s), oligonucleotide products, and antibodies for binding to target cells, all covalently linked on the conjugates of the invention, for example, by using oligomeric or polymeric backbones of the invention.

[0234] The present invention also provides a method of treating cancer, the method comprising administering to a patient in need thereof a medicament comprising a therapeutic agent conjugate according to the invention, preferably administering an effective dose of said medicament to a patient in need thereof, preferably a human cancer patient.

[0235] Considerations regarding suitable forms for administration are known in the art and include toxicological effects, solubility, route of administration, and maintaining activity. For example, a pharmacological composition injected into the blood stream should be soluble.

[0236] The appropriate dosage form will depend in part on the use or route of entry, for example, transdermal or injection. Such a dosage form should allow the compound to reach the target cells, whether or not the target cells are present in a multicellular host. Other factors are known in the art and include considerations such as dosage forms that delay the compound or composition from exerting its effect and toxicity.

[0237] One 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.

[0238] [Table 1]

[0239] [Table 2]

[0240]

Table 3

[0241]

Table 4

[0242]

Table 5

[0243]

Table 6

[0244]

Table 7

[0245]

Table 8

[0246]

Table 9

[0247]

Table 10

[0248]

Table 11

[0249]

Table 12

[0250]

Table 13

[0251]

Table 14

[0252]

Table 15

[0253] Table 16

[0254]

Table 17

[0255]

Table 18

[0256]

Table 19

[0257] Table 20

[0258] Table 21

[0259] Table 22

[0260] Table 23

[0261] Table 24

[0262] Table 25

[0263] Table 26

[0264] Table 27

[0265] Table 28

[0266] Table 29

[0267] Table 30

[0268] Table 31

[0269] Table 32

[0270]

Table 33

[0271] Table 34

[0272] Table 35

[0273] Table 36

[0274] Table 37

[0275] It is part of the present invention that the therapeutic agent conjugate may be further combined with a covalent conjugate (complex) of a binding molecule or binding moiety with a saponin, or further combined with a pharmaceutical compound, antibody, etc., thereby providing a composition comprising two or even three or more enhancers, drug substances, etc. For example, a conjugate of the present invention combined with a binding moiety complexed with an effector molecule, and optionally further combined with a pharmaceutical agent, which may or may not be linked to a saponin, and which may or may not be coupled to a ligand, such as a targeting immunoglobulin, domain, or fragment thereof. Furthermore, one embodiment is a therapeutic agent conjugate of the present invention, wherein the cell surface molecule targeting molecule is provided with two or more effector moieties, such as toxins or immunotoxins, and the two or more effector moieties may be the same or different.

[0276] Exemplary Embodiments One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position 23, the saponin is preferably a saponin that can be isolated from a Gypsophila or Saponaria species, and more preferably the saponin is saponin SO1861 or any of its diastereomers.

[0277] One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the cell surface molecule targeting molecule is at least a ligand, such as an immunoglobulin, having at least an effector moiety attached thereto.

[0278] In one embodiment, the endosomal escape-enhancing conjugate of the invention is characterized in that the cell surface molecule targeting molecule is an immunoglobulin or at least a binding domain thereof for binding to a cell surface molecule, preferably the cell surface molecule is selected from HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L, PSMA, CanAg, integrin-alpha V, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD33, CD239, CD70, CD123, CD352, DLL3, CD25, ephrin A4, MUC1, Trop2, CEACAM5, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, and CD71.

[0279] In one embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the linker is coupled to the saponin via a cleavable bond and the cell surface molecule targeting molecule is an immunoglobulin. Preferably, the cleavable bond undergoes cleavage under acidic, reductive, enzymatic, or photoinduced conditions, and preferably, the cleavable bond is a covalent bond, preferably an imine bond, a hydrazone bond, an oxime bond, a 1,3-dioxolane bond, or an ester bond, and preferably, the cleavable bond is a disulfide bond or a peptide bond.

[0280] One embodiment is an endosomal escape-enhancing conjugate of the invention, wherein the saponin moiety is a terminal saponin, preferably saponin SO1861, the linker is a chemical linker that covalently links the saponin to a cell surface molecule targeting molecule of the conjugate of the invention, wherein the cell surface molecule targeting molecule is an immunoglobulin such as trastuzumab or cetuximab, and the linker preferably provides a cleavable bond between the terminal saponin moiety and the cell surface molecule targeting molecule included in the conjugate.

[0281] In one embodiment is a conjugate according to the invention, wherein the saponin is a bisdesmosidic triterpene.

[0282] In one embodiment is a conjugate according to the invention, wherein the saponin is a bisdesmosidic triterpene saponin.

[0283] One embodiment is a conjugate according to the invention, wherein the saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde functional group at position 23.

[0284] An embodiment is a conjugate according to the invention, wherein the saponin is a saponin that can be isolated from a Gypsophila or Saponaria species.

[0285] In one embodiment is a conjugate according to the invention, wherein the saponin is SO1861 or any of its diastereomers.

[0286] One embodiment is a conjugate according to the invention, wherein at least one saponin is attached to a cell surface molecule targeting molecule (a binding site for an epitope on a cell surface molecule) via a cleavable bond. Preferably, the cleavable bond is susceptible to cleavage under acidic, reductive, enzymatic, or light-induced conditions, and the cleavable bond is preferably a disulfide bond or a peptide bond.

[0287] One embodiment is a conjugate according to the invention, wherein the cleavable bond is a covalent bond, preferably an imine bond, a hydrazone bond, an oxime bond, a 1,3-dioxolane bond, or an ester bond.

[0288] In one embodiment is a conjugate according to the invention, wherein the endosomal escape-enhancing conjugate comprises a defined number of glycosides or a defined range.

[0289] In one embodiment is a conjugate according to the invention, the stated range is 1 to 30 glycoside(s), preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 6, more preferably 2 to 6, more preferably 2 to 5, more preferably 3 to 5, more preferably 3 to 4 glycosides.

[0290] In one embodiment is a conjugate according to the invention, wherein the effector moiety is a drug substance, eg, a toxin, eg, a proteinaceous toxin, a drug, a polypeptide, or a polynucleotide.

[0291] One embodiment is a conjugate according to the invention, wherein the target cell is a diseased cell or a disease-associated cell, preferably a tumor cell or a tumor-associated cell (e.g., a tumor vascular cell), or an immune cell (e.g., a regulatory T cell), or an autoimmune cell.

[0292] In one embodiment is a conjugate according to the invention, wherein at least one effector moiety is attached to a cell surface targeting molecule included in the conjugate via a cleavable bond, preferably the cleavable bond is susceptible to cleavage under acidic, reductive, enzymatic, or photoinduced conditions, and / or the cleavable bond is a disulfide bond or a peptide bond.

[0293] One embodiment is a conjugate according to the invention, wherein the saponin is capable of increasing endosomal escape of the effector molecule.

[0294] One embodiment is a conjugate according to the invention for use as a medicament.

[0295] One embodiment is a pharmaceutical composition, comprising a conjugate according to the invention (previous embodiment) and a pharmaceutically acceptable excipient.

[0296] An embodiment is a pharmaceutical composition according to the invention, further comprising at least one additional drug substance, for example an additional immunoglobulin.

[0297] An embodiment is a conjugate for use according to the invention or a pharmaceutical composition according to the invention for use in a method of treating cancer or an autoimmune disease.

[0298] An embodiment is a method of treating cancer, the method comprising administering a conjugate according to the invention to a patient in need thereof.

[0299] One embodiment is a method of treating cancer, the method comprising administering to a patient in need thereof a pharmaceutical composition according to the present invention.

[0300] One embodiment is a kit comprising a container containing an endosomal escape-enhancing conjugate according to the invention, the kit further comprising instructions for using the binding molecule.

[0301] A scaffold comprising at least one saponin according to the present invention is suitable for use as a semi-finished product for the preparation of a functionalized ADC or functionalized AOC, the functionalized ADC or functionalized OAC comprising at least one covalently coupled saponin of the present invention and at least one effector moiety of the present invention. One embodiment is a scaffold of the present invention further comprising a payload or effector moiety of the present invention, such as a toxin or oligonucleotide, covalently attached to the scaffold of the present invention, either directly or via a linker of the present invention, preferably a cleavable linker of the present invention. Such functionalized scaffolds can be used to prepare ADC-saponin conjugates or AOC-saponin conjugates comprising the scaffold with a covalently attached scaffold and an effector moiety attached thereto. For example, such a functionalized ADC or OAC may comprise two to four saponins, e.g., covalently coupled to cysteine ​​side chains on a conjugate according to the present invention, which are then covalently coupled, e.g., directly or via a (cleavable) linker, to a cell surface targeting molecule, e.g., a ligand or antibody (fragment). Alternatively, such functionalized ADC or OAC comprises a dendron, e.g., a G4-dendron, which comprises 1 to 16 covalently coupled saponins attached thereto, the dendron being, for example, covalently coupled to cysteine ​​and / or lysine side chains of a cell surface targeting molecule comprised in a conjugate in accordance with the invention.

[0302] One 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.

[0303] An aspect of the invention relates to a conjugate of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0304] Certain aspects of the invention relate to a conjugate of the invention or a pharmaceutical composition of the invention for use in the treatment or prevention of cancer or autoimmune disease.

[0305] The present invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way. [Example]

[0306] Example A - Treatment of mammalian tumor-bearing animals with a conjugate of the invention in combination with an ADC results in survival and tumor regression. Female Balb / c nude mice were injected subcutaneously with a suspension of human A431 tumor cells. Human epidermal carcinoma was developed under the skin of the mice in a xenograft animal tumor model. After tumor cell injection, the xenograft tumors grew to approximately 170-180 mm. 3 The A431 tumor cells were allowed to develop to a size of 100 μg / cm. The A431 tumor cells have the following characteristics: high EGFR expression, intermediate CD71 expression, and low HER2 expression. The tumor model based on A431 tumor cells is an aggressive model, particularly because the tumors grow rapidly.

