Saponin derivatives with improved therapeutic concentration ranges

Abstract

This invention provides saponin derivatives with improved therapeutic concentration ranges. [Solution] The present invention relates to a saponin-based saponin derivative comprising a triterpene aglycone and a first glycan and / or a second glycan, wherein the saponin derivative comprises: an aglycone core structure containing a derivatized aldehyde group; and / or the first glycan, wherein the first glycan contains a derivatized carboxyl group; and / or the second glycan, wherein the second glycan contains at least one derivatized acetoxy group. The present invention also provides use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to a saponin-based saponin derivative comprising a triterpene aglycone and a first glycan and / or a second glycan, wherein the saponin derivative comprises: an aglycone core structure containing a derivatized aldehyde group; and / or the first glycan, wherein the first glycan contains a derivatized carboxyl group; and / or the second glycan, wherein the second glycan contains at least one derivatized acetoxy group. The present invention also relates to a first pharmaceutical composition comprising the saponin derivative of the present invention. In addition, 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 the antibody-toxin conjugate, receptor-ligand-toxin conjugate, antibody-drug conjugate, receptor-ligand-drug conjugate, antibody-oligonucleotide conjugate, or receptor-ligand-oligonucleotide conjugate. The present invention also relates to the first pharmaceutical composition or the pharmaceutical combination of the present invention for use as a pharmaceutical or for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases. Furthermore, the present invention relates to an in vitro or ex vivo method for transferring a molecule from outside a cell into the cell, comprising contacting the cell with the molecule and the saponin derivative of the present invention. [Background technology]

[0002] Targeted oncology is a cancer therapy that uses drugs to target specific genes and proteins involved in the proliferation and survival of cancer cells. Immunotoxins are highly promising because they are targeted toxins containing antibodies as the targeting moiety, possessing the specificity of antibodies against tumor-specific antigens, thereby directing the toxin to the desired site of action, and potentially introducing additional cell-killing mechanisms such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. For this effect to be observed, the toxin needs to be released into the cytosol after internalization. The main drawback is that the target moiety carrying the payload is often not sufficiently internalized and is either recycled directly to the surface after internalization or degraded in lysosomes, thereby hindering sufficient delivery of the payload into the cell cytosol. High serum levels of targeted toxins are required to ensure toxic payload concentrations for tumor cells and to overcome insufficient cytosolic entry, often resulting in serious side effects, particularly immunogenicity and vasoleakage syndrome. Therefore, when treating cancer patients with antibody-drug conjugates (ADCs), a sufficiently broad therapeutic concentration range remains important.

[0003] To address the drawback of insufficient cytosolic entry, several strategies have been developed, for example, involving redirection of toxins into endogenous cell membrane transport complexes in biosynthetic pathways, endosomal disruption, attenuation of endosomal membrane integrity, or the use of cell-permeable peptides.

[0004] For example, glycosylated triterpenes such as saponins have been found to act as endosomal escape promoters for targeted toxins in tumor therapy, such as ribosome-inactivating proteins (RIPs). Analysis of the structure-activity relationship of saponins has shown that the presence of the following core structural elements appears to be beneficial for the saponin's ability to enhance the cytotoxicity of RIPs (see Equation (I), X). 1 =H or OH, and X 2=Polysaccharide moiety): - A branched trisaccharide at C-3 containing glucuronic acid - An aldehyde at C-4 - A carboxyl group at C-28 - A polysaccharide moiety attached to the C-28 position (R) consisting of at least four sugar moieties containing an acetyl group 2 ).

[0005] [ka]

[0006] In particular, the triterpenoid saponin, SO1861 (formula II, sometimes also referred to as SPT001), was identified as a potent molecule for promoting the endosomal exodus of tumor cell-targeting toxins. A dual mechanism of action is hypothesized for the promoter: firstly, a direct increase in endosomal exodus results in caspase-dependent apoptosis, which, secondly, combines with lysosome-mediated cell death pathways, triggered after the release of cathepsins and other hydrolytic enzymes following the disruption of the lysosomal membrane.

[0007] [ka]

[0008] The application of saponins as endosomal escape promoters is based on the recognition that these saponins have the ability to rupture red blood cell membranes. However, precisely at the same time, the cell-rupture activity of saponins contributes to the risk of side effects when patients are treated with such saponins, thereby affecting the optimal therapeutic range in terms of limiting the therapeutic index. In fact, the toxicity of such saponins is a concern when considering, for example, the optimal administration plan, route of administration, and frequency when they are administered extracellularly and / or intracellularly to patients requiring antitumor therapy.

[0009] All characteristics of the chemical composition of the saponin itself, including the structure of the triterpene backbone, the pentacyclic C30 terpene skeleton (also known as sapogenin or aglycone), the number and length of the sugar side chains, and the type and binding variants of the sugar residues bound to the backbone, contribute to the hemolytic index and / or cytotoxicity of such saponins.

[0010] Saponins, when considering cellular endosomes and cytosols, lack target specificity in themselves, and even with the same administration route, saponins are distributed within (human) patients, as expected and most often, by kinetics other than those of targeted toxins. Therefore, after administering a therapeutic combination, for example, ADC and saponin, to patients in need, it is implied that saponin molecules are found in all organs, and specificity is mediated solely by targeted toxins. Systemic distribution of saponins requires higher concentrations for successful treatment compared to specific accumulation in target cells. Therefore, to achieve an appropriate therapeutic concentration range, the toxicity of modified saponins must be sufficiently low for successful application from the perspective of systemic saponin administration in the body.

[0011] Therefore, if, for example, co-administration of saponins with ADCs is considered, it is still necessary to improve the therapeutic index: if the enhancement of the cytotoxic effect of ADCs is considered, it is necessary to better control (or better: lower) the cytotoxicity of saponins while simultaneously maintaining sufficient efficacy. [Overview of the Initiative] [Means for solving the problem]

[0012] To our surprise, the inventors have found that modified saponins, namely, - A branched trisaccharide moiety containing a modified glucuronic acid bonded at C-3 of the saponin aglycone; and / or - A modified aldehyde at C-4 of the saponin aglycone; and / or - A polysaccharide moiety bonded at C-28 of the saponin aglycone, and containing a modified acetoxy group in the said polysaccharide moiety. Saponin derivatives having the following characteristics: We have found that the saponin derivative has reduced toxicity when the cell viability of cells in contact with it is considered, has activity (although we do not wish to be bound by any particular theory: in relation to the similar or improved endosomal escape-promoting activity of modified saponins) when the enhancement of cytotoxicity or BNA-mediated gene silencing is considered, and / or has reduced hemolytic activity compared to the toxicity, activity, and hemolytic activity of unmodified saponins. Thus, we provide a saponin derivative having an improved therapeutic concentration range because the ratio between cytotoxicity and IC50 values ​​for, for example, toxin enhancement or gene silencing is increased, and / or because the ratio between saponin hemolytic activity and IC50 values ​​for, for example, toxin enhancement or gene silencing is increased.

[0013] A first aspect of the present invention is a saponin-based saponin derivative comprising a triterpene aglycone core structure and at least one of a first glycan and a second glycan bonded to the aglycone core structure, wherein: i. the saponin derivative comprises an aglycone core structure containing a derivatized aldehyde group; or ii. the saponin derivative comprises the first glycan, wherein the first glycan contains a carboxyl group, preferably a derivatized carboxyl group of a glucuronic acid moiety; or iii. the saponin derivative comprises the second glycan, wherein the second glycan contains at least one derivatized acetoxy (Me(CO)O-) group; or iv. The saponin derivative comprises any combination of derivatization i.ii. and iii., preferably any two combinations of derivatization i.ii. and iii., wherein the first glycan and the second glycan are independently selected from monosaccharides, linear oligosaccharides, and branched oligosaccharides.

[0014] One embodiment of the present invention is a saponin derivative in which the saponin derivative is a monodesmoside triterpene glycoside or a bisdesmoside triterpene glycoside, more preferably a bisdesmoside triterpene glycoside.

[0015] A second aspect of the present invention relates to a first pharmaceutical composition comprising the saponin derivative of the present invention and optionally a pharmaceutically acceptable excipient and / or diluent.

[0016] A third aspect of the present invention is a combination of pharmaceuticals: · The first pharmaceutical composition of the present invention; and The present invention relates to a pharmaceutical combination comprising a second pharmaceutical composition comprising any one or more of the following: 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 pharmaceutically acceptable excipients and / or diluents.

[0017] A fourth aspect of the present invention relates to a third pharmaceutical composition comprising the saponin derivative of the present invention, and further comprising any one or more of the following: antibody-toxin conjugates, receptor-ligand-toxin conjugates, antibody-drug conjugates, receptor-ligand-drug conjugates, antibody-nucleic acid conjugates, or receptor-ligand-nucleic acid conjugates, and optionally comprising pharmaceutically acceptable excipients and / or diluents.

