Saponin derivatives improving the therapeutic window

Abstract

The present invention relates to a saponin derivative based on a saponin, the saponin derivative comprising a triterpenoid aglycone and a first sugar chain and / or a second sugar chain, and comprising: an aglycone core structure comprising an aldehyde group which has been derivatized; and / or a first sugar chain, wherein the first sugar chain comprises a carboxyl group which has been derivatized; and / or a second sugar chain, wherein the second sugar chain comprises at least one acetoxy group which has been derivatized. The present invention further relates to a first pharmaceutical composition comprising the saponin derivative of the present invention. Furthermore, the present invention relates to a pharmaceutical combination comprising the first pharmaceutical composition of the present invention and a second pharmaceutical composition comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate. The present invention further relates to the first pharmaceutical composition or the pharmaceutical combination of the present invention for use as a medicament or for use in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or an autoimmune disease. Furthermore, the present invention relates to an in vitro or ex vivo method for transferring a molecule from the outside of a cell to the inside of said cell, comprising contacting said cell with said molecule and the saponin derivative of the present invention.

Description

TECHNICAL FIELD

[0001] The present invention relates to a saponin derivative based on a saponin, said saponin derivative comprising a triterpenoid aglycone and a first sugar chain and / or a second sugar chain, and comprising: an aglycone core structure comprising an aldehyde group which has been derivatized; and / or a first sugar chain, wherein the first sugar chain comprises a carboxyl group which has been derivatized; and / or a second sugar chain, wherein the second sugar chain comprises at least one acetoxy group which has been derivatized. The present invention further relates to a first pharmaceutical composition comprising the saponin derivative of the present invention. Furthermore, the present invention relates to a pharmaceutical combination comprising the first pharmaceutical composition of the present invention and a second pharmaceutical composition, said second pharmaceutical composition comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate or a receptor-ligand-oligonucleotide conjugate. The present invention further relates to the first pharmaceutical composition or the pharmaceutical combination of the present invention for use as a medicament or for use in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis or an autoimmune disease. Furthermore, the present invention relates to an in vitro or ex vivo method for transferring a molecule from the outside of a cell to the inside of said cell, comprising contacting said cell with said molecule and a saponin derivative of the present invention. BACKGROUND

[0003] Targeted tumor therapy is the treatment of cancer using drugs that target specific genes and proteins involved in the growth and survival of cancer cells. Immunotoxins are targeted toxins containing an antibody as targeting moiety and are very promising as they combine the specificity of an antibody directed against a tumor-specific antigen, which enables them to direct the toxin to the point of action, and can additionally introduce cell-killing mechanisms such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. To show their effect, the toxin needs to be released into the cytosol after internalization. A major drawback is that the targeting moiety carrying the payload is often not fully internalized, is recycled directly to the surface after internalization, or is degraded in the lysosome, thereby hampering sufficient delivery of the payload into the cytosol of the cell. To ensure a toxic payload concentration in the tumor cell and to overcome insufficient cytosolic entry, high serum levels of the targeted toxin are often required, leading to severe side effects, in particular including immunogenicity and vascular leak syndrome. Therefore, when treating cancer patients with antibody-drug conjugates (ADCs), there is still a focus on a sufficiently wide therapeutic window.

[0004] To address the drawback of insufficient cytosolic entry, several strategies have been developed, involving for example redirection of the toxin to endogenous cell membrane transport complexes of the biosynthetic pathway, disruption of endosomes, weakening of the membrane integrity of the endosomal membrane, or the use of cell-penetrating peptides.

[0005] For example, in tumor therapy, glycosylated triterpenes (e.g. saponins) were found to be potent endosomal escape enhancers of targeted toxins (e.g. ribosome inactivating proteins (RIPs)). Structure-activity relationship analysis of saponins revealed that the presence of the following core structural elements seems to favor the ability of saponins to enhance the cytotoxicity of RIPs (see Formula (I), wherein X 1 = H or OH and X 2 = a polysaccharide moiety):

[0006] - a branched trisaccharide containing a glucuronic acid at the C-3 position

[0007] - an aldehyde at the C-4 position

[0008] - a carboxyl group at the C-28 position

[0009] - a polysaccharide moiety attached to the C-28 position of at least four sugar moieties containing acetyl groups (R 2 ).

[0010]

[0011] In particular, SO1861 (Formula II, sometimes also referred to as SPT001), a triterpenoid saponin, was identified as an effective molecule to enhance endosomal escape of tumor cell targeted toxins. The dual action of the enhancer mechanism was postulated: first, direct increase of endosomal escape leads to caspase-dependent apoptosis, its second, in combination with the lysosome-mediated cell death pathway, which is triggered after the release of cathepsins and other hydrolytic enzymes after the disruption of the lysosomal membrane.

[0012]

[0013] The use of saponins as endosomal escape enhancers is based on the recognition that these saponins have the ability to disrupt red blood cell membranes. However, at the same time, the cell-disrupting activity of saponins contributes to side effects (risks) when a subject is treated with such saponins, thus affecting the optimal therapeutic window in terms of limiting the therapeutic index. Indeed, the extracellular and / or intracellular toxicity of such saponins when administered to a patient in need of an anti-tumor treatment is of concern when, for example, the optimal dosing regimen and route and frequency of administration are considered.

[0014] All features of the chemical composition of saponins per se, including the structure of the triterpenoid backbone, the pentacyclic C30 terpene skeleton (also referred to as sapogenin or aglycone), the number and length of the sugar side chains and the type and linkage variants of the sugar residues attached to the backbone, contribute to the hemolytic potential and / or cytotoxicity of such saponins.

[0015] When considering the endosomes and cytosol of cells, the saponin itself is not target specific and the saponin is unexpectedly and most often distributed in (human) subjects with other kinetics than the targeted toxin, even when the same route of administration is considered. Therefore, after administration of a therapeutic combination comprising e.g. an ADC and a saponin to a patient in need thereof, the saponin molecule can be found in any organ, which means that the specificity is only mediated by the targeted toxin. The distribution of the saponin in the body requires a higher concentration for a successful treatment when compared to the specific accumulation in the target cells. Therefore, considering the systemic application of saponins in vivo, the toxicity of the modified saponin needs to be low enough for a successful application in order to obtain a suitable therapeutic window.

[0016] Therefore, there is still a need to improve the therapeutic index when considering the co-administration of saponins with e.g. ADCs: when considering the enhanced cytotoxic effect of the ADC, there is a need to better control (or better: reduce) the cytotoxicity of the saponin while maintaining sufficient efficacy. SUMMARY

[0017] Surprisingly, the inventors have found that modified saponins, i.e. saponin derivatives, have

[0018] - a branched trisaccharide moiety bound at the C-3 position of the aglycone of the saponin and containing a modified glucuronic acid; and / or

[0019] - a modified aldehyde at the C-4 position of the aglycone of the saponin; and / or

[0020] - a polysaccharide moiety bound at the C-28 position of the aglycone of the saponin and comprising a modified acetoxy group in said polysaccharide moiety;

[0021] which have a reduced toxicity when considering the cell viability of cells in contact with the saponin derivative, an activity (without wishing to be bound by any theory: related to an enhanced endosomal escape of the modified saponin) when considering the enhancement of e.g. toxin cytotoxicity or BNA-mediated gene silencing and / or a reduced hemolytic activity when compared to the toxicity, activity and hemolytic activity of the unmodified saponin. Therefore, the inventors provide saponin derivatives with an improved therapeutic window as the ratio between the IC50 value of the cytotoxicity and e.g. the toxin enhancement or gene silencing is increased and / or as the ratio between the IC50 value of the hemolytic activity of the saponin and e.g. the toxin enhancement or gene silencing is increased.

[0022] A first aspect of the present invention relates to a saponin derivative based on a saponin comprising a triterpene aglycone core structure and at least one of a first saccharide chain and a second saccharide chain linked to said aglycone core structure, wherein:

[0023] i. the saponin derivative comprises an aglycone core structure comprising an aldehyde group which has been derivatized; or

[0024] ii. the saponin derivative comprises the first sugar chain, wherein the first sugar chain comprises a carboxyl group that has been derivatized, preferably a carboxyl group of a glucuronic acid moiety; or

[0025] iii. the saponin derivative comprises the second sugar chain, wherein the second sugar chain comprises at least one acetoxy (Me(CO)O-) group that has been derivatized; or

[0026] iv. the saponin derivative comprises any combination of derivatizations i., ii. and iii., preferably any combination of two derivatizations i., ii. and iii.

[0027] wherein the first sugar chain and the second sugar chain are independently selected from the group consisting of monosaccharides, linear oligosaccharides and branched oligosaccharides.

[0028] One embodiment is the saponin derivative of the present application, wherein the saponin derivative is a monosaccharide chain triterpene glycoside or a disaccharide chain triterpene glycoside, more preferably a disaccharide chain triterpene glycoside.

[0029] A second aspect of the present application relates to a first pharmaceutical composition comprising a saponin derivative according to the present application and optionally a pharmaceutically acceptable excipient and / or diluent.

[0030] A third aspect of the present application relates to a pharmaceutical combination comprising:

[0031] • the first pharmaceutical composition of the present application; and

[0032] • a second pharmaceutical composition comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate or a receptor-ligand-oligonucleotide conjugate, and optionally a pharmaceutically acceptable excipient and / or diluent.

[0033] A fourth aspect of the present application relates to a third pharmaceutical composition comprising a saponin derivative of the present application and further comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-nucleic acid conjugate or a receptor-ligand-nucleic acid conjugate, and optionally a pharmaceutically acceptable excipient and / or diluent.

[0034] A fifth aspect of the present application relates to the first pharmaceutical composition of the present application, the pharmaceutical combination of the present application or the third pharmaceutical composition of the present application for use as a medicament.

[0035] The sixth aspect of the present application relates to the first pharmaceutical composition of the present application, the pharmaceutical combination of the present application or the third pharmaceutical composition of the present application for use in the treatment or prevention of cancer, an infectious disease, a viral infection, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, thyroid hormone-mediated amyloidosis or an autoimmune disease.

[0036] The seventh aspect of the present application relates to an in vitro or ex vivo method for transferring a molecule from the outside of a cell into the interior of said cell, preferably into the cytosol of said cell, said method comprising the following steps:

[0037] a) providing a cell;

[0038] b) providing a molecule for transfer from the outside of a cell into the cell provided in step a);

[0039] c) providing a saponin derivative according to the present application;

[0040] d) contacting the cell of step a) in vitro or ex vivo with the molecule of step b) and the saponin derivative of step c), thereby establishing a transfer of said molecule from the outside of the cell into said cell.

[0041] Definitions

[0042] The term "saponin" has its conventional scientific meaning and refers herein to a group of amphipathic glycosides comprising one or more hydrophilic glycosyl moieties bound to a lipophilic aglycone core, the lipophilic aglycone core being a sapogenin. Saponins can be naturally occurring or synthetic (i.e. non-naturally occurring). The term "saponin" includes naturally occurring saponins, derivatives of naturally occurring saponins and saponins synthesized de novo by chemical and / or biotechnological synthetic routes.

[0043] The term "modified saponin" has its conventional scientific meaning and refers herein to a saponin, i.e. a saponin derivative, which prior to the chemical modification to provide the modified saponin has one or more chemical modifications at the position of any one of the aldehyde group, carboxyl group, acetate group and / or acetyl group which was present in the non-derivatized saponin prior to the chemical modification to provide the modified saponin. For example, the modified saponin is provided by chemical modification of any one or more of the aldehyde group, carboxyl group, acetate group and / or acetyl group in the saponin on which the modified saponin is based, i.e. providing the saponin, and chemically modifying any one of the aldehyde group, carboxyl group, acetate group and / or acetyl group, thereby providing the modified saponin. For example, the saponin which is modified to provide the modified saponin is a naturally occurring saponin. Typically, the modified saponin is a synthetic saponin, typically the modified saponin is a modification of a natural saponin and is thus derived from a natural saponin, although the modified saponin can also be derived from a synthetic saponin which can or can not have a natural counterpart. Typically, the modified saponin does not have a natural counterpart, i.e. the modified saponin is not naturally produced by, for example, a plant or tree.

[0044] The term "aglycone core structure" has its conventional scientific meaning and refers herein to the aglycone core of a saponin which does not have one or two carbohydrate antennae or sugar chains (glycans) bound thereto. For example, quillaic acid is the aglycone core structure of SO1861, QS-7 and QS21. Typically, the glycan of a saponin is a monosaccharide or oligosaccharide, e.g. a linear or branched glycan.

[0045] Unless otherwise indicated, the term "QS21" refers to any one isomer of QS21 having the structural formula shown in Figure 41 and mixtures of two or more, e.g. all isomers shown in Figure 41 As will be appreciated by the skilled person, a typical natural extract comprising QS21 will comprise a mixture of different isomers of QS21. However, by purification or (semi-)synthetic routes, a single isomer can be isolated.

[0046] The term "sugar chain" has its conventional scientific meaning and refers herein to any one of a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide) or a chain comprising multiple sugar moieties (oligosaccharide, polysaccharide). The sugar chain can consist only of sugar moieties or can also comprise other moieties, e.g. any one of 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid and 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, e.g. as present in QS-21.

[0047] The term "chemically modified" has its conventional scientific meaning and refers herein to a chemical modification of a first chemical group or a first chemical moiety, thereby providing a second chemical group or a second chemical moiety. Examples are the chemical modification of a carbonyl group to a -(H)C-OH group, the chemical modification of an acetate group to a hydroxyl group, the formation of a conjugate of a saponin with an N-epsilon-maleimide hexanoic acid hydrazide (EMCH) moiety at the aldehyde group of the saponin by a chemical reaction, etc.

[0048] The term "chemically modified aldehyde group" has its conventional scientific meaning and refers herein to a chemical reaction product obtained by a chemical reaction involving an aldehyde group of a saponin, which chemical reaction results in the initial aldehyde group being replaced by a new chemical group. For example, the formation of a -(H)C-OH group from an initial aldehyde group of a saponin.

[0049] The term "chemically modified carboxyl group" has its conventional scientific meaning and refers herein to a chemical reaction product obtained by a chemical reaction involving a carboxyl group of a saponin, e.g. a carboxyl of a glucuronic acid moiety, and another molecule, which chemical reaction results in the initial carboxyl group being replaced by a new chemical group. For example, the formation of a conjugate between a saponin and any one of 2-amino-2-methyl-1,3-propanediol (AMPD), N-(2-aminoethyl)maleimide (AEM), or 1 -[bis(dimethylamino)methylene]-1 H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), involves a carboxyl of a glucuronic acid of a saponin.

[0050] The term "Api / Xyl-" or "Api- or Xyl-" in the context of the name of a sugar chain has its conventional scientific meaning and refers herein to a sugar chain comprising a Api moiety or comprising a Xyl moiety.

[0051] The term "saponin on which the modified saponin is based" has its conventional scientific meaning and refers herein to a saponin which is modified in order to provide the modified saponin. Typically, the saponin on which the modified saponin is based is a naturally occurring saponin which is chemically modified to provide the modified saponin.

[0052] The term "modified saponin based on a saponin" has its conventional scientific meaning and refers herein to a saponin which has been subjected to a chemical modification step thereby providing the modified saponin, wherein the saponin from which the modified saponin is made is typically a naturally occurring saponin.

[0053] The term "oligonucleotide" has its conventional scientific meaning and refers herein to encompass any natural or synthetic nucleic acid string, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acid, which is presented as a single-stranded molecule or double-stranded molecule, e.g. BNA, antisense oligonucleotide (ASO), short or small interfering RNA (siRNA; silencing RNA), antisense DNA, antisense RNA, etc.

[0054] The term "antibody-drug conjugate" or "ADC" has its conventional scientific meaning and refers herein to any conjugate of an antibody (such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single domain antibody, V HH Horse, Camelid V H Horse, Camelid V etc.), and any molecule (e.g. active pharmaceutical ingredient, toxin, oligonucleotide, enzyme, small molecule drug compound, etc.) capable of exerting a therapeutic effect when in contact with cells of a subject, such as a human patient.

[0055] The term "antibody-oligonucleotide conjugate" or "AOC" has its conventional scientific meaning and refers herein to any conjugate of an antibody (such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single domain antibody, V HH Horse, Camelid V H Horse, Camelid V etc.), and any oligonucleotide molecule (such as oligonucleotide) capable of exerting a therapeutic effect when in contact with cells of a subject, such as a human patient, selected from the group consisting of natural or synthetic nucleic acid string, DNA, modified DNA, RNA, mRNA, modified RNA, synthetic nucleic acid, which is presented as a single-stranded molecule or double-stranded molecule, e.g. BNA, antisense oligonucleotide (ASO), short or small interfering RNA (siRNA; silencing RNA), antisense DNA, antisense RNA, etc.

[0056] The term "effector molecule" or "effector moiety" when referring to an effector molecule as part of, for example, a covalent conjugate, has its regular scientific meaning and refers herein to a molecule that can selectively bind to, for example, any one or more of the following target molecules: a protein, a peptide, a carbohydrate, a sugar (e.g. a glycan), a (phospho)lipid, a nucleic acid (e.g. DNA), RNA, an enzyme, and modulate the biological activity of these one or more target molecules. The effector molecule is, for example, any one or more of a molecule selected from a small molecule (e.g. a drug molecule), a toxin (e.g. a protein toxin), an oligonucleotide (e.g. a BNA), a xeno nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof. Thus, for example, the effector molecule or effector moiety is any one or more of a molecule selected from a small molecule (e.g. a drug molecule), a toxin (e.g. a protein toxin), an oligonucleotide (e.g. a BNA), a xeno nucleic acid or siRNA, an enzyme, a peptide, a protein, or any combination thereof that can selectively bind to any one or more of the following target molecules: a protein, a peptide, a carbohydrate, a sugar (e.g. a glycan), a (phospho)lipid, a nucleic acid (e.g. DNA), RNA, an enzyme, and modulate the biological activity of these one or more target molecules when bound to said target molecule. Typically, the effector molecule can exert a biological effect within a cell, for example a mammalian cell, for example a human cell, for example in the cytosol of said cell. Thus, typical effector molecules are drug molecules, plasmid DNA, toxins such as the toxins comprised by antibody-drug conjugates (ADCs), oligonucleotides such as siRNAs, BNAs, nucleic acids comprised by antibody-oligonucleotide conjugates (AOCs). For example, the effector molecule is a molecule that can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression or cell signaling.

[0057] The term "HSP27" relates to a BNA molecule silencing the expression of HSP27 in a cell.

[0058] The term "bridged nucleic acid", or simply "BNA", or "locked nucleic acid" or simply "LNA", has its regular scientific meaning and refers herein to a modified RNA nucleotide. A BNA is also referred to as a "constrained RNA molecule" or "unreachable RNA molecule". A BNA monomer can contain a five-, six- or even seven-membered bridged structure with a "fixed" C3'-endo sugar puckering. The bridge is synthetically incorporated at the 2', 4'-position of the ribose to provide a 2', 4'-BNA monomer. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. A BNA is a structurally rigid oligonucleotide with increased binding affinity and stability.

[0059] The terms first, second, third, etc. in the description and in the claims are used for distinguishing between for example similar elements, compositions, components, or steps in a method and are not necessarily used to describe a sequence or an order of steps or a sequence of composition. Such terms are interchangeable under appropriate circumstances and the embodiments of the application can operate in other sequences than described or illustrated herein, unless otherwise specified.

[0060] Unless otherwise indicated, the embodiments of the application described herein can operate in combination and cooperation.

[0061] Furthermore, various embodiments, although being referred to as "preferred" or "for example" or "such as" or "in particular" or the like, should be interpreted as exemplary ways of implementing the application, but not as limiting the scope of the application.

[0062] The term "comprising", as used in the claims, should not be interpreted as being restricted to comprising only those elements or steps listed in the specific claims section, or comprising all of the elements or steps of a method or composition as set forth in the specification. It is possible that other elements or steps are added, or that some elements or steps are not present, without changing the basic underlying inventive concept. The term "comprising" therefore is used, in the broadest sense, to mean the inclusion of any of the specified elements or steps, but not to the exclusion of any other elements or steps. The term "comprising" is used in the claims to mean that the claims are not limited to the elements or steps listed in the claims, but that other elements or steps can be added, or that some elements or steps can be removed, without changing the basic underlying inventive concept. Thus, the scope of the claims should not be limited to the elements or steps recited in the claims section but should be given the full breadth of the claims below.

[0063] Furthermore, the indefinite articles "a" or "an", as used in the specification and in the claims, are not intended to exclude the possibility that more than one element or component can be present. Thus, the indefinite articles "a" or "an" are used in the sense that "one or more".

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Synthesis of molecule 3A.

[0066] Figure 2 Synthesis of molecule 6.

[0067] Figure 3 Synthesis of molecule 8.

[0068] Figure 4 Synthesis of molecule 9.

[0069] Figure 5 Synthesis of molecule 10.

[0070] Figure 6 Synthesis of molecule 11.

[0071] Figure 7 Synthesis of molecule 12.

[0072] Figure 8 Synthesis of molecule 14.