[0307] Table A presents the results of treatment of control and tumor-bearing mice. Tumor-bearing mice were treated with the indicated antibodies directed against either human Her2 / neu, human EGFR, or human CD71, cell surface receptors on xenograft tumors. Cetuximab was covalently conjugated to the saponin SO1861. SO1861 was first provided with the linker EMCH (N-ε-maleimidocaproic acid hydrazide), a maleimide and hydrazide crosslinker used to covalently conjugate a sulfhydryl (reduced cysteine ​​of an antibody) to a carbonyl (aldehyde or ketone; in this case, the carbonyl of the aldehyde at position C-23 of the saponin). Saponin-EMCH was covalently coupled to the reduced cysteine ​​of cetuximab, forming a covalent thioether bond between EMCH and the cysteine ​​side chain. The ADCs trastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9, IgG)-saporin (covalent conjugate) were tested for their tumor-attacking efficacy in mice. Tumor volume was measured over time after the initiation of treatment with the ADC. The dose of the ADC was suboptimal in this tumor model. Previous experiments established that at a suboptimal dose of the ADC, tumor regression or cessation of tumor growth would not be observable.

[0308] [Table 38]

[0309] These results demonstrate that combination therapy of tumor-bearing mice with an ADC at a dose that is ineffective when treatment of the ADC alone is considered (tumor growth, mouse death (euthanasia) is not prevented) with a conjugate of the present invention consisting of an antibody targeting a tumor cell-specific receptor covalently linked to a saponin, i.e., SO1861, provides an efficient and effective treatment regimen, expressed as regressing tumors and prolonged survival of treated animals (over the duration of the experiment). The covalent conjugate is administered to cancer-bearing mice at a dose that is ineffective when administered alone (tumor growth, mouse death (euthanasia) is not prevented). Therefore, a suboptimal dose of an ADC combined with a conjugate containing a covalently linked saponin of the present invention that has no antitumor activity when administered alone provides an effective treatment option for cancer patients, and a relatively low dose of the ADC is effective. A lower dose of the ADC holds the promise of a lower risk of adverse events, or even no side effects at all. Additionally, the stimulatory effect of conjugates bearing saponins of the present invention on the efficacy of ADCs indicates that ADCs previously found to lack efficacy when treating tumor patients may gain renewed attention and value because their efficacy is improved in combination therapy settings, as demonstrated in this example. Reference is made to Tables A2 and A3, which summarize ADCs that have previously been investigated in human clinical settings, but some ADCs have since been withdrawn from further clinical investigation. In particular, ADCs whose clinical development has been terminated due to an observed lack of efficacy and / or the occurrence of unacceptable adverse events are ADCs that may gain renewed value for cancer patients when combined with conjugates containing covalently attached saponins of the present invention, such as the tested cetuximab-saponin.

[0310] Example B - Quillaja saponaria saponin mixture containing QS-21 with endosomal / lysosomal escape enhancing activity. Scheme I shows the common molecular structure of the QS-21 series of saponins (adapted from Conrado Pedebos, Laercio Pol-Fachin, Ramon Pons, Cilaine V. Teixeira Hugo Verli, Atomic Model and Micelle Dynamics of QS-21 Saponin, Molecules 2014, 19, 3744-3760). A mixture of water-soluble saponins obtained from Quillaja saponaria (Sigma-Aldrich, Product No. S4521; Roth, Item No. 6857; InvivoGen, Product "Quil-A") may be applied to the endosomal / lysosomal escape-enhancing conjugates, compositions, and combinations of the present invention based on the endosomal / lysosomal escape-enhancing properties of at least one individual saponin present in the mixture, e.g., QS-21, or based on the combination of two or more saponins comprised by the mixture, e.g., QS-21 and QS-7.

[0311] The present inventors demonstrated that a 2.5 microgram / ml dose of a mixture of saponins from Quillaja saponaria can enhance the endosomal escape of dianthine when tested in mammalian tumor cells using a cell-based bioassay. The effector moiety exposed to the cells was dianthine covalently coupled to the ligand EGF: EGF-dianthine. The cells tested were the tumor cell lines HeLa for free saponins, and A431, MDA-MB-468, CaSki, and A2058 for testing saponins when covalently coupled to cetuximab.

[0312] Example 1: Cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) was conjugated with SO1861 via a cysteine ​​residue (Cys) (unstable) and dianthin (a protein toxin) via a lysine residue (Lys) (stable), resulting in cetuximab-(Cys-L-SO1861). 3,9 (Lys-S-dianthine) 2 This resulted in the production of cetuximab (Cys-L-SO1861). 3,9 (Lys-S-dianthine) 2 The conjugates were tested in the A431 (EGFR) xenograph mouse tumor model for EGFR tumor-targeted cell killing as illustrated in Figure 9. Tumors were ~150 mm 3 Dosing began on day 12 when tumors reached a size of 0.5 mg / kg on day 12, and tumor volumes were determined after each dose. Mice (n=3) were treated with cetuximab-(Cys-L-SO1861) at 0.5 mg / kg on day 12, 1 mg / kg on day 15, and 1.5 mg / kg on day 24. 3,9 (Lys-S-dianthine) 2 or cetuximab-(Lys-S-dianthine) 1,6 On day 26, tumor volume reduction was significantly greater in the cetuximab-(Cys-L-SO1861) group than in the control group. 3,9 (Lys-S-dianthine) 2 This could be observed in tumor-bearing mice treated with SO1861 (Figure 1A). This indicates that unstable conjugation of SO1861 to an antibody-protein toxin (stable) conjugate can improve the targeted therapeutic efficacy of tumor-targeting antibody-protein toxin, thereby leading to more effective tumor-targeting therapy.

[0313] Next, SO1861 was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) (unstable), and dianthin (a protein toxin) was conjugated via a lysine residue (Lys) (unstable), resulting in cetuximab-(Cys-L-SO1861). 3,9 (Lys-L-dianthine) 2The conjugate was tested in the A431 (EGFR) xenograph mouse tumor model for EGFR tumor-targeted cell killing as illustrated in Figure 9. When tumors were ~150 mm 3 Dosing began on day 12 when tumors reached a size of 0.5 mg / kg, and tumor volumes were determined after each dose. Mice (n=3) were treated (intraperitoneally; i.p.; dose escalation) on day 12 with cetuximab-(Cys-L-SO1861) at 0.5 mg / kg; day 15: 1 mg / kg; day 24: 1.5 mg / kg. 3,9 (Lys-L-Gianthin)2 or Cetuximab-(Lys-L-Gianthin) 1,6 (intraperitoneal; ip; dose escalation), which showed a significant improvement in Cetuximab-(Cys-L-SO1861) after 35 days compared to controls. 3,9 (Lys-L-dianthine) 2 The results showed that tumor-bearing mice treated with cetuximab-(Cys-L-SO1861) according to the present invention exhibited tumor growth inhibition (Figure 1B). Mice (n=3) were administered (intravenously, dose escalation) on day 12 at 0.5 mg / kg; day 15 at 1 mg / kg, day 18 at 2 mg / kg, and day 24 at 2.5 mg / kg. 3,9 (Lys-L-dianthine) 2 Tumor growth inhibition was also observed when mice were treated with SO1861 (intravenously; dose escalation) compared to controls (data represent one mouse because two mice died during treatment). This indicates that labile conjugation of SO1861 to an antibody-protein toxin (labile) conjugate can improve the targeted therapeutic efficacy of tumor-targeting antibody-protein toxins, thereby leading to more effective tumor-targeting therapy.

[0314] Next, SO1861-EMCH was conjugated to cetuximab (a monoclonal antibody that recognizes and binds to human EGFR) via a cysteine ​​residue (Cys) at DAR 3,9, and an antisense HSP27 BNA oligonucleotide (which targets the cancer target hsp27 mRNA in cancer cells and induces its degradation (gene silencing)) via a labile (L) linker to the lysine residue (Lys) of the antibody at DAR 1,8, forming cetuximab-(Cys-L-SO1861). 3,9 (Lys-L-HSP27BNA) 1,8 This resulted in the production of cetuximab (Cys-L-SO1861). 3,9 (Lys-L-HSP27BNA) 1,8 was tested in the A431 xenographic "nude" mouse tumor model for EGFR-mediated tumor-targeted HSP27 gene silencing according to the present invention as illustrated in Figure 10. 3 Dosing was initiated on day 12, when tumors reached a size of 100 mg / kg, and HSP27 mRNA expression was determined. To do this, tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression levels compared to the cellular control mRNA expression level (reference gene). 30 mg / kg cetuximab-(Cys-L-SO1861) 3,9 (Lys-L-HSP27BNA) 1,8 Tumor-bearing mice (n=3) treated with (intraperitoneal; i.p.) cetuximab-(Cys-L-SO1861) were 3,8 or cetuximab-(Lys-L-HSP27BNA) 1,5 A 40% reduction in HSP27 mRNA expression in tumors was observed after one dose compared to vehicle control tumors (Figure 2). A 25% reduction in HSP27 gene expression was observed compared to vehicle control tumors. This demonstrates and enables the conjugation of SO1861 and HSP27 BNA to the same targeting antibody in accordance with the present invention to efficiently induce SO1861-mediated enhanced cytoplasmic delivery of therapeutic antisense oligonucleotides in solid tumors in tumor-bearing mice, leading to tumor-targeted gene silencing.

[0315] In another example, a trifunctional linker scaffold was designed and produced with three specific chemical terminal groups for conjugation with SO1861 on one arm and HSP27BNA on the other arm to produce SO1861-L-trifunctional linker-L-HSP27BNA. SO1861-L-trifunctional linker-L-HSP27BNA was then conjugated through its third arm to a cysteine ​​residue (Cys) of the anti-EGFR antibody cetuximab (cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)). 3,7 ), was tested in accordance with the present invention for EGFR-mediated tumor-targeted gene silencing activity in the A431 xenographic "nude" mouse tumor model, as illustrated in Figure 11. 3 Dosing was initiated on day 12, when the tumors reached a size of 100 μg / mL, and HSP27 mRNA expression was determined. To do this, tumor samples were collected 72 h after the first dose and analyzed for HSP27 gene expression levels compared to the cellular control mRNA expression level (reference gene). This was achieved by administering 30 mg / kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 3,7 One dose was 25 mg / kg cetuximab-(Cys-L-SO1861) 3,8 or 25 mg / kg cetuximab-(Lys-L-HSP27BNA) 4 We found that cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) resulted in a 40% reduction in HSP27 gene expression in tumors compared to a single dose of monotherapy (Figure 3). Compared to vehicle control tumors, cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) resulted in a 25% reduction in HSP27 gene expression. 3,7 This was observed in tumor-bearing mice treated with a single dose of cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA). 3,7 demonstrates and enables efficient induction of SO1861-mediated enhanced cytoplasmic delivery of therapeutic antisense oligonucleotides and induces targeted gene silencing in vivo in solid tumors in tumor-bearing mice.