[0018] A fifth aspect of the present invention relates to the first pharmaceutical composition of the present invention, the pharmaceutical combination of the present invention, or the third pharmaceutical composition of the present invention for use as a pharmaceutical.

[0019] A sixth aspect of the present invention relates to the first pharmaceutical composition, the pharmaceutical combination, or the third pharmaceutical composition of the present invention for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

[0020] A seventh aspect of 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 the cell, preferably into the cytosol of the cell, comprising: a) providing a cell; b) providing a molecule to be transferred from the outside of the cell to the inside of the cell provided in step a); c) providing a saponin derivative of the present invention; and d) contacting the cell from step a) with the molecule from step b) and the saponin derivative from step c) in vitro or ex vivo, thereby establishing the transfer of the molecule from the outside of the cell to the inside of the cell.

[0021] definition The term "saponin" has its usual scientific meaning and, as used herein, refers to a group of amphiphilic glycosides comprising one or more hydrophilic glycone moieties bound to a lipophilic aglycone core that is a sapogenin. Saponins may be naturally occurring or synthetic (i.e., non-natural). The term "saponin" encompasses naturally occurring saponins, derivatives of naturally occurring saponins, and saponins synthesized de novo via chemical and / or biotechnological synthetic pathways.

[0022] The term "modified saponin" has its usual scientific meaning, and in this specification, it refers to a saponin, i.e., a saponin derivative, that has one or more chemical modifications at the positions where an aldehyde group, carboxyl group, acetate group, and / or acetyl group was already present in the non-derivativeized saponin before being subjected to chemical modification for the provision of a modified saponin. For example, a modified saponin is provided by any one or more chemical modifications of an aldehyde group, carboxyl group, acetate group, and / or acetyl group in the saponin on which the modified saponin is based; that is, the saponin is provided, and then one of the aldehyde group, carboxyl group, acetate group, and / or acetyl group is chemically modified, thereby providing a modified saponin. For example, the saponin modified for the provision of a modified saponin is a naturally occurring saponin. Typically, modified saponins are synthetic saponins, and typically, modified saponins are modified versions of natural saponins and therefore derived from natural saponins; however, it is also possible that modified saponins may or may not have natural counterparts derived from synthetic saponins. Typically, modified saponins have no natural counterparts; that is, modified saponins are not naturally produced by, for example, grasses or trees.

[0023] The term "aglycone core structure" has its usual scientific meaning and, as used herein, refers to the aglycone core of a saponin that does not have one or two carbohydrate antennas or sugar chains (glycans) attached to it. For example, chiral acids SO1861, QS-7, and QS21 are aglycone core structures. Typically, the glycans of saponins are monosaccharides or oligosaccharides, such as linear or branched glycans.

[0024] The term "QS21," unless further specified, refers to any one isomer of QS21 having the structural formula shown in Figure 41, as well as a mixture of two or more isomers, including all of the isomers shown in Figure 41. As those skilled in the art will understand, a typical natural extract containing QS21 will contain a mixture of various isomers of QS21. However, a single isomer can be isolated via purification or (semi-)synthetic routes.

[0025] The term "glycan" has its usual scientific meaning and, as used herein, refers to any of the following: a glycan, a carbohydrate antenna, a single sugar moiety (monosaccharide), or a chain containing multiple sugar moieties (oligosaccharide, polysaccharide). A glycan may consist solely of sugar moieties, or it may also include further moieties such as 4E-methoxycinnamic acid, 4Z-methoxycinnamic acid, and any one of 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, as found in, for example, QS-21.

[0026] The term "chemically modified" has its usual scientific meaning and, as used herein, refers to the chemical modification of a first chemical group or first chemical moiety to provide a second chemical group or second chemical moiety. Examples include the chemical modification of a carbonyl group to a -(H)C-OH group, the chemical modification of an acetate group to a hydroxyl group, and the provision of a saponin in which the aldehyde group is conjugated with an N-ε-maleimidocaproic acid hydrazide (EMCH) moiety by a chemical reaction.

[0027] The term "chemically modified aldehyde group" has its usual scientific meaning and, as used herein, refers to a chemical reaction product obtained by a chemical reaction involving the aldehyde group of a saponin, resulting in the substitution of the original aldehyde group with a new chemical group. An example is the formation of a -(H)C-OH group from the original aldehyde group of a saponin.

[0028] The term "chemically modified carboxyl group" has its usual scientific meaning and, as used herein, refers to a chemical reaction product obtained by a chemical reaction involving a carboxyl group of a saponin, for example, the carboxyl group of the glucuronic acid moiety, and a further molecule, resulting in the substitution of the original carboxyl group with a new chemical group. An example is the formation of a conjugate between a saponin and one of the following: 2-amino-2-methyl-1,3-propanediol (AMPD), N-(2-aminoethyl)maleimide (AEM), or 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), involving the carboxyl group of the glucuronic acid of the saponin.

[0029] In the context of glycan nomenclature, the terms "Api / Xyl-" or "Api- or Xyl-" have their usual scientific meanings and, as used herein, refer to glycans that contain either an apiose (Api) moiety or a xylose (Xyl) moiety.

[0030] The term "saponin based on modified saponins" has its usual scientific meaning and, as used herein, refers to a saponin that has been modified for the purpose of providing modified saponins. Typically, the saponin based on modified saponins is a naturally occurring saponin that is subjected to chemical modification for the purpose of providing modified saponins.

[0031] The term “saponin-based modified saponin” has its usual scientific meaning and, as used herein, refers to a saponin subjected to a chemical modification process that provides a modified saponin, where the modified saponin is typically a naturally occurring saponin.

[0032] The term "oligonucleotide" has its ordinary scientific meaning and, as used herein, includes, among other things, synthetic nucleic acids such as BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNA), antisense DNA, antisense RNA, etc., provided as DNA, modified DNA, RNA, mRNA, modified RNA, single-stranded molecules or double-stranded molecules, and refers to any natural or synthetic series of nucleic acids, including DNA, modified DNA, RNA, mRNA, modified RNA, single-stranded molecules or double-stranded molecules, and includes synthetic nucleic acids such as BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNA), antisense DNA, antisense RNA, etc.

[0033] The term "antibody-drug conjugate" or "ADC" has its ordinary scientific meaning and, as used herein, refers to any conjugate of an antibody, such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single-domain antibodies, V HH , camelid V H etc., with any molecule that can have a therapeutic effect when contacted with the cells of a patient, such as a human patient, for example, a pharmaceutical active ingredient, a toxin, an oligonucleotide, an enzyme, a small molecule drug compound, etc.

[0034] The term "antibody-oligonucleotide conjugate" or "AOC" has its ordinary scientific meaning and, as used herein, refers to any conjugate of an antibody, such as IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more V H domains, single-domain antibodies, V HH , camelid V H etc., with an oligonucleotide molecule selected from any natural or synthetic series of nucleic acids that can have a therapeutic effect when contacted with the cells of a patient, such as a human patient, for example, DNA, modified DNA, RNA, mRNA, modified RNA, single-stranded molecules or double-stranded molecules, and includes synthetic nucleic acids such as BNA, antisense oligonucleotides (ASO), short or small interfering RNAs (siRNA; silencing RNA), antisense DNA, antisense RNA, etc.

[0035] The terms “effector molecule” or “effector portion” have their usual scientific meaning, for example, when referring to an effector molecule as a portion of a covalent conjugate, and herein, they refer to a molecule that can selectively bind to any one or more target molecules, such as proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, and enzymes, and modulate the biological activity of such one or more target molecules. An effector molecule is a molecule selected from any one or more of the following: small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, xenonucleotides or siRNA, enzymes, peptides, proteins, or any combination thereof. Therefore, for example, an effector molecule or effector moiety is a molecule or moiety selected from any one or more of the following: small molecules such as drug molecules, toxins such as protein toxins, oligonucleotides such as BNA, xenonucleotides or siRNA, enzymes, enzymes, peptides, proteins, or any combination thereof, which can selectively bind to any one or more target molecules: proteins, peptides, carbohydrates, sugars such as glycans, (phospho)lipids, nucleic acids such as DNA, RNA, or enzymes, and which, upon binding to the target molecules, modulate the biological activity of such one or more target molecules. Typically, an effector molecule can exert a biological effect inside a cell, such as a mammalian cell, such as a human cell, for example, in the cytosol of said cell. Therefore, typical effector molecules are drug molecules, plasmid DNA, toxins such as toxins contained in antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, and nucleic acids contained in antibody-oligonucleotide conjugates (AOCs). For example, an effector molecule is a molecule that can act as a ligand that can increase or decrease enzyme activity, gene expression, or cellular signaling.