[0073] Figure 9 Synthesis of molecule 15.

[0074] Figure 10 Synthesis of molecule 16.

[0075] Figure 11 Synthesis of molecule 18.

[0076] Figure 12 Synthesis of molecule 19.

[0077] Figure 13 Synthesis of molecule 20.

[0078] Figure 14 Synthesis of molecule 21.

[0079] Figure 15 Mass chromatogram of molecule 6.

[0080] Figure 16 Details of the mass chromatogram of molecule 6 synthesized starting from SO1861.

[0081] Figure 17 Details of the mass chromatogram of molecule 9, starting from molecule 6.

[0082] Figure 18A IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-dianthin.

[0083] Figure 18B IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0084] Figure 19A IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0085] Figure 19BIC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0086] Figure 20A IC50 curves of the toxicity of saponin derivatives to EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0087] Figure 20B IC50 curves of the toxicity of saponin derivatives to EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0088] Figure 21A IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (HeLa).

[0089] Figure 21B IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (A431).

[0090] Figure 22 Hemolytic activity of saponin derivatives as determined by human erythrocyte hemolysis assay.

[0091] Figure 23A IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0092] Figure 23B IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0093] Figure 24A IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0094] Figure 24B IC50 curve of saponin derivatives enhancing endosome escape activity in EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0095] Figure 25A IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (HeLa).

[0096] Figure 25BIC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (A431).

[0097] Figure 26A IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (HeLa).

[0098] Figure 26B IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (A431).

[0099] Figure 27 Hemolytic activity of saponin derivatives as determined by human erythrocyte hemolysis assay.

[0100] Figure 28 Hemolytic activity of saponin derivatives as determined by human erythrocyte hemolysis assay.

[0101] Figure 29 Hemolytic activity of saponin derivatives as determined by human erythrocyte hemolysis assay.

[0102] Figure 30A IC50 curves of the activity of saponin derivatives against EGFR-expressing cells (HeLa) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0103] Figure 30B IC50 curves of the activity of saponin derivatives against EGFR-expressing cells (A431) in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin.

[0104] Figure 31A IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (HeLa).

[0105] Figure 31B IC50 curves showing the toxicity of saponin derivatives to EGFR-expressing cells (A431).

[0106] Figure 32 Hemolytic activity of saponin derivatives as determined by human erythrocyte hemolysis assay.

[0107] Figure 33A IC50 curves of various QS saponin fractions enhancing endosome escape activity in EGFR-expressing cells (HeLa) in the presence of 5 pM cetuximab-saporin.

[0108] Figure 33B IC50 curves of various QS saponin fractions enhancing endosome escape activity in EGFR-expressing cells (A431) in the presence of 5 pM cetuximab-caryophyllein.

[0109] Figure 34A IC50 curves of the toxicity of QS saponin fractions to EGFR-expressing cells (HeLa).

[0110] Figure 34B IC50 curves of the toxicity of QS saponin fractions to EGFR-expressing cells (A431).

[0111] Figure 35 The hemolytic activity of QS saponin fractions was determined by human erythrocyte hemolysis assay.

[0112] Figure 36 Synthesis of molecule 23.

[0113] Figure 37 Synthesis of molecule 25.

[0114] Figure 38 Synthesis of molecule 27.

[0115] Figure 39 Synthesis of molecule 28.

[0116] Figure 40A Synthesis of molecule 29.

[0117] Figure 40B :QS21-Ald-EMCH (Molecular 30).

[0118] Figure 40C :QS21-Glu-AMPD (molecule 31).

[0119] Figure 40D :QS21-(Ald-EMCH)-(Glu-AMPD)(Molecular 32).

[0120] Figure 40E :QS21-(Ald-OH)-(Glu-AMPD)(Molecular 33).

[0121] Figure 41 The structures of four QS-21 isomers.

[0122] Figure 42 Determination of critical micelle concentration: ANS fluorescence yield of SO1861 with single modification.

[0123] Figure 43 Determination of critical micelle concentration: ANS fluorescence yield of double-modified SO1861.

[0124] Figure 44 Determination of critical micelle concentration: ANS fluorescence yield of trimodified SO1861.

[0125] Figure 45Determination of critical micelle concentration: ANS fluorescence yield of QS saponins.

[0126] Figure 46 Determination of critical micelle concentration: ANS fluorescence yield of QS21.

[0127] Figure 47A Determination of critical micelle concentration: ANS fluorescence yield of modified QS21.

[0128] Figure 47B Determination of critical micelle concentration: ANS fluorescence yield of single-modified QS21.

[0129] Figure 47C Determination of critical micelle concentration: ANS fluorescence yield of double-modified QS21.

[0130] Figure 48 Cell viability assay (MTS) of A431 cells using SO1861 or SO1861-EMCH+10pM cetuximab-caryophyllin.

[0131] Figure 49 Cell viability assay (MTS) of A431 cells using cetuximab-caryophyllin + 300 nM and 4000 nM SO1861-EMCH.

[0132] Figure 50 Cell viability assay (MTS) of A431 cells using cetuximab-caryophyllin + 300 nM and 1500 nM SO1861 or 4000 nM SO1861-EMCH.

[0133] Figure 51 Cell viability assay (MTS) of A431 cells using SO1861 or SO1861-EMCH+10pM EGF caryophyllin.

[0134] Figure 52A , Figure 52B Cell viability assay (MTS) of A431 cells using EGF caryophyllin + 10 nM, 300 nM and 1500 nM SO1861 or 4829 nM SO1861-EMCH.

[0135] Figure 53A , Figure 53B : Trastuzumab-caryophyllin or trastuzumab-saponin + 1500nM SO1861 or 4000nM SO1861-EMCH for the measurement of cell viability (MTS) of A431 cells.

[0136] Figure 54Analysis of HSP27 mRNA gene silencing in A431 cells using SO1861-EMCH+100nM HSP27BNA and 100nM cetuximab-HSP27BNA.

[0137] Figure 55 Analysis of HSP27 mRNA gene silencing in A431 cells using cetuximab-HSP27 BNA conjugate (DAR1.5 or DAR4) + 100 nM SO1861-EMCH or 4000 nM SO1861-EMCH.

[0138] Figure 56 Analysis of HSP27 mRNA gene silencing in SK-BR-3 cells using trastuzumab-HSP27BNA conjugate (DAR4.4) + 100 nM SO1861-EMCH or 4000 nM SO1861-EMCH.

[0139] Figure 57A , Figure 57B Analysis of HSP27 mRNA gene silencing in A431 and A2058 cells using HSP27BNA+4000nM SO1861-EMCH.

[0140] Figure 58 Analysis of HSP27 mRNA gene silencing in SK-BR-3 cells using HSP27BNA or HSP27LNA+4829nM SO1861-EMCH.

[0141] Figure 59 Synthesis of molecule 26.

[0142] Figure 60 The general reaction scheme for the Michael addition reaction of maleimide group with thiol (if R = CH2-CH2-OH, then...) Figure 60 The synthesis of SO1861-Ald-EMCH-blocked (SO1861-Ald-EMCH-mercaptoethanol) is described.

[0143] Figure 61(A) MALDI-TOF-MS spectrum of SO1861-Ald-EMCH and (B) SO1861-Ald-EMCH-mercaptoethanol. (A) RP mode: m / z 2124 Da ([M+K]+, saponin-Ald-EMCH), m / z 2109 Da ([M+K]+, SO1861-Ald-EMCH), m / z 2094 Da ([M+Na]+, SO1861-EMCH). (B) RP mode: m / z 2193 Da ([M+K]+, saponin-Ald-EMCH-mercaptoethanol), m / z 2185 Da ([M+K]+, SO1861-Ald-EMCH-mercaptoethanol), m / z 2170 Da ([M+Na]+, SO1861-Ald-EMCH-mercaptoethanol).

[0144] Figure 62 MALDI-TOF-MS spectra of SO1861-EMCH (A) before and after hydrolysis in HCl solution at pH 3 and (B).

[0145] Figures 63A-63D Unconjugated saponins mediate enhanced endosome escape and target cell killing. A) HeLa cells (EGFR) treated with SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904 with or without 1.5 pM EGF caryophyllin. + A) Cell viability analysis. B) HeLa cells (EGFR cells) treated with EGF caryophyllin and fixed concentrations of SO1861, SO1832, SO1862 (an isomer of SO1861), or SO1904. + C) Cell viability analysis. HeLa cells (EGFR) treated with SO1861 or GE1741 with or without 1.5 pM EGF caryophyllin. + D) Cell viability analysis. HeLa cells (EGFR) treated with various QSmix (a mixture of saponins from Quiillaia Saponaria) with or without 1.5 pM EGF caryophyllin. + Cell viability analysis.

[0146] Figures 64A-64E Unconjugated SO1861 versus SO1861-Ald-EMCH activity. According to the present invention, EGFR-targeted antisense BNA oligomer delivery and gene silencing in cancer cells. A, B, C) A431 (EGFR+) treated with SO1861 or SO1861-Ald-EMCH with or without 1.5 pM EGF caryophyllin. + ), HeLa(EGFR+ ) or A2058 (EGFR - Cell viability analysis. D, E) A431 cells (EGFR) treated with SO1861 or SO1861-L-N3 (also known as SO1861-N3 or SO1861-N3 / azide) with or without 1.5 pM EGF caryophyllin. ++ Cell viability analysis of HeLa (EGFR+) cells or HeLa (EGFR+) cells.

[0147] Figure 65 Unconjugated SO1861 versus SO1861-Ald-EMCH (unstable hydrazone bond) versus SO1861-HATU (also known as SO1861-(S) (stable) and SO1861-Glu-HATU). HeLa cells (EGFR) treated with SO1861, SO1861-Glu-HATU (also known as SO1861-(S) (S=HATU) and SO1861-Ald-EMCH (the hydrazone bond between the SO1861 aglycone core and the EMCH linker is also called the "unstable linker") with or without EGF caryophyllein. + Cell viability analysis.

[0148] Detailed description

[0149] The invention will be described with reference to specific embodiments, but is not limited thereto, but is defined only by the claims.

[0150] Surprisingly, the inventors have discovered modified saponins, i.e. saponin derivatives, that have the following groups:

[0151] -A branched-chain trisaccharide moiety bound at the C-3 position of the saponin aglycone and containing a modified glucuronic acid; and / or

[0152] - An aldehyde modified at the C-4 position of the aglycone of the saponin; and / or

[0153] - A polysaccharide moiety, which is bound at the C-28 position of the aglycone of the saponin, and the polysaccharide moiety contains a modified acetyl group;

[0154] It exhibits reduced toxicity when considering cell viability of cells in contact with saponin derivatives; and activity when considering, for example, toxin cytotoxicity or enhanced BNA-mediated gene silencing (not wishing to be bound by any theory: related to similar or improved endosome escape enhancement activity of modified saponins) if one or both of the aforementioned groups of the modified saponin are derivatized (i.e., one or both of the following groups: an aldehyde group in the aglycone, a carboxyl group of glucuronic acid bound to the polysaccharide chain at C-3 of the aglycone, and an acetyl group bound to the polysaccharide chain at C-28 of the aglycone); and / or reduced hemolytic activity compared to the toxicity, activity, and hemolytic activity of unmodified saponins. Therefore, the inventors have provided saponin derivatives with improved therapeutic windows because, for the saponin derivatives, cytotoxicity is lower than that measured for their naturally occurring counterparts, hemolytic activity is lower than that measured for the naturally occurring counterparts of the saponin derivatives, and for single-derived saponins and for double-derived saponins, the ratio between cytotoxicity and, for example, toxic enhancement or gene silencing IC50 values ​​is similar or increased, and / or because the ratio between saponin hemolytic activity and, for example, toxic enhancement or gene silencing IC50 values ​​is similar or increased. An overview of exemplary saponin derivatives is provided in conjunction with... Figures 1-14 and Figures 36-40E Refer to Table A2 for an overview of cytotoxicity, hemolytic activity and endosome escape enhancement activity (“activity”), and the ratio between the IC50 of cytotoxicity and the IC50 of activity, and the ratio between the IC50 of hemolytic activity and the IC50 of activity, as determined on various cell types.

[0155] A first aspect of the present invention relates to saponin-based saponin derivatives comprising a triterpenoid aglycone core structure (also referred to as 'aglycone') and at least one of a first sugar chain and a second sugar chain connected to said aglycone core structure, wherein:

[0156] i. The saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises a derivatized aldehyde group; or

[0157] ii. The saponin derivative comprises the first sugar chain, wherein the first sugar chain comprises a derivatized carboxyl group, preferably a carboxyl group of the glucuronic acid moiety; or

[0158] iii. The saponin derivative comprises the second sugar chain, wherein the second sugar chain comprises at least one derivatized acetoxy (Me(CO)O-) group; or

[0159] iv. The saponin derivative comprises any combination of derivatizations i., ii. and iii., preferably any combination of two derivatizations i., ii. and iii.

[0160] The first sugar chain and the second sugar chain are independently selected from monosaccharides, linear oligosaccharides, and branched oligosaccharides.

[0161] One embodiment is a saponin derivative of the present invention, wherein the saponin derivative is a monosaccharide chain triterpenoid glycoside or a disaccharide chain triterpenoid glycoside, more preferably a disaccharide chain triterpenoid glycoside.

[0162] Surprisingly, modifications (derivatives) of any one, two, or three of the following: the aldehyde group at C-23 of the aglycone, the carboxyl group in the sugar moiety at C-3 of the aglycone (i.e., the carboxyl group in the glucuronic acid moiety), and the acetyl group in the sugar unit bound to the (oligosaccharide) moiety at C-28 of the aglycone, result in reduced cytotoxicity when such saponin derivatives come into contact with cells, i.e., various types of cells. The inventors have identified Tables A2 and A3 as... Figures 1-14 and Figures 36-40E The present invention describes a series of different saponin derivatives with reduced cytotoxicity. Therefore, part of the invention is to provide these series of saponin derivatives with reduced cytotoxicity, wherein the reduction in cytotoxicity is measured relative to unmodified naturally occurring saponin counterparts. The saponin derivatives can be formed from these naturally occurring saponins. Typically, the saponin derivatives of the present invention contain one, two, or three derivatizations when compared to naturally occurring counterparts such as SO1861 and QS-21 (isotypes). When considering the reduction in cytotoxicity, it is equally suitable to include one, two, or three modified (derivatively modified) saponin derivatives at the sites described above in the saponin molecule when providing cytotoxic saponins. Furthermore, the inventors have surprisingly determined that various modifications are suitable for reducing cytotoxicity, reducing hemolytic activity, and maintaining and preserving a sufficient degree of endosome escape enhancement activity. When considering hemolytic activity, similar to the reduction in cytotoxicity, hemolytic activity is reduced when one, two, or three of the specified chemical groups of the saponin are derivatized. These derivationalizations can have various properties, such as Table A2, Table A3, and Figures 1-14 and Figures 36-40E The derivatizations listed herein, as small as the derivatization of an aldehyde group to a hydroxyl group and as large as the derivatization of an aldehyde group with EMCH, along with the derivatization of the carboxyl group of glucuronic acid with AEM, all reduce cytotoxicity and hemolytic activity. Clearly, in order to provide saponin derivatives with improved cytotoxicity in terms of reduced cytotoxicity and improved hemolytic activity in terms of reduced hemolytic activity, any one or more of the three chemical groups in the saponin, for example one, two, or a wide variety of different chemical groups with different sizes and / or different chemical properties, can be derivatized compared to naturally occurring saponin counterparts.

[0163] Without being bound by any theory, it is assumed that the aldehyde group at the C-3 atom of the saponin aglycone involves and / or contributes to the enhanced endosome escape activity of the disaccharide-chain triterpenoid glycoside saponin. For example, the toxicity of such toxins increases when exposed to cells in the presence of such saponins compared to when the same dose of (protein) toxins is exposed in vitro and in vivo in the absence of such saponins. In fact, the inventors have determined that saponin derivatives having a derivatized carboxyl group in the glucuronic acid unit and / or a derivatized acetyl group in the polysaccharide chain and containing a free aldehyde group in the aglycone have enhanced endosome escape activity. These derivatives exhibit reduced hemolytic activity and reduced cytotoxicity. For example, as molecules 3A, 8, 11, 18, 19, and 28 (Tables A2, A3, A5, A6, ...), ... Figure 1 , 3 Saponin derivatives of (6, 11, 12, 39) have a free, unmodified aldehyde group in the aglycone core and do indeed exhibit activity when considering the enhanced cytotoxicity of antibody-drug conjugates (in contact with various (tumor) cells expressing receptors to which the antibodies bind). Therefore, these saponin derivatives are explicitly conceived as embodiments of the present invention.

[0164] Surprisingly, when the cytotoxicity of the effector molecule is delivered to (tumor) cells in the form of a ligand-toxin conjugate (e.g., ADC), saponin derivatives with derivatized aldehyde groups in the aglycone (so that the saponin derivative does not contain free aldehyde groups) still exhibit characteristic endosome escape-enhancing activity, provided that neither the acetyl group in the C-28 polysaccharide chain nor the carboxyl group in the C-23 polysaccharide chain is derivatized, or only one of them is. For example, see Tables A2, A3, and... Figure 2 , Figure 4 , Figure 5 , Figure 8 , Figure 9 , Figure 13 , Figure 38 and Figure 40A Molecules 6, 9, 10, 14, 15, 20, 27, and 29, shown in the table, possess modified aldehyde groups and, with or without a single further derivatized saponin derivative, have the ability to enhance the cytotoxic effects of effector molecules that contact tumor cells in the presence of such saponin derivatives having derivatized aldehyde groups in the aglycone. All of these saponin derivatives exhibit reduced cytotoxicity and reduced hemolytic activity, and are therefore explicitly contemplated as embodiments of the present invention.

[0165] The inventors have also discovered that certain modifications lead to an increase in the critical micelle concentration (CMC) compared to the corresponding unmodified saponins. For example, saponin derivatives denoted as molecules 2, 6, 8, 10, 15, 27, and 28, preferably those denoted as molecules 2, 6, 8, 10, and 15, have increased CMCs compared to their corresponding underivative saponins and are therefore explicitly conceived as embodiments of the invention. Without wishing to be bound by any theory, it is believed that the increased CMC is advantageous for several reasons. For example, the increased CMC can facilitate the use of the modified saponins in subsequent conjugation reactions, since free molecules are generally more sensitive to conjugation reactions than ordered molecules in a micelle structure. Furthermore, in cases where saponin derivatives are required to exert biological functions (e.g., in in vivo therapy or in vitro methods), such as when saponin derivatives are used as is, or even when they are released in situ after cleavage from a carrier or another entity, increased CMC is advantageous compared to unmodified saponins because free saponin molecules will interact more readily with their biological targets than if these saponin derivatives were ordered in a micellar structure. Increased CMC can also be used to facilitate the large-scale production and concentration of saponin derivatives, since at concentrations exceeding (above) the critical micelle concentration, saponins form micelles that hinder separation (e.g., using preparative HPLC). Surprisingly, for the saponin derivatives of the present invention, the observed increased CMC was not associated with increased cytotoxicity or hemolytic activity. The relationship between CMC and cytotoxicity is unpredictable and complex, as can be seen, for example, from the data in Table 2 of deGroot et al. (“Saponin interactions with model membrane systems-Langmuirmonolayer studies, hemolysis and formation of ISCOMs”, Planta medica 82.18(2016): 1496-1512), which shows that, with α-Hederin as a reference point, the increase in CMC may be associated with an increase in general cytotoxicity (as is often the case with digoxin), but it may also be associated with a decrease in cytotoxicity only (as is often the case with glycyrrhizin and hederacoside C). Furthermore, for saponin derivatives denoted as molecules 2, 6, 10, and 15, the increased CMC compared to the corresponding free saponins is also associated with an increased ratio: IC50 hemolysis / IC50 activity, making these saponin derivatives particularly preferred embodiments of the present invention.

[0166] Therefore, the inventors have provided saponin derivatives with improved therapeutic windows, taking into account cytotoxicity and / or hemolytic activity, as well as, for example, the enhancement of toxins and / or an increased CMC compared to the corresponding underived saponins. Such saponin derivatives of the present invention are particularly suitable for therapeutic regimens involving, for example, ADCs or AOCs for the prevention or treatment of cancer. The safety of these saponin derivatives is improved when considering cytotoxicity and / or hemolytic activity, particularly when administered to patients requiring treatment with, for example, ADCs or AOCs.

[0167] One embodiment is a saponin derivative according to the invention, wherein the saponin derivative comprises a first sugar chain, wherein the first sugar chain comprises a derivatized carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, and / or wherein the saponin derivative comprises a second sugar chain, wherein the second sugar chain comprises at least one derivatized acetoxy (Me(CO)O-) group (also referred to herein as a derivatized acetate group), preferably, the saponin derivative comprises both the derivatized first sugar chain and the derivatized second sugar chain, more preferably, the saponin derivative comprises both the derivatized first sugar chain and the derivatized second sugar chain, and the saponin derivative comprises an aglycone core structure containing an aldehyde group or a derivatized aldehyde group, most preferably, the saponin derivative comprises both the derivatized first sugar chain and the derivatized second sugar chain, and the saponin derivative comprises an aglycone core structure containing an aldehyde group. Also preferred are all other possible combinations of these two derivatizations, where, when considering naturally occurring saponins, one of the three chemical groups of the saponin remains unchanged. In addition, one, two, or three, preferably one or two, chemical groups of the saponin are derivatized according to any one or more derivatizations listed in Tables A2 and A3.