[0316] Example 2 In another example according to the present invention, SO1861 (unstable) and the protein toxin dianthin (unstable or stable) were conjugated to the HER2-targeting antibody trastuzumab. Trastuzumab-(Cys-L-SO1861) 3,8 (Lys-L-dianthine) 1,7 or trastuzumab-(Cys-L-SO1861) 3,8 (Lys-S-dianthine) 1,7 , and as illustrated in Figure 9, SK-BR-3 (HER2) and MDA-MB-468 (HER2) - ) cells. Trastuzumab-(Cys-L-SO1861) 3,8 (Lys-L-dianthine) 1,7 (IC50=0.8nM) and trastuzumab-(Cys-L-SO1861) 3,8 (Lys-S-dianthine) 1,7 Both trastuzumab and trastuzumab-(Lys-L-dianthine) efficiently induced cell killing of SK-BR-3 cells (HER2) (Figure 4A). 1,7 , trastuzumab-(Lys-S-dianthine) 1,7 , or trastuzumab-(L-SO1861) 3,8 This was not observed in SK-BR-3 cells treated alone (Figure 4A). - ), no cell-killing activity can be observed for any of the conjugates according to the present invention (FIG. 4B). This indicates that conjugation of SO1861 to the HER-targeting antibody-protein toxin conjugate efficiently induces enhanced cytoplasmic delivery of the protein toxin mediated by SO1861 in target cells, resulting in target cell death.

[0317] In another example according to the present invention, SO1861 (unstable) and the protein toxin dianthin (unstable or stable) were conjugated to the EGFR-targeting antibody cetuximab. Cetuximab-(Cys-L-SO1861) 3,9(Lys-L-dianthine) 2 or cetuximab-(Cys-L-SO1861) 3,9 (Lys-S-dianthine) 2 As illustrated in Figure 9, A431 cells (EGFR) and A2058 cells (EGFR - ) was tested for improved cell killing. Cetuximab-(Cys-L-SO1861) 3,9 (Lys-L-dianthine) 2 (IC50=0.3nM) and cetuximab-(Cys-L-SO1861) 3,8 (Lys-S-dianthine) 1,7 (IC50=0.3nM) Both cetuximab-(Lys-L-dianthine) 1,6 (IC50=2pM), Cetuximab-(Lys-S-Dianthin) 1,6( The IC5 of 1000 was 2 pM (Figure 4C). - ), the combination according to the present invention did not exhibit any cell-killing activity (IC50>200 nM; FIG. 4D). This indicates that conjugation of SO1861 to the EGFR-targeting antibody-protein toxin conjugate efficiently improves SO1861-mediated cytoplasmic delivery of the protein toxin in target cells, resulting in improved target cell death.

[0318] Example 3 In another example according to the present invention, SO1861 (unstable) and HSP27BNA oligo (unstable) were conjugated to the EGFR-targeting antibody cetuximab. Cetuximab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 3,8 were cultured in A431 cells (EGFR) and A2058 cells (EGFR) according to the present invention as illustrated in FIG. - ) cells. Cetuximab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 3,8 Cetuximab, Cetuximab-(Lys-L-HSP27BNA) 3,9or cetuximab-(Cys-L-SO1861) 3,8 Compared with EGFR alone, it efficiently induces HSP27 gene silencing in A431 cells (IC50 = 3 nM) (Figure 5A). - ), gene silencing activity was significantly enhanced by cetuximab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 3,8 (IC50>100 nM; Figure 5B). This demonstrates and enables that conjugation of SO1861 and HSP27BNA to the same targeting antibody in accordance with the present invention efficiently induces enhanced SO1861-mediated cytoplasmic delivery of therapeutic antisense oligonucleotides in target cells, leading to targeted gene silencing.

[0319] In another example according to the present invention, SO1861 (unstable) and HSP27BNA oligo (unstable) were conjugated to the HER2-targeting antibody trastuzumab. Trastuzumab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 3,5 were tested for enhanced HSP27 gene silencing in SK-BR-3 cells (HER2) according to the present invention as illustrated in Figure 10. Trastuzumab-(Cys-L-SO1861) 3,8 (Lys-L-HSP27BNA) 3,5 Trastuzumab-(Lys-L-HSP27BNA) 4,4 Compared to SO1861 alone, it efficiently induced HSP27 gene silencing in SK-BR-3 cells (IC50=9nM) (Figure 6). This demonstrates and enables that conjugation of SO1861 and HSP27BNA to a HER2-targeting antibody according to the present invention can efficiently induce improved cytoplasmic delivery of therapeutic antisense oligonucleotides mediated by SO1861 in target cells, thereby inducing target gene silencing.

[0320] In another example, cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 were cultured in A431 (EGFR) cells and A2058 (EGFR) cells according to the present invention as illustrated in FIG. - Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) was tested for enhanced HSP27 gene silencing in IL-16 cells. 3,7 is cetuximab-(Lys-L-HSP27BNA) 4 or cetuximab-(Cys-L-SO1861) 3,7 Compared with EGFR alone, it efficiently induces HSP27 gene silencing in A431 cells (IC50 = 2 nM) (Figure 7A). - ), gene silencing activity was observed at high (>80 nM) concentrations of cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA) 3,7 This was observed only in cells with high EGFR expression (IC50 = 100 nM; Figure 7B). This indicates and enables that in highly EGFR-expressing cells, cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)3,7 efficiently induces SO1861-mediated enhanced cytoplasmic delivery of therapeutic antisense oligonucleotides in target cells, leading to targeted gene silencing.

[0321] Example 4, Figures 8A-D, display the relative cell viability when trastuzumab (Figure 8A), cetuximab (Figure 8B), or T-DM1 (Figure 8C), the unconjugated protein toxin, saporin, dianthin, and saporin conjugated to a (non-cell-binding) IgG antibody (Figure 8D) are administered to various cancer cell lines: SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, and A2058.

[0322] Trastuzumab and cetuximab have no or little effect on cell viability when exposed to most cell lines. When trastuzumab is exposed to SK-BR-3 cells at relatively high doses, it has some effect on cell growth inhibition by blocking the function of the HER2 growth factor receptor. When cetuximab is exposed to MDA-MB-468 cells at relatively high doses, it has some effect on cell growth inhibition by blocking the function of the EGFR growth factor receptor.

[0323] TDM-1, or ado-trastuzumab emtansine, is a targeted therapy approved by the U.S. Food and Drug Administration for the treatment of HER2-positive metastatic breast cancer previously treated with Herceptin (chemical name: trastuzumab) and taxane chemotherapy; and for early-stage HER2-positive breast cancer after surgery when residual disease is found after neoadjuvant (preoperative) treatment with Herceptin and taxane chemotherapy. TDM-1 is a combination of Herceptin (trastuzumab) and the chemotherapy drug emtansine. Figure 8C shows that TDM-1 results in decreased cell viability for all cell lines tested at concentrations >1000 pM. The free toxins saporin and dianthin, as well as the toxin saporin coupled to a control IgG that has no affinity for any of the cell surface molecules on the cell lines tested, had little or no effect on cell viability over the wide range of toxin concentrations tested, up to 100,000 pM (Figure 8D).

[0324] Example 5 Dendron (-L-SO1861) nSynthesis (Figures 13, 14, 15) Materials and Methods Abbreviations DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMF N,N-Dimethylformamide EDCI·HCl 3-((ethylimino)methyleneamino)-N,N-dimethylpropan-1-aminium chloride EMCH.TFA N-(ε-Maleimidocaproic acid) hydrazide, trifluoroacetate min min rt Retention time TCEP Tris(2-carboxyethyl)phosphine hydrochloride Temp Temperature TFA Trifluoroacetic acid THF Tetrahydrofuran

[0325] Analysis method LC-MS method 1, 1 Equipment: Agilent 1200 Bin. Pump: G1312A, degasser; autosampler, ColCom; DAD: Agilent G1316A, 210, 220, and 220-320 nm; PDA: 210-320 nm; MSD: Agilent LC / MSD G6130B ESI, pos / neg 100-1000; ELSD Alltech 3300 gas flow 1.5 ml / min, gas temp: 40°C; Column: Waters XSelect™ CSH C18, 30 x 2.1 mm, 3.5 μm, temp: 35°C, flow rate: 1 mL / min, gradient: t0 = 5% A, t 1.6min = 98%A, t 3min = 98% A, post time: 1.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water

[0326] LC-MS method 2, 2 Equipment: Agilent 1260 Bin. Pump: G7112B, Multisampler, Column Comp. DAD: Agilent G7115A, 210, 220, and 220-320 nm. PDA: 210-320 nm. MSD: Agilent LC / MSD G6130B ESI. Mass range depends on the molecular weight of the product: A pos / neg 100-1000 Bpos / neg 100-1400; ELSD Alltech 3300 gas flow rate 1.5 ml / min, gas temp: 40°C; column: Waters XSelect™ C18, 30 x 2.1 mm, 3.5 μm, temp: 40°C, flow rate: 1 mL / min, gradient: t0 = 5% A, t 1.6min = 98%A, t 3min = 98% A, post time: 1.3 min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% formic acid in water

[0327] LC-MS method 3, 3 Equipment: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm; SQD: ACQ-SQD2 ESI, pos / neg 800-1500; ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Waters XSelect™ CSH C18, 50 x 2.1 mm, 2.5 μm, Temp: 25°C, Flow rate: 0.6 mL / min, Gradient: t0 = 5% A, t 2.0min = 98%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0328] LC-MS method 4, 4 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm; SQD: ACQ-SQD2 ESI, pos / neg 1500-2500; ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Waters XSelect™ CSH C18, 50 x 2.1 mm, 2.5 μm, Temp: 25°C, Flow rate: 0.6 mL / min, Gradient: t0 = 15% A, t 2.0min = 60%A, t 2.7min = 98% A, post time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0329] LC-MS method 5, 5 Equipment: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN, SO; UPCMA, PDA: UPPDATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight: A pos / neg 1500-2500 B neg 2000-3000; ELSD: Gas pressure 40 psi, Drift tube temp: 50°C; Column: Acquity C18, 50 x 2.1 mm, 1.7 μm, Temp: 60°C, Flow rate: 0.6 mL / min, Gradient: t0 = 2% A, t 5.0min =50%A, t 6.0min = 98% A, post time: 1.0 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate in water (pH = 9.5).