[0036] The term "HSP27" refers to the BNA molecule that silences HSP27 expression in cells.

[0037] The terms “crosslinked nucleic acid” or simply “BNA,” or “locked nucleic acid” or simply “LNA,” have their usual scientific meanings and, as used herein, refer to modified RNA nucleotides. BNA is also referred to as “bound RNA molecule” or “inaccessible RNA molecule.” BNA monomers may contain five-membered, six-membered, or even seven-membered crosslinked structures with “fixed” C3'-endo sugar puckering. The crosslinking is synthetically incorporated at the 2',4'-position of ribose to produce 2',4'-BNA monomers. BNA monomers can be incorporated into oligonucleotide polymer structures using standard phosphoramidite chemistry known in the art. BNA is a structurally rigid oligonucleotide with increased binding affinity and stability.

[0038] The terms used herein and in the claims, such as 1st, 2nd, 3rd, etc., are used, for example, to distinguish between similar elements, compositions, components in a composition, or individual method steps, and are not necessarily used to indicate a sequence or chronological order. These terms are interchangeable under appropriate circumstances, and embodiments of the present invention may be operated in an order other than those described or illustrated herein, unless otherwise specifically specified.

[0039] The embodiments described herein may be operated in combination and in conjunction, unless otherwise specified.

[0040] Furthermore, even if various embodiments are referred to as “preferred,” “for example,” “as an example,” or “especially,” they should be interpreted as exemplary ways in which the present invention can be carried out, rather than as limiting the scope of the invention.

[0041] The term “comprising” as used in the claims should not be interpreted as being limited to, for example, elements or components in a process or composition listed later; it does not exclude other elements or components in a process or composition. It should be interpreted as identifying the presence of the described feature, integer, process, or component as it is written, but not as excluding the presence or addition of one or more other features, integers, processes, or components, or groups thereof. Accordingly, the scope of the expression “a method comprising processes A and B” should not be limited to a method consisting only of processes A and B, but rather, with respect to the present invention, A and B are merely listed among the processes of a method, and furthermore, the claims should be interpreted as including equivalents of these process processes. Accordingly, the scope of the expression “a composition comprising components A and B” should not be limited to a composition consisting only of components A and B, but rather, with respect to the present invention, A and B are merely listed among the components of a composition, and furthermore, the claims should be interpreted as including equivalents of these components.

[0042] In addition, the indefinite article “a” or “an” does not rule out the possibility of two or more elements or components existing, unless the context clearly requires that there be only one and sole element or component. Therefore, the indefinite article “a” or “an” usually means “at least one.” [Brief explanation of the drawing]