[0168] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative comprises an aglycone core structure selected from the following:

[0169] 2α-hydroxyoleanolic acid;

[0170] 16α-hydroxyoleanolic acid;

[0171] Ivy saponin (23-hydroxyoleanolic acid);

[0172] 16α,23-Dihydroxyoleanolic acid;

[0173] Carnation saponins;

[0174] Soapylic acid;

[0175] Proaescinogen-21(2-methylbut-2-enoate)-22-acetate;

[0176] 23-Oxo-Yaussaponin C-2l,22-bis(2-methylbut-2-enoate);

[0177] 23-Oxo-Yaussaponin C-21(2-methylbut-2-enoate)-16,22-diacetate;

[0178] Digitalis saponin glycosides;

[0179] 3,16,28-Trihydroxyolean-12-ene;

[0180] Carnation acid,

[0181] as well as

[0182] The above derivatives,

[0183] Preferably, the saponin derivative comprises an aglycone core structure selected from saponin acid and caryophyllene saponin or derivatives thereof; more preferably, the aglycone core structure of the saponin derivative is saponin acid or a derivative thereof. Since the inventors have now discovered that, based on triterpenoid glycoside saponins, saponin derivatives can provide improvements in reducing cytotoxicity and hemolysis in cells in contact with these derivatives, any saponin with such enhanced endosome escape activity tested by the inventors can be substantially improved accordingly, for example, saponins having the aglycones of the above embodiments listed in Table A1. When considering the enhancement of toxins and, for example, BNA, the reduction of toxicity and hemolytic activity while maintaining sufficiently high activity is an important achievement of the inventors when considering the expansion of the therapeutic window of saponin derivatives alone or in combination with, for example, ADCs or AOCs. Sufficiently high doses of the derivatized saponins can be applied, for example, in the treatment of tumors in cancer patients in need, with reduced risks of cytotoxic side effects and undesirable hemolytic activity exerted or induced by the saponin derivatives compared to the application of natural saponin counterparts. Improvements to the therapeutic window of the saponin derivatives of the present invention are apparent, for example, from the exemplary saponin derivatives in Tables A5 and A6, which list the ratio between the IC50 of cytotoxic or hemolytic activity and the IC50 of endosome escape-enhancing activity, as well as hemolytic activity, cytotoxicity, and activity.

[0184] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative comprises an aglycone core structure selected from the following: saponin acid, caryophyllene saponin, and their derivatives; preferably, the saponin derivative comprises an aglycone core structure selected from the following: saponin acid, and their derivatives, wherein the first sugar chain, when present, is associated with the C3 atom (also referred to as 'C-3' atom) or C... 28 Atoms (also referred to as 'C-28' atoms), preferably linked to C3 atoms, and / or wherein the second sugar chain, when present, is linked to the C3 atom of the aglycone core structure.28 Atomic linkage. Preferred are those saponin derivatives based on saponins having two sugar chains bound to the aglycone, but generally any saponin exhibiting enhanced endosome escape activity is suitable for derivatization according to the invention, with the aim of providing single, double, or triple, preferably single or double, derivatized saponins with lower cytotoxicity, lower hemolytic activity, and sufficiently high enhanced endosome escape activity.

[0185] One embodiment is a saponin derivative according to the present invention, wherein the first sugar chain, if present, is selected from (list S1):

[0186] GlcA-、

[0187] Glc-、

[0188] Gal-、

[0189] Rha-(1→2)-Ara-、

[0190] Gal-(1→2)-[Xyl-(1→3)]-GlcA-,

[0191] Glc-(1→2)-[Glc-(1→4)]-GlcA-,

[0192] Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-,

[0193] Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-,

[0194] Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-,

[0195] Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-,

[0196] Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-,

[0197] Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-,

[0198] Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-,

[0199] Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-,

[0200] Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-,

[0201] Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-,

[0202] Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-,

[0203] Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-,

[0204] Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-,

[0205] Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-,

[0206] Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-,

[0207] Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-,

[0208] Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-,

[0209] Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-,

[0210] Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-,

[0211] Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and its derivatives,

[0212] And / or the second sugar chain thereon, if present, selected from (List S2):

[0213] Glc-、

[0214] Gal-、

[0215] Rha-(1→2)-[Xyl-(1→4)]-Rha-,

[0216] Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-,

[0217] Ara-、

[0218] Xyl-、

[0219] Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc- where R1 is 4E-methoxycinnamic acid,

[0220] Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc- where R2 is 4Z-methoxycinnamic acid,

[0221] Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-、

[0222] Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-,

[0223] Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc- where R3 is 4E-methoxycinnamic acid,

[0224] Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-,

[0225] Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-、

[0226] (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-),

[0227] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-,

[0228] Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-,

[0229] Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-,

[0230] Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-,

[0231] Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-,

[0232] Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc- where R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid).

[0233] Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc-where R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid),

[0234] Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-,

[0235] Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-,

[0236] 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-,

[0237] Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-,

[0238] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-,

[0239] Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-,

[0240] Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-,

[0241] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3, 4-di-OAc-Qui-(1→4)]-Fuc-,

[0242] Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-,

[0243] 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-,

[0244] Glc-(1→3)-[Xyl-(1→3)-Xy1-(1→4)]-Rha-(1→2)-Fuc-,

[0245] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-,

[0246] Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-,

[0247] Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-,

[0248] Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid),

[0249] Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid).

[0250] Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- where R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid).

[0251] Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc-where R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid),

[0252] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc-where R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid),

[0253] Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc-where R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid),

[0254] Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid)

[0255] Glc-(1→3)-[Glc-(1→6)]-Gal-, and its derivatives.

[0256] Typically, when cells come into contact with saponins and toxins, saponins that enhance the cytotoxicity of the toxins have one or two such monosaccharide or polysaccharide chains bound to an aglycone. Preferred are those saponins containing two sugar chains selected for derivatization. Table A1 provides an overview of particularly preferred saponins that are mono-, di-, or tri-derivatively derived, preferably mono- or di-derivative, when the endosome escape-enhancing activity should be maintained to a sufficiently high level. Of course, structural variations of such saponins are equally applicable to the derivatization according to the invention if the saponin exhibits endosome escape-enhancing activity against, for example, toxins, BNA, etc.

[0257] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative comprises the first sugar chain and the second sugar chain, wherein the first sugar chain comprises one or more sugar moieties and the second sugar chain comprises one or more sugar moieties, and wherein the aglycone core structure is saponin acid or caryophyllene saponin, wherein one, two, or three of the following are preferred, preferably one or two:

[0258] i. The aldehyde group in the core structure of the aglycone has been derivatized.

[0259] ii. The first sugar chain contains a carboxyl group of a derivatized glucuronic acid moiety, and

[0260] iii. The second sugar chain contains at least one derivatized acetoxy (Me(CO)O-) group.

[0261] The following overview illustrates appropriate derivatization:

[0262]

[0263] According to the present invention, saponins can contain three derivatizations and still exhibit sufficiently high endosome escape-enhancing activity. Specifically, when the reduction in cytotoxicity and / or hemolytic activity is greater than the (potential or apparent) reduction in the ability to enhance the action and activity of the effector molecule within cells, such as toxins or BNAs in tumor cells in contact with the effector molecule and the derivatized saponin, the present invention provides saponins containing one, two, or three derivatizations, taking into account the aldehyde group of the aglycone, the carboxyl group (if present) in the glucuronic acid unit of the polysaccharide at C-3, and the acetyl group (if present) in the polysaccharide chain at C-28. Saponin derivatives having one or two modifications are preferred. Saponin derivatives, for example, having a free aldehyde group and one or two derivatizations in the sugar chain are suitable for improving endosome escape of effector molecules such as toxins or BNAs. As previously mentioned, saponin derivatives having a derivatized aldehyde group are also suitable. Such saponin derivatives, which do not possess a free aldehyde group in the aglycone upon derivatization, still exhibit sufficient and effective endosome escape-enhancing activity. Without being bound by any theory, due to the acidic conditions in endosomes and lysosomes (mammalian) cells such as human cells, the aldehyde group can be re-formed intracellularly when the portion initially bound to the saponin is pH-driven cleaved, providing a saponin derivative with a derivatized aglycone at C-23. Examples of saponin derivatives with modified aldehyde groups that can be re-formed in endosomes or lysosomes are saponin derivatives containing hydrazone bonds, which are formed between the carbonyl group of the aldehyde and, for example, the hydrazide moiety in a chemical group bound to the aglycone (e.g., N-ε-maleimide hexanoic acid hydrazide (EMCH)), or EMCH and mercaptoethanol bound to the maleimide group to form a thioether bond. Examples of such saponin derivatives are... Figure 8 and Figure 9 Provided in, and shown below as molecule 2 and molecule 3.

[0264] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative is a derivative of a saponin selected from the following: bark saponin, dipsacus saponin B, bupleurum saponin A, bupleurum saponin D, honeysuckle saponin A, phytolacca saponin A, phytolacca saponin genistein, aesculin, AS6.2, NP-005236, AMA-1, AMR, α-Hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AEX 55. NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, SO1861, GE17 41. SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, SO1862, QS-7, QS1861, QS-7api, QS1862, QS-17, QS-18, QS-21A-apio, QS-21A-xylo, QS-21 B-apio, QS-21 B-xylo, β-Aescin, Aescin Ia, tea seed saponin I, tea seed saponin J, assam saponin F, digitalis saponin, primrose acid 1, and AS64R, stereoisomers of the above and combinations thereof, preferably, the saponin derivatives are selected from QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, and GE1741 derivatives; more preferably, the saponin derivatives are selected from QS-21 derivatives and SO1861 derivatives; most preferably, the saponin derivatives are SO1861 derivatives. These saponins are essentially saponins exhibiting enhanced endosome escape activity as determined by the inventors, or are structurally highly similar to saponins with already determined enhanced endosome escape activity. A structural summary of these saponins is summarized in Table A1.

[0265] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative is a derivative of saponin or caryophyllein represented by molecule 1:

[0266]

[0267] in

[0268] The first sugar chain A1 represents a hydrogen, monosaccharide, or straight-chain or branched oligosaccharide. Preferably, A1 represents a sugar chain as defined above for certain embodiments of the present invention (list S1). More preferably, A1 represents a sugar chain as defined above for certain embodiments of the present invention (list S1), and A1 contains or is composed of a glucuronic acid portion.

[0269] The second sugar chain A2 represents hydrogen, a monosaccharide, or a straight-chain or branched oligosaccharide. Preferably, A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2). More preferably, A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2). A2 contains at least one acetoxy (Me(CO)O-) group, such as one, two, three, or four acetoxy groups, preferably one. At least one of A1 and A2 is not hydrogen. Preferably, both A1 and A2 are oligosaccharide chains.

[0270] Furthermore, R is either a hydrogen aglycone in carnation saponin or a hydroxyl group in saponin acid;

[0271] The saponin derivatives thereon correspond to the saponin represented by molecule 1, wherein at least one of the following derivatizations is present:

[0272] i. C of saponin acid or carnation saponin 23 The aldehyde group at the position has been derivatized;

[0273] ii. When A1 represents a sugar chain as defined above for certain embodiments of the invention (list S1) and A1 comprises or is composed of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 has been derivatized; and

[0274] iii. When A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2) and A2 contains at least one acetoxy group, one or more (preferably all) acetoxy groups of one or more sugar moieties of A2 have been derivatized.

[0275] One embodiment is a saponin derivative according to the invention, wherein A1 represents a sugar chain as defined above for certain embodiments of the invention (list S1) and comprises or is composed of a glucuronic acid moiety, and wherein the carboxyl group of the glucuronic acid moiety of A1 has been derivatized and / or wherein A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2), and A2 comprises at least one acetoxy group, and wherein at least one acetoxy group of A2 has been derivatized.

[0276] One embodiment is a saponin derivative according to the present invention, wherein the saponin represented by molecule 1 is a disaccharide-chain triterpenoid saponin.

[0277] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative corresponds to a saponin represented by molecule 1, and wherein at least one of the following derivatizations is present, preferably one or two of the following derivatizations, more preferably one:

[0278] i. C of saponin acid or carnation saponin 23 The aldehyde group at the position has been derivatized through the following methods;

[0279] - Reduced to alcohol;

[0280] - Converted into hydrazone bonds, preferably through reaction with acylhydrazide;

[0281] ii. When A1 represents a sugar chain as defined above for certain embodiments of the invention (list S1) and A1 comprises or is composed of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 has been derivatized by conversion to an amide bond, preferably by reaction with an amine; and

[0282] iii. When A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2) and A2 contains at least one acetoxy group, one or more of the sugar moieties of A2, preferably all of the acetoxy groups, have been derivatized to hydroxyl groups (HO-) by deacetylation.

[0283] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative corresponds to a saponin represented by molecule 1, and wherein at least one of the following derivatizations is present, preferably one or two of the following derivatizations, more preferably one:

[0284] i. C of saponin acid or carnation saponin 23 The aldehyde group at the position has been derivatized through the following methods;

[0285] - Reduced to alcohol;

[0286] - Saponins are provided by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form hydrazone bonds, such as SO1861-Ald-EMCH or QS-21-Ald-EMCH, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol.

[0287] - Converted to a hydrazone bond via reaction with N-[β-maleimide propionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by forming a thioether bond with mercaptoethanol; or

[0288] - It is converted to a hydrazone bond by reacting with N-[κ-maleimide undecanoic acid] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derived by forming a thioether bond with mercaptoethanol;

[0289] ii. When A1 represents a sugar chain as defined above for certain embodiments of the invention (list S1) and A1 comprises or is composed of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 has been derivatized into an amide bond by reacting with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby providing saponin-Glu-AMPD such as QS-21-Glu-AMPD or SO1861-Glu-AMPD or saponin-Glu-AEM such as QS-21-Glu-AEM or SO1861-Glu-AEM; and

[0290] iii. When A2 represents a sugar chain as defined above for certain embodiments of the invention (list S2) and A2 contains at least one acetoxy group, one or more of the sugar moieties of A2, preferably all of the acetoxy groups, have been derivatized to hydroxyl groups (HO-) by deacetylation.

[0291] One embodiment is a saponin derivative according to the present invention, wherein A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA and / or A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc, preferably, the saponin represented by molecule 1 is 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylanopyranosyl-(1→2)-[1→2]-[Xyl-D-xylanopyranosyl-(1→2) ... 3)]-β-D-glucuronyl pyranoside 28-O-β-D-glucuronyl-(1→3)-β-D-xylopyranyl-(1→4)-α-L-rhamnopyranyl-(1→2)-[β-D-xylopyranyl-(1→3)-4OAc-β-D-quinolyl-(1→4)]-β-D-fucoside, more preferably SO1861, GE1741, SA1641 and / or QS-21, most preferably SO1861.

[0292] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative is selected from the following derivatives: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, carnation saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, stereoisomers thereof, and combinations thereof. Preferably, the saponin derivative is selected from SO1861 derivative, GE1741 derivative, SA1641 derivative, QS-21 derivative, and combinations thereof. More preferably, the saponin derivative is SO1861 derivative or QS21 derivative. Most preferably, the saponin derivative is SO1861 derivative.

[0293] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative is a SO1861 derivative comprising a single derivatization, wherein the single derivatization is a conversion of the carboxyl group of the glucuronic acid moiety of SO1861, for example by incorporating 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridonium 3-oxide hexafluorophosphate (HATU) to the carboxyl group of the glucuronic acid moiety of SO1861 (see [link to previous embodiment]). Figure 59 ), or by incorporating (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) to the carboxyl group of the glucuronic acid moiety of SO1861, or wherein the saponin derivative is an SO1861 derivative represented by molecule 2, which indicates the C-position of the saponin aglycone core structure. 23 SO1861 derivatives containing an aldehyde group, which has been derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond:

[0294]

[0295] Alternatively, the saponin derivative described herein is an SO1861 derivative represented by molecule 3, which indicates the C-position of the saponin aglycone core structure. 23The SO1861 derivative contains an aldehyde group, which has been derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond, wherein the maleimide group of the EMCH is derivatized with mercaptoethanol, thereby forming a thioether bond:

[0296]

[0297] The saponin represented by molecule 2 is suitable as a precursor for conjugation reactions with another molecule containing a free thiol group. The maleimide group of the saponin derivative shown as molecule 2 can form a thioether bond with such a free thiol group. For example, the saponin derivative of molecule 2 can be covalently coupled to a peptide or protein containing a free thiol group, such as cysteine ​​with a free thiol group. Such proteins are, for example, antibodies or their binding fragments or binding domains, such as Fab, scFv, and single-domain antibodies, such as V. HH For example, camel family V H The use of the saponin derivative of molecule 2 in coupling reactions with antibodies, for example, those containing free thiol groups, provides a conjugate for targeted delivery of saponins into cells and intracellularly when the antibody (or its binding domain or fragment) is an antibody that specifically binds to target cell surface molecules (e.g., receptors present on tumor cells). Preferably, the saponin derivative is coupled with an antibody or V capable of binding to tumor cell-specific surface molecules (e.g., receptors, such as HER2, EGFR, CD71). HH Couplet.

[0298] One embodiment is a saponin derivative according to the present invention, provided that the saponin derivative does not comprise a single derivatized SO1861 derivative, wherein the single derivatization is achieved by reacting 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridonium 3-oxide hexafluorophosphate (HATU) with the carboxyl group of the glucuronic acid of SO1861 to convert the carboxyl group of SO1861, or wherein the saponin derivative is an SO1861 derivative represented by molecule 2, which indicates the C-position of the saponin acid aglycone core structure. 23 The SO1861 derivative contains an aldehyde group, which has been converted into a hydrazone bond by reacting with N-ε-maleimide hexanoylhydrazide (EMCH):

[0299]

[0300] One embodiment is a saponin derivative according to the present invention, wherein...

[0301] i. The saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by:

[0302] - Reduced to alcohol;

[0303] - Converted to a hydrazone bond by reacting with N-ε-maleimide hexanoylhydrazide (EMCH), wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol;

[0304] - Converted to a hydrazone bond via reaction with N-[β-maleimide propionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by forming a thioether bond with mercaptoethanol; or

[0305] - It is converted to a hydrazone bond by reacting with N-[κ-maleimide undecanoic acid] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

[0306] ii. The first sugar chain contains a carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, which is derivatized by reacting with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM) to form an amide bond;

[0307] iii. The second sugar chain contains an acetoxy group (Me(CO)O-), which has been derivatized to a hydroxyl group (HO-) by deacetylation; or

[0308] iv. The saponin derivative comprises any combination of two or three derivatizations i., ii. and iii., preferably any combination of two derivatizations i., ii. and iii.

[0309] Preferably, the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by reacting with EMCH to form a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol.

[0310] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the first sugar chain comprises a carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, which has been derivatized by reacting with N-(2-aminoethyl)maleimide (AEM) to form an amide bond.

[0311] One embodiment is a saponin derivative according to the present invention, wherein when the aldehyde group in the aglycone core structure is derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond and the saponin is SO1861, at least one of glucuronic acid and acetoxy group (Me(CO)O-) is also derivatized, and when the saponin is SO1861 and the carboxyl group of the glucuronic acid moiety of SO1861 is derivatized by reacting 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) with the carboxyl group of the glucuronic acid moiety of SO1861, at least one of the aldehyde group and acetoxy group (Me(CO)O-) is also modified.

[0312] One embodiment is a saponin derivative according to the invention, wherein when the aldehyde group in the aglycone core structure of the saponin derivative is derivatized by reaction with EMCH and the saponin is SO1861, at least one of glucuronic acid and acetoxy group (Me(CO)O-) is also derivatized, and when the saponin is SO1861 and the carboxyl group of the glucuronic acid moiety of SO1861 is derivatized by binding HATU, at least one of the aldehyde group and acetoxy group (Me(CO)O-) is also derivatized.

[0313] The embodiment referred to herein as Embodiment D1 is a saponin derivative according to the present invention, characterized in that the saponin derivative is not SA1641, wherein the aglycone core structure comprises an aldehyde group, the aldehyde group having been derivatized by reacting with a compound of formula (A1) to an amine (e.g., by reductive amination):

[0314]

[0315] In other words, the embodiment referred to herein as Embodiment D1 is a saponin derivative according to the invention, characterized in that it is not the result of a reaction between SA1641 and a compound of formula (A1). Therefore, Embodiment D1 is a saponin derivative according to the invention, characterized in that it is not the result of coupling via the reductive amination of at least one SA1641 molecule to a compound of formula (A1).

[0316] The embodiment referred to herein as Embodiment D2 is a saponin derivative according to the invention, characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group, the aldehyde group being derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form an hydrazone bond, wherein the maleimide group of the EMCH is optionally derivatized by forming a thioether bond with a thiol, and wherein no other derivatization is present on the saponin, preferably characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form an hydrazone bond, wherein the maleimide group of the EMCH is optionally derivatized by forming a thioether bond with a thiol.