[0330] Preparation method Preparative MP-LC method 1, 1 Instrument type: Revelis™ prep MPLC; Column: Waters XSelect™ CSH C18 (145 x 25 mm, 10µ); Flow rate: 40 mL / min; Column temp: room temperature; Eluent A: 10 mM ammonium bicarbonate in water pH = 9.0; Eluent B: 99% acetonitrile 1% 10 mM ammonium bicarbonate in water; Gradient: t 0min =5%B, t 1min =5%B, t 2min =10%B, t 17min =50%B, t 18min =100%B, t 23min = 100%B; detection UV: 210, 225, 285 nm.

[0331] Preparative MP-LC method 2, 2 Instrument type: Revelis™ prep MPLC; Column: Phenomenex LUNA C18(3) (150 × 25 mm, 10 μ); Flow rate: 40 mL / min; Column temp: room temperature; Eluent A: 0.1% (v / v) formic acid in water, Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: t 0min =5%B, t 1min =5%B, t2min =10%B, t 17min= 50%B,t 18min =100%B, t 23min = 100%B; detection UV: 210, 225, 285 nm.

[0332] Preparative LC-MS method 1, 3 MS instrument type: Agilent Technologies G6130B quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect™ CSH (C18, 100 x 30 mm, 10μ); Flow rate: 25 ml / min; Column temp: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; lin. gradient depending on product polarity: A t0=20%A, t 2min = 20%A, t 8.5min = 60%A, t 10min = 100%A, t 13min =100%A B t 0= 5%A, t 2min =5%A, t 8.5min = 40%A, t 10min = 100%A, t 13min =100%A C t 0= 10% A, t 2min = 10%A, t 8.5min =50%A, t 10min = 100%A, t 13min = 100% A; detection: DAD (220-320 nm); detection: MSD (ESI pos / neg) mass range: 100-800; fraction collection based on DAD.

[0333] Preparative LC-MS method 2, 4MS instrument type: Agilent Technologies G6130B quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; column: Waters XBridge Shield (C18, 150 × 19 mm, 5 μm); flow rate: 25 ml / min; column temp: room temperature; eluent A: 100% acetonitrile; eluent B: 10 mM ammonium bicarbonate in water pH = 9.0; gradient: t0 = 5% A, t 2.5min =5%A, t 11min = 40%A, t 13min = 100%A, t 17min = 100% A; Detection: DAD (220-320 nm); Detection: MSD (ESI pos / neg) mass range: 100-800; DAD based on fraction collection

[0334] Flash chromatography Grace Reveleris X2® C-815 Flash; solvent delivery system: 3 piston pump, auto-priming, 4 independent channels in one run, up to 4 solvents, automatic line switching when solvent runs out; maximum pump flow rate 250 mL / min; maximum pressure 50 bar (725 psi); detection: UV 200-400 nm, combination of up to 4 UV signals, scanning of the entire UV range, ELSD; column size: 4-330 g on instrument, Luer type, 750 g to 3000 g with optional holder.

[0335] Synthesis of SO1861-EMCH (Figure 13). To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 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 analyzed by 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 r.t.(min):1.08 4 Dendron (-L-SO1861) 4 Synthesis (Figure 14)

[0336] Intermediate 1: Di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate. 6-Azidohexanoic acid (0.943 g, 6.00 mmol), EDCI.HCl (1.21 g, 6.30 mmol), and OxymaPure (0.938 g, 6.60 mmol) were dissolved in DMF (10.0 mL), and the mixture was stirred for 5 minutes. Next, a solution of di-tert-butyl (azanediylbis(ethane-2,1-diyl))dicarbamate (1.82 g, 6.00 mmol) in DMF (5.00 mL) was added, and the reaction mixture was stirred at room temperature. After 5 hours, the reaction mixture was evaporated in vacuo, and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (2 × 50 mL), and brine (50 mL), dried over NaSO, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (ethyl acetate-heptane gradient, 10:90 increasing to 100:0) to give the title compound (2.67 g, 100%) as a white solid. Purity based on LC-MS: 98%. LRMS(m / z):287 / 343 / 465[M-155 / M-99 / M23] 1 LC-MS r.t.(min):2.02 2A

[0337] Intermediate 2: N,N-bis(2-aminoethyl)-6-azidohexanamide dihydrochloride. To di-tert-butyl (((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))dicarbamate (2.66 g, 6.00 mmol) was added HCl in isopropanol (5-6 N, 20.0 mL, 110 mmol), and the reaction mixture was stirred at room temperature. After 4 h, the reaction mixture was evaporated in vacuo, and the resulting crude product was co-evaporated with DCM (3 × 20 mL) to give the crude title product (1.49 g, 79%) as a white solid. LRMS(m / z):243[M1] 1

[0338] Intermediate 3: Tetra-tert-butyl ((5S,5S)-((((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(6-oxohexane-6,1,5-triyl))tetracarbamate. To a solution of N,N-bis(2-aminoethyl)-6-azidohexanamide dihydrochloride (1.19 g, 3.76 mmol) in DMF (30.0 mL) and DIPEA (2.62 mL, 15.1 mmol), Boc-Lys(Boc)-ONp (3.69 g, 7.90 mmol) was added, and the mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the residue was dissolved in ethyl acetate (100 mL). The resulting solution was washed with 1N potassium bisulfate solution (100 mL) and saturated sodium bicarbonate solution (5 × 100 mL), dried over NaSO, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (3.07 g, 91%) as a slightly yellowish solid. Purity based on LC-MS: 94%. LRMS(m / z):800 / 900 / 922[M-99 / M1 / M23] 1 LC-MS r.t.(min): 2.17 2A

[0339] Intermediate 4: 4-Nitrophenyl 3-(acetylthio)propanoate 4-Nitrophenyl trifluoroacetate (5.17 g, 22.0 mmol) and 3-(acetylthio)propionic acid (2.96 g, 20.0 mmol) were dissolved in DCM (50.0 mL). DIPEA (6.97 mL, 40.0 mmol) was then added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the residue was dissolved in ethyl acetate (50 mL). The resulting solution was washed with 1N potassium bisulfate solution (50 mL), saturated sodium bicarbonate solution (5 × 50 mL), and brine (50 mL), dried over Na2SO4, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (4.90 g, 91%) as a slightly yellowish solid. Purity based on LC-MS 99%. LRMS(m / z):292[M23] 1 LC-MS r.t.(min):1.94 2A

[0340] Intermediate 5: (S)-2,6-Diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide tetrahydrochloride. Tetra-tert-butyl ((5S,5′S)-((((6-azidohexanoyl)azanediyl)bis(ethane-2,1-diyl))bis(azanediyl))bis(6-oxohexane-6,1,5-triyl))tetracarbamate (1.80 g, 2.00 mmol) was dissolved in HCl in isopropanol (5-6N, 50.0 ml, 275 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the resulting crude product was co-evaporated with DCM (3×20 mL) to give the crude title product as a white solid. LRMS(m / z):250[M2] 2 ,500[M1] 1

[0341] Intermediate 6: (2S)-2,6-Bis[3-(acetylsulfanyl)propanamido]-N-[2-(6-azido-N-{2-[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]ethyl}hexanamido)ethyl]hexanamide. To a solution of (S)-2,6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide) tetrahydrochloride (1.29 g, 2.00 mmol) in DMF (30 mL) and DIPEA (3.48 mL, 20.0 mmol) was added 4-nitrophenyl 3-(acetylthio)propanoate (2.26 g, 8.40 mmol) and the reaction mixture was stirred at room temperature over the weekend. The reaction mixture was evaporated in vacuo, and the residue was dissolved in DCM / methanol (95:5 v / v, 100 mL). The resulting solution was washed with 1N potassium bisulfate solution (100 mL), 1N sodium hydroxide solution (3 × 100 mL), and brine (100 mL), dried over NaSO, filtered, and evaporated in vacuo. The residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (1.33 g, 65%) as a white solid. LC-MS revealed an impurity (15%) with an m / z value corresponding to the product with one deprotected thioacetate group. The impurity was formed during or after workup. Purity based on LC-MS: 85%. LRMS(m / z):510[M2] 2 ,1019 / 1041[M1 / M23] 1 LC-MS r.t.(min):1.86 2B

[0342] Intermediate 7: N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamido) formate framework 2 (102 mg, 0.100 mmol) was dissolved in methanol (1.00 mL). Freshly prepared 1 N sodium hydroxide solution (0.440 mL, 0.440 mmol) was then added, and the reaction mixture was stirred at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (0.500 mL, 0.500 mmol) was added, and the resulting mixture was stirred at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and co-evaporated with methanol (2 × 10 mL). The residue was dissolved in a mixture of methanol / water (9:1 v / v, 1.00 mL), and the resulting solution was subjected to preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (75.6 mg, 87%) as a colorless sticky oil. Purity based on LC-MS: 96%. LRMS(m / z):513[M2] 2 ,825[M1] 1 LC-MS r.t.(min):1.42 2A

[0343] Intermediate 8: Dendron (-L-SO1861) 4 The amine N,N'-((9S,19S)-14-(6-aminohexanoyl)-1-mercapto-9-(3-mercaptopropanamido)-3,10,18-trioxo-4,11,14,17-tetraazatricosane-19,23-diyl)bis(3-mercaptopropanamido) formate (2.73 mg, 3.13 μmol) was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TECEP / acetonitrile (3:1 v / v, 3.00 mL). Next, SO1861-EMCH (29.2 mg, 0.014 mmol) was added, and the reaction mixture was stirred at room temperature. After 1.5 h, the reaction mixture was analyzed by preparative LC-MS. 3BThe fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (12.3 mg, 43%) as a white fluffy solid. Purity based on LC-MS: 97%. LRMS(m / z):1517[M-6] 6- ,1821[M-5] 5- ,2276[M-4] 4- LC-MS r.t.(min):4.39 5A

[0344] Intermediate 9: Dendron (-L-SO1861) 4 A mixture of 1-azidodendron (SO1861) 4-amine (6.81 mg, 0.748 μmol) and 2,5-dioxopyrrolidin-1-yl 1-azido-3,6,9,12-tetraoxapentadecan-15-oate (2.90 mg, 7.48 μmol) was dissolved in DMF (1.00 mL). DIPEA (1.302 μL, 7.48 μmol) was then added, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (5.86 mg, 84%) as a white fluffy solid. Purity based on LC-MS: 90%. LRMS(m / z):2344[M-4] 4- LC-MS r.t.(min):4.78 5B