[0043] [Figure 1] This is a diagram showing the synthesis of molecule 3A. [Figure 2] This is a diagram showing the synthesis of molecule 6. [Figure 3] This is a diagram showing the synthesis of molecule 8. [Figure 4] This is a diagram showing the synthesis of molecule 9. [Figure 5] This is a diagram showing the synthesis of molecule 10. [Figure 6]This is a diagram showing the synthesis of molecule 11. [Figure 7] This is a diagram showing the synthesis of molecule 12. [Figure 8] This diagram shows the synthesis of molecule 14. [Figure 9] This is a diagram showing the synthesis of molecule 15. [Figure 10] This is a diagram showing the synthesis of molecule 16. [Figure 11] This is a diagram showing the synthesis of molecule 18. [Figure 12] This diagram shows the synthesis of molecule 19. [Figure 13] This is a diagram showing the synthesis of molecule 20. [Figure 14] This is a diagram showing the synthesis of molecule 21. [Figure 15] This figure shows the mass chromatogram of molecule 6. [Figure 16] This figure shows the detailed mass chromatogram of the synthesis of molecule 6, starting from SO1861. [Figure 17] This figure shows the details of the mass chromatogram of molecule 9, starting from molecule 6. [Figure 18A] This figure shows the IC50 curves of the endosome escape-promoting activity of saponin derivatives in EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 18B] This figure shows the IC50 curves of the endosomal escape-promoting activity of saponin derivatives in EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 19A] This figure shows the IC50 curves of the endosome escape-promoting activity of saponin derivatives in EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 19B] This figure shows the IC50 curves of the endosomal escape-promoting activity of saponin derivatives in EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 20A]This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-dianthine. [Figure 20B] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-dianthine. [Figure 21A] This figure shows the IC50 curves of toxicity for saponin derivatives in EGFR-expressing cells (HeLa). [Figure 21B] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (A431). [Figure 22] This figure shows the hemolytic activity of saponin derivatives as measured by a human erythrocyte hemolysis assay. [Figure 23A] This figure shows the IC50 curves of the endosome escape-promoting activity of saponin derivatives in EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 23B] This figure shows the IC50 curves of the endosomal escape-promoting activity of saponin derivatives in EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 24A] This figure shows the IC50 curves of the endosome escape-promoting activity of saponin derivatives in EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 24B] This figure shows the IC50 curves of the endosomal escape-promoting activity of saponin derivatives in EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantin. [Figure 25A] This figure shows the IC50 curves of toxicity for saponin derivatives in EGFR-expressing cells (HeLa). [Figure 25B] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (A431). [Figure 26A]This figure shows the IC50 curves of toxicity for saponin derivatives in EGFR-expressing cells (HeLa). [Figure 26B] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (A431). [Figure 27] This figure shows the hemolytic activity of saponin derivatives as measured by a human erythrocyte hemolysis assay. [Figure 28] This figure shows the hemolytic activity of saponin derivatives as measured by a human erythrocyte hemolysis assay. [Figure 29] This figure shows the hemolytic activity of saponin derivatives as measured by a human erythrocyte hemolysis assay. [Figure 30A] This figure shows the IC50 curves of the activity of saponin derivatives against EGFR-expressing cells (HeLa) in the presence of a non-effective constant concentration of 5 pM EGF-diantine. [Figure 30B] This figure shows the IC50 curves of the activity of saponin derivatives against EGFR-expressing cells (A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantine. [Figure 31A] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (HeLa). [Figure 31B] This figure shows the IC50 toxicity curves of saponin derivatives in EGFR-expressing cells (A431). [Figure 32] This figure shows the hemolytic activity of saponin derivatives as measured by a human erythrocyte hemolysis assay. [Figure 33A] This figure shows the IC50 curves of the endosomal escape-promoting activity of various QS saponin fractions in EGFR-expressing cells (HeLa) in the presence of cetuximab-saporin (Saporin) at a concentration of 5 pM. [Figure 33B] This figure shows the IC50 curves of the escape-promoting activity of various QS saponin fractions in EGFR-expressing cells (A431) in the presence of cetuximab-saporin at a concentration of 5 pM. [Figure 34A]This figure shows the IC50 toxicity curves of the QS saponin fraction in EGFR-expressing cells (HeLa). [Figure 34B] This figure shows the IC50 toxicity curves of the QS saponin fraction in EGFR-expressing cells (A431). [Figure 35] This figure shows the hemolytic activity of the QS saponin fraction as measured by a human erythrocyte hemolysis assay. [Figure 36] This is a diagram showing the synthesis of molecule 23. [Figure 37] This is a diagram showing the synthesis of molecule 25. [Figure 38] This is a diagram showing the synthesis of molecule 27. [Figure 39] This is a diagram showing the synthesis of molecule 28. [Figure 40A] This is a diagram showing the synthesis of molecule 29. [Figure 40B] This is a diagram of QS21-Ald-EMCH (molecule 30). [Figure 40C] This is a diagram of QS21-Glu-AMPD (molecule 31). [Figure 40D] This is a diagram of QS21-(Ald-EMCH)-(Glu-AMPD) (molecule 32). [Figure 40E] This is a diagram of QS21-(Ald-OH)-(Glu-AMPD) (molecule 33). [Figure 41] This figure shows the structures of the QS21 isomers. [Figure 42] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of 1-modified SO1861. [Figure 43] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of 2-modified SO1861. [Figure 44] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of 3-modified SO1861. [Figure 45] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of QS saponin. [Figure 46] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of QS21. [Figure 47A]Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of modified QS21. [Figure 47B] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of 1-modified QS21. [Figure 47C] Measurement of critical micelle concentration: This figure shows the ANS fluorescence yield of 2-modified QS21. [Figure 48] This figure shows the cell viability assay (MTS) of SO1861 or SO1861-EMCH+10pM cetuximab-saporine in A431 cells. [Figure 49] This figure shows the cell viability assay (MTS) results for cetuximab-dianthin + 300 nM and 4000 nM SO1861-EMCH in A431 cells. [Figure 50] This figure shows the cell viability assay (MTS) results for cetuximab-saponin + 300 nM and 1500 nM SO1861 or 4000 nM SO1861-EMCH in A431 cells. [Figure 51] This figure shows the cell viability assay (MTS) of SO1861 or SO1861-EMCH+10pM EGF dianthine in A431 cells. [Figure 52A] This figure shows the cell viability assay (MTS) results for EGF dianthin + 10 nM, 300 nM, and 1500 nM SO1861 in A431 cells. [Figure 52B] This figure shows the cell viability assay (MTS) results for EGF dianthin + 300 nM, 1500 nM SO1861, or 4829 nM SO1861-EMCH in A431 cells. [Figure 53A] This figure shows the cell viability assay (MTS) of trastuzumab-dianthin + 1500 nM SO1861 or 4000 nM SO1861-EMCH in A431 cells. [Figure 53B] This figure shows the cell viability assay (MTS) of trastuzumab-saporin + 1500 nM SO1861 or 4000 nM SO1861-EMCH in A431 cells. [Figure 54]This figure shows the HSP27 mRNA gene silencing analysis in A431 cells using SO1861-EMCH + 100nM HSP27BNA and 100nM cetuximab-HSP27BNA. [Figure 55] This figure shows the HSP27 mRNA gene silencing analysis in A431 cells using cetuximab-HSP27 BNA conjugate (DAR1.5 or DAR4) + 100nM SO1861-EMCH or 4000nM SO1861-EMCH. [Figure 56] This figure shows the HSP27 mRNA gene silencing analysis in SK-BR-3 cells using trastuzumab-HSP27 BNA conjugate (DAR4.4) + 100nM SO1861-EMCH or 4000nM SO1861-EMCH. [Figure 57A] This figure shows the HSP27 mRNA gene silencing analysis of HSP27BNA+4000nM SO1861-EMCH in A431 cells. [Figure 57B] This figure shows the HSP27 mRNA gene silencing analysis of HSP27BNA+4000nM SO1861-EMCH in A2058 cells. [Figure 58] This figure shows the HSP27 mRNA gene silencing analysis of HSP27BNA or HSP27LNA+4829nM SO1861-EMCH in SK-BR-3 cells. [Figure 59] This is a diagram showing the synthesis of molecule 26. [Figure 60] This figure shows a general reaction scheme for the Michael addition reaction of the EMCH maleimide group with a thiol (Figure 60 describes the synthesis of block SO1861-Ald-EMCH (SO1861-Ald-EMCH-mercaptoethanol) when R=CH2-CH2-OH). [Figure 61]This figure shows the MALDI-TOF-MS spectra of (A) 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). [Figure 62A] This figure shows the MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl at pH 3. [Figure 62B] This figure shows the MALDI-TOF-MS spectra of SO1861-EMCH before (A) and after (B) hydrolysis in HCl at pH 3. [Figure 63A] This figure shows non-conjugate saponin-mediated endosomal escape and enhanced target cell killing. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904 with and without 1.5 pM EGF dianthin. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthin and constant concentrations of SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741 with and without 1.5 pM EGF dianthin. D) Analysis of cell viability of HeLa cells (EGFR+) treated with various QSmixes (saponin mixtures derived from Quillaia saponaria) with and without 1.5 pM EGF dianthine. [Figure 63B]This figure shows non-conjugate saponin-mediated endosomal escape and enhanced target cell killing. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904 with and without 1.5 pM EGF dianthin. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthin and constant concentrations of SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741 with and without 1.5 pM EGF dianthin. D) Analysis of cell viability of HeLa cells (EGFR+) treated with various QSmixes (saponin mixtures derived from Quillaia saponaria) with and without 1.5 pM EGF dianthine. [Figure 63C] This figure shows non-conjugate saponin-mediated endosomal escape and enhanced target cell killing. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904 with and without 1.5 pM EGF dianthin. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthin and constant concentrations of SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741 with and without 1.5 pM EGF dianthin. D) Analysis of cell viability of HeLa cells (EGFR+) treated with various QSmixes (saponin mixtures derived from Quillaia saponaria) with and without 1.5 pM EGF dianthine. [Figure 63D]This figure shows non-conjugate saponin-mediated endosomal escape and enhanced target cell killing. A) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904 with and without 1.5 pM EGF dianthin. B) Cell viability analysis of HeLa cells (EGFR+) treated with EGF dianthin and constant concentrations of SO1861, SO1832, SO1862 (isomer of SO1861), or SO1904. C) Cell viability analysis of HeLa cells (EGFR+) treated with SO1861 or GE1741 with and without 1.5 pM EGF dianthin. D) Analysis of cell viability of HeLa cells (EGFR+) treated with various QSmixes (saponin mixtures derived from Quillaia saponaria) with and without 1.5 pM EGF dianthine. [Figure 64A] This figure shows the activity of unconjugated SO1861 versus SO1861-Ald-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-Ald-EMCH with and without 1.5 pM EGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-L-N3 (also referred to as SO1861-N3 or SO1861-N3 / azide) with and without 1.5 pM EGF dianthine. [Figure 64B]This figure shows the activity of unconjugated SO1861 versus SO1861-Ald-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-Ald-EMCH with and without 1.5 pM EGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-L-N3 (also referred to as SO1861-N3 or SO1861-N3 / azide) with and without 1.5 pM EGF dianthine. [Figure 64C] This figure shows the activity of unconjugated SO1861 versus SO1861-Ald-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-Ald-EMCH with and without 1.5 pM EGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-L-N3 (also referred to as SO1861-N3 or SO1861-N3 / azide) with and without 1.5 pM EGF dianthine. [Figure 64D]This figure shows the activity of unconjugated SO1861 versus SO1861-Ald-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-Ald-EMCH with and without 1.5 pM EGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-L-N3 (also referred to as SO1861-N3 or SO1861-N3 / azide) with and without 1.5 pM EGF dianthine. [Figure 64E] This figure shows the activity of unconjugated SO1861 versus SO1861-Ald-EMCH. EGFR-targeted antisense BNA oligo delivery and gene silencing in cancer cells according to the present invention. A, B, C) Cell viability analysis of A431 (EGFR++), HeLa (EGFR+), or A2058 (EGFR-) cells treated with SO1861 or SO1861-Ald-EMCH with and without 1.5 pM EGF dianthine. D, E) Cell viability analysis of A431 (EGFR++) or HeLa (EGFR+) cells treated with SO1861 or SO1861-L-N3 (also referred to as SO1861-N3 or SO1861-N3 / azide) with and without 1.5 pM EGF dianthine. [Figure 65] This figure shows unconjugated SO1861 vs. SO1861-Ald-EMCH (unstable hydrazone binding) vs. SO1861-HATU (also referred to as SO1861-(S) (stable) and SO1861-Glu-HATU). Cell viability analysis of HeLa cells (EGFR+) treated with SO1861, SO1861-Glu-HATU (also referred to as SO1861-(S)(S=HATU)), and SO1861-Ald-EMCH (hydrazone binding between the SO1861 aglycone core and EMCH linker is also referred to as the "unstable linker") with and without EGF dianthine. [Modes for carrying out the invention]

[0044] The present invention is described in relation to specific embodiments, but is not limited thereto, and is limited only by the claims.

[0045] To our surprise, the inventors have found that modified saponins, namely, - A branched trisaccharide moiety containing a modified glucuronic acid bonded at C-3 of the saponin aglycone; and / or - A modified aldehyde at C-4 of the saponin aglycone; and / or - A polysaccharide moiety bonded at C-28 of the saponin aglycone, and containing a modified acetoxy group in the said polysaccharide moiety. Saponin derivatives having the following characteristics: We found that the saponin derivatives exhibit reduced toxicity when the cell viability of cells in contact with them is considered; when one or two of the aforementioned groups in the modified saponin (i.e., the aldehyde group of the aglycone, the carboxyl group of glucuronic acid in the polysaccharide chain linked to C-3 of the aglycone, and the acetyl group in the polysaccharide chain linked to C-28 of the aglycone) are derivatized, they exhibit activity (although we do not wish to be bound to any particular theory: in relation to the similar or improved endosomal escape activity of the modified saponin); and / or, they exhibit reduced hemolytic activity compared to the toxicity, activity, and hemolytic activity of the unmodified saponin. The present inventors thereby provide saponin derivatives having an improved therapeutic concentration range, for the following reasons: the cytotoxicity of the saponin derivatives is lower than that measured for their natural counterparts; the hemolytic activity is lower than that measured for their natural counterparts; and for single-derivative saponins and double-derivative saponins, the ratio of IC50 values ​​between cytotoxicity and, for example, toxin enhancement or gene silencing is similar or increased, and / or the ratio of IC50 values ​​between saponin hemolytic activity and, for example, toxin enhancement or gene silencing is similar or increased. For an overview of exemplary saponin derivatives, see Table A2 in combination with Figures 1-14 and 36-40. For an overview of cytotoxicity, hemolytic activity, and endosomal escape-promoting activity ("activity") measured in various cells, as well as the ratio of IC50 for cytotoxicity to IC50 for activity, and the ratio of IC50 for hemolytic activity to IC50 for activity, see Tables A5 and A6.