[0317] The embodiment referred to herein as Embodiment D3 is a saponin derivative according to the invention, characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group, the aldehyde group being derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond, wherein the maleimide group of the EMCH is derivatized by forming a thioether bond with a thiol selected from one, preferably all of the following:

[0318] ·Mercaptoethanol,

[0319] • A poly(amide) dendritic polymer having an ethylenediamine core, wherein the ethylenediamine core has been derivatized with at least 2-iminothiophene.

[0320] A conjugate of ·-anthocyanin-3 and a poly(amidoamine) dendritic polymer having an ethylenediamine core, wherein the ethylenediamine core is further derivatized with at least 2-iminothiophene.

[0321] • G4 dendrite, which has been derivatized with at least 2-iminothiophene.

[0322] • A conjugate of anthocyanin-5 and G4 dendritic molecules, wherein the G4 dendritic molecules are further derivatized with at least 2-iminothiophene.

[0323] Bovine serum albumin (BSA), and

[0324] • A peptide having the sequence SESDDAMFCDAMDESDSK[SEQ ID NO: 1].

[0325] Furthermore, there are no other derivatizations on the saponins. As those skilled in the art will understand, the expression "G4 dendritic molecule" should be interpreted as referring to the compound of formula (A2):

[0326]

[0327] The embodiment referred to herein as Embodiment D4 is a saponin derivative according to the invention, characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the carboxyl group is derivatized into an amide by reacting with an anthocyanin-3 and a poly(amide) dendritic polymer having an ethylenediamine core, optionally further derivatized. Furthermore, no other derivatization occurs on the saponin. Preferably, the saponin derivative is not a saponin, particularly SO1861, wherein the carboxyl group is derivatized into an amide by reacting with an anthocyanin-3 and a poly(amide) dendritic polymer having an ethylenediamine core, optionally further derivatized.

[0328] The embodiment referred to herein as embodiment D5 is a saponin derivative according to the invention, characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group, the aldehyde group having been derivatized to an amine by conversion (e.g., by reductive amination) through a conjugate reaction with anthocyanin-3 and a poly(amide) dendritic polymer having an ethylenediamine core, and wherein no other derivatization is present on the saponin, preferably characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group, the aldehyde group having been derivatized to an amine by conversion (e.g., by reductive amination) through a conjugate reaction with anthocyanin-3 and a poly(amide) dendritic polymer having an ethylenediamine core.

[0329] The embodiment referred to herein as embodiment D6 is a saponin derivative according to the present invention, characterized in that the saponin derivative does not contain toxins, microRNAs or polynucleotides encoding proteins, preferably characterized in that the saponin derivative does not contain pharmaceutically active substances, such as toxins, drugs, polypeptides and / or polynucleotides, more preferably characterized in that the saponin derivative does not contain effector molecules.

[0330] The embodiment referred to herein as embodiment D7 is a saponin derivative according to the present invention, characterized in that the saponin derivative does not contain a polymeric or oligomeric structure selected from the following:

[0331] • Poly(amine) or oligo(amine), such as polyethyleneimine and poly(amidoamine),

[0332] Polyethylene glycol,

[0333] • Poly(ester) or oligo(ester), such as poly(lactide),

[0334] Poly(lactam),

[0335] • Poly(lactic acid)-co-lactic acid copolymer

[0336] • Polysaccharides or oligosaccharides, such as cyclodextrins and polydextroses,

[0337] Poly(amino acids) or oligo(amino acids), such as proteins and peptides, and

[0338] Nucleic acids and their analogues, such as DNA, RNA, LNA (locked nucleic acid), and PNA (peptide nucleic acid);

[0339] Preferably, the saponin derivative does not contain a polymeric or oligomeric structure, wherein the polymeric or oligomeric structure is a structurally ordered form, such as a polymer, oligomer, dendritic polymer, dendritic oligomer, or it is an assembled polymeric structure, such as a hydrogel, microgel, nanogel, stable polymeric micelle, or liposome. More preferably, the saponin derivative does not contain a polymeric or oligomeric structure.

[0340] The embodiment referred to herein as embodiment D8 is a saponin derivative according to the present invention, characterized in that the saponin derivative does not contain a molecular structure consisting mainly or entirely of at least two identical or similar bonded units.

[0341] The embodiment referred to herein as Embodiment D9 is a saponin derivative according to the present invention, characterized in that the saponin derivative is not a compound of formula (A3), but is a reaction product of SO1861 and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethylammonium hexafluorophosphate N-oxide (HATU):

[0342]

[0343] Preferably, the saponin derivative is not an activated ester. See also Figure 59 .

[0344] The embodiment referred to herein as embodiment D10 is a saponin derivative according to the invention, characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the carboxyl group is derivatized by conversion to an amide bond or an ester bond, and wherein no other derivatization is present on the saponin, preferably characterized in that the saponin derivative is not a saponin, particularly SO1861, wherein the carboxyl group is derivatized by conversion to an amide bond or an ester bond.

[0345] The embodiment referred to herein as embodiment D11 is a saponin derivative according to the present invention, characterized in that the saponin derivative does not contain the caryophyllin portion.

[0346] The preferred embodiment referred to herein as embodiment D12 is a saponin derivative according to the present invention, characterized in that the saponin derivative comprises a single saponin moiety.

[0347] The preferred embodiment referred to herein as embodiment D13 is a saponin derivative according to the present invention, characterized in that the saponin derivative has a molecular weight of less than 2500 g / mol, preferably less than 2300 g / mol, and more preferably less than 2150 g / mol.

[0348] The preferred embodiment referred to herein as Embodiment D14 is a saponin derivative according to the invention, characterized in that the molecular weight of the saponin derivatization is less than 400 g / mol, preferably less than 300 g / mol, and more preferably less than 270 g / mol. The molecular weight of the saponin derivatization corresponds to the molecular weight of a saponin derivative excluding the aglycone core and one (for monosaccharide aglycones) or two (for disaccharide aglycones) glycosyl (glycan) chains. Those skilled in the art will understand that when the saponin derivative has a lower molecular weight than its corresponding underivatized saponin (e.g., this is generally the case for SO1861-Ac-OH derivatized by deacetylation of SO1861), saponin derivatization does not result in any increase in molecular weight, thus meeting the requirement of the saponin derivatization having a molecular weight of less than 400 g / mol, preferably less than 300 g / mol, and more preferably less than 270 g / mol as in Embodiment D14.

[0349] As those skilled in the art will understand, embodiments D1-D14 can be combined with each other, as well as with other embodiments described in this application. For example, in embodiments of the present invention, the following combinations of embodiments D1-D14 are provided:

[0350] • D12 and one or more of D1-D11 and D13;

[0351] • D13 and one or more of D1-D12;

[0352] • One or more of D12, D13, and D1-D11;

[0353] • D1, D2, D10, and D12;

[0354] • D1, D3, D7, D9 and preferably D13; or

[0355] • D3, D9, D12 and D13.

[0356] Those skilled in the art will understand that these combinations of embodiments D1-D14 can be combined again with, for example, other embodiments according to the invention, and preferably with embodiment D14.

[0357] Particularly preferred embodiments correspond to embodiments D3, D9, D12, and one or a combination of D13 and D14. In other words, particularly preferred embodiments are saponin derivatives according to the invention, wherein the saponin derivative comprises a single saponin moiety, wherein the molecular weight of the saponin derivative is less than 2500 g / mol, preferably less than 2300 g / mol, more preferably less than 2150 g / mol, and wherein the saponin derivative…

[0358] • Not a saponin, particularly SO1861, wherein the aglycone core structure comprises an aldehyde group, which is derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol, and wherein preferably no other derivatization is present on the saponin; and

[0359] It is not an activated ester; it is a reaction product of SO1861 and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridin-1-ylmethylene]-N-methylmethylammonium hexafluorophosphate N-oxide (HATU).

[0360] A second aspect of the invention relates to a first pharmaceutical composition comprising a saponin derivative according to the invention and optionally a pharmaceutically acceptable excipient and / or diluent.

[0361] One embodiment is a first pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention, preferably a pharmaceutically acceptable diluent, and further comprising:

[0362] • Pharmaceutically acceptable salts, preferably pharmaceutically acceptable inorganic salts, such as ammonium, calcium, copper, iron, magnesium, manganese, potassium, sodium, strontium, or zinc salts, preferably NaCl; and / or

[0363] • Pharmaceutically acceptable buffer systems, such as those containing phosphates, borates, citrates, carbonates, histidines, lactates, tromethorphan, gluconates, aspartates, glutamates, tartrates, succinates, malates, fumarates, acetates, and / or ketoglutarates.

[0364] One embodiment is a first pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is a liquid at a temperature of 25°C and has a pH in the range of 2-11, preferably in the range of 4-9, more preferably in the range of 6-8.

[0365] One embodiment is a first pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is liquid at a temperature of 25°C, and wherein the concentration of the saponin derivative is 10. -12 In the range of up to 1 mol / L, preferably in the range of 10 -9 In the range of up to 0.1 mol / L, more preferably in the range of 10 -6 Within the range of 0.1 mol / L.

[0366] Typically, such a first pharmaceutical composition is suitable for use in combination with, for example, an ADC or an AOC. For example, the first pharmaceutical composition may be administered to a patient requiring ADC or AOC therapy before, together with, or shortly after (after) administration of, an ADC or AOC to a patient requiring such ADC or AOC therapy. For example, the first pharmaceutical composition may be mixed with a pharmaceutical composition containing an ADC or AOC, and an appropriate dose of the resulting mixture may be administered to a patient requiring ADC or AOC therapy. According to the invention, when the saponin derivative and the ADC or AOC are co-localized within target cells such as tumor cells, the saponin derivative contained in the first pharmaceutical composition enhances the efficacy and potency of the effector molecules contained in the ADC or AOC. Compared to contacting the same cells with the same dose of ADC or AOC in the absence of the saponin derivative, the effector molecules are released into the cytosol of the target cells to a greater extent under the influence of the saponin derivative. Therefore, when the effector molecule is co-localized with the saponin derivative of the first pharmaceutical composition within the target cell, similar efficacy can be achieved at a lower dose of ADC or AOC compared to the dose required to achieve the same efficacy in cells where the saponin derivative is absent when delivering the effector molecule containing the ADC or AOC.

[0367] One embodiment is the first pharmaceutical composition of the present invention, wherein the saponin derivative is a saponin derivative represented by molecule 2:

[0368]

[0369] Or a SO1861 derivative comprising a single derivatization, wherein the single derivatization is achieved by reacting 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) with the carboxyl group of the glucuronic acid moiety of SO1861 to convert the carboxyl group of SO1861, or the saponin derivative is a saponin derivative represented by molecule 3:

[0370]

[0371] A third aspect of the present invention relates to a pharmaceutical combination comprising:

[0372] The first pharmaceutical composition of the present invention; and

[0373] The second pharmaceutical composition comprises any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate, and optionally comprises a pharmaceutically acceptable excipient and / or diluent.

[0374] A fourth aspect of the invention relates to a third pharmaceutical composition comprising the saponin derivative of the invention and further comprising any one or more of the following: antibody-toxin conjugate, receptor-ligand-toxin conjugate, antibody-drug conjugate, receptor-ligand-drug conjugate, antibody-nucleic acid conjugate, or receptor-ligand-nucleic acid conjugate, and optionally comprising a pharmaceutically acceptable excipient and / or diluent.

[0375] One embodiment is the pharmaceutical composition of the present invention or the third pharmaceutical composition of the present invention, wherein the second pharmaceutical composition or the third pharmaceutical composition comprises any one or more of an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate, wherein the drug is, for example, a toxin, such as saponin and caryophyllein, and wherein the oligonucleotide is, for example, siRNA or BNA, which, for example, is used for gene silencing of apolipoprotein B or HSP27.

[0376] One embodiment is the pharmaceutical composition of the present invention or the third pharmaceutical composition of the present invention, wherein the saponin derivative is a saponin derivative selected from the following derivatives: 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, Gypl, carnation saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, stereoisomers thereof, and combinations thereof. Preferably, the saponin derivative is selected from SO1861 derivative, GE1741 derivative, SA1641 derivative, QS-21 derivative, and combinations thereof. More preferably, the saponin derivative is an SO1861 derivative or a QS21 derivative. Most preferably, the saponin derivative is an SO1861 derivative. Even more preferably, the saponin derivative is a saponin derivative represented by molecule 2 or molecule 3.

[0377] One embodiment is a third pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention, preferably a pharmaceutically acceptable diluent, and further comprising:

[0378] • Pharmaceutically acceptable salts, preferably pharmaceutically acceptable inorganic salts, such as ammonium, calcium, copper, iron, magnesium, manganese, potassium, sodium, strontium, or zinc salts, preferably NaCl; and / or

[0379] • Pharmaceutically acceptable buffer systems, such as those containing phosphates, borates, citrates, carbonates, histidines, lactates, tromethorphan, gluconates, aspartates, glutamates, tartrates, succinates, malates, fumarates, acetates, and / or ketoglutarates.

[0380] One embodiment is a third pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is a liquid at a temperature of 25°C and has a pH in the range of 2-11, preferably in the range of 4-9, more preferably in the range of 6-8.

[0381] One embodiment is a third pharmaceutical composition according to the invention, comprising a saponin derivative according to the invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is liquid at a temperature of 25°C, and wherein the concentration of the saponin derivative is 10.-12 In the range of up to 1 mol / L, preferably in the range of 10 -9 In the range of up to 0.1 mol / L, more preferably in the range of 10 -6 Within the range of 0.1 mol / L.

[0382] The fifth aspect of the invention relates to a first pharmaceutical composition, a pharmaceutical combination, or a third pharmaceutical composition of the invention used as a medicament. In a preferred embodiment, a first pharmaceutical composition of the invention is provided for use as a medicament, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU; a pharmaceutical combination of the invention is provided for use as a medicament, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU; or a third pharmaceutical composition of the invention is provided for use as a medicament, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU.

[0383] In another aspect of the invention, saponin derivatives described herein are provided for use as pharmaceuticals, preferably SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU.

[0384] The sixth aspect of the invention relates to a first pharmaceutical composition, a pharmaceutical combination, or a third pharmaceutical composition of the invention for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, α-1 antitrypsin-associated liver disease, acute hepatic porphyria, thyroxine-mediated amyloidosis, or autoimmune diseases. In a preferred embodiment, the first pharmaceutical composition of the invention is provided, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU. A pharmaceutical combination of the invention is provided, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU. Preferably composed of, or provided with, the third pharmaceutical composition of the present invention, wherein the saponin derivative comprises SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3 or SO1861-Glu-HATU, preferably composed of, for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, α-1 antitrypsin-associated liver disease, acute hepatic porphyria, thyroxine-mediated amyloidosis or autoimmune diseases.

[0385] A seventh aspect of the invention relates to an in vitro or ex vivo method for transferring molecules from outside a cell to inside the cell, preferably to a cytosol of the cell, the method comprising the steps of:

[0386] a) Provide cells;

[0387] b) Provide molecules for transfer from outside the cell to the cells provided in step a);

[0388] c) Provide a saponin derivative according to the invention;

[0389] d) Contact the cells of step a) in vitro or in vitro with the molecules of step b) and the saponin derivatives of step c) to establish the transfer of the molecules from the outside of the cells into the cells.

[0390] One embodiment is the method of the present invention, wherein the cell is a human cell, such as a T cell, NK cell, or tumor cell, and / or wherein the molecule in step b) is any of the following: an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate, wherein the drug is, for example, a toxin, and wherein the oligonucleotide is, for example, siRNA or BNA, and / or wherein the saponin derivative is a derivative selected from the following: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21B-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, carnation saponin A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, stereoisomers thereof, and combinations thereof. Preferably, the saponin derivative is selected from SO1861 derivative, GE1741 derivative, SA1641 derivative, QS-21 derivative, and combinations thereof. More preferably, the saponin derivative is an SO1861 derivative or a QS21 derivative. Most preferably, the saponin derivative is an SO1861 derivative; or the saponin derivative is a single-derived SO1. The 861 derivative, wherein the single derivatization is achieved by reacting with an amine, for example by attaching 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridonium 3-oxide hexafluorophosphate (HATU) to the carboxyl group of the glucuronic acid moiety of SO1861, or by attaching (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) to the carboxyl group of the glucuronic acid moiety of SO1861, to convert the carboxyl group of the glucuronic acid moiety of SO1861 into an amide bond, or wherein the saponin derivative is an SO1861 derivative represented by molecule 2, which indicates the C-position of the saponin aglycone core structure. 23 SO1861 derivatives containing an aldehyde group, which has been derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond:

[0391]

[0392] Alternatively, the saponin derivative described herein is an SO1861 derivative represented by molecule 3, which indicates the C-position of the saponin aglycone core structure. 23 The SO1861 derivative contains an aldehyde group, which has been derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond, wherein the maleimide group of the EMCH is derivatized with mercaptoethanol to form a thioether bond.

[0393]

[0394] Or the saponin derivative is a derivative provided that it does not comprise a single derivatized SO1861 derivative, wherein the single derivatization is achieved by reacting 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridonium 3-oxide hexafluorophosphate (HATU) with the carboxyl group of the glucuronic acid of SO1861 to convert the carboxyl group of the glucuronic acid of SO1861, or wherein the saponin derivative is an SO1861 derivative represented by molecule 2, which indicates the C-position of the core structure of the saponin acid aglycone. 23 The SO1861 derivative contains an aldehyde group, which has been converted into a hydrazone bond by reacting with N-ε-maleimide hexanoylhydrazide (EMCH):

[0395]

[0396] ; or the saponin derivative therein is a derivative, wherein

[0397] i. The saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by:

[0398] - Reduced to alcohol;

[0399] - Converted to a hydrazone bond by reacting with N-ε-maleimide hexanoylhydrazide (EMCH), wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol;

[0400] - Converted to a hydrazone bond via reaction with N-[β-maleimide propionic acid]hydrazide (BMPH), wherein the maleimide group of BMPH is optionally derivatized by forming a thioether bond with mercaptoethanol; or

[0401] - It is converted to a hydrazone bond by reacting with N-[κ-maleimide undecanoic acid] hydrazide (KMUH), wherein the maleimide group of KMUH is optionally derivatized by forming a thioether bond with mercaptoethanol;

[0402] ii. The first sugar chain contains a carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, which is derivatized by reacting with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM) to form an amide bond;

[0403] iii. The second sugar chain contains an acetoxy group (Me(CO)O-), which has been derivatized to a hydroxyl group (HO-) by deacetylation; or

[0404] iv. The saponin derivative comprises any combination of two or three derivatizations i., ii. and iii., preferably any combination of two derivatizations i., ii. and iii.

[0405] Preferably, the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group that has been derivatized by reacting with EMCH to form a hydrazone bond, wherein the maleimide group of EMCH is optionally derivatized by forming a thioether bond with mercaptoethanol; or wherein the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure comprises an aldehyde group, and wherein the first sugar chain comprises a carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, wherein the carboxyl group has been derivatized by reacting with N-(2-aminoethyl)maleimide (AEM) to form an amide bond; or wherein the saponin derivative is a derivative provided that when the aldehyde group in the aglycone core structure is derivatized by reacting with N-ε-maleimide hexanoylhydrazide (EMCH) to form a hydrazone bond and the saponin is SO1861, at least one of the glucuronic acid and the acetoxy group (Me(CO)O-) is also derivatized, and the condition is met. The condition is that when the saponin is SO1861 and the carboxyl group of the glucuronic acid moiety of SO1861 is derivatized by reacting 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridonium 3-oxide hexafluorophosphate (HATU) with the carboxyl group of the glucuronic acid of SO1861, at least one of the aldehyde group and the acetoxy group (Me(CO)O-) is also modified; or the saponin is a derivative provided that when the aldehyde group in the aglycone core structure of the saponin derivative is derivatized by reacting with EMCH and the saponin is SO1861, at least one of the glucuronic acid and the acetoxy group (Me(CO)O-) is also derivatized, and when the saponin is SO1861 and the carboxyl group of the glucuronic acid moiety of SO1861 is derivatized by the bound HATU, at least one of the aldehyde group and the acetoxy group (Me(CO)O-) is also derivatized.

[0406] In specific embodiments, an in vitro or ex vivo method as described herein is provided for transferring molecules from outside the cell to inside the cell, preferably to the cytosol of the cell, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU.

[0407] Although the invention has been described according to several embodiments, it will be apparent to those skilled in the art upon reading the specification and studying the accompanying drawings that substitutions, modifications, replacements, and equivalents of the invention will be apparent to them. The invention is not limited in any way to the described embodiments. Changes may be made without departing from the scope defined by the appended claims.