[0345] Intermediate 10: Dendron (-L-SO1861) 4 The α-maleimide 1-dendron (SO1861) 4-amine (8.12 mg, 0.891 μmol) and 2,5-dioxopyrrolidin-1-yl 1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12-tetraoxapentadecan-15-oate (3.94 mg, 8.91 μmol) were dissolved in DMF (1.00 mL). DIPEA (1.55 μL, 8.91 μmol) was then added, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 3 h, the reaction mixture was analyzed by preparative LC-MS. 3CThe fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (6.76 mg, 80%) as a white fluffy solid. Purity based on LC-MS: 66%. LRMS(m / z):2358[M-4] 4- LC-MS r.t.(min): 2.13 6C

[0346] Intermediate 11: Dendron (-L-SO1861) 4 -Maleimide 2 framework 2 (5.10 mg, 5.00 μmol) was dissolved in methanol (100 μL). Freshly prepared 1 N sodium hydroxide solution (22.0 μL, 22.0 μmol) was then added, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (25.0 μL, 25.0 μmol) was added, and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was evaporated in vacuo and coevaporated with methanol (2 × 5 mL). The resulting residue was dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TCEP / acetonitrile (3:1 v / v, 3.242 mL). Directly from this solution, 1000 μL was added to SO1861-EMCH (14.4 mg, 6.94 μmol, 4.5 equivalents relative to the backbone), and the mixture was shaken for 1 min and allowed to stand at room temperature. After 10 min, the reaction mixture was lyophilized overnight. To the resulting residue, 2,5-dioxopyrrolidin-1-yl 3-(2-(2-(3-(2,5-dioxo-2h-pyrrol-1(5h)-yl)propanamido)ethoxy)ethoxy)propanoate (5.84 mg, 0.014 mmol) and DMF (1.00 mL) were added. Next, DIPEA (2.39 μL, 0.014 mmol) was added, and the suspension was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative LC-MS. 3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (10.9 mg, 85%) as a white fluffy solid. Purity based on LC-MS: 80%. LRMS(m / z):2354[M-4] 4-LC-MS r.t.(min):4.16 5B Dendron (-L-SO1861) 8 Synthesis (Figure 15)

[0347] Intermediate 1: tert-Butyl N-[(1S)-1-{[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis({[(tert-butoxy)carbonyl]amino})hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis({[(tert-butoxy)carbonyl]amino})hexanamido]pentyl] {carbamoyl}-5-{[(tert-butoxy)carbonyl]amino}pentyl]carbamate (S)-2-6-diamino-N-(2-(6-azido-N-(2-((S)-2,6-diaminohexanamido)ethyl)hexanamido)ethyl)hexanamide tetrahydrochloride (964 mg, 1.50 mmol) was dissolved in DMF (25.0 mL) and triethylamine (2.08 mL, 15.0 mmol). Boc-Lys(Boc)-ONp (3.36 g, 7.18 mmol) was then added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was evaporated in vacuo, and the residue was purified by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) to give the title product (2.71 g, 100%) as a white solid. 97% purity based on LC-MS. LRMS(m / z):807[M-198] 2 LC-MS r.t.(min):2.35 2B

[0348] Intermediate 2: (2S,2'S)-N,N'-((5S,15S,22S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamide)-6,14,21-trioxo-7,10,13,20-tetraazahexacosane-1,5-diyl)bis(2,6-diaminohexanamide) octahydrochloride. Intermediate 1 (2.71 g, 1.50 mmol) was dissolved in HCl in isopropanol (5-6N, 25.0 ml, 138 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction mixture was then evaporated in vacuo, and the resulting crude product was co-evaporated with DCM (3 x 20 mL) to give the crude title product as a white solid. LRMS(m / z):203 / 254[M-200 / M4] 4 ,338[M3] 3 ,507[M2] 2 ,1012[M1] 1

[0349] Intermediate 3: (2S)-2,6-bis[3-(acetylsulfanyl)propanamido]-N-[(1S)-1-{[2-(6-azido-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis[3-(acetylsulfanyl)propanamido]hexanamido]pentyl]hexanamido (2S,2'S)-N,N'-((5S,15S,22 To (S)-22,26-diamino-10-(6-azidohexanoyl)-15-((S)-2,6-diaminohexanamide)-6,14,21-trioxo-7,10,13,20-tetraazahexacosane-1,5-diyl)bis(2,6-diaminohexanamide) octahydrochloride (300 mg, 0.230 mmol) was added DMF (20.0 mL), triethylamine (320 μL, 2.30 mmol), and 4-nitrophenyl 3-(acetylthio)propanoate (595 mg, 2.21 mmol). The resulting suspension was sonicated at 60° C. for 30 min and then stirred at room temperature overnight. The reaction mixture was evaporated in vacuo and the residue was purified first by flash chromatography (DCM-methanol / DCM (1 / 9 v / v) gradient, 100:0 increasing to 0:100) and then by preparative MP-LC. 2 to give the title product (70 mg, 15%) as a white solid. 100% purity based on LC-MS. LRMS(m / z):685[M3] 3 LC-MS r.t.(min):1.91 2A

[0350] Intermediate 4: (2S)-N-[(1S)-1-{[2-(6-amino-N-{2-[(2S)-2,6-bis[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]hexanamido]ethyl}hexanamido)ethyl]carbamoyl}-5-[(2S)-2,6-bis(3-sulfanylpropanamido)hexanamido]pentyl]-2,6-bis(3-sulfanylpropanamido)hexanamide formate. Scaffold 4 (10.0 mg, 4.87 μmol) was dissolved in methanol (200 μL). Freshly prepared 1 N sodium hydroxide solution (42.9 μL, 0.043 mmol) was then added, and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, 1.0 M trimethylphosphine solution in THF (24.4 μL, 0.024 mmol) was added, and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was diluted with water (1 mL) and directly analyzed by preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (4.02 mg, 48%) as a white fluffy solid. LRMS(m / z):564[M3] 3 ,846[M2] 2 LC-MS r.t.(min):1.54 2C

[0351] Intermediate 5: Dendron (-L-SO1861) 8 The amine scaffold 5 (0.52 mg, 0.299 μmol) and SO1861-EMCH (29.2 mg, 0.014 mmol) were dissolved in a mixture of 20 mM NH4HCO3 with 0.5 mM TCEP / acetonitrile (3:1 v / v, 1.00 mL), and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, TCEP (0.30 mg, 1.05 μmol) was added, and the reaction mixture was shaken for 1 min. The mixture was then directly analyzed by preparative LC-MS. 3B The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (2.17 mg, 40%) as a white fluffy solid. Purity based on LC-MS: 97%. LRMS(m / z):2282[M-8] 8- ,2607[M-7] 7- LC-MS r.t.(min):4.41 5A

[0352] Example 6 SO1861-Trifunctional Linker-BNA Oligo Synthesis (Figure 16) Materials and Methods Trifunctional Linker Trifunctional linkers (DBCO, TCO, maleimide) were ordered from Bio-Synthesis Inc. (Lewisville, Texas). HSP27BNA oligo HSP27BNA(-thiol) oligo (sequence 5′-GGCacagccagtgGCG-3′) ( Zhang et al., 2011 ) was purchased from Bio-synthesis Inc. (Lewisville, Texas).

[0353] Intermediate 1: To SO1861-azide (SO1861 60 mg, 0.032 mmol) and 1-azido-3,6,9,12-tetraoxapentadecane-15-hydrazide (39.3 mg, 0.129 mmol), methanol (extra dry, 1.00 mL) and TFA (9.86 μL, 0.129 mmol) were added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS: 100%. LRMS(m / z):2150[M-1] 1- LC-MS r.t.(min):1.10 3B

[0354] Intermediate 2: SO1861-trifunctional linker. SO1861-azide (45 mg, 0.021 mmol) and trifunctional linker (26.5 mg, 0.022 mmol) were dissolved in DMF (2.50 mL), and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was analyzed by preparative LC-MS. 3CThe fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS: 89%. LRMS(m / z):1677[M-2] 2- LC-MS r.t.(min):2.54 6A

[0355] Intermediate 3: (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide 1-(4-formylbenzamide)-N-(4-(6 To a mixture of (28.0 mg, 0.048 mmol)-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide and EMCH.TFA (24.5 mg, 0.072 mmol) was added methanol (extra dry, 2.00 mL) and TFA (11.1 μL, 0.145 mmol), and the reaction mixture was stirred at 50° C. After 30 min, the reaction mixture was evaporated in vacuo, and the resulting residue was analyzed by MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (33.4 mg, 88%) as a bright purple fluffy solid. Purity based on LC-MS: 92%. LRMS(m / z):394[M2] 2 ,789[M1] 1 LC-MS r.t.(min):1.28 7A

[0356] Intermediate 4: Methyltetrazine-BNA Oligo HSP27 BNA oligodisulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). TCEP (14.3 mg, 0.050 mmol) was then added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. The reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 30 min). Next, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residual solution, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (30.0 mL), and the resulting mixture was added to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (14.8 mg, 18.8 μmol) in acetonitrile (10.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was frozen and lyophilized over the weekend to give the crude title product as a pink, fluffy solid. To the crude product, 20 mM NH4HCO3 (20.0 mL) was added, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter under the same conditions as described above. Next, 20 mM NH4HCO3 solution (20.0 mL) was added to the residual solution again, and the resulting mixture was filtered again using a centrifugal filter under the same conditions as described above. The residual solution was diluted with 20 mM NH4HCO3 (20.0 mL), and the resulting mixture was lyophilized overnight to yield the title product (90.0 mg, 115%) as a pink, fluffy solid. Purity based on LC-MS: 91%. LRMS(m / z):1631[M-4] 4- ,2174[M-3] 3- LC-MS r.t.(min):0.73 7B

[0357] Intermediate 5: SO1861-trifunctional linker-BNA oligomethyltetrazine-BNA oligo (90.0 mg, 0.014 mmol) and SO1861-trifunctional linker (48.6 mg, 0.014 mmol) were dissolved in a mixture of water / acetonitrile (4:1 v / v, 12.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 15 min, the mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. Purity based on LC-MS was 92% (two peaks, both m / z values ​​corresponding to the title compound). LRMS(m / z):1641[M-6] 6- ,1970[M-5] 5- LC-MS r.t. (min): 3.24 and 3.40 6B