[0046] A first aspect of the present invention is a saponin-based saponin derivative comprising a triterpene aglycone core structure (also referred to as "aglycone core") and at least one of a first glycan and a second glycan bonded to the aglycone core structure, wherein: i. the saponin derivative comprises an aglycone core structure containing a derivatized aldehyde group; or ii. the saponin derivative comprises the first glycan, wherein the first glycan contains a carboxyl group, preferably a derivatized carboxyl group of a glucuronic acid moiety; or iii. the saponin derivative comprises the second glycan, wherein the second glycan contains at least one derivatized acetoxy (Me(CO)O-) group; or iv. The saponin derivative comprises any combination of derivatization i.ii. and iii., preferably any two combinations of derivatization i.ii. and iii., wherein the first sugar chain and the second sugar chain are independently selected from monosaccharides, linear oligosaccharides, and branched oligosaccharides.

[0047] One embodiment of the present invention is a saponin derivative in which the saponin derivative is a monodesmoside triterpene glycoside or a bisdesmoside triterpene glycoside, more preferably a bisdesmoside triterpene glycoside.

[0048] Surprisingly, any one, two, or three modifications (derivativeization) of the aldehyde group at C-23 of the saponin aglycone, the carboxyl group at the sugar moiety at C-3 of the aglycone, i.e., the glucuronic acid moiety, and the acetyl group at the sugar unit of the (oligo-)sugar moiety linked at C-28 of the saponin aglycone core result in reduced cytotoxicity when such saponin derivatives come into contact with cells, i.e., various types of cells. The reduction in cytotoxicity has been confirmed by the inventors for a series of various saponin derivatives listed in Tables A2, A3, and Figures 1-14 and 36-40. Therefore, the provision of these series of saponin derivatives with reduced cytotoxicity is part of the present invention, where reduced cytotoxicity is compared to the cytotoxicity measured for the unmodified natural saponin counterparts. Saponin derivatives can be formed from such natural saponins. Typically, the saponin derivatives of the present invention involve one, two, or three derivatizations compared to their naturally occurring counterparts, such as SO1861 and QS-21 (isomers). When reduction of cytotoxicity is considered, saponin derivatives containing one, two, or three modifications (derivativeizations) at the positions within the saponin molecule outlined herein are equally suitable when a saponin with reduced cytotoxicity is to be provided. Furthermore, the inventors have surprisingly confirmed that a wide variety of different modifications are suitable for reducing cytotoxicity, reducing hemolytic activity, and maintaining and preserving a sufficient degree of endosomal escape-promoting activity. When hemolytic activity is considered, as with the reduction of cytotoxicity, hemolytic activity is reduced when one, two, or three of the indicated chemical groups of the saponin are derivatized. Such derivatizations may be of various properties, for example, outlined in Tables A2 and A3, and Figures 1-14 and 36-40. Both small-scale derivatization, such as the derivatization of an aldehyde group to a hydroxyl group by reduction, and large-scale derivatization, such as the derivatization of an aldehyde group by EMCH in combination with the derivatization of the carboxyl group of glucuronic acid by AEM, reduce cytotoxicity and hemolytic activity.It is clear that, in order to provide saponins that exhibit improved cytotoxicity in terms of reduced cytotoxicity and improved hemolytic activity in terms of reduced hemolytic activity compared to naturally occurring saponin counterparts, any one or more of the three chemical groups of a saponin, for example, one, two, or three, can be derivatized with a wide variety of chemical groups of different sizes and / or different chemical properties.

[0049] While we do not wish to be bound by any theory, it is hypothesized that the aldehyde group at the C-3 atom of the saponin aglycone is related to and / or contributes to the endosomal escape-promoting activity of bisdesmoside triterpene glycoside saponins, i.e., the increased toxicity of the (protein) toxin when contacted with cells in the presence of such saponin, for example, both in vitro and in vivo, compared to the toxicity of such toxin when the same dose is contacted with the same cells in the absence of such saponin. In fact, we have confirmed that saponin derivatives having a carboxyl group derivatized in the glucuronic acid unit and / or an acetyl group derivatized in the polysaccharide chain, and containing a free aldehyde group in the aglycone, possess endosomal escape-promoting activity. These derivatives exhibit reduced hemolytic activity and reduced cytotoxicity. For example, saponin derivatives such as molecules 3A, 8, 11, 18, 19, and 28 (Tables A2, A3, A5, A6, Figures 1, 3, 6, 11, 12, 39) have a free, unmodified aldehyde group in their aglycone core and are indeed active when considering the enhancement of cytotoxicity of antibody-drug conjugates, where the antibody comes into contact with various (tumor) cells expressing receptors to which it binds. Therefore, these saponin derivatives are clearly assumed embodiments of the present invention.

[0050] Surprisingly, even saponin derivatives that have a derivatized aldehyde group in the aglycone and do not contain a free aldehyde group still exhibit cytotoxicity of effector molecules delivered to (tumor) cells in the form of ligand-toxin conjugates, such as ADCs. ), and as a prerequisite, if only zero or one of the acetyl groups in the polysaccharide chain at C-28 and the carboxyl groups in the polysaccharide chain at C-23 are derivatized, they still exhibit characteristic endosomal escape-promoting activity. For example, saponin derivatives having modified aldehyde groups and zero or a single further derivatization, as shown as molecules 6, 9, 10, 14, 15, 20, 27, and 29 in Tables A2, A3, and Figures 2, 4, 5, 8, 9, 13, 38, and 40, have the ability to enhance the cytotoxicity of effector molecules when contacted with tumor cells in the presence of such saponin derivatives having aldehyde groups derivatized in the aglycone. All of these saponin derivatives exhibit reduced cytotoxicity and reduced hemolytic activity and are therefore clearly embodiments envisioned by the present invention.

[0051] The inventors have also found that certain modifications result in an increased critical micelle concentration (CMC) compared to the corresponding unmodified saponin. For example, the saponin derivatives shown as molecules 2, 6, 8, 10, 15, 27, and 28, preferably those shown as molecules 2, 6, 8, 10, and 15, have an increased CMC compared to their corresponding underivativeized saponins and are therefore clearly embodiments of the present invention. While we do not wish to be bound by any theory, an increased CMC is considered advantageous for several reasons. For example, since free molecules are generally more susceptible to the effects of conjugation reactions than molecules correctly arranged in a micelle structure, an increased CMC may facilitate the use of the modified saponin in subsequent conjugation reactions. Furthermore, when saponin derivatives need to exert a biological function (e.g., in vivo, ex vivo, or in vitro), for example, whether the saponin derivatives are used as is or released in vitro after being cleaved from a carrier or another entity, an increased CMC is advantageous compared to unmodified saponins because free saponin molecules are more readily available to interact with their biological targets than when these saponin derivatives are properly aligned in a micelle structure. Since saponins form micelles that interfere with isolation (e.g., using preparative HPLC) at concentrations above the critical micelle concentration, an increased CMC can also be useful for promoting the large-scale production and concentration of saponin derivatives. Surprisingly, for the saponin derivatives of the present invention, the observed increased CMC is not associated with increased cytotoxicity or hemolytic activity.The interaction between CMC and cytotoxicity is unpredictable and complex, as can be seen from the data in Table 2 of Groot et al. ("Saponin interactions with model membrane systems - Langmuir monolayer studies, hemolysis and formation of ISCOMs," Planta medica, 2016, Vol. 82, No. 18, pp. 1496-1512), which shows that, for example, using α-hederin as a baseline, CMC can be associated with an increase in general cytotoxicity (similar to the case of digitonin), but also in exactly the same way with a decrease in cytotoxicity (similar to the case of glycyrrhizin and hederacoside C). Furthermore, for the saponin derivatives shown as molecules 2, 6, 10, and 15, these saponin derivatives are particularly preferred embodiments of the present invention because the increased CMC is also related to the increased ratio:IC50 hemolysis / IC50 activity compared to the corresponding free saponin.

[0052] The inventors therefore provide saponin derivatives having an improved therapeutic concentration range when cytotoxicity and / or hemolytic activity are considered, and when, for example, toxin enhancement and / or increased CMC compared to the corresponding non-derivative saponin are considered. Such saponin derivatives of the present invention are particularly suitable for application in treatment plans, for example, ADCs or AOCs, for the prevention or treatment of cancer. The safety of such saponin derivatives is improved when cytotoxicity and / or hemolytic activity are considered, in particular when such saponin derivatives are administered, for example, to patients requiring treatment with ADCs or AOCs.