[0408] The invention has been described above with reference to several exemplary embodiments. Modifications are possible and are included within the scope of protection defined by the appended claims. The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] Examples and Exemplary Implementations

[0416] Material:

[0417] SO1861, SO1832, SO1862 (isomers), and SO1904 were isolated and purified from crude plant extracts of Saponaria officinalis L. (QS21, pure, QS18, QS17, and QS7 fractions) obtained by Analyticon Discovery GmbH. QS21 (fraction) was purchased from Desert King International, San Diego. Trastuzumab (Tras, Roche), cetuximab (Cet), Merck KGaA was purchased from a pharmacy. EGF caryophyllin was produced from E. coli according to standard procedures. Cetuximab-saponin conjugate was produced and purchased from Advanced Targeting Systems (San Diego, CA). Tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, 99%, Sigma-Aldrich), Zeba TM Spin desalting column (2 mL, Thermo-Fisher), NuPAGE TM 4-12% Bis-Tris protein gel (Thermo-Fisher), NuPAGE TM MES SDS Run Buffer (Thermo-Fisher), Novex TM Sharp pre-stained protein standards (Thermo-Fisher), PageBlue TM Protein staining solution (Thermo-Fischer), Pierce TMBCA Protein Assay Kit (Thermo-Fisher), N-Ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-Dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50 M (GE Healthcare), Superdex 200P (GE Healthcare), Isopropanol (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 dehydrated sorbitan monolaurate (TWEEN) 20, Sigma-Aldrich), Dubke's phosphate-buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), disodium EDTA-Na2 dihydrate (99%, Sigma-Aldrich), sterile filters 0.2μm and 0.45μm (Sartorius), succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200PG (GE Healthcare), tetra(ethylene glycol) succinimide 3-(2-pyridyl dithio)propionate (PEG4-SPDP, Thermo-Fisher), [O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethylureonium 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) fresh from an ultrapure laboratory water system (MilliQ, Merck), Ni-NTA 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 (99.5%).7%, Sigma-Aldrich); sodium carbonate (99.9%, Sigma-Aldrich); PD MiniTrap desalting column with Sephadex G-25 resin (GE Healthcare); PD10 G25 desalting column (GE Healthcare); 0.5, 2, 5, and 10 mL Zeba Spin desalting columns (Thermo-Fisher); Vivaspin centrifugal filters T4 10kDa MWCO, T4 100kDa MWCO, and T15 (Sartorius); Biosep s3000 aSEC column (Phenomenex); Vivacell ultrafiltration units 10 and 30kDa MWCO (Sartorius); Nalgene rapid flow filter (Thermo-Fisher).

[0418] abbreviations

[0419] AEM: N-(2-aminoethyl)maleimide trifluoroacetate

[0420] AMPD: 2-Amino-2-methyl-1,3-propanediol

[0421] BOP: (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

[0422] DIPEA: N,N-Diisopropylethylamine

[0423] DMF: N,N-dimethylformamide

[0424] EMCH.TFA: N-(ε-maleiminohexanoic acid) hydrazide, trifluoroacetate

[0425] HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate

[0426] Min: minutes

[0427] NMM: 4-Methylmorpholine

[0428] rt: Retention time

[0429] TCEP: Tris(2-carboxyethyl)phosphonic acid hydrochloride

[0430] Temp: Temperature

[0431] TFA: Trifluoroacetic acid

[0432] Analytical methods

[0433] LC-MS Method 1

[0434] Equipment: Waters I-Class; Bin. Pump: UPIBSM, SM: UPISM™ with SO; UPCMA, PDA: UPPTATC, 210-320 nm; SQD: ACQ-SQD2 ESI, mass range depends on the molecular weight of the product (neg or neg / pos) in the range of 1500-2400 or 2000-3000; ELSD: gas pressure 40 psi, drift tube temperature: 50 °C; Column: Acquity C18, 50 × 2.1 mm, 1.7 μm; Temp: 60 °C; Flow rate: 0.6 mL / min.

[0435] The gradient depends on the polarity of the product:

[0436] At0=2%A, t5.0min=50%A, t6.0min=98%A

[0437] Bt0=2%A, t5.0min=98%A, t6.0min=98%A

[0438] Post-release time: 1.0 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate aqueous solution (pH = 9.5).

[0439] LC-MS Method 2 2

[0440] Equipment: Waters iClass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPTATC, 210-320nm, SQD: ACQ-SQD2 ESI, mass range depending on product molecular weight: pos / neg 100-800 or neg 2000-3000; ELSD: gas pressure 40psi, drift tube temperature: 50℃; Column: Waters XSelect™ CSH C18, 50×2.1mm, 2.5μm, Temp: 25℃, flow rate: 0.5mL / min, gradient: t0min=5%A, t2.0min=98%A, t2.7min=98%A, post-release time: 0.3min, eluent A: acetonitrile, eluent B: 10mM ammonium bicarbonate aqueous solution (pH=9.5).

[0441] LC-MS Method 3

[0442] Equipment: Waters iClass; Bin. Pump: UPIBSM, SM: UPISM™ with SO; UPCMA, PDA: UPPTATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depending on the molecular weight of the product pos / neg 105-800, 500-1200 or 1500-2500; ELSD: 40 psi, drift tube temperature: 50 °C; Column: Waters XSelect™ CSHC18, 50 × 2.1 mm, 2.5 μm, Temp: 40 °C, flow rate: 0.5 mL / min, gradient: t0 min = 5% A, t2.0 min = 98% A, t2.7 min = 98% A, post-release time: 0.3 min, eluent A: 0.1% formic acid in acetonitrile solution, eluent B: 0.1% formic acid in aqueous solution.

[0443] LC-MS Method 4

[0444] Equipment: Waters iClass; Bin. Pump: UPIBSM, SM: UPISM™ with SO; UPCMA, PDA: UPPTATC, 210-320 nm, SQD: ACQ-SQD2 ESI, mass range depends on product molecular weight: POS / neg 100-800 or neg 2000-3000; ELSD: gas pressure 40 psi, drift tube temperature: 50 °C; column: Waters Acquity Shield RP18, 50 × 2.1 mm, 1.7 μm, Temp: 25 °C, flow rate: 0.5 mL / min, gradient: t0 min = 5% A, t2.0 min = 98% A, t2.7 min = 98% A, post-release time: 0.3 min, eluent A: acetonitrile, eluent B: 10 mM ammonium bicarbonate aqueous solution (pH = 9.5).

[0445] Preparation method

[0446] Preparative MP-LC Method 1

[0447] Instrument type: Reveleris TM Preparative MPLC; Column: Waters XSelect™ CSH C18 (145×25mm, 10μm); Flow rate: 40mL / min; Column temperature: room temperature; Eluent A: 10mM ammonium bicarbonate aqueous solution, pH=9.0); Eluent B: 99% acetonitrile + 1% 10mM ammonium bicarbonate aqueous solution; Gradient:

[0448] At0min=5%B, tlmin=5%B, t2min=10%B, t17min=50%B, t18min=100%B, t23min=100%B

[0449] B t0min=5%B, t1min=5%B, t2min=20%B, t17min=60%B, t18min=100%B, t23min=100%B

[0450] Detection of UV: 210, 235, 254nm and ELSD.

[0451] Preparative MP-LC Method 2

[0452] Instrument type: Reveleris TM Preparative MPLC; Column: Phenomenex LUNA C18(3) (150×25mm, 10μm); Flow rate: 40mL / min; Column temperature: room temperature; Eluent A: 0.1% (v / v) formic acid aqueous solution, Eluent B: 0.1% (v / v) formic acid acetonitrile solution; Gradient:

[0453] A t0min=5%B, t1min=5%B, t2min=20%B, t17min=60%B, t18min=100%B. t23min=100%B

[0454] B t0min=2%B, t1min=2%B, t2min=2%B, t17min=30%B, t18min=100%B, t23min=100%B

[0455] C t0min=5%B, t1min=5%B, t2min=10%B, t17min=50%B, t18min=100%B, t23min=100%B

[0456] D t0min=5%B, t1min=5%B, t2min=5%B, t17min=40%B, t18min=100%B, t23min=100%B

[0457] Detection of UV: 210, 235, 254nm and ELSD.

[0458] Preparative LC-MS Method 3

[0459] MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect™ CSH (C18, 150×19mm, 10μm); Flow rate: 25ml / min; Column temperature: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10mM ammonium bicarbonate aqueous solution, pH=9.0; Gradient:

[0460] A t0=20%A, t2.5min=20%A, t11min=60%A, t13min=100%A, t17min=100%A

[0461] B t0=5%A, t2.5min=5%A, t11min=40%A, t13min=100%A, t17min=100%A

[0462] Detection: DAD (210nm); Detection: MSD (ESI pos / neg) quality range: 100-800; DAD-based fractional collection.

[0463] Preparative LC-MS Method 4

[0464] MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Protein (C4, 150×19mm, 10μm); Flow rate: 25ml / min; Column temperature: room temperature; Eluent A: 100% acetonitrile; Eluent B: 10mM ammonium bicarbonate aqueous solution, pH=9.0; Gradient:

[0465] A t0=2%A, t2.5min=2%A, t11min=30%A, t13min=100%A, t17min=100%A

[0466] B t0=10%A, t2.5min=10%A, t11min=50%A, t13min=100%A, t17min=100%A

[0467] C t0=5%A, t2.5min=5%A, t11min=40%A, t13min=100%A, t17min=100%A

[0468] Detection: DAD (210nm); Detection: MSD (ESI pos / neg) quality range: 100-800; DAD-based fractional collection

[0469] Rapid chromatography

[0470] Grace Reveleris C-815 Flash; Solvent delivery system: 3-plunger pump with automatic initiation, 4 independent channels, up to 4 solvents in a single run, automatic line switching when solvent is depleted; maximum pump flow rate 250 mL / min; maximum pressure 50 bar (725 psi); detection: UV 200-400 nm, combining up to 4 UV signals and scanning the entire UV range, ELSD; column size: 4-330 g on instrument, luer type, 750 g, up to 3000 g, with optional retainer.

[0471] Example 1: Synthesis of saponin derivatives

[0472] Based on naturally occurring SO1861, the following modified SO1861 saponins, i.e. saponin derivatives, were synthesized, as outlined in Table A2:

[0473]

[0474]

[0475]

[0476]

[0477] Refer to the following description of the synthesis of SO1861 derivatives, and refer to Table A2 and the accompanying figures.

[0478] Synthesis of SO1861-Ald-EMCH (Molecule 2); see Figure 60 , Figure 61 A in SO1861-Ald-EMCH (Molecule 2)

[0479] SO1861 (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) from soapwort were placed in a round flask equipped with a stirrer and dissolved in 13 mL of methanol. Approximately 400 μL of TFA was added to the solution, and the reaction mixture was stirred at 800 rpm and room temperature for 3 h on an RCT B magnetic stirrer (IKA Labortechnik). After stirring for 3 h, the mixture was diluted with MilliQ water or PBS and thoroughly dialyzed against MilliQ water or PBS for 24 h using a regenerated cellulose membrane tube (Spectra / Por7) with a MWCO of 1 kDa. After dialyzing, the solution was lyophilized to obtain a white powder. Yield: 62.4 mg (95%). The dried aliquots were further used for... 1 Characterized by H NMR and MALDI-TOF-MS.

[0480] 1 ¹H NMR (400 MHz, methanol-D₄)(SO₁₈₆₆): δ = 0.50–5.50 (m, protons of saponin triterpenoids and glycoskeletal structures), 9.43 (¹H, s, aldehyde protons of saponins, H₂). a ).

[0481] 1 ¹H NMR (400 MHz, methanol-D₄) (SO₁₈₆₆₁₆-AlD-EMCH, PBS treated): δ = 0.50–5.50 (m, protons of saponin triterpenoids and glycoskeletal structures), 6.79 (2H, s, maleimide protons, H₂). c ), 7.62-7.68 (1H, m, hydrazone proton, H) b ).

[0482] MALDI-TOF-MS (RP mode): m / z21 24Da([M+K]) + (Saponin-EMCH), m / z 2109Da ([M+K)) + , SO1861-AlD-EMCH), m / z2094Da([M+Na] + (SO1861-Ald-EMCH). See also Figure 61 A in the middle.

[0483] MALDI-TOF-MS (RN mode): m / z 2275Da ([MH) - (Saponin-EMCH conjugate), 2244Da ([MH)) - (Saponin-EMCH conjugate), 2222Da([MH)) - (Saponin-EMCH conjugate), 2178Da ([MH)) -Saponin-EMCH conjugate), 2144 Da ([MH]-, saponin-EMCH conjugate), 2122 Da ([MH]-, saponin-EMCH conjugate), 2092 Da ([MH]-, saponin-EMCH conjugate), 2070 Da ([MH]-, saponin-EMCH conjugate) - , SO1861-A1D-EMCH), 2038Da([MH]-, SO1832-EMCH), 1936Da([MH] - SO1730-EMCH), 1861Da ([MH) - SO1861). SO1861-ALD-EMCH is composed of molecule 2 (chemical formula: C 93 H 143 N3O 48 (Precise mass: 2069.88) indicates:

[0484]

[0485] To test the pH-dependent hydrolysis of hydrazone bonds, SO1861-Ald-EMCH was dissolved in an HCl solution at pH 3, and MALDI-TOF-MS spectra were recorded at two different time points. Figure 62 ).like Figure 62 As shown in A in Figure 62 and B in Figure 62, the significant decreasing trend of the peak corresponding to SO1861-Ald-EMCH at m / z 2070 Da is evident. Figure 61 As can be seen in B. Due to the generation of SO1861 during hydrolysis, an increase in the peak at m / z 1861 Da is recorded, accompanied by a decreasing trend at m / z 2070 Da. These results show that hydrazone bonds respond to hydrolysis and are broken even when they are attached to SO1861.

[0486] SO1861-Ald-EMCH-mercaptoethanol (molecule 3; SO1861-Ald-EMCH blocked); see Figure 60 andB in Figure 61 ​

[0487] When carried out in a pH range of 6.5–7.5, the maleimide group of SO1861-Ald-EMCH undergoes a rapid and specific Michael addition reaction with thiols.

[0488] Add 200 μL of mercaptoethanol (18 mg, 230 μmol) to SO1861-Ald-EMCH (0.1 mg, 48 nmol) and shake the solution at 800 rpm and room temperature for 1 h on a ThermoMixer C (Eppendorf). After shaking for 1 h, dilute the solution with methanol and dialyze thoroughly against methanol for 4 h using a regenerated cellulose membrane tube (Spectra / Por 7) with a MWCO of 1 kDa. After dialyzing, provide SO1861-Ald-EMCH-mercaptoethanol (molecule 3), aliquot the sample, and analyze by MALDI-TOF-MS.

[0489] MALDI-TOF-MS (RP mode): m / z 2193Da([M+K]) + SO1861-Ald-EMCH-mercaptoethanol), m / z 2185 Da ([M+K) + SO1861-Ald-EMCH-mercaptoethanol), m / z 2170 Da ([M+Na) + (SO1861-Ald-EMCH-mercaptoethanol). See also Figure 61 B. SO1861-Ald-EMCH-mercaptoethanol is composed of molecule 3 (chemical formula: C 95 H 149 N3O 49 S, precise mass: 2147.90) indicates:

[0490]

[0491] Synthesis of SO1861-Glu-AMPD (Molecule 3A); see Figure 1

[0492] SO1861 (28.8 mg, 0.015 mmol), AMPD (8.11 mg, 0.077 mmol), and HATU (17.6 mg, 0.046 mmol) were dissolved in a mixture of DMF (1.00 mL) and NMM (8.48 μL, 0.077 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled, frozen, and lyophilized overnight. Then, the fractions were analyzed using preparative LC-MS. 3 The product was further purified. The fractions corresponding to the product were immediately pooled, freeze-dried overnight, and the title compound (20.2 mg, 67%) was obtained as a white, fluffy solid. Purity based on LC-MS = 93% (Chemical formula: C). 87 H 139 NO 47 Precision quality: 1949,85.

[0493] LRMS (m / z): 1949 [M-1] 1- .

[0494] LC-MS rt(min): 2.45 1B

[0495] Synthesis of SO1861-Ald-OH (Molecule 6); see Figure 2

[0496] SO1861 (20.0 mg, 10.7 μmol) was dissolved in methanol (1.00 mL). Next, sodium borohydride (4.06 mg, 0.107 mmol; NaBH4) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was diluted with water (0.50 mL) and 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 (15.9 mg, 79%) as a white, fluffy solid. The purity based on LC-MS was 97% (chemical formula: C). 83 H 132 O 46 Precise quality: 1864, 80).

[0497] LRMS (m / z): 1865 [M⁻¹] 1- (see Figure 15 and 16 )

[0498] LC-MS rt(min): 1.95 1B

[0499] Synthesis of SO1861-Ac-OH (Molecule 8); see Figure 3

[0500] A solution of sodium hydroxide (2.00 mg, 0.050 mmol) in water (0.25 mL) and methanol (0.25 mL) was added to SO1861 (9.30 mg, 4.99 μmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture 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 (8.86 mg, 97%) as a white, fluffy solid. Purity based on LC-MS = 97% (Chemical Formula: C81H). 128 O 45 Precision quality: 1820,77).

[0501] LRMS (m / z): 1820 [M⁻¹] 1- .

[0502] LC-MS rt(min): 1.83 1B

[0503] Synthesis of SO1861-(Ald-OH)-(Glu-AMPD) (Molecule 9); see Figure 4

[0504] SO1861-Ald-OH (9.37 mg, 5.02 μmol), AMPD (2.64 mg, 0.025 mmol), and BOP (6.66 mg, 0.015 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (5.52 μL, 0.050 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture 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 (6.32 mg, 64%) as a white, fluffy solid. Purity based on LC-MS = 95% (Chemical formula: C). 87 H 141 NO 47 Precision quality: 1951,87.

[0505] LRMS (m / z): 1952 [M-1] 1- (see Figure 17 )

[0506] LC-MS rt(min): 2.45 1B

[0507] Synthesis of SO1861-(Ald-OH)-(Ac-OH) (Molecule 10); see Figure 5

[0508] A solution of sodium hydroxide (5.74 mg, 0.144 mmol) in water (0.50 mL) and methanol (0.50 mL) was added to SO1861-Ald-OH (26.8 mg, 0.014 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled, frozen, and lyophilized overnight to give the title compound (24.2 mg, 92%) as a white, fluffy solid. Purity based on LC-MS = 98%. (Chemical formula: C) 81 H 130 O 45 Precise quality: 1822,79

[0509] LRMS (m / z): 1822 [M⁻¹] 1- .

[0510] LC-MS rt(min): 1.81 1B

[0511] Synthesis of SO1861-(Ac-OH)-(Glu-AMPD) (Molecule 11); see Figure 6

[0512] SO1861-Ac-OH (14.3 mg, 7.84 μmol), AMPD (4.12 mg, 0.039 mmol), and BOP (10.4 mg, 0.024 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (8.62 μL, 0.078 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC2. The fractions corresponding to the product were immediately collected, frozen, and lyophilized overnight. Then, the product was analyzed by first using preparative MP-LC2. 2 Subsequently, preparative LC-MS was used. 3 The product was then purified. The fractions corresponding to the product were immediately pooled, freeze-dried overnight, and the title compound (9.47 mg, 63%) was obtained as a white, fluffy solid. Purity based on LC-MS = 98% (Chemical formula: C). 85 H 137 NO 46 Precision quality: 1907, 84).

[0513] LRMS (m / z): 1908 [M-1] 1- .

[0514] LC-MS rt(min): 2.31 1B

[0515] Synthesis of SO1861-(Ald-OH)-(Ac-OH)-(Glu-AMPD) (Molecule 12); see Figure 7

[0516] SO1861-(Ald-OH)-(Ac-OH) (8.57 mg, 4.70 μmol), AMPD (42.58 mg, 0.025 mmol), and BOP (6.57 mg, 0.015 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (5.17 μL, 0.047 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2 The fractions corresponding to the product were immediately collected, frozen, and lyophilized overnight. Then, the fractions were analyzed again using preparative MP-LC. 2 The product was further purified. The fractions corresponding to the product were immediately pooled, freeze-dried overnight, and the title compound (7.21 mg, 80%) was given as a white, fluffy solid. Purity based on LC-MS = 97.8%, chemical formula: C 85 H 139 NO 46 Precise quality: 1909, 86)

[0517] LRMS (m / z): 1910 [M⁻¹] 1- .

[0518] LC-MS rt(min): 2.21 1B

[0519] Synthesis of SO1861-(Ald-EMCH)-(Glu-AMPD) (Molecule 14); see Figure 8

[0520] SO1861-Glu-AMPD (10.6 mg, 5.43 μmol) and EMCH.TFA (9.22 mg, 0.027 mmol) were dissolved in methanol (ultra-dry, 0.50 mL). Then, TFA (1.66 μL, 0.022 mmol) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled, frozen, and lyophilized overnight. Then, the fractions were analyzed using preparative LC-MS. 3 The product was further purified. Fractions corresponding to the product were pooled together. The resulting solution was neutralized with formic acid, frozen, and lyophilized overnight to give the title compound (2.61 mg, 22%) as a white, fluffy solid. Purity based on LC-MS = 95% (Chemical formula: C 97 H 152 N4O 49 Precision quality: 2156.95.

[0521] LRMS (m / z): 2156 [M⁻¹] 1- .