[0358] Intermediate 1: To SO1861-azide (SO1861 60 mg, 0.032 mmol) and 1-azido-3,6,9,12-tetraoxapentadecane-15-hydrazide (39.3 mg, 0.129 mmol), methanol (extra dry, 1.00 mL) and TFA (9.86 μL, 0.129 mmol) were added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 h, the reaction mixture was analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS: 100%. LRMS(m / z):2150[M-1] 1- LC-MS r.t.(min):1.10 3B

[0359] Intermediate 2: SO1861-trifunctional linker. SO1861-azide (45 mg, 0.021 mmol) and trifunctional linker (26.5 mg, 0.022 mmol) were dissolved in DMF (2.50 mL), and the resulting mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was analyzed by preparative LC-MS.3C The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (58.4 mg, 84%) as a white fluffy solid. Purity based on LC-MS: 89%. LRMS(m / z):1677[M-2] 2- LC-MS r.t.(min):2.54 6A

[0360] Intermediate 3: (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamide)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide 1-(4-formylbenzamide)-N-(4-(6 To a mixture of (28.0 mg, 0.048 mmol)-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide and EMCH.TFA (24.5 mg, 0.072 mmol) was added methanol (extra dry, 2.00 mL) and TFA (11.1 μL, 0.145 mmol), and the reaction mixture was stirred at 50° C. After 30 min, the reaction mixture was evaporated in vacuo, and the resulting residue was analyzed by MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (33.4 mg, 88%) as a bright purple fluffy solid. Purity based on LC-MS: 92%. LRMS(m / z):394[M2] 2 ,789[M1] 1 LC-MS r.t.(min):1.28 7A

[0361] Intermediate 4: Methyltetrazine-BNA Oligo HSP27 BNA oligodisulfide (70.0 mg, 0.012 mmol) was dissolved in 20 mM NH4HCO3 (20.0 mL). TCEP (14.3 mg, 0.050 mmol) was then added, and the reaction mixture was shaken for 1 min and allowed to stand at room temperature. The reaction mixture was filtered using a centrifugal filter with a molecular weight cutoff of 3000 Da (5000 × g, 30 min). Next, a solution of 20 mM NH4HCO3 with 2.5 mM TCEP (20.0 mL) was added to the residual solution, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 (30.0 mL), and the resulting mixture was added to a solution of (E)-1-(4-((2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)hydrazinylidene)methyl)benzamido)-N-(4-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl)-3,6,9,12-tetraoxapentadecan-15-amide (14.8 mg, 18.8 μmol) in acetonitrile (10.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was frozen and lyophilized over the weekend to give the crude title product as a pink, fluffy solid. To the crude product, 20 mM NH4HCO3 (20.0 mL) was added, and the resulting suspension was filtered through a 0.45 μm syringe filter. The filtrate was filtered using a centrifugal filter under the same conditions as described above. Next, 20 mM NH4HCO3 solution (20.0 mL) was added to the residual solution again, and the resulting mixture was filtered again using a centrifugal filter under the same conditions as described above. The residual solution was diluted with 20 mM NH4HCO3 (20.0 mL), and the resulting mixture was lyophilized overnight to yield the title product (90.0 mg, 115%) as a pink, fluffy solid. Purity based on LC-MS: 91%. LRMS(m / z):1631[M-4] 4- ,2174[M-3] 3- LC-MS r.t.(min):0.73 7B

[0362] Intermediate 5: SO1861-trifunctional linker-BNA oligomethyltetrazine-BNA oligo (90.0 mg, 0.014 mmol) and SO1861-trifunctional linker (48.6 mg, 0.014 mmol) were dissolved in a mixture of water / acetonitrile (4:1 v / v, 12.0 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 15 min, the mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (82.0 mg, 60%) as a white fluffy solid. Purity based on LC-MS was 92% (two peaks, both m / z values ​​corresponding to the title compound). LRMS(m / z):1641[M-6] 6- ,1970[M-5] 5- LC-MS r.t. (min): 3.24 and 3.40 6B

[0363] Example 7 SO1861-BNA Oligoconjugation HSP27BNA oligodisulfide (1.10 mg, 0.187 μmol) was dissolved in 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was filtered (14000 × g, 30 min) using a centrifugal filter with a molecular weight cutoff of 3000 Da. The residual solution was diluted with 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 1.00 mL), and the resulting mixture was added to SO1861-EMCH (3.54 mg, 0.375 μmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 10 min, the reaction mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (1.25 mg, 85%) as a white fluffy solid. 100% purity based on LC-MS. LRMS(m / z):1561[M-5]5- ,1951[M-4] 4- LC-MS r.t.(min):2.46 6B

[0364] Dendron (SO1861)4-BNA oligoconjugate (Figure 17). HSP27 BNA oligodisulfide (1.1 mg, 0.187 μmol) was dissolved in 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the mixture was shaken for 1 min and allowed to stand at room temperature. After 1 h, the reaction mixture was filtered (14,000 × g, 30 min) using a centrifugal filter with a molecular weight cutoff of 3,000 Da. The residual solution was diluted with 20 mM NH4HCO3 (500 μL) with 1.0 mM TCEP, and the resulting mixture was filtered again under the same conditions described above. The residual solution was diluted with 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 1.0 mL), and the resulting mixture was added to dendron (SO1861) 4-maleimide 1 (3.54 mg, 0.375 μmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 10 min, the reaction mixture was analyzed by preparative LC-MS. 4A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (1.25 mg, 85%) as a white fluffy solid. Purity based on LC-MS 94% LRMS (m / z): 1896 [M-8] 8- ,2167[M-7] 7- LC-MS r.t.(min):3.77 6B Dendron (NEM) 4 Synthesis (Figure 18) To benzyl bis(2-((S)-2,6-bis(3-mercaptopropanamido)hexanamido)ethyl)carbamate (1.69 mg, 2.00 μmol) and N-ethylmaleimide (1.05 mg, 8.40 μmol) was added a mixture of 20 mM NH4HCO3 / acetonitrile (3:1 v / v, 2.00 mL), and the reaction mixture was stirred at room temperature. After 2 hours, the reaction mixture was lyophilized overnight. The resulting residue was analyzed by preparative LC-MS. 3Ato give the title compound (1.53 mg, 57%) as a white fluffy solid. 98% purity based on LC-MS. LRMS(m / z):1346 [M1] 1 LC-MS r.t.(min): 1.43 3A

[0365] Example 8: A431 Mouse Tumor Mouse Model and Vitro and Vivo Gene Silencing Studies Materials and Methods: HSP27 BNA (sequence 5'-GGCacagccagtgGCG-3') with linker oligo (Zhang et al., 2011) was purchased from Bio-Synthesis Inc. (Lewisville, Texas). In vitro RNA isolation and gene expression analysis: RNA from cells was isolated and analyzed according to standard protocols (Biorad). In vivo mouse tumor model: Mouse studies were performed at CrownBio (China) according to standard protocols and procedures. Model: Subcutaneous A431 human epidermoid carcinoma xenograft model in female BALB / c nude mice. Tumor volume was monitored, and tumor samples were collected for gene silencing analysis (see below). RNA isolation and gene expression analysis of tumor samples from mice: Total RNA was isolated from tumors using TriZol (Thermo Fisher) according to the manufacturer's instructions. The isolated RNA was resuspended in nuclease-free water (NFW). RNA samples were checked for their RNA integrity on a gel. To prepare cDNA, 100 ng of total RNA was first mixed with random hexamers (Qiagen; final concentration 2 μM) in a final volume of 12.5 μl of NFW, denatured at 70°C for 5 min, and immediately chilled on ice. Next, 7.5 μl of cDNA synthesis mix was added, consisting of 4 μl of 5x RT buffer (Promega), 0.4 μl of 25 mM dNTPs (Promega), 1 μl of 200 U / μL MMLV RT enzyme (Promega), 0.5 μL of 40 U / μL RNAse inhibitor (Promega), and 1.6 μL NFW. The following cDNA synthesis protocol was used: 1) 10 min at 25°C, 2) 60 min at 37°C, 3) 5 min at 85°C, and 4) indefinitely at 4°C. For a single qPCR reaction, the following mix was prepared: 1 μL cDNA, 0.05 μL forward primer (250 μM), 0.05 μL reverse primer (250 μM), 8.9 μL LNFW, and 10 μL SYBR Green (Bio-Rad). The following qPCR protocol was used: 1 cycle: 5 min at 95°C, 40 cycles: 15 s at 95°C, 30 s at 60°C.

[0366] HSP27 gene expression was measured using a -(Ct HSP27 -GEOMEAN(Ct ref1 ;Ct ref2 ;Ct ref3 ;Ct ref4 )) The formula was used to calculate the geNORM stability. where ref1, ref2, ref3, and ref4 are the reference genes IMMT, EIF2S2, GUSB, and UBC for tumor analysis. To select the most ideal and stable reference genes for this tumor sample, four of the nine tested reference genes were selected based on their performance in GeNORM analysis. To do so, the qPCR results were imported into the Qbase software program. This allows for the calculation of two quality measures: the coefficient of variation (V) of the normalized reference gene expression levels; and the geNORM stability M value (M). Reference genes with M<0.2 and V<0.15 are considered highly stable. Based on this analysis, IMMT and EIF2S2 were selected as the most stable reference genes. However, UBC and GUSB were also added to the group of reference genes to further improve the accuracy of normalization. Each sample was analyzed in technical triplicates using a CFX96 real-time qPCR machine (Bio-Rad).

[0367] [Table 39]

[0368] Example 9: Conjugated synthetic monoclonal antibodies, SO1861, QS saponins trastuzumab (Herceptin®), cetuximab (Erbitux®), and T-DM1 (Kadcyla®) were purchased from a pharmacy (Charite, Berlin). CD71 monoclonal antibody was purchased from BioCell (Okt9, #BE0023). SO1861 was isolated and purified by Analyticon Discovery GmbH from a raw plant extract obtained from Saponaria officinalis L. Quillaja Saponaria saponin extract (QS mix) was purchased (Sigma Aldrich, #S4521).