[0053] One embodiment is a saponin derivative of the present invention, wherein the saponin derivative comprises the first sugar chain, wherein the first sugar chain contains a carboxyl group, preferably a derivatized carboxyl group of a glucuronic acid moiety, and / or wherein the saponin derivative comprises the second sugar chain, wherein the second sugar chain contains at least one derivatized acetoxy (Me(CO)O-) group (also referred to herein as a derivatized acetate group), preferably the saponin derivative is derivatized The saponin derivative comprises both the first glycan being derivatized and the second glycan being derivatized, more preferably the saponin derivative comprises both the first glycan being derivatized and the second glycan being derivatized, 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 first glycan being derivatized and the second glycan being derivatized, and the saponin derivative comprises an aglycone core structure containing an aldehyde group. Equally preferred are, when natural saponins are considered, all other possible combinations of such derivatizations that leave one of these three chemical groups in the saponin unchanged. Furthermore, one, two, or three, preferably one or two, chemical groups in the saponin are derivatized by any one or more of the derivatizations listed in Tables A2 and A3.

[0054] One embodiment is a saponin derivative according to the present invention, wherein the saponin derivative is: 2-alpha-hydroxyoleanolic acid; 16-alpha-hydroxyoleanolic acid; Hederagenin (23-hydroxyoleanolic acid); 16-alpha,23-dihydroxyoleanolic acid; Gypsogenin; Chiral acid; Protoescigenin-21(2-methylbuta-2-enoate)-22-acetate; 23-Oxo-Baringtogenol C-21,22-Bis(2-methylbuta-2-enoate); 23-Oxo-Baringtogenol C-21(2-methylbuta-2-enoate)-16,22-diaacetate; Digitogenin; 3,16,28-trihydroxyoleanane-12-ene; Gypsogenic acid; and their derivatives, It includes an aglycon core structure selected from the group consisting of the following: Preferably, the saponin derivative comprises an aglycone core structure selected from chiric acid and gypsogenin or derivatives thereof, and more preferably, the saponin derivative aglycone core structure is chiric acid or a derivative thereof. We have now found that improved saponin derivatives can be provided with respect to reduced cytotoxicity and reduced hemolysis of cells in contact with such derivatives, based on triterpene glycoside-type saponins. Therefore, any saponin having endosomal escape-promoting activity as tested by us, such as the saponins having the aglycones of the embodiments described above and listed in Table A1, is therefore fundamentally improveable. When enhancement of toxins and, for example, BNA is considered, reducing toxicity and hemolytic activity while maintaining sufficiently high levels of activity is an important achievement by us when considering the expansion of the therapeutic concentration range of saponin derivatives, either alone or in combination with, for example, ADCs or AOCs. While sufficiently high doses of derivatized saponins are applicable, for example, in tumor therapy for cancer patients requiring them, the risks of cytotoxic side effects and undesirable hemolytic activity, which are exerted or induced by saponin derivatives, are reduced compared to the application of natural saponin counterparts. The improvement in the therapeutic concentration range of the saponin derivatives of the present invention is evident, for example, in the ratio between the IC50 of either cytotoxicity or hemolytic activity and the IC50 of endosomal escape-promoting activity, as well as in the exemplary saponin derivatives listed in Tables A5 and A6, where hemolytic activity, cytotoxicity, and activity are listed.

[0055] One embodiment is a saponin derivative of the present invention, wherein the saponin derivative comprises an aglycone core structure selected from the group consisting of chiric acid, gypsogenin, and derivatives thereof, preferably the saponin derivative comprises an aglycone core structure selected from the group consisting of chiric acid and derivatives thereof, wherein, if the first sugar chain is present, the C3 atom (also referred to as "C-3 atom") or C of the aglycone core structure 28 Atoms (also referred to as "C-28 atoms"), preferably C3 atoms, are bonded to and / or the second sugar chain, if present, of the C of the aglycone core structure. 28 The sugar chains are bonded to the atom. Preferably, the saponin derivatives are based on saponins having both sugar chains bonded to the aglycone; however, any saponin exhibiting endosomal escape activity is generally suitable for derivatization according to the present invention to provide single, double, or triple, preferably single or double derivatized saponins having lower cytotoxicity, lower hemolytic activity, and sufficiently high endosomal escape activity.

[0056] One embodiment is a saponin derivative of the present invention, wherein the first sugar chain is present, as follows (list S1): GlcA-, Glc-, Gal-, Rha-(1→2)-Ara-, Gal-(1→2)-[Xyl-(1→3)]-GlcA-, Glc-(1→2)-[Glc-(1→4)]-GlcA-, Glc-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Xyl-(1→2)-Ara-(1→3)-[Gal-(1→2)]-GlcA-, Glc-(1→3)-Gal-(1→2)-[Xyl-(1→3)]-Glc-(1→4)-Gal-, Rha-(1→2)-Gal-(1→3)-[Glc-(1→2)]-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-,Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Ara-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Rha-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Glc-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Rha-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, Xyl-(1→4)-Fuc-(1→2)-Gal-(1→2)-Fuc-(1→2)-GlcA-, and Selected from those derivatives, and / or if the second sugar chain is present, the following (List S2): Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc- where R1 is 4E-methoxycinnamic acid, Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc- where R2 is 4Z-methoxycinnamic acid, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-,Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-,Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc- where R3 is 4E-methoxycinnamate, Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-,Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc- where R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc- where R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-,Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- where R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- where R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- where R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid, Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- where R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid Glc-(1→3)-[Glc-(1→6)]-Gal-, and Selected from those derivatives.

[0057] Typically, when cells come into contact with saponins and toxins, saponins that enhance the cytotoxicity of the toxins have one or two such monosaccharides or polysaccharide chains attached to the aglycone. Preferred are saponins selected for derivatization containing two sugar chains. A summary of saponins particularly preferred for single, double, or triple derivatization, preferably single or double derivatization, when endosomal escape-promoting activity should be maintained to a sufficiently high degree, is provided in Table A1. Naturally, structural variants of such saponins are equally suitable for derivatization according to the present invention if such saponins exhibit endosomal escape-promoting activity against, for example, toxins, BNA, etc.

[0058] In one embodiment, the saponin derivative comprises the first sugar chain and the second sugar chain, wherein the first sugar chain comprises two or more sugar moieties, and the second sugar chain comprises two or more sugar moieties, and the aglycone core structure is chiric acid or gypsogenin, where: i. The aldehyde group of the aglycone core structure is derivatized. ii. The first sugar chain contains a carboxyl group of the glucuronic acid portion that has been derivatized; and iii. The second sugar chain contains at least one acetoxy (Me(CO)O-) group that has been derivatized. The saponin derivative of the present invention is one, two, or three, preferably one or two of the above.

[0059] The following summary illustrates a suitable derivatization:

[0060] [Table 1]

[0061] According to the present invention, saponins can exhibit sufficiently high endosomal escape-promoting activity even with three derivatizations. In particular, when the reduction in cytotoxicity and / or hemolytic activity outweighs the (potential or apparent) reduction in the ability to enhance the effects and activity of intracellular effector molecules, such as toxins or BNA, in tumor cells contacted with the effector molecules and the derivatized saponin. Accordingly, the present invention provides derivatized saponins comprising one, two, or three derivatizations, where an aldehyde group of the aglycone is considered, where a carboxyl group of the glucuronic acid unit in the polysaccharide at C-3 is considered if present, and where an acetyl group in the polysaccharide chain at C-28 is considered if present. Saponin derivatives having one or two modifications are preferred. Saponin derivatives having, for example, a free aldehyde group and one or two derivatizations in the glycan are suitable for improving endosomal escape of effector molecules such as toxins and BNA. As mentioned earlier, saponin derivatives with a derivatized aldehyde group are also equally suitable. Such saponin derivatives that do not have a free aldehyde group in the aglycone during derivatization still exhibit sufficient and efficient endosomal escape-promoting activity. While we do not wish to be bound by any theory, as a result of the acidic conditions inside the endosomes and lysosomes of (mammalian) cells such as human cells, the aldehyde group may be reformed inside the cell during pH-driven cleavage of the moiety initially bound to the aldehyde group of the saponin, which provides a saponin derivative with a derivatized aglycone at position C-23. Examples of saponin derivatives having modified aldehyde groups that can be reformed in endosomes or lysosomes are saponin derivatives containing a hydrazone bond, which is formed between the carbonyl group of the aldehyde and, for example, a hydrazide moiety in a chemical group bonded to the aglycone, such as N-ε-maleimidocaproic acid hydrazide (EMCH), or EMCH having mercaptoethanol bonded to a maleimide group to form a thioether bond. Examples of such saponin derivatives are shown in Figures 8 and 9 and are shown below as molecules 2 and 3.