[0522] LC-MS rt(min): 2.64 1B

[0523] Synthesis of SO1861-(Ald-EMCH)-(Ac-OH) (Molecule 15); see Figure 9

[0524] SO1861-Ac-OH (9.05 mg, 4.97 μmol) and EMCH.TFA (8.43 mg, 0.025 mmol) were dissolved in methanol (ultra-dry, 0.50 mL). Then, TFA (1.52 μL, 0.022 mmol) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (6.58 mg, 65%) as a white, fluffy solid. Purity based on LC-MS = 97% (Chemical formula: C). 91 H 141 N3O 47 Precision quality: 2027,87).

[0525] LRMS (m / z): 2028 [M-1] 1- .

[0526] LC-MS rt(min): 1.96 1B

[0527] Synthesis of SO1861-(Ald-EMCH)-(Ac-OH)-(Glu-AMPD) (Molecule 16); see Figure 10

[0528] SO1861-(Ac-OH)-(Glu-AMPD) (6.00 mg, 3.14 μmol) and EMCH.TFA (5.33 mg, 0.016 mmol) were dissolved in methanol (ultra-dry, 0.50 mL). Then, TFA (0.96 μL, 0.013 mmol) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled, frozen, and lyophilized overnight. Then, the fractions were analyzed using preparative LC-MS. 3 The product was further purified. Fractions corresponding to the product were pooled together. The resulting solution was neutralized with formic acid, frozen, and lyophilized overnight to give the title compound (1.04 mg, 16%) as a white, fluffy solid. Purity based on LC-MS = 94% (Chemical formula: C 95 H 150 N4O 48 Precision quality: 2114.94).

[0529] LRMS (m / z): 2115 [M-1] 1- .

[0530] LC-MS rt(min): 2.55 1B

[0531] Synthesis of SO1861-Glu-AEM (Molecule 18); see Figure 11

[0532] SO1861 (10.4 mg, 5.58 μmol), AEM (7.10 mg, 0.028 mmol), and HATU (6.36 mg, 0.017 mmol) were dissolved in a mixture of DMF (1.00 mL) and NMM (6.13 μL, 0.056 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture 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 (7.82 mg, 71%) as a white, fluffy solid. Purity based on LC-MS = 95% (Chemical formula: C). 89 H 136N2O 47 Precision quality: 1984, 83).

[0533] LRMS (m / z): 1985 [M-1] 1-

[0534] LC-MS rt(min): 2.62 1B

[0535] Synthesis of SO1861-(Glu-AEM)-(Ac-OH) (Molecule 19); see Figure 12

[0536] SO1861-Ac-OH (9.02 mg, 4.95 μmol), AEM (7.10 mg, 0.028 mmol), and HATU (5.65 mg, 0.015 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (5.44 μL, 0.050 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled, frozen, and lyophilized overnight to give the title compound (7.16 mg, 74%) as a white, fluffy solid. Purity based on LC-MS = 96% (Chemical formula: C). 87 H 134 N2O 46 Precision quality: 1942, 82).

[0537] LRMS (m / z): 1944 [M-1] 1- .

[0538] LC-MS rt(min): 2.47 1B

[0539] Synthesis of SO1861-(Glu-AEM)-(Ald-OH) (Molecule 20); see Figure 13

[0540] SO1861-Ald-OH (9.38 mg, 5.03 μmol), AEM (6.39 mg, 0.025 mmol), and HATU (5.73 mg, 0.015 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (5.53 μL, 0.050 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture 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 (8.63 mg, 86%) as a white, fluffy solid. Purity based on LC-MS = 95% (Chemical formula: C). 89 H 138 N2O 47Precision quality: 1986, 85).

[0541] LRMS (m / z): 1987 [M-1] 1-

[0542] LC-MS rt(min): 2.62 1B

[0543] Synthesis of SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) (Molecule 21); see Figure 14

[0544] SO1861-(Ald-OH)-(Ac-OH) (8.92 mg, 4.89 μmol), AEM (6.54 mg, 0.026 mmol), and HATU (5.65 mg, 0.015 mmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (5.38 μL, 0.049 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture 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 (8.92 mg, 94%) as a white, fluffy solid. Purity based on LC-MS = 97% (Chemical formula: C). 87 H 136 N2O 46 Precision quality: 1944, 84).

[0545] LRMS (m / z): 1944 [M-1] 1- .

[0546] LC-MS rt(min): 2.46 1B

[0547] Synthesis of SO1861-L-N3 (molecule 23); see Figure 36

[0548] Chemical formula: C 94 H 151 N5O 50 Precision quality: 2149.94

[0549] Synthesis of SO1861-L-NHS (Molecule 25); see Figure 37

[0550] SO1861-L-N3 (7.71 mg, 3.58 μmol) and DBCO-NHS (2.88 mg, 7.17 μmol) were dissolved in anhydrous DMF (0.50 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was added dropwise to diethyl ether (40 mL). After centrifugation (7800 RPM, 5 min), the supernatant was discarded, and the precipitate was resuspended in diethyl ether (20 mL) and centrifuged again. After discarding the supernatant, the residue was dissolved in water / acetonitrile (3:1, v / v, 3 mL), and the resulting solution was directly frozen and lyophilized overnight to give the title compound (8.81 mg, 96%) as a white, fluffy solid. The purity was 84% ​​based on LC-MS. It contained 14% hydrolyzed NHS ester (chemical formula: C 117 H 169 N7O 55 Precision quality: 2552,06).

[0551] LRMS (m / z): 2551 [M-1] 1- .

[0552] LC-MS rt(min): 2.76 / 2.78 2 (Due to the bimodal nature of the isomer)

[0553] Synthesis of SO1861-Glu-HATU (molecule 26); see Figure 59

[0554] To generate SO1861-Glu-HATU, the carboxyl group of SO1861 was activated by a reagent used in peptide coupling chemistry to produce the active ester, namely 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU). The resulting active ester of SO1861 is shown in... Figure 59 middle.

[0555] Based on naturally occurring QS-21, the following modified QS-21 saponins, i.e., saponin derivatives, were synthesized:

[0556]

[0557]

[0558] Synthesis of QS21-Ald-OH (molecule 27); see Figure 38

[0559] QS21 (9.41 mg, 4.73 μmol) was dissolved in methanol (0.50 mL). Then, sodium borohydride (1.79 mg, 0.047 mmol) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 30 min, the reaction mixture was diluted with water (0.50 mL) and subjected to preparative MP-LC.2A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (4.68 mg, 50%) as a white, fluffy solid. The purity based on LC-MS was 99% (exact mass: 1990, 4 isomers: Api / Xyl (2:1)).

[0560] LRMS (m / z): 1990 [M-1] 1- .

[0561] LC-MS rt(min): 1.25 / 2.31 1B (Bimodal, 17 / 83 UV-area%, due to QS21 being a mixture)

[0562] Synthesis of QS21-Glu-AEM (molecule 28); see Figure 39

[0563] QS-21 (2.42 mg, 1.22 μmol); Figure 41 AEM (1.68 mg, 6.61 μmol) and HATU (1.48 mg, 3.89 μmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (1.34 μL, 0.012 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (1.80 mg, 70%) as a white, fluffy solid. The purity based on LC-MS was 92% (exact mass: 2110, 4 isomers: Api / Xyl (2:1)).

[0564] LRMS(m / z): 2110 [M-1] 1- .

[0565] LC-MS rt(min): 2.84 / 2.93 1B (Bimodal, 10 / 90 UV-area%, due to QS21 being a mixture)

[0566] Synthesis of OS21-(Ald-OH)-(Glu-AEM) (Molecule 29); see Figure 40A

[0567] QS-21-Ald-OH (1.92 mg, 0.964 μmol), AEM (1.29 mg, 5.08 μmol), and HATU (1.10 mg, 2.89 μmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (1.06 μL, 9.64 μmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2AThe fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (1.46 mg, 72%) as a white, fluffy solid. The purity based on LC-MS was 92% (exact mass: 2112, 4 isomers: Api / Xyl (2:1)).

[0568] LRMS(m / z): 2112[M-1] 1- .

[0569] LC-MS rt(min): 2.83 / 2.92 1B (Bimodal, 7 / 93 UV area%, due to QS21 being a mixture)

[0570] Synthesis of QS21-Ald-EMCH Figure 40B ; molecule 30)

[0571] QS21 (4.82 mg, 2.42 μmol) and EMCH.TFA (4.11 mg, 0.012 mmol) were dissolved in methanol (ultra-dry, 0.25 mL). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately collected, frozen, and lyophilized overnight. Then, preparative MP-LC was used. 2A The product was further purified. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (2.78 mg, 52%) as a white, fluffy solid. The purity was 96% based on LC-MS.

[0572] LRMS (m / z): 2196 [M⁻¹] 1- .

[0573] LC-MS rt(min): 2.44 1B (Multiple peaks are generated because QS21 is a mixture)

[0574] Synthesis of QS21-Glu-AMPD Figure 40C ; molecule 31)

[0575] QS21 (4.89 mg, 2.46 μmol), AMPD (1.29 mg, 0.012 mmol), and BOP (3.26 mg, 7.37 μmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (2.70 μL, 0.025 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (3.76 mg, 74%) as a white, fluffy solid. The purity was 94% based on LC-MS.

[0576] LRMS (m / z): 2076 [M⁻¹] 1- .

[0577] LC-MS rt(min): 2.78 1B (Multiple peaks are generated because QS21 is a mixture)

[0578] Synthesis of QS21-(Ald-EMCH)-(Glu-AMPD) Figure 40D ; molecule 32)

[0579] QS21-Glu (2.47 mg, 1.19 μmol) and EMCH.TFA (2.02 mg, 5.95 μmol) were dissolved in methanol (ultra-dry, 100 μL). Then, TFA (0.36 μL, 4.76 μmol) was added. The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (2.25 mg, 83%) as a white, fluffy solid. The purity was 95% based on LC-MS.

[0580] LRMS (m / z): 2283 [M-1] 1- .

[0581] LC-MS rt(min): 2.88 1B (Multiple peaks are generated because QS21 is a mixture)

[0582] Synthesis of OS21-(Ald-OH)-(Glu-AMPD) Figure 40E ; molecule 33)

[0583] QS21-(Ald-OH) (4.90 mg, 2.46 μmol), AMPD (1.29 mg, 0.012 mmol), and BOP (3.26 mg, 7.37 μmol) were dissolved in a mixture of DMF (0.50 mL) and NMM (2.70 μL, 0.025 mmol). The reaction mixture was shaken for 1 min and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (2.16 mg, 42%) as a white, fluffy solid. The purity was 96% based on LC-MS.

[0584] LRMS (m / z): 2077 [M-1] 1- .

[0585] LC-MS rt(min): 2.77 1B (Multiple peaks are generated because QS21 is a mixture)

[0586] Example 2: Activity of Saponin Derivatives - Preliminary Study

[0587] The saponin modifications described in this paper were found to have virtually no interference with the ability of saponins to enhance endosome escape (modified saponins or saponins released from conjugates that do not contain endosomes). The experimental results are summarized in Table Ex2 below.

[0588] Chemically modified saponin SO1861 did show reactivity in cell-based bioassays, with relative cell viability as the reading. HeLa cells were incubated with the following construct for 72 hours, and cell viability was assessed before and after 72 hours of incubation. In the experiments, cells were exposed to a 1.5 pM caryophyllin-EGF conjugate. The negative control was cells incubated with a buffer medium and 10 μg / ml of saponin, without caryophyllin-EGF. Cell viability of the control (where saponin and EGF-caryophyllin were omitted) was set at 100%. The positive control was 10 μg / ml of unmodified saponin SO1861 + caryophyllin-EGF. Cell viability was essentially 0% after 72 hours. For the chemically modified saponin variant, 10 μg / ml of saponin was tested in combination with 1.5 pM caryophyllin-EGF. SO1861-Ald-EMCH reduced cell viability at 10 μg / ml.

[0589] These data indicate that saponins can be modified on free aldehyde groups or free carbonyl groups without losing endosome escape and enhancing activity.

[0590] Table Ex2. Cytotoxic activity (+ or -) of SO1861 and its derivatives when co-administered with the targeted toxin (EGF caryophyllin). Co-administration resulted in enhanced cytotoxicity compared to untreated EGFR-expressing cells (e.g., A431, HeLa).

[0591]

[0592] Example 3: Activity of Saponin Derivatives - Detailed Study

[0593] Applying various saponins (e.g., SO1861, QS-21) as “free” unconjugated molecules to cells together with ligand toxin fusions (e.g., EGF-caryophyllin) or antibody-protein toxin conjugates leads to enhanced cytotoxic activity in target expression cells.

[0594] The inventors chemically modified SO1861 (isolated and purified from the root extract of *Saponaria officinalis*) and QS21 (isolated and purified from *Quillaja saponaria*, Desert King) at different positions (single, double, or triple modification) within the molecules, thereby providing a series of saponin derivatives listed in Tables A2 and A3. The saponin derivatives were tested for: 1) enhanced ligand toxin activity (modified SO1861 / QS21 titration + 5 pM EGF caryophyllene) against EGFR-expressing cells (HeLa and A431) to enhance endosome escape; 2) inherent cytotoxicity against HeLa and A431 (modified SO1861 / QS21 titration); and 3) hemolytic activity against human erythrocytes (modified SO1861 / QS21 titration against human erythrocytes).

[0595] To determine the endosome escape enhancement activity, modified SO1861 cells (see [reference needed]) were titrated in the presence of a non-effectively fixed concentration of 5 pM EGF-caryophyllin in EGFR-expressing cells (HeLa and A431). Figures 18A-18B and Figures 19A-19B Furthermore, the endosome escape enhancement activity of saponin derivatives titrated in the presence of a non-effective fixed concentration of 5 pM EGF-caryophyllin was determined (for a comparison of SO1861 with SO1861-Ald-EMCH and SO1861-Ald-EMCH-blocking, see [link to documentation]). Figures 23A-23B For a comparison of SO1861 with SO1861-(Ald-EMCH)-(Ac-OH), SO1861-(Ald-EMCH)-(Glu-AMPD), and SO1861-(Ald-EMCH)-(Ac-OH)-(Glu-AMPD), see [link to documentation]. Figures 24A-24BThis revealed that modified saponins with single modifications compared to SO1861 exhibited activity at the following concentrations: SO1861-Ald-OH against HeLa: IC50 = 600 nM and against A431: IC50 = 600 nM; SO1861-Glu-AMPD against HeLa: IC50 = 600 nM and against A431: IC50 = 600 nM; SO1861-Ac-OH against HeLa: IC50 = 1000 nM and against A431: IC50 = 1000 nM. C50 = 800 nM, SO1861-Glu-AEM IC50 = 1500 nM for HeLa and IC50 = 2000 nM for A431, and SO1861-Ald-EMCH IC50 = 2000 nM for HeLa and IC50 = 2000 nM for A431, and double modification showed the following activity with IC50 values: SO1861-(Ac-OH)-(Glu-AMPD) IC50 for HeLa: =3000nM and for A431: IC50 = 3000nM, SO1861-(Ald-OH)-(Glu-AMPD) for HeLa: IC50 = 4000nM and for A431: IC50 = 4000nM, SO1861-(Ald-OH)-(Ac-OH) for HeLa: IC50 = 4000nM and for A431: IC50 = 5000nM, SO1861-(Glu-AEM)-(Ac The IC50 values ​​for SO1861-(Ald-EMCH)-(Ac-OH) against HeLa were 8000 nM and 4000 nM, respectively. For SO1861-(Ald-EMCH)-(Ac-OH), the IC50 values ​​for HeLa were 8000 nM and 10.000 nM, respectively. For SO1861-(Glu-AEM)-(Ald-OH), the IC50 values ​​for HeLa were 40.000 nM and 20.000 nM, respectively. The three modifications tested did not show activity at the current concentrations. Using unmodified SO1861 as a control, the following IC50 values ​​were obtained: IC50 = 100 nM in HeLa and IC50 = 200 nM in A431. Enhanced endosome escape of modified QS21 was determined by titrating a saponin derivative in the presence of a non-effectively fixed concentration of 5pMEGF-caryophyllin on EGFR-expressing cells (see [link]). Figure 30A and 30BThis revealed that, compared to unmodified QS21, the following modified QS21 exhibited activity at the following concentrations: QS21 against HeLa: IC50 = 200 nM and against A431: IC50 = 200 nM; QS21-Ald-OH against HeLa: IC50 = 600 nM and against A431: IC50 = 600 nM; QS21-Glu-AEM against HeLa: IC50 = 600 nM and against A431: IC50 = 700 nM; and QS21-(Ald-OH)-(Glu-AEM) against HeLa: IC50 = 1500 nM and against A431: IC50 = 3000 nM. In summary, both unmodified SO1861 and QS21 were effective in HeLa and A431 cells at IC50 = 200 nM. Single SO1861 / QS21 modifications (SO1861-Ald-EMCH, SO1861-Ald-EMCH blocked, SO1861-Glu-AMPD, SO1861-Ald-OH, SO1861-Ac-OH, SO1861-Glu-AEM, QS21-Ald-OH, QS21-Glu-AEM) showed IC50 activity of 600 nM–2000 nM in HeLa or A431 cells (Tables A5 and A6), while dual SO1861 / QS21 modifications showed IC50 activity of 1500–40,000 nM in HeLa or A431 cells (Tables A5 and A6). For trimodification of SO1861, no activity was observed up to 20,000 nM.

[0596] As described above, to determine the endosome escape enhancement activity, SO1861 derivatives, QS21 derivatives, and their underivative counterparts were titrated in EGFR-expressing cells (HeLa and A431) in the presence of a non-effectively fixed concentration of 5 pM EGF caryophyllin. This revealed that underivative SO1861, underivative QS21, and the QS21 derivative QS21-Glu-AMPD were effective in HeLa and A431 cells at IC50 = 200 nM. Single SO1861 / QS21 modifications (SO1861-Ald-EMCH, SO1861-Ald-EMCH (blocked)) (SO1861-Ald-EMCH (mercaptoethanol), SO1861-Glu-AMPD, SO1861-(Ald-OH), SO1861-Ac-OH, SO1861-Glu-AEM, QS21-Ald-EMCH, QS21-(Ald-OH), QS21-Glu-AEM) showed IC50 activity of 600 nM-2000 nM in HeLa or A431 cells (Tables A5 and A6), while dual SO1861 modifications and dual QS21 modifications showed IC50 activity of 1500-40,000 nM in HeLa or A431 cells (Tables A5 and A6). No activity was observed for the three modifications of SO1861 up to 20,000 nM.

[0597] For toxicity assays, modified SO1861 was titrated in HeLa cells (see [link to toxicity assay]). Figure 20A and 21A ) and A431 cells (see Figure 20B and 21B )superior. Figure 25A and 25B Details of the toxicity tests for SO1861, SO1861-Ald-EMCH, and SO1861-Ald-EMCH-closed are described. Figure 26A and 26BDetails of SO1861, SO1861-(Ald-EMCH)-(Ac-OH), SO1861-(Ald-EMCH)-(Glu-AMPD), and SO1861-(Ald)-EMCH)-(Ac-OH)-(Glu-AMPD) are shown. This reveals that unmodified SO1861 exhibits the strongest intrinsic toxicity to HeLa cells: IC50 = 2000 nM, while the monomodified SO1861-Ac shows toxicity to HeLa cells with an IC50 of 10,000 nM. For all other SO1861-derives, the intrinsic toxicity (IC50) to HeLa cells is greater than 20,000 nM. In A431 cells, the toxicity of unmodified SO1861 was observed at an IC50 of 1000 nM, while the single-modified SO1861-Ac-OH, SO1861-Ald-OH, SO1861-Glu-AMPD, and SO1861-Ald-EMCH showed IC50 values ​​of 2000 nM, 7000 nM, 20.000 nM, and 30.000 nM, respectively. To determine the toxicity of modified QS21, it was tested in HeLa cells (see...). Figure 31A ) and A431 cells ( Figure 31BThe saponin derivatives were titrated. This revealed that QS21 showed toxicity to HeLa cells (IC50 = 6000 nM) and A431 cells (IC50 = 3000 nM), QS21-Ald-OH showed toxicity to HeLa cells (IC50 = 20,000 nM) and A431 cells (IC50 = 20,000 nM), and QS21-Glu-AEM showed toxicity to HeLa cells (IC50 > 100,000 nM). QS21-(Ald-OH)-(Glu-AEM) showed toxicity to HeLa cells at nM and IC50 > 100.000 nM to A431 cells. No toxicity was observed at up to 100.000 nM for SO1861 or QS21 double-modified and SO1861 triple-modified cells. As described above, unmodified or modified SO1861 or QS21 was titrated on HeLa and A431 cells. This reveals that unmodified SO1861 exhibits toxicity at IC50 = 1000 nM (HeLa) and IC50 = 2000 nM (A431), while QS21 shows toxicity at IC50 = 6000 nM (HeLa) and IC50 = 3000 nM (A431), and QS21-Glu-AMPD shows toxicity at IC50 = 9000 nM (HeLa) and IC50 = 5000 nM (A431) (Tables A5 and A6). For HeLa cells, SO1861-Al showed toxicity for both single SO1861 modification and single QS21 modification. d-EMCH, SO1861-Ald-EMCH (blocked), SO1861-(Ald-OH), SO1861-Glu-AEM, QS21-Ald-EMCH, and QS21-Glu-AEM showed no toxicity up to 100.000 nM, while SO1861-Glu-AMPD, SO1861-Ac-OH, and QS21-(Ald-OH) showed toxicity at IC50 = 20.000 nM, IC50 = 10.000 nM, and IC50 = 20.000 nM, respectively (Tables A5 and A6).In A431 cells, SO1861-Glu-AEM and QS21-Glu-AEM showed no toxicity at concentrations up to 100,000 nM, while toxicity was observed for SO1861-Ald-EMCH (IC50 = 30,000 nM), SO1861-Ald-EMCH (blocking) (IC50 = 30,000 nM), SO1861-(Ald-OH) (IC50 = 7000 nM), SO1861-Ac-OH (IC50 = 2000 nM), SO1861-Glu-AMPD (IC50 = 20,000 nM), QS21-Ald-EMCH (IC50 = 30,000 nM), and QS21-(Ald-OH) (IC50 = 20,000 nM) (Tables A5 and A6). No toxicity was observed up to 100,000 nM for SO1861 double-modified or QS21 double-modified and SO1861 tri-modified (Tables A5 and A6).