[0369] Materials: trastuzumab (Tras, Herceptin®, Roche), cetuximab (Cet, Erbitux®, Merck KGaA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, 99%, Sigma-Aldrich), Zeba™ spin desalting columns (2 mL, Thermo-Fisher), NuPAGE™ 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE™ MES SDS running buffer (Thermo-Fisher), Novex™ Sharp prestained protein standards (Thermo-Fisher), PageBlue™ protein staining solution (Thermo-Fisher), Pierce™ BCA protein assay kit (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50M (GE Healthcare), Sephadex 200P (GE Healthcare), isopropyl alcohol (IPA, 99.6%, VWR), tris(hydroxymethyl)aminomethane (Tris, 99%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane hydrochloride (Tris·HCl, Sigma-Aldrich), L-histidine (99%, Sigma-Aldrich), D-(-)-trehalose dehydrate (99%, Sigma-Aldrich), polyethylene glycol sorbitan monolaurate (TWEEN 20, Sigma-Aldrich), Dulbecco's phosphate-buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), ethylenediaminetetraacetic acid disodium dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), sterile filtered 0.2 μm and 0.45 μm (Sartorius), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), dianthine-Cys (Dia-Cys, a dianthine variant with a single C-terminal cysteine ​​functional group, Proteogenix), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP, Thermo-Fisher), HSP27 oligonucleotide (Biosynthesis), [O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium-hexafluorophosphate] (HATU, 97%, Sigma-Aldrich), dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich), N-(2-aminoethyl)maleimide trifluoroacetate (AEM, 98%, Sigma-Aldrich), L-cysteine ​​(98.5%, Sigma-Aldrich), deionized water (DI Ultrapure Lab Water) Freshly prepared from the Systems (MilliQ, Merck) was nickel-nitrilotriacetic acid agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5-dithiobis(2-nitrobenzoic acid) (Ellman's reagent, DTNB, 98%, Sigma-Aldrich), S-acetylmercaptosuccinic anhydride fluorescein (SAMSA reagent, Invitrogen), sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), PD MiniTrap desalting column - Sephadex G-25 resin (GE Healthcare), PD10 G25 desalting column (GE Healthcare), and Zeba spin desalting column 0.1.5, 2, 5, and 10 mL (Thermo-Fisher), Vivaspin centrifugal filters T4 10 kDa MWCO, T4 100 kDa MWCO, and T15 (Sartorius), Biosep s3000 aSEC columns (Phenomenex), Vivacell ultrafiltration units 10 and 30 kDa MWCO (Sartorius), and Nalgene Rapid-Flow filters (Thermo-Fisher).

[0370] Methods: MALDI-TOF-MS matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) spectra were recorded on a MALDI-mass spectrometer (Bruker Ultrafex III). Typically, samples dissolved in MilliQ water in the nanomolar to micromolar range were spotted onto a target (MTP384 target plate, polished steel TF, Bruker Daltons) by the droplet drying method using either super-DHB (99%, Fluka) or sinapinic acid (SA, 99%, Sigma-Aldrich) as the matrix dissolved in acetonitrile (MADLI-TOF-MS tested, Sigma) / 0.1%...

Claims

1. A conjugate comprising a cell surface molecule targeting molecule and at least one effector moiety, and further comprising at least one covalently attached saponin, wherein the at least one saponin is a triterpenoid saponin.

2. 2. The conjugate of claim 1, wherein at least one saponin is a bisdesmosidic triterpene saponin belonging to the 12,13-dehydrooleanane type having an aldehyde function at the C-23 position.

3. 3. The conjugate of claim 1 or 2, wherein at least one saponin comprises a glucuronic acid functional group in the carbohydrate substituent at the C-3 beta-OH group of the saponin.

4. 4. The conjugate according to any one of claims 1 to 3, wherein at least one saponin is isolated from any one or more of Gypsophila species and / or Saponaria species and / or Agrostenma species and / or Quillaja species such as Quillaja Saponaria and / or is a single specific saponin or is a mixture of two or more different saponins and / or at least one saponin is less than or equal to 3.000 Daltons.

5. At least one saponin is a saponin of Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereoisomers, and / or combinations thereof; and / or the saponin is SO1861 and / or GE1741 and / or SA1641 and / or QS-21 and / or a saponin having a quilacic acid aglycone core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3β-OH group, and a Glc-(1→3)-Xyl-(1→4)-R carbohydrate substituent at the C-28-OH group.

5. The conjugate of any one of claims 1 to 4, which has a ha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc carbohydrate substituent and / or is 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronopyranosylquila acid 28-O-β-D-glucopyranosyl-(1→3) β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)-4-OAc-β-D-quinovopyranosyl-(1→4)]-β-D-fucopyranoside.

6. At least one saponin has a molecular weight of 1,500 daltons or greater, comprises an oleanane-type triterpene containing an aldehyde group at the C-23 position and / or a hydroxyl group at the C-16 position, and has a first branched carbohydrate side chain at the C-3 position, which first branched carbohydrate side chain may or may not contain glucuronic acid. The saponin also comprises an ester group with a second branched carbohydrate side chain at the C-28 position, or the second branched carbohydrate side chain comprises at least four sugar units, or at least one acetyl residue, e.g., two acetyl residues, or a deoxy sugar, or a quinovose, or a glucose, or a 4-methoxycinnamic acid, or a 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, or a 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid linked to the sugar through an ester bond.

6. The conjugate of any one of claims 1 to 5, wherein the at least one saponin is QS-21 or any of 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, QS-18, QS1861, cationized QS1861 (QS1862), or Quil-A.

7. A conjugate according to any one of claims 1 to 6, wherein the cell surface molecule targeting molecule comprises or consists of a ligand or a proteinaceous ligand or a proteinaceous binding molecule for binding to a cell surface molecule.

8. 8. The conjugate according to any one of claims 1 to 7, wherein the cell surface molecule targeting molecule is a non-proteinaceous and / or proteinaceous ligand for binding to a cell surface molecule, such as EGF or a cytokine, or is capable of binding to a cell surface molecule, such as an immunoglobulin, at least one binding domain of an immunoglobulin and / or at least one binding fragment of an immunoglobulin, such as an antibody, an IgG, a molecule comprising a Vhh domain or a Vh domain, a Fab, scFv, Fv, dAb, F(ab)2, Fcab fragment.

9. The cell surface molecule targeting molecule may be a tumor cell surface molecule or a tumor cell specific receptor or CD71, CA125, EpCAM (17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg, integrin alphaV, CA6, CD33, mesothelin, 9. The conjugate of any one of claims 1 to 8, capable of binding to a receptor selected from Crypto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25, EphrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2.

10. The conjugate of claim 9 , wherein the cell surface molecule targeting molecule is capable of binding to CD71.

11. 10. The conjugate of claim 9, wherein after binding to a cell surface molecule targeting molecule according to any one of claims 1 and 7 to 10 and a conjugate according to any one of claims 1 to 10, the tumor cell receptor is internalized by the tumor cell and / or binding of the conjugate to the tumor cell receptor is followed by tumor cell receptor mediated internalization, e.g. internalization of the complex of the conjugate and the tumor cell receptor via endocytosis, e.g. via endocytosis.

12. The cell surface molecule targeting molecule is a monoclonal antibody or at least one cell surface molecule binding fragment or domain thereof, or is selected from the group consisting of the anti-EGFR monoclonal antibody cetuximab, the anti-CD38 monoclonal antibody daratumumab, the anti-CD33 monoclonal antibody gemtuzumab, the anti-HER2 monoclonal antibody trastuzumab, the anti-EGFR monoclonal antibody panitumumab, the anti-CD30 monoclonal antibody brentuzumab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, the IgG type OKT-9 anti-CD71 monoclonal antibody, the anti-HER2 monoclonal antibody patutuzumab, the anti-CD20 monoclonal antibody rituzumab, the anti-CD20 monoclonal antibody ofatuzumab, the anti-CD52 monoclonal antibody alemtuzumab, pinatuzumab, OKT-10 Anti-CD38 monoclonal antibody, anti-CD20 monoclonal antibody tositutuzumab, anti-CD20 monoclonal antibody ibritutuzumab, anti-CA125 monoclonal antibody oregovomab, anti-EpCAM (17-1A) monoclonal antibody edrecolomab, anti-EGFR monoclonal antibody nimotuzumab, anti-CD33 monoclonal antibody huMy9-6, anti-vascular integrin alpha-V beta-3 monoclonal antibody etaracizumab, anti-CD22 monoclonal antibody epratuzumab, anti-CEA monoclonal antibody labetuzumab, anti-CD44v6 monoclonal antibody bitutuzumab, anti-FAP monoclonal antibody sibrotuzumab, anti-CD19 monoclonal antibody huB4, anti-CanAg monoclonal antibody huC242, anti-CD56 monoclonal antibody huN901, anti-CA6 monoclonal antibody DS6, anti-IGF-IR monoclonal antibody cixutuzumab, anti-IGF-IR monoclonal antibody 3B7, anti-integrin monoclonal antibody CNTO 95 or anti-syndecan-1 monoclonal antibody B-B4, or at least one tumor cell receptor binding fragment and / or at least one tumor cell receptor binding domain thereof, or the antibody-drug conjugate is Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, polatuzumabvedotin and Affilutin, IMGN-779, Neuradiab, AGS-67E, ASG-15ME, vandortuzumab vedotin, CDX-014, AGS-16M18, vorsetuzumab mafodotin, denintuzumab mafodotin, SGN-CD70A, RG-7636, SC-006, MM-310, PF-06647263, PF-06263507, PF-06650808, XMT-1522, AMG-595, pinatuzumab vedotin, cantuzumab ravtansine, AVE-9633, BIWI-1, RG-7882, ASG-5ME, DCDS-0780A, SC-004, RG-7600, sofituzumab vedotin, IMGN-289, SAR-428926, SGNCD-19B, SGNCD-123A, SGNCD-352A, RG-7841, IMGN-388, lorvotuzumab mertansine, BAY-794620, RG-7598, Oncolysin B, ADCT-502, AMG-172, ImmuRAIT-LL2, indusatumab vedotin, clivatuzumab tetraxetan, depatuxizumab mafodotin, vadastuximab talirine, MLN-2704, coltuximab ravtansine, lifastuzumab vedotin, Bismab-A, Avicidin, SGN-15, ASP-6183, SAR-566658, Oncolysin S, glembatumumab vedotin, MM-302, HTI-1511, ZW-33, HuMax-CD74-ADC, sacituzumab govitecan, ibritumomab tiuxetan, rovalpituzumab tesirine, or wherein the ligand-drug conjugate comprises at least one non-proteinaceous ligand and / or at least one proteinaceous ligand for binding to a cell surface molecule such as EGF or a cytokine, or is at least one cell surface molecule binding fragment thereof or a domain thereof.