[0062] One embodiment of the saponin derivative of the present invention is wherein the saponin derivative is: Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, maculantoidin A, esculentside A, phytolaccagenin, esinate, AS6.2, NP-005236, AMA-1, AMR, alpha-hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017 775, SA1657, AG2, SO1861, GE1741, SO1542, SO1584, SO1658, SO1674, SO1832, SO1904, SO1862, QS-7,QS1861,QS-7 api, QS1862, QS-17, The saponin derivative is selected from the group consisting of QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-escin, escin Ia, tea seed saponin I, tea seed saponin J, Assam saponin F, digitonin, primula acid I, and AS64R, as well as their stereoisomers and combinations thereof. Preferably, the saponin derivative is selected from the group consisting of QS-21 derivatives, SO1861 derivatives, SA1641 derivatives, and GE1741 derivatives. More preferably, the saponin derivative is selected from the group consisting of QS-21 derivatives and SO1861 derivatives. Most preferably, the saponin derivative is an SO1861 derivative. These saponins essentially exhibit endosomal escape-promoting activity as demonstrated by the inventors, or are structurally very similar to saponins for which endosomal escape-promoting activity has been established. The structures of these saponins are summarized in Table A1.

[0063] One embodiment of the saponin derivative of the present invention is wherein the saponin derivative is molecular 1:

[0064] [ka]

[0065] [In the formula, The first sugar chain A1 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A1 represents a sugar chain as defined above for a particular embodiment of the present invention (list S1), more preferably A1 represents a sugar chain as defined above for a particular embodiment of the present invention (list S1) and A1 contains or consists of a glucuronic acid moiety; The second sugar chain A2 represents hydrogen, a monosaccharide, or a linear or branched oligosaccharide, preferably A2 represents a sugar chain as defined above for a particular embodiment of the present invention (list S2), more preferably A2 represents a sugar chain as defined above for a particular embodiment of the present invention (list S2), and A2 contains at least one acetoxy (Me(CO)O-) group, for example one, two, three, or four, preferably one acetoxy group, where at least one of A1 and A2 is not hydrogen, and preferably both A1 and A2 are oligosaccharide chains; And R is either a hydrogen atom in gypsogenin or a hydroxyl atom in chiral acid. A derivative of chiral saponin or gypsogenin saponin, represented by Here, the saponin derivative is derivatized as follows: i. Position C of chiral acid or gypsogenin 23 The aldehyde group in is derivatized; ii. If A1 represents a sugar chain as defined above for a particular embodiment of the present invention (list S1), and A1 includes or consists of a glucuronic acid moiety, then the carboxyl group of the glucuronic acid moiety of A1 is derivatized; and iii. If A2 represents a sugar chain as defined above for a particular embodiment of the present invention (list S2) and A2 has at least one acetoxy group, then one or more, preferably all, acetoxy groups of one or more sugar moieties of A2 are derivatized. This corresponds to a saponin represented by molecule 1, which contains at least one of the following.

[0066] One embodiment is a saponin derivative of the present invention in which A1 represents a sugar chain (list S1) as defined above for a particular embodiment of the present invention and includes or consists of a glucuronic acid moiety, wherein the carboxyl group of the glucuronic acid moiety of A1 is derivatized, and / or A2 represents a sugar chain (list S2) as defined above for a particular embodiment of the present invention and A2 contains at least one acetoxy group, wherein at least one acetoxy group of A2 is derivatized.

[0067] One embodiment is a saponin derivative of the present invention in which the saponin represented by molecule 1 is a bisdesmoside triterpene saponin.

[0068] One embodiment is a saponin derivative of the present invention, wherein the saponin derivative corresponds to a saponin represented by molecule 1, and the following derivatization: i. Position C of the chiric acid or gypsogenin 23 The aldehyde group in is - Reduction to alcohol; - Preferably, conversion to a hydrazone bond via reaction with a hydrazide. It is derivatized by; ii. If A1 represents a sugar chain as defined above for a particular embodiment of the present invention (list S1), and A1 includes or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized by conversion to an amide bond, preferably via reaction with an amine; and iii. If A2 represents a sugar chain as defined above for a particular embodiment of the present invention (list S2) and A2 has at least one acetoxy group, then one or more, preferably all, acetoxy groups of one or more sugar moieties of A2 are derivatized by conversion to a hydroxyl group (HO-) by deacetylation. The saponin derivative is characterized by the presence of at least one of the following, preferably one or two, and more preferably one.

[0069] In one embodiment, the saponin derivative corresponds to a saponin represented by molecule 1, and in this embodiment, the following derivatization occurs: i. Position C of chiral acid or gypsogenin 23 The aldehyde group in - Reduction to alcohol; - This involves conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby providing saponin-Ald-EMCH, e.g., SO1861-Ald-EMCH or QS-21-Ald-EMCH, where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), in which the maleimide group of BMPH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; or - This involves conversion to a hydrazone bond via a reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), in which the maleimide group of KMUH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; ii. If A1 represents a sugar chain as defined above for a particular embodiment of the present invention (list S1) and A1 includes or consists of a glucuronic acid moiety, the carboxyl group of the glucuronic acid moiety of A1 is derivatized by conversion to an amide bond via reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby providing saponin-Glu-AMPD, e.g., QS-21-Glu-AMPD or SO1861-Glu-AMPD, or saponin-Glu-AEM, e.g., QS-21-Glu-AEM or SO1861-Glu-AEM; and iii. A saponin derivative of the present invention wherein A2 represents a sugar chain as defined above for a particular embodiment of the present invention (list S2), and A2 has at least one acetoxy group, and where at least one, preferably one or two, and more preferably one, of one or more acetoxy groups of one or more sugar moieties of A2 is derivatized by conversion to a hydroxyl group (HO-) by deacetylation.

[0070] In one embodiment, 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, and preferably the saponin represented by molecule 1 is 3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylchylic acid-28 -O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4OAc-beta-D-quinopopyranosyl-(1→4)]-beta-D-fucopyranoside, more preferably SO1861, GE1741, SA1641 and / or QS-21, most preferably SO1861, is the saponin derivative of the present invention.

[0071] In one embodiment, the saponin derivative is: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum album The saponin derivative of the present invention is selected from the group consisting of derivatives of SO1861,

[0072] In one embodiment, the saponin derivative is an SO1861 derivative that includes single derivatization, where the single derivatization is, for example, by the bonding of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) to the carboxyl group of the glucuronic acid moiety of SO1861 (see Figure 59), or by the bonding of (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) to the carboxyl group of the glucuronic acid moiety of SO1861, thereby converting the carboxyl group of the glucuronic acid of SO1861, or in which the saponin derivative is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), at position C shown in the chiral acid aglycone core structure. 23 It contains an aldehyde group; it is an SO1861 derivative represented by molecule 2:

[0073] [ka]

[0074] Alternatively, in this case, the saponin derivative is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH) at position C of the chiral acid aglycone core structure. 23 The SO1861 derivative containing an aldehyde group, wherein the maleimide group of EMCH is derivatized with mercaptoethanol, thereby forming a thioether bond; the saponin derivative of the present invention is represented by molecule 3:

[0075] [ka]

[0076] The saponin derivative represented by molecule 2 is suitable for use as a precursor for conjugation reactions with further molecules containing free sulfhydryl groups. The maleimide group of the saponin derivative shown as molecule 2 can form thioether bonds with free sulfhydryl groups, etc. For example, the saponin derivative of molecule 2 can be covalently bound to a peptide or protein containing free sulfhydryl groups, such as cysteine ​​with free sulfhydryl groups. Such a protein can be, for example, an antibody or its binding fragment or binding domain, e.g., Fab, scFv, single-domain antibody, e.g., V HH For example, camels V H For example, the application of a saponin derivative of molecule 2 in a coupling reaction with an antibody having a free sulfhydryl group provides a conjugate for targeted delivery of the saponin to the cell and into the cell, when the antibody (or binding domain or fragment thereof) is an antibody for specific binding to target cell surface molecules, such as receptors present on tumor cells. Preferably, the saponin derivative is an antibody or V that can bind to tumor cell-specific surface molecules, such as receptors such as HER2, EGFR, and CD71. HH It is combined with.