[0598] In addition, the hemolytic activity of unmodified and modified SO1861 was determined by the human erythrocyte hemolysis assay (see [reference]). Figure 22 , Figure 27 , Figure 28 and Figure 32This reveals that unmodified SO1861 exhibits activity at IC50 = 8000 nM, while unmodified QS21 exhibits activity at IC50 = 3000 nM. Single-modified SO1861-Ald-EMCH showed no hemolytic activity at concentrations up to 1,000,000 nM, while hemolytic activity against human erythrocytes was observed with SO1861-Ald-EMCH (IC50 = 300,000 nM), SO1861-Ald-OH (IC50 = 30,000 nM), SO1861-Ac-OH (IC50 = 20,000 nM), SO1861-Glu-AMPD (IC50 = 20,000 nM), SO1861-Glu-AEM (IC50 = 30,000 nM), QS21-Ald-OH (IC50 = 20,000 nM), and QS21-Glu-AEM (IC50 = 10,000 nM) (Tables A5 and A6). For double modification with SO1861 or QS21, no hemolytic activity was observed (up to 1,000,000 nM) for SO1861-(Ald-EMCH)-(Glu-AMPD), SO1861-(Ald-EMCH)-(Ac-OH), SO1861-(Glu-AEM)-(Ald-OH), and QS21-(Ald-OH)-(Glu-AEM), while hemolytic activity was observed for SO1861-(Ald-OH)-(Glu-AMPD) (IC50 = 100,000), SO1861-(Ald-OH)-(Ac-OH) (IC50 = 200,000), SO1861-(Ac-OH)-(Glu-AMPD) (IC50 = 140,000), and SO1861-(Glu-AEM)-(Ac-OH) (IC50 = 100,000). No hemolysis was observed at up to 100,000 nM for SO1861 trimodification. Hemolysis assays showed hemolytic activity of unmodified SO1861 at IC50 = 8000 nM, unmodified QS21 at IC50 = 3000 nM, and modified QS21-Glu-AMPD at IC50 = 3000 nM.Monomodified SO1861-Ald-EMCH showed no hemolytic activity up to 1,000,000 nM, while SO1861-Ald-EMCH (blocked) (IC50 = 300,000 nM), SO1861-(Ald-OH) (IC50 = 30,000 nM), SO1861-Ac-OH (IC50 = 20,000 nM), and SO1861-Glu-AMP showed activity. Hemolytic activity of human erythrocytes was observed in D (IC50 = 20.000 nM), SO1861-Glu-AEM (IC50 = 30.000 nM), QS21-Ald-EMCH (IC50 = 30.000 nM), QS21-(Ald-OH) (IC50 = 20.000 nM), and QS21-Glu-AEM (IC50 = 10.000 nM) (Tables A5 and A6). For SO1861 double modification or QS21 double modification, no hemolytic activity was observed (up to 1.000-000 nM) for SO1861-Ald-EMCH-(Glu-AMPD), SO1861-(Ac-OH)-EMCH, SO1861-(Ald-OH)-(Glu-AEM), QS21-Ald-EMCH-(Glu-AMPD), and QS21-(Ald-OH)-(Glu-AEM), while no hemolytic activity was observed for SO1861-(Ald-OH)-(Glu-AMPD). Hemolytic activity was observed with SO1861-(Ald-OH)-(Ac-OH) (IC50 = 200.000 nM), SO1861-(Ac-OH)-(Glu-AMPD) (IC50 = 140.000 nM), SO1861-(Ac-OH)-(Glu-AEM) (IC50 = 100.000 nM), and QS21-(Ald-OH)-(Glu-AMPD) (IC50 = 40.000 nM) (Tables A5 and A6). No hemolysis was observed with SO1861 trimodification up to 100.000 nM (Tables A5 and A6).

[0599] Testing the endosome escape enhancement activity of various QS saponin fractions (titration of saponin + 5pM cetuximab-saponin on HeLa and A431 cells, see [reference]). Figures 33A-33B ), toxicity (titration of saponins on HeLa and A431 cells, see Figures 34A-34B ) and hemolytic activity (titration of saponins on human erythrocytes, see Figure 32 and Figure 35This revealed that QS21 (fraction), QS17 (fraction), and QS18 (fraction) showed activity at 200 nM in HeLa and A431 cells, while QS7 (fraction) showed activity at IC50 = 6000 nM (HeLa) and 10,000 nM (A431). When toxicity was observed during the assay, QS21 (fraction), QS17 (fraction), and QS18 (fraction) showed toxicity at IC50 = 10,000 nM in HeLa and A431 cells, while QS7 (fraction) showed toxicity at IC50 = 20,000 nM. Figures 34A-34B Next, a hemolysis assay was performed, which revealed the hemolytic activity of QS21 (fraction) at IC50 = 3000 nM, QS17 (fraction) and QS18 (fraction) at IC50 = 5000 nM, while for QS7 (fraction), no hemolytic activity was detected up to 20,000 nM. Figure 35 ).

[0600] The hemolytic activity of various SO saponins (SO1862 (isomer), SO1832, SO1904) and antibody-SO1861 conjugates (cetuximab-SO1861(DAR4), trastuzumab-SO1861(DAR4)) was tested. This revealed that the hemolytic activity of SO1862 (isomer), SO1832, and SO1904 was comparable to that of SO1861 (IC50 = 10.000 nM). Figure 29 The cetuximab-SO1861(DAR4) conjugate showed no hemolytic activity up to 60,000 nM, while trastuzumab-SO1861(DAR4) showed initial hemolytic activity from 60,000 nM upwards (IC50 = 200,000 nM). Figure 29 ).

[0601] When comparing the cytotoxicity, hemolytic activity, and endosome escape enhancement activity of SO1861, SO1861-Ald-EMCH, and SO1861-Ald-EMCH-mercaptoethanol (SO1861-Ald-EMCH-blocked), the latter two showed similar or substantially the same cytotoxicity, hemolytic activity, and activity in the endosome escape enhancement activity bioassay, and both exhibited lower cytotoxicity and lower hemolytic activity than SO1861. See also Tables A5 and A6.

[0602] Cell viability assay

[0603] According to the manufacturer's instructions ( The Aqueous One Solution Cell Proliferation Assay (Promega) determined cell viability using MTS analysis. The MTS solution was diluted 20× in DMEM (PAN-Biotech GmbH) supplemented with 10% FBS (PAN-Biotech GmbH) without phenol red. Cells were washed once with 200 μL of PBS in each well, followed by the addition of 100 μL of diluted MTS solution to each well. The plate was incubated at 37°C for approximately 20–30 minutes. Subsequently, the optical density at 492 nm was measured using a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the 'culture medium only' wells was subtracted from all other wells before calculating the untreated / treated cell ratio by dividing the background correction signal of the untreated wells by the background correction signal of the treated wells.

[0604] FACS analysis

[0605] In 10 cm culture dishes, cells were seeded at 500,000 cells / plate in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (PAN-Biotech GmbH) and incubated for 48 hours (5% CO2, 37°C) until confluence reached 90%. Next, the cells were digested with trypsin (TryplEExpress, Gibco Thermo Scientific) to form single cells. 0.75 × 10⁶ cells were then cultured. 6 Transfer the cells to 15 mL Falcon tubes and centrifuge (1,400 rpm, 3 min). Discard the supernatant while allowing the cell pellet to remain submerged. Separate the pellet by gently tapping the Falcon tube on a vortex mixer and rinse with 4 mL of cold PBS (Mg-free). 2+ and Ca 2+ Wash cells with 2% FBS. After washing, resuspend cells in 3 mL of cold PBS (Mg2+-free). 2+ and Ca 2+ The cells were centrifuged again and resuspended in 200 μL of cold PBS (2% FBS) and aliquoted into three round-bottom FACS tubes (1 mL / tube). The cells were then centrifuged again and resuspended in 200 μL of cold PBS (MgCl2-free). 2+ and Ca 2+ In 2% FBS or 200 μL antibody solution; in 195 μL cold PBS (Mg2+-free) 2+ and Ca 2+The cells contained 5 μL of antibody in 2% FBS. APC mouse IgG1,κ isotype CtrlFC (#400122, Biolegend) was used as an isotype control, and APC anti-human EGFR (#352906, Biolegend) was used. Samples were incubated on a drum mixer at 4°C for 30 min. Then, the cells were rinsed with cold PBS (MgCl2-free). 2+ and Ca 2+ Cells were washed 3× with 2% FBS and fixed for 20 min at room temperature with 2% PFA in PBS. Cells were washed 2× with cold PBS and resuspended in 250–350 μL of cold PBS for FACS analysis. Samples were analyzed using the BD FACSCantoII flow cytometry system (BD Biosciences) and FlowJo software. The results of the FACS analysis are summarized in Table A4.

[0606] Table A4. Cell surface expression levels of EGFR and HER2 in various cell lines (mean fluorescence intensity (MFI))

[0607]

[0608] Hemolysis test

[0609] Red blood cells (RBCs) were separated from the erythrocyte sedimentation rate (ESR) amber layer using a Ficoll gradient. The obtained RBC pellet (approximately 4-5 ml) was then rinsed with 50 ml of DPBS (calcium-free). 2+ / Mg 2+ Wash twice with PAN-Biotech GmbH. Cell pellet was obtained by centrifugation at 800 x g for 10 min at room temperature. RBCs were counted and resuspended in DPBS (calcium-free) at 500,000,000 c / ml based on the total cell count. 2+ / Mg 2+ )middle.

[0610] In DPBS (with Ca 2+ / Mg 2+ Saponin dilutions were prepared at a final strength of 1.11X in PAN-Biotech GmbH. For the positive lysis control, saponin was prepared in DPBS. + / +Prepare a 0.02% Triton-X100 solution. Dispense 135 μL of each of the 135 μL RBC suspensions into a 96-well V-bottom plate. Add the RBC suspension to this 15 μL and mix immediately (10 sec – 600 rpm). Incubate the plate at room temperature for 30 min with gentle stirring. Then, rotate the plate at 800 x g for 10 min to precipitate the RBCs, and transfer 100–120 μL of the supernatant to a standard 96-wp (96-well plate). Subsequently, measure the OD at 405 nm using a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, subtract “DPBS only” from all other wells. + / + "The background signal of the well was then used to calculate the percentage of hemolysis before dividing the background correction signal of the processed well by the background correction signal of the 0.02% Triton-X100 well (x100) compared with the background correction signal of the 0.02% Triton-X100 well."

[0611] Table A5. Erythrocytes and HeLa cells treated with SO1861, SO1861 derivatives, QS-21 derivatives and QS-21.

[0612]

[0613]

[0614]

[0615] See molecule 3, also known as SO1861-Ald-EMCH-mercaptoethanol or SO1861-Ald-EMCH (mercaptoethanol).

[0616] Table A6. Erythrocytes and A431 cells treated with SO1861, SO1861 derivatives, QS-21 derivatives, and QS-21

[0617]

[0618]

[0619] Example 4: Critical micelle concentration (CMC) of saponin derivatives

[0620] Materials and methods

[0621] The critical micelle concentrations (CMCs) of saponins derived from soapwort (SO) (Table A7) and soap bark (QS) (Tables A8 and A9) were determined using the method of DeVendittis et al. (A fluorimetric method for the estimation of the critical micelle concentration of surfactants, Analytical Biochemistry, Vol. 115, No. 2, August 1981, pp. 278-286).

[0622] Emission spectra of 8-anilinonaphthalene-1-sulfonic acid (ANS) in purified water (MQ) or PBS (Dubbk PBS+ / +) were determined at saponin dry weight concentrations ranging from 1 to 1400 μM, covering the range below and above the concentration cutoff point (CMC). Above the CMC, the fluorescence yield of ANS increased and the maximum emission wavelength decreased due to the partitioning of the fluorescent dye into the micelles. Fluorescence yield was recorded at an excitation wavelength of 355 nm and an emission wavelength of 460 nm on a Fluoroskan Ascent FL (Thermo Scientific). 6 μg of ANS was used for each sample and measurement at a concentration of 75.86 μM.

[0623] result

[0624] SO1861 saponins

[0625] Chemical modification of functional groups such as aldehydes (Ald) and glucuronic acid (Glu), and removal of acetyl groups (Ac), have shown an impact on the micellar properties of the corresponding saponins. For example... Figure 42 As shown, single modifications of each functional group on SO1861 saponins significantly affected the micelle-forming ability indicated by the slope of the relative fluorescence values ​​of the obtained ANS. Modification with glucuronic acid (SO1861-Glu-AMPD, SO1861-Glu-AEM) clearly resulted in a steeper slope. Figure 42 This resulted in lower CMC values ​​for natural SO1861 at 185 μM. Similar observations have been obtained for SO1861-Ald-EMCH blocked samples. However, modifications to the aldehyde and acetyl groups (SO1861-Ald-OH, SO1861-Ald-EMCH, SO1861-Ac-OH) resulted in significantly flatter slopes. Figure 42 This results in a higher CMC value relative to natural SO1861. SO1861-Ald-EMCH samples are of particular interest because the obtained slope is almost flat and the CMC cannot be determined even at concentrations up to 800 μM.

[0626] For the dual modification of SO1861 saponins ( Figure 43 ) and three modifiers ( Figure 44 Similar observations have been obtained regarding the modification sites. While modifications to glucuronic acid (SO1861-(Ald-OH)-(Glu-AMPD), SO1861-(Ac-OH)-(Glu-AMPD), SO1861-(Glu-AEM)-(Ac-OH), SO1861-(Glu-AEM)-(Ald-OH), SO1861-(Ald-EMCH)-(Glu-AMPD)) resulted in steeper ANS fluorescence yield slopes and therefore lower CMC values, modifications to the aldehyde and acetyl positions (SO1861-(Ald-OH)-(Ac-OH), SO1861-(Ald-EMCH)-(Ac-OH)) resulted in flatter ANS fluorescence yield slopes and therefore increased CMC values ​​relative to native SO1861 (Table A7).

[0627] When comparing the trimodified saponins SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) and SO1861-(Ald-OH)-(Ac-OH)-(Glu-AMPD), modification of glucuronic acid at SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) results in a flatter slope for the corresponding ANS fluorescence yield, while modification of glucuronic acid at SO1861-(Ald-OH)-(Ac-OH)-(Glu-AMPD) results in a steeper slope for the corresponding ANS fluorescence yield relative to natural SO1861. Figure 44 These results demonstrate the importance of modification at the aldehyde and / or acetoxy positions when CMC is taken into account, because even in Glu-modified derivatives (which have a lower CMC than free saponins), the aldehyde and / or acetoxy modifications can increase CMC when CMC is taken into account, at least partially mitigating the negative effects of Glu modification.

[0628] Table A7. CMC values ​​of SO saponins determined in PBS

[0629]

[0630] QS saponins

[0631] The CMC values ​​for saponins derived from the soapberry tree (QS), specifically QS7, QS17, QS18, QS21 Frac, and QS21 SP, have been determined as shown in Table A8. Figure 45As shown, the slope of the ANS fluorescence yield of the corresponding QS saponins corresponds to the obtained CMC values. The obtained CMC values ​​show a decreasing trend from the highest CMC value of QS21 SP (49 μM) to QS-17, QS-18, and QS-21Frac, all exhibiting similar CMC values ​​at approximately 70 μM. Finally, for QS-7, a CMC value of 230 μM was obtained.

[0632] When comparing the ANS fluorescence yield of QS21 SP measured in purified water (MQ) and PBS, the slope in purified water (MQ) was slightly steeper, resulting in a slightly higher expected CMC value in purified water. Figure 46 (Table A9).

[0633] For the single-modified QS21 saponin QS21-Ald-EMCH (molecule 30; Figure 40B ), QS21-Glu-AEM, QS21-(Ald-OH) and QS21-Glu-AMPD( Figure 47A , Figure 47B ), AMPD modification only on glucuronic acid (QS21-Glu-AMPD, Figure 47B This results in a steeper slope for ANS fluorescence production compared to native QS21, leading to a lower CMC value of 40 μM (Table A9). All other QS21 monomodifications at the glucuronic acid (QS21-Glu-AEM) and aldehyde positions (QS21-(Ald-OH), QS21-Ald-EMCH) result in a flatter slope for ANS fluorescence production than native QS21. Figure 47B ).

[0634] Similar to the discovery of double modification of soapwort saponin SO1861, the AldGlu modification of QS21 saponin (QS21-(Ald-OH)-(Glu-AMPD)) was also observed. Figure 40E , molecule 33, Figure 47C This also results in a steeper slope in ANS fluorescence yield relative to native QS21, leading to a lower CMC value of 39 μM (Table A9). All other QS21 double modifications at the glucuronic acid and aldehyde positions (QS21-(Ald-OH)-(Glu-AEM), QS21-Ald-EMCH-(Glu-AEM), ...) Figure 47C It produces a flatter ANS fluorescence yield slope than the natural QS21.

[0635] Table A8. CMC values ​​of QS saponins determined in purified water (MQ).

[0636] Saponin CMC (μM) QS7 230±25 QS21 (Frac) 75±8 QS17 70±7 QS18 68±7 QS21 (SP) 49±5

[0637] Table A9. CMC values ​​of modified QS21 saponins determined in PBS

[0638]

[0639]

[0640] Example 5: Endosomal escape enhancement activity of SO1861 and SO1861-Ald-EMCH

[0641] Testing SO1861 and SO1861-Ald-EMCH (also known as SO1861-EMCH, for example in...) Figures 48-58 The ability of SO1861 to enhance the endosome escape of target protein toxins was investigated. To this end, SO1861 or SO1861-Ald-EMCH was titrated onto fixed concentrations of 10 pM cetuximab-saponin (a cetuximab conjugated with the protein toxin, saponin, and containing DAR4) on EGFR-expressing cells (A431). This revealed that SO1861 (IC50 = 800 nM) and SO1861-Ald-EMCH (IC50 = 2000 nM) in combination with 10 μM cetuximab-saponin induced effective cytotoxicity in A431 cells, while SO1861 or SO1861-Ald-EMCH alone did not show cytotoxic activity. Figure 48 ).

[0642] Next, cetuximab-caryophyllin or cetuximab-saponin were titrated on various fixed concentrations of SO1861 or SO1861-Ald-EMCH. This revealed that cetuximab-caryophyllin at low pM concentrations (IC50 = 1 pM) could be titrated in the presence of 4000 nM SO1861-Ald-EMCH, 4829 nM SO1861-Ald-EMCH, or 1500 nM SO1861. Figure 49 ) or cetuximab-saponin (IC50 = 0.5 pM, Figure 50 Effective cell killing. This cell-killing effect was not observed with 300 nM SO1861 or 300 nM SO1861-Ald-EMCH. Figure 49 and 50 ).

[0643] Next, SO1861 or SO1861-Ald-EMCH was titrated onto a fixed concentration of 10 pM EGF caryophyllin (an EGFR-targeting fusion protein toxin) on EGFR-expressing cells (A431). This revealed that the combination of SO1861 (IC50 = 800 nM) and SO1861-Ald-EMCH (IC50 = 2000 nM) with 10 μM EGF caryophyllin induced effective cell killing in A431 cells, while SO1861 or SO1861-Ald-EMCH alone did not show any cell-killing activity. Figure 51 ).

[0644] Next, EGF caryophyllin was titrated on various fixed concentrations of SO1861 or SO1861-Ald-EMCH. This revealed that, in the presence of 4829 nM SO1861-Ald-EMCH or 1500 nM SO1861, EGF caryophyllin at low pM concentrations (IC50 = 0.1 pM) was effective. Figure 52A , Figure 52B Effective cell killing. No such cell-killing effect was observed with 10 nM SO1861 or 300 nM SO1861. Figure 52A , Figure 52B ).