13. 13. The conjugate of any one of claims 1 to 12, wherein the at least one effector moiety is one or more of an oligonucleotide, a nucleic acid, a xenonucleic acid, or is one or more of a vector, a gene, a transgene inducing cell suicide, a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), an antisense oligonucleotide (ASO, AON), a short interfering RNA (siRNA), a microRNA (miRNA), a DNA aptamer, an RNA aptamer, an mRNA, a minicircle DNA, a peptide nucleic acid (PNA), a phosphoramidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a bridged nucleic acid (BNA), a 2'-deoxy-2'-fluoroarabinonucleic acid (FANA), a 2'-O-methoxyethyl-RNA (MOE), a 2'-O,4'-aminoethylene bridged nucleic acid, a 3'-fluorohexitol nucleic acid (FHNA), a plasmid, a glycol nucleic acid (GNA) and a threose nucleic acid (TNA) or a derivative thereof.

14. The conjugate of claim 13, wherein at least one effector moiety consists of any one or more of antisense oligonucleotides (ASO, AON).

15. The conjugate according to any one of claims 1 to 14, wherein at least one effector moiety is covalently attached to the cell surface molecule targeting molecule via at least one linker or directly.

16. At least one effector moiety is covalently attached to a molecule that targets a cell surface molecule, such that the antibody-drug conjugate is an antibody that targets a cell surface molecule, such as gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, moxetumomab pasudotox, and polatuzumab vedotin, and Affilutin, ADCT-502, AGS-16M18, AGS-67E, AMG-172, AMG-595, ASG-15ME, ASG-5ME, ASP-6183, AVE-9633, Avicidin, BAY-794620, Bismab-A, BIWI-1, cantuzumab ravtansine, CDX-014, coltuximab ravtansine, DCDS-0780A, denintuzumab mafodotin, HTI-1511, HuMax-CD74-ADC, IMGN-289, IMGN-388, IMGN-779, ImmuRAIT-LL2, indusatumab vedotin, lifastuzumab vedotin, lorvotuzumab mertansine, MLN-2704, MM-3inotuzumab10, Oncolysin S, PF-06263507, PF-06647263, PF-06650808, pinatuzumab vedotin, RG-7598, RG-7600, RG-7636, RG-7841, RG-7882, sacituzumab govitecan, SAR-428926, SAR-566658, SC-004, SC-006, SGN-15, SGNCD-123A, SGNCD-19B, SGNCD-352A, SGN-CD70A, sofituzumab vedotin, vandortuzumab vedotin, vorsetuzumab 16. The conjugate of any one of claims 1 to 15, which is any one of mafodotin, XMT-1522, ZW-33, trastuzumab emtansine, MM-302, ibritumomab tiuxetan, inotuzumab ozogamicin, rovalpituzumab tesirin, neuradiab, clivatuzumab tetraxetan, depatuxizumab mafodotin, vadastoximab taliline, glembatatumab vedotin, oncolysin B, or an antibody-drug conjugate thereof.

17. 17. A conjugate according to any one of claims 1 to 16, comprising an aldehyde functional group at the C-23 position of a bisdesmosidic triterpene saponin of the 12,13-dehydrooleanol type, wherein at least one saponin is covalently attached to a cell surface molecule targeting molecule via an aldehyde functional group or to at least one effector moiety via an aldehyde functional group, preferably via an amino acid residue.

18. 18. The conjugate of claim 17, wherein an aldehyde functional group in at least one saponin is covalently linked to a linker N-ε-maleimidocaproic acid hydrazide, which is thioether linked to a sulfhydryl group of the cell surface molecule targeting molecule and / or at least one effector moiety, e.g. a sulfhydryl group of cysteine.

19. 19. The conjugate of any one of claims 1 to 18, wherein at least one saponin is of the 12,13-dehydrooleanane type and has a glucuronic acid functional group on a carbohydrate substituent at the C-3 beta-OH group of the saponin, and the saponin is covalently attached to an amino acid residue of a cell surface molecular targeting molecule and / or to at least one effector moiety via the glucuronic acid functional group.

20. 20. The conjugate of claim 19, wherein a glucuronic acid functional group of a carbohydrate substituent in at least one saponin is covalently attached to the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, which is amide bonded to an amine group of the cell surface molecule targeting molecule and / or at least one effector moiety (e.g. an amine group of a lysine or the N-terminus of the cell surface molecule targeting molecule and / or at least one effector moiety).

21. 21. The conjugate of any one of claims 1 to 20, wherein at least one saponin is covalently attached to the cell surface molecule targeting molecule and / or to at least one effector moiety, either directly or via at least one linker (e.g. based on N-ε-maleimidocaproic acid hydrazide or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate).

22. 22. The conjugate of claim 21, comprising a trifunctional linker, to which at least one saponin is covalently attached, the trifunctional linker comprising a third chemical group for covalently attaching to a cell surface targeting molecule and a first chemical group for covalently attaching to at least one effector moiety.

23. 19. The conjugate of any one of claims 1 to 18, wherein at least one saponin is covalently attached to the cell surface molecule targeting molecule and to the at least one effector moiety via at least one linker comprising a trifunctional linker to which both the cell surface molecule targeting molecule and the at least one effector moiety are attached.

24. The conjugate of any one of claims 18, 20 to 23, comprising at least one cleavable linker.

25. 25. The conjugate according to any one of claims 18, 20 to 24, wherein at least one linker comprises at least one cleavable linker, said cleavable linker undergoes cleavage in vivo under acidic conditions present in endosomes and / or lysosomes of a mammalian cell.

26. 26. The conjugate of any one of claims 1 to 25, wherein at least one saponin forms an amide bond with a lysine side chain or a thio-ether or disulfide bond with a cysteine ​​side chain, the lysine and / or cysteine ​​being comprised in a cell surface molecule targeting molecule and / or at least one effector moiety, and the saponin is attached to the lysine and / or cysteine ​​directly or via at least one linker.

27. 27. The conjugate of claim 26, wherein the linker comprises one or more of N-ε-maleimidocaproic acid hydrazide and / or 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, a trifunctional linker, a cleavable linker, and / or a disulfide bond, a thioether bond, an amide bond, and a hydrazide bond.

28. 28. The conjugate of any one of claims 1 to 27, wherein at least one saponin is covalently attached to the cell surface molecule targeting molecule and / or to the at least one effector moiety via at least one linker, the linker being or comprising a scaffold comprising a polymeric or oligomeric structure and further comprising at least one fourth chemical group for covalently attaching the scaffold to the cell surface molecule targeting molecule and / or to the at least one effector moiety.

29. 29. The conjugate of claim 28, wherein at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via a cleavable and / or non-cleavable bond.

30. 30. The conjugate of claim 29, wherein the cleavable bond is cleaved under any of acidic, reducing, enzymatic, and light-induced conditions.

31. 31. The conjugate of claim 29 or 30, wherein the cleavable bond undergoes cleavage in vivo under acidic conditions present within the endosomes and / or lysosomes of a mammalian cell.

32. 32. The conjugate of any one of claims 28 to 31, wherein at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via any one or more of imine, hydrazone, hydrazide, oxime, 1,3-dioxolane, disulfide, thioether, amide, peptide, or ester bonds.

33. 32. The conjugate of any one of claims 28 to 31, wherein at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via any one or more of an imine bond, a hydrazone bond, and a hydrazide bond.

34. 34. The conjugate of claim 33, wherein the aldehyde functional group at position C-23 of at least one saponin is covalently linked to a linker N-ε-maleimidocaproic acid hydrazide, which is covalently linked to a sulfhydryl group of the backbone polymeric or oligomeric structure (such as a sulfhydryl group of cysteine) via a thioether bond.

35. A conjugate according to any one of claims 28 to 31, characterized in that at least one saponin is covalently attached to the backbone polymeric or oligomeric structure via an amide bond.

36. 36. The conjugate of claim 35, wherein the glucuronic acid functionality of the carbohydrate substituent of the C-3 beta-OH group of at least one saponin is covalently linked to an amine group of the backbone polymeric or oligomeric structure (such as an amine group of a lysine or the N-terminus of the backbone) via an amide bond with the linker 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate.

37. 37. The conjugate of any one of claims 28 to 36, wherein the at least one saponin comprises in covalent bond an aldehyde functionality at position C-23 of the at least one saponin, if present, and a glucuronic acid functionality of a carbohydrate substituent at the C-3 beta-OH group of the at least one saponin.

38. The conjugate according to any one of claims 28 to 37, wherein at least one fourth chemical group of the scaffold for covalently linking the scaffold to a cell surface molecule targeting molecule and / or to at least one effector moiety is a click chemistry group.

39. 39. The conjugate of any one of claims 28 to 38, wherein the backbone polymeric or oligomeric structure consists of a linear, branched and / or cyclic polymer, oligomer, dendrimer, dendron, dendronized polymer, dendronized oligomer, DNA, polypeptide, polylysine, polyethylene glycol, or an assembly of these polymeric or oligomeric structures.

40. 40. The conjugate of any one of claims 1 to 39, wherein the at least one saponin is a defined number of saponins or a defined range of saponins.

41. 41. The conjugate of any one of claims 1 to 40, wherein the conjugate comprises more than one saponin and is covalently attached directly to an amino acid residue of the cell surface molecule targeting molecule and / or via at least one effector moiety and / or via at least one linker of any one of claims 28 to 40 and / or via at least one cleavable linker and / or via at least one polymeric or oligomeric backbone, wherein 1 to 32 saponins are covalently attached to the at least one backbone.

42. 42. The conjugate of any one of claims 1 to 41, wherein at least one saponin is covalently attached to the cell surface molecule targeting molecule and at least one effector moiety via a trifunctional linker, the trifunctional linker comprising a second chemical group to which the at least one saponin is covalently attached either directly or via a linker such as a cleavable linker and / or via a backbone comprising a polymeric or oligomeric structure, and a fourth chemical group according to any one of claims 28 to 41 for covalently attaching the backbone to the trifunctional linker, the trifunctional linker further comprising a third chemical group for covalently attaching to the cell surface molecule targeting molecule and comprising a first chemical group for covalently attaching to at least one effector moiety, the cell surface molecule targeting molecule being attached to the third chemical group and / or the at least one effector moiety being attached to the first chemical group.

43. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 42.

44. A conjugate according to any one of claims 1 to 42, or a pharmaceutical composition according to claim 43, for use as a medicament.

45. A conjugate according to any one of claims 1 to 42 or claim 44, or a pharmaceutical composition according to claim 43 or claim 44, for use in the treatment or prevention of cancer or an autoimmune disease.

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