[0077] One embodiment is based on the premise that the saponin derivative is not an SO1861 derivative involving single derivatization, wherein the single derivatization is a conversion of the carboxyl group of the glucuronic acid moiety of SO1861 by the reaction of 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, or where the saponin derivative is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), at position C shown in the chiral acid aglycone core structure. 23 The present invention is a saponin derivative represented by molecule 2, which is an SO1861 derivative containing an aldehyde group; represented by molecule 2:

[0078] [ka]

[0079] One embodiment is a saponin derivative of the present invention, wherein, i. The saponin derivative comprises an aglycone core structure, where the aglycone core structure is - Reduction to alcohol; - This involves conversion to a hydrazone bond via a reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; or - This involves conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; It contains an aldehyde group that has been derivatized by; ii. The first sugar chain contains a carboxyl group of the glucuronic acid moiety, which is derivatized by conversion to an amide bond via reaction with a carboxyl group, preferably 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); and iii. The second sugar chain contains an acetoxy (Me(CO)O-) group that has been derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. The saponin derivative comprises any combination of two or three of derivatizations i, ii, and iii, preferably any combination of two of derivatizations i, ii, and iii; Preferably, the saponin derivative includes an aglycone core structure, where the aglycone core structure contains an aldehyde group that has been derivatized by conversion to a hydrazone bond via reaction with EMCH, where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol.

[0080] One embodiment of the present invention is a saponin derivative comprising an aglycone core structure, wherein the aglycone core structure contains an aldehyde group, and wherein the first sugar chain contains a carboxyl group, preferably a carboxyl group of the glucuronic acid moiety, which is derivatized by conversion to an amide bond via reaction with N-(2-aminoethyl)maleimide (AEM).

[0081] One embodiment of the present invention is a saponin derivative of the present invention, provided that the aldehyde group in the aglycone core structure is derivatized by conversion to a hydrazide bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), and if the saponin is SO1861, then at least one of the glucuronic acid and the acetoxy group (Me(CO)O-) is also derivatized, and if the saponin is SO1861 and the carboxyl group of the glucuronic acid portion of SO1861 is derivatized by reaction between 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and the carboxyl group of the glucuronic acid portion of SO1861, then at least one of the aldehyde group and the acetoxy group (Me(CO)O-) is also derivatized.

[0082] One embodiment of the present invention is a saponin derivative of the present invention, provided that the aldehyde group in the aglycone core structure of the saponin derivative is derivatized via reaction with EMCH, and if the saponin is SO1861, at least one of the glucuronic acid and the acetoxy group (Me(CO)O-) is also derivatized, and if the saponin is SO1861 and the carboxyl group of the glucuronic acid portion of SO1861 is derivatized by the bonded HATU, at least one of the aldehyde group and the acetoxy group (Me(CO)O-) is also derivatized.

[0083] One embodiment referred to herein as Example D1 is a saponin derivative of the present invention, characterized in that the saponin derivative is not SA1641, wherein the aglycone core structure contains an aldehyde group that has been derivatized by conversion, for example by reductive amination to an amine by reaction with a compound of formula (A1):

[0084] [ka]

[0085] In other words, one embodiment referred to herein as Embodiment D1 is a saponin derivative of the present invention characterized in that it is not the result of a reaction between SA1641 and a compound of formula (A1). Accordingly, Embodiment D1 is a saponin derivative of the present invention characterized in that it is not the result of coupling by reductive amination of at least one SA1641 molecule with a compound of formula (A1).

[0086] One embodiment, referred to herein as Embodiment D2, is a saponin derivative of the present invention characterized in that the saponin derivative is not a saponin, particularly SO1861, and therein the aglycone core structure contains an aldehyde group that has been derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), wherein the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with thio, and therein the saponin is not otherwise derivatized. Preferably, the saponin derivative of the present invention is characterized in that the saponin derivative is not a saponin, particularly SO1861, and therein the aglycone core structure contains an aldehyde group that has been derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), wherein the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with thio.

[0087] One embodiment referred to herein as D3 is characterized in that the saponin derivative is not a saponin, particularly SO1861, and in which the aglycone core structure contains an aldehyde group that is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is: • Mercaptoethanol, • Poly(amidoamine) dendrimers having an ethylenediamine core derivatized with at least 2-iminothiolane, • A conjugate of cyanine-3 and a poly(amidoamine) dendrimer having an ethylenediamine core further derivatized with at least 2-iminothiolane. • G4 dendrons derivatized with at least 2-iminothiolane, • A conjugate of cyanine-5 and a G4 dendron further derivatized with at least 2-iminothiolane. • Bovine serum albumin (BSA), and • Peptide with sequence SESDDAMFCDAMDESDSK[SEQ ID NO:1] The present invention relates to a saponin derivative characterized by being derivatized by the formation of a thioether bond with one, preferably selected from all, thiols, and wherein no other derivatizations are present in the saponin. As those skilled in the art will understand, the expression "G4 dendron" should be interpreted as meaning the compound of formula (A2):

[0088] [ka]

[0089] One embodiment referred to herein as D4 is characterized in that the saponin derivative is not saponin, particularly SO1861, and therein the carboxyl group is derivatized by conversion to an amide through a reaction with an optionally further derivatized conjugate consisting of cyanine-3 and a poly(amidoamine) dendrimer having an ethylenediamine core, and therein there is no other derivatization of the saponin, preferably the saponin derivative of the present invention in which the saponin derivative is not saponin, particularly SO1861, and therein the carboxyl group is derivatized by conversion to an amide through a reaction with an optionally further derivatized conjugate consisting of cyanine-3 and a poly(amidoamine) dendrimer having an ethylenediamine core.

[0090] One embodiment referred to herein as D5 is characterized in that the saponin derivative is not a saponin, particularly SO1861, and the aglycone core structure contains an aldehyde group that has been derivatized by a conversion such as reductive amination to an amine through a reaction with a conjugate consisting of cyanine-3 and a poly(amidoamine) dendrimer having an ethylenediamine core, and in which no other derivatization is present in the saponin. Preferably, the saponin derivative of the present invention is characterized in that the saponin derivative is not a saponin, particularly SO1861, and the aglycone core structure contains an aldehyde group that has been derivatized by a conversion such as reductive amination to an amine through a reaction with a conjugate consisting of cyanine-3 and a poly(amidoamine) dendrimer having an ethylenediamine core.

[0091] One embodiment referred to herein as D6 is a saponin derivative of the present invention characterized in that the saponin derivative does not contain toxins, microRNAs, or polynucleotides encoding proteins, preferably the saponin derivative does not contain pharmaceutically active substances, such as toxins, drugs, polypeptides and / or polynucleotides, and more preferably the saponin derivative does not contain effector molecules.

[0092] One embodiment referred to herein as D7 is in which the saponin derivative is: • Poly- or oligo(amine), e.g., polyethyleneimine and poly(amideamine), • Polyethylene glycol, • Poly- or oligo(ester), e.g., poly(lactide), • Poly(lactam), • Polylactide-co-glycolide copolymer, • Poly- or oligosaccharides, such as cyclodextrins and polydextroses, • Poly- or oligo(amino acids), such as proteins and peptides, and • Nucleic acids and their analogues, such as DNA, RNA, LNA (Lock nucleic acid), and PNA (Peptide nucleic acid); It is characterized by not containing polymer or oligomer structures selected from the group consisting of the following: Preferably, the saponin derivative of the present invention is characterized in that it does not contain a polymer or oligomer structure that is a structurally regular form, such as a polymer, oligomer, dendrimer, dendronized polymer, or dendronized oligomer, or that it is an aggregated polymer structure such as a hydrogel, microgel, nanogel, stabilized polymer micelle, or liposome, and more preferably, the saponin derivative is characterized in that it does not contain a polymer or oligomer structure.

[0093] One embodiment referred to herein as D8 is a saponin derivative of the present invention characterized in that the saponin derivative does not contain a molecular structure that is primarily or entirely constructed from at least two equal or similar units bonded together.

[0094] One embodiment referred to herein as D9 is characterized in that the saponin derivative is not a compound of formula (A3), which is a reaction product of SO1861 and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]-N-methylmethaneaminium hexafluorophosphate N-oxide (HATU):

[0095] [ka]

[0096] Preferably, the saponin derivative of the present invention is characterized in that the saponin derivative is not an activated ester. See Figure 59.

[0097] In one embodiment referred to as D10 herein, the saponin derivative is not saponin, particularly SO1861, in which the carboxyl group is derivatized by conversion to an amide bond or an ester bond, and in which there is no other derivatization in the saponin. Preferably, the saponin derivative is not saponin, particularly SO1861, in which the carboxyl group is derivatized by conversion to an amide bond or an ester bond, which is a saponin derivative of the present invention.

[0098] In one embodiment referred to as D11 herein, the saponin derivative is a saponin derivative of the present invention, characterized in that the saponin derivative does not contain a dianthin moiety.

[0099] A preferred embodiment referred to as D12 herein is a saponin derivative of the present invention, characterized in that the saponin derivative contains a single saponin moiety.

[0100] A preferred embodiment referred to as D13 herein is a saponin derivative of 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, more preferably less than 2150 g / mol.

[0101] One preferred embodiment, referred to herein as D14, is a saponin derivative of