[0645] Next, trastuzumab-caryophyllin or trastuzumab-saponin (trastuzumab conjugated with the protein toxin saponin, possessing DAR4) was titrated onto fixed concentrations of 1500 nM SO1861 or 4000 nM SO1861-Ald-EMCH on HER2-expressing cells (SK-BR-3). This revealed effective cell killing with low pM concentrations of trastuzumab-caryophyllin (IC50 = 0.1 pM) or trastuzumab-saponin (IC50 = 0.1 pM) in the presence of 1500 nM SO1861 or 4000 nM SO1861-Ald-EMCH. Figure 53A , Figure 53B ).

[0646] Figures 48-53B All of these results outlined in the paper demonstrate that SO1861-Ald-EMCH effectively enhances endosome escape and cytoplasmic delivery of the targeted protein toxin, thereby significantly reducing the effective concentration of the targeted protein toxin from the nM range to a low pM range.

[0647] The ability of SO1861-Ald-EMCH to enhance endosome escape from antisense oligonucleotides (BNAs, bridged nucleic acids) targeting HSP27 mRNA was tested. To this end, SO1861-Ald-EMCH was titrated onto EGFR / HER2-expressing cells (A431) at fixed concentrations of 100 nM HSP27BNA, 100 nM cetuximab-HSP27BNA (a cetuximab conjugated with HSP27BNA, possessing DAR4), or 100 nM trastuzumab-HSP27BNA (a trastuzumab conjugated with HSP27BNA, possessing DAR4). This revealed the effectiveness of SO1861-Ald-EMCH (IC50 = 700 nM) in enhancing endosome escape from antisense oligonucleotides (BNAs, bridged nucleic acids) targeting HSP27 mRNA. Figure 54 The combination of 100 nM trastuzumab-HSP27BNA (not shown) induced effective HSP27 gene silencing in A431 cells. SO1861-Ald-EMCH alone did not show HSP27 gene silencing activity. Figure 54 ).

[0648] Next, cetuximab-HSP27BNA (DAR1.5 or DAR4) and trastuzumab-HSP27BNA (DAR4.4) were titrated into various fixed concentrations of SO1861-Ald-EMCH in cells expressing EGFR (A431) or HER2 (SK-BR-3). This revealed that in the presence of 4000 nM SO1861-Ald-EMCH, lower nM concentrations of cetuximab-HSP27BNA (IC50 = 0.5 nM) were effective. Figure 55 In A431 cells, cetuximab-HSP27BNA alone or cetuximab-HSP27BNA+100nM SO1861-Ald-EMCH showed no gene silencing activity or only slight activity (IC50>100nM) at very high concentrations. Figure 55 In SKBR-3 cells, trastuzumab-HSP27BNA (IC50 = 0.5 nM) was effective. Figure 56 In the presence of 4000 nM SO1861-Ald-EMCH, it showed effective HSP27 gene silencing activity, while trastuzumab-HSP27BNA alone or trastuzumab-HSP27BNA+100 nM SO1861-Ald-EMCH showed only slight gene silencing activity (IC50>100 nM). Figure 56 ).

[0649] Next, untargeted HSP27BNA was titrated on fixed concentrations of SO1861-Ald-EMCH in various cell lines. This revealed that HSP27 gene silencing was effective in A431, A2058, and SK-BR3 cells with low nM concentrations of HSP27BNA (IC50(SK-BR3) = 2 nM; IC50(A431) = 10 nM; IC50(A2058) = 10 nM) in the presence of 4000 nM or 4829 nM SO1861-Ald-EMCH, while HSP27BNA alone induced gene silencing only at much higher concentrations (IC50(SK-BR3) = 300 nM; IC50(A431) = 1000 nM; IC50(A2058) > 1000 nM). Figure 57A , Figure 57B and Figure 58 When comparing the activity of HSP27BNA (with and without SO1861-Ald-EMCH) to that of HSP27LNA (LNA, locked nucleic acid), the inventors observed that the endosome escape / gene silencing enhancer was comparable to that of HSP27BNA, but at higher HSP27LNA concentrations ( Figure 58 ).

[0650] All of these demonstrate that SO1861-Ald-EMCH effectively enhances endosome escape and cytoplasmic delivery of both targeted and non-targeted BNA / LNA oligomers, thereby significantly reducing the effective concentrations of both targeted and non-targeted antisense oligomers from the μM range to the low nM range.

[0651] Material

[0652] Trastuzumab (Tras, Roche), Cetuximab (CET), MerckKGaA). Caryophyllin-cys is produced and purchased from Proteogenix, France, and EGF caryophyllin is produced from E. coli according to standard procedures. Cetuximab-saponin and trastuzumab-saponin conjugates are produced and purchased from Advanced Targeting Systems (San Diego, CA).

[0653] method

[0654] Flash Chromatography

[0655] Grace Reveleris C-815 Flash; Solvent delivery system: 3-plunger pump with automatic initiation, 4 independent channels, up to 4 solvents in a single run, automatic line switching when solvent is depleted; maximum pump flow rate 250 mL / min; maximum pressure 50 bar (725 psi); detection: UV 200-400 nm, combining up to 4 UV signals and scanning the entire UV range, ELSD; column size: 4-330 g on the instrument, luer type, 750 g, up to 3000 g, with optional retainer.

[0656] HSP27BNA oligomer sequence

[0657] HSP27 BNA oligomer (5′-GGCacagccagtgGCG-3′), ordered from Bio-Synthesis Inc. (Lewisville, Texas) with or without a 5′-thiol C6 linker, according to Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B Liaol, P Kraft, R Bandaru, Y Wu, LM Greenberger and ID Horak, Down-modulation of cancer targets using locked nucleic acid (LNA)-based antisense oligonucleotiddes without transfection, Gene Therapy (2011) 18, 326-333] ([SEQ-ID NO: 2]).

[0658] RNA isolation and gene expression analysis

[0659] RNA from cells was isolated and analyzed according to the standard protocol (Biorad). The qPCR primers used are shown in Table A10.

[0660] Table A10. Primers used for qPCR are shown below.

[0661]

[0662] Trastuzumab-Saponin and Cetuximab-Saponin Synthesis

[0663] Customized mAb-saponin conjugates are generated and purchased from Advanced Targeting Systems (San Diego, CA).

[0664] Synthesis of trastuzumab-caryophyllin and cetuximab-caryophyllin

[0665] Caryophyllin-Cys (17.0 ml, ~9.6 mg) was concentrated by ultrafiltration using a Vivaspin T15 filter tube (3,000 g, 20 °C, 10 min). The resulting 3.25 ml aliquots were filtered through a zeba 10 ml rotary column gel filter and eluted with TBS at pH 7.5.

[0666] Trastuzumab (mAb) or cetuximab (mAb) (0.30 ml, ~10 mg) was diluted to 10 mg / ml with DPBS pH 7.5, eluted and desalted with DPBS pH 7.5 using a 5 ml Zeba spin column, and normalized to 2.50 mg / ml. Freshly prepared SMCC solution (1.00 mg / ml, 4.20 molar equivalents, 13.9 × 10⁻⁶ mcg) in DMSO was added to the aliquots of the mAb. -5 Aliquots of sample (mmol) were briefly vortexed and then incubated at 20°C for 60 minutes using a roller mixer. Subsequently, a freshly prepared glycine solution (2.0 mg / ml, 5.0 molar equivalent, 69.5 × 10⁻⁶ mmol) in DPBS at pH 7.5 was added. -5 The reaction was quenched by aliquots of sample (mmol). A 10 mL Zeba rotary column was used, eluted with TBS pH 7.5, and gel filtered to obtain mAb-SMCC (4.27 mg, 2.80 × 10⁻⁶ mmol). -5 (mmol, 1.514 mg / ml).

[0667] Caryophyllin-Cys (7.54 mg, 25.3 × 10⁻⁶) -5 Freshly prepared TCEP solution (1.00 mg / ml, 0.5 molar equivalent, 12.6 × 10⁻⁶ mmol, 2.258 mg / ml) at TBS pH 7.5 was added to the solution. -5 Aliquots of the sample (mmol) were briefly vortexed and then incubated at 20°C for 60 minutes using a roller mixer. The mixture was then eluted with TBS pH 7.5 using a 10 mL Zeba rotating column and obtained caryophyllin-SH (6.0 mg, 20.2 × 10⁻⁶ mmol) by gel filtration. -5 mmol, 1.722 mg / ml, caryophyllin: SH = 1.1).

[0668] An aliquot of caryophyllin-SH (7.20 molar equivalents) was added to the main mAb-SMCC, the mixture was briefly vortexed, and then incubated overnight at 20°C. After 16 hours, the mixture was further incubated by adding freshly prepared NEM solution (2.50 mg / ml, 5.0 molar equivalents, 101 × 10⁻⁶) at TBS pH 7.5. -5 The reaction was quenched by aliquots of mmol. The reaction mixture was filtered to 0.45 μm and then concentrated to <2 ml by ultrafiltration using a Vivaspin T15 filter tube (3,000 g, 20 °C, 15 min). The conjugate was purified by gel filtration using a 1.6 × 35 cm Superdex 200 PG column, eluted with DPBS pH 7.5.

[0669] Antibody-(L-HSP27 BNA) n [On HSP27BNA disulfide]

[0670] Trastuzumab (L-HSP27) 4 Cetuximab-(L-HSP27) 4 Synthesized via PEG4-SPDP (containing DAR4) and cetuximab-(L-HSP27). 2 Synthesized via PEG4-SPDP (with DAR2)

[0671] Trastuzumab, cetuximab, is referred to below as "Ab". Ab is conjugated to the HSP27 BNA disulfide via a tetra(ethylene glycol)succinimide-3-(2-pyridyl dithio)propionate (PEG4-SPDP) linker, thereby forming an unstable (L) disulfide bond between Ab and HSP27 BNA. This procedure provides an exemplary description for trastuzumab-(L-HSP27 BNA)4:

[0672] HSP27 BNA disulfide oligomer (2.7 mg, 470 nmol, 6.10 mg / ml) was reacted with TCEP (10 molar equivalents, 4.7 μmol, 1.34 mg, 50 mg / ml) at 20 °C for 30 min while being mixed by roller mixing. The oligomer-SH was then purified by elution to TBS pH 7.5 using a PD10 G25 desalting column and used rapidly. Oligomer-SH (2.48 mg, 90%, 1.24 mg / ml, SH to oligomer ratio = 0.8) was obtained.

[0673] Trastuzumab (1.5 mg, 10.3 nmol, 2.50 mg / ml) was reacted with an aliquot of freshly prepared PEG4-SPDP solution (6.81 molar equivalents, 70.1 nmol, 39 μg) in DMSO (1 mg / ml) at 20 °C for 60 min, while being mixed by roller mixing. The reaction was then quenched with glycine (15.1 μl 2 mg / ml freshly prepared solution in TBS pH 7.5), followed by desalting via a Zeba desalting column and elution with TBS pH 7.5. The resulting Tras-S-PEG4-SPDP aliquots were collected and analyzed by UV-Vis. Pyridine-2-thione (PDT) was released using TCEP, and SPDP incorporation was determined by UV-Vis analysis at 343 nm (SPDP to Ab ratio: 4). The remaining Tras-(S-PEG4-SPDP)4 was reacted with an aliquot of freshly prepared HSP27 oligonucleotide (oligomer-SH) (8 molar equivalents, 82.4 nmol, 1.24 mg / ml) and incubated overnight at 20°C with roller mixing. After 17 hours, the conjugate was analyzed by UV-vis analysis to determine the incorporation of HSP27 by displacing pyridine-2-thione (PDT) at 343 nm. The crude conjugate was purified using a 1.6 × 33 cm Sephadex G50 column with elution in DPBS pH 7.5. The resulting trastuzumab-(L-HSP27)4 was a single fraction. Yield: nd. Purity: 96%, HSP27 BNA to Ab ratio = 4.4.

[0674] Example 6 - Enhanced endosome escape activity of saponins

[0675] Previously, the efficacy of various saponins (SO1861, SA1642) was assessed by co-administering them as "free" unconjugated molecules with ligand toxin fusions (e.g., EGF caryophyllin) or antibody-protein toxin conjugates to cells, resulting in enhanced cytotoxic activity in cells expressing the target. Here, in HeLa (EGFR... + On cells, three different saponin molecules (SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904) isolated from the root extract of *Sapindus mukorossi* were titrated in the presence and absence of a non-effectively fixed concentration of 1.5 pM EGF caryophyllin. This revealed a strong enhancement of cytotoxic activity (IC50 = 300 nM) in all tested saponin variants compared to treatments without EGF caryophyllin. Figure 63ANext, EGF caryophyllin was titrated with a fixed concentration of saponin (~1000 nM), and this revealed a strong enhancement of targeted cell killing (IC50 = 0.4 pM) observed at low concentrations of EGF caryophyllin for all the saponins used: SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904. Figure 63B EGF-caryophyllin alone can only induce cell killing at very high concentrations (IC50 = 10,000 pM). This indicates that these specific types of saponins have an inherent ability to effectively induce endosome escape even with very small amounts of available targeted toxins.

[0676] To extend this assay, saponins from other sources were analyzed. A saponin (GE1741) purified from the root extract of *Gypsophila elegans* M. Bieb. was titrated on HeLa cells in the presence and absence of 1.5 pM EGF caryophyllin and compared with purified SO1861. GE1741 also enhanced EGF caryophyllin-induced HeLa cell killing, but showed slightly lower efficacy compared to SO1861. (GE1741 IC50 = 800 nM;) Figure 63C It also showed high general toxicity (IC50 = 5.000 nM in the absence of EGF caryophyllin); Figure 63C In a similar experiment, co-administration of different partially purified soapberry saponin mixtures (QSmix1-3) with 1.5 pM EGF caryophyllin on HeLa cells showed that two of the three (QSmix1 and QSmix3) had similar activities to SO1861 (IC50 QSmix1 / QSmix3 = 300 nM). Figure 63D QSmix(2) showed low efficiency in enhancing 1.5 pM EGF caryophyllin-induced cell killing (IC50 = 2000 nM). Figure 63D However, no general toxicity was observed. This indicates that specific types of saponins can also be obtained from the QS extract, which effectively induce endosome escape from the target ligand toxin EGF caryophyllin. Therefore, the saponins described in this embodiment, such as saponins GE1741, SO1861, SO1862, SO1832, and SO1904, are particularly attractive saponins derived according to the present invention.

[0677] Example 7 - Endosomal escape enhancement activity of saponins and saponin derivatives

[0678] Unstable / acid-sensitive derivatizations (Ald-EMCH or SO1861-L-N3 (also known as SO1861-N3 and SO1861-azide or SO1861-N3 / azide) are applied to SO1861 via an aldehyde group, producing SO1861-Ald-EMCH or SO1861-L-N3. To verify the activity of SO1861-Ald-EMCH, the molecule was titrated in EGFR-expressing (A431, HeLa) and non-expressing (A2058) cells with and without fixed, ineffective (1.5 pM) concentrations of EGF caryophyllin. In all three cell lines, SO1861 alone showed a strong reduction in cell viability, while SO1861-Ald-EMCH as a single compound showed toxicity up to 25,000 nM. Figures 64A-64C When SO1861-Ald-EMCH is combined with 1.5 pM EGF caryophyllin, in EGFR + A strong reduction in target-specific cell viability was observed in A431 and HeLa cells (IC50 = 3.000 nM); Figure 64A , Figure 64B ), while EGFR-A2058 cells were completely unaffected. Figure 64C Similar results were obtained for SO1861-L-N3. Co-administration of SO1861-L-N3 with 1.5 pM EGF caryophyllin also showed effective cytotoxicity against A431 and HeLa cells (IC50 = 3.000 nM), but without EGF caryophyllin, general toxicity was observed above 10.000 nM. Figure 64D , Figure 64E ).

[0679] HATU is conjugated to SO1861 via its carboxylic acid group, producing SO1861-(S), also known as SO1861-HATU and SO1861-Glu-HATU. To determine activity, different concentrations of SO1861-(S) were co-administered with 1.5 pM EGF caryophyllin, and its cytotoxic activity in EGFR-expressing HeLa cells was tested. SO1861-(S) showed similar activity to SO1861, indicating that the conjugation to the carboxylic acid group does not affect the endosome escape enhancement efficacy of this molecule, similar to what was observed with SO1861-Ald-EMCH. Figure 65 ). sequence list <110> SAPREME TECHNOLOGIES BV Charité University of Berlin Medical School <120> Saponin derivatives that improve the therapeutic window <130> P6092645PCT <150> NL2025904 <151> June 24, 2020 <150> NL2023568 <151> 2019-07-25 <150> PCT / EP2019 / 084210 <151> 2019-12-09 <150> PCT / EP2019 / 084290 <151> 2019-12-09 <150> PCT / EP2019 / 084292 <151> 2019-12-09 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Customized peptides <400> 1 Ser Glu Ser Asp Asp Ala Met Phe Cys Asp Ala Met Asp Glu Ser Asp 1 5 10 15 Ser Lys <210> 2 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> HSP27 BNA oligomer <400> 2 ggcacagcca gtggcg 16 <210> 3 <211> 16 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> HSP27 LNA oligomer <400> 3 ggcacagcca gtggcg 16 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> HSP27 primer forward direction <400> 4 gcagtccaac gagatcacca 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> HSP27 primer reverse <400> 5 taaggcttta cttggcggca 20

Claims

1. A chemically modified saponin based on SO1861 saponin comprising a triterpene aglycone core structure and a first sugar chain and a second sugar chain attached to the aglycone core structure, wherein the chemically modified saponin corresponds to the chemically modified saponin represented by molecule 1: (molecule 1) wherein the aglycone core structure of molecule 1 is quillaic acid and R is defined as hydroxyl; wherein the first sugar chain A1 is Gal-(1→2)-[Xyl-(1→3)]-GlcA-, and the second sugar chain A2 is Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-; and wherein the saponin is selected from the group consisting of: molecule 3 molecule 3A molecule 6 molecule 8 molecule 9 molecule 10 molecule 11 molecule 14 molecule 15 molecule 18 molecule 19 molecule 20.

2. A first pharmaceutical composition comprising the chemically modified saponin of claim 1 and optionally a pharmaceutically acceptable excipient and / or diluent.

3. A pharmaceutical combination comprising: the first pharmaceutical composition of claim 2; and a second pharmaceutical composition comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate, and optionally comprising a pharmaceutically acceptable excipient and / or diluent.

4. A third pharmaceutical composition comprising the chemically modified saponin of claim 1 and further comprising any one or more of an antibody-toxin conjugate, a receptor-ligand-toxin conjugate, an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-nucleic acid conjugate or a receptor-ligand-nucleic acid conjugate, and optionally a pharmaceutically acceptable excipient and / or diluent.

5. The pharmaceutical combination according to claim 3 or the third pharmaceutical composition according to claim 4, wherein the second pharmaceutical composition or the third pharmaceutical composition comprises any one or more of an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate or a receptor-ligand-oligonucleotide conjugate, wherein the drug is a toxin, a saporin or a bryodin, and wherein the oligonucleotide is an siRNA or a BNA, or an siRNA or BNA for gene silencing of apolipoprotein B or HSP27.

6. The pharmaceutical composition according to claim 3 or claim 5, or the third pharmaceutical composition according to claim 4, wherein the antibody-drug conjugate is an antibody, IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more VH domains, a single-domain antibody, or V HH Or camel family V H Conjugates.

7. Use of the first pharmaceutical composition of claim 2, the pharmaceutical combination of any one of claims 3 or 5-6 or the third pharmaceutical composition according to any one of claims 4-6 for the manufacture of a medicament.

8. Use of the first pharmaceutical composition of claim 2, the pharmaceutical combination of any one of claims 3 or 5-6 or the third pharmaceutical composition according to any one of claims 4-6 for the manufacture of a medicament for the treatment or prevention of a cancer, an infectious disease, a viral infection, a hypercholesterolemia, a primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis or an autoimmune disease.

9. An in vitro or ex vivo method for the transfer of a molecule from the outside of a cell to the inside of the cell, or to the cytosol of the cell, for non-therapeutic or diagnostic purposes, comprising the steps of: a) providing a cell; b) providing said molecule for transfer from outside of said cell into said cell provided in step a); c) providing a chemically modified saponin according to claim 1 ; d) contacting said cell of step a) in vitro or ex vivo with said molecule of step b) and said chemically modified saponin of step c), thereby establishing transfer of said molecule from outside of said cell into said cell.

10. The method according to claim 9, wherein said cell is a human cell, and / or wherein said chemically modified saponin is a chemically modified saponin according to claim 1, and / or wherein the molecule of step b) is any one of an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate or a receptor-ligand-oligonucleotide conjugate, wherein said drug is a toxin, and wherein said oligonucleotide is an siRNA or a BNA.

11. The method according to claim 9, wherein said cell is a T cell, a NK cell or a tumor cell, and / or wherein said chemically modified saponin is a chemically modified saponin according to claim 1, and / or wherein the molecule of step b) is any one of an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate or a receptor-ligand-oligonucleotide conjugate, wherein said drug is a toxin, and wherein said oligonucleotide is an siRNA or a BNA.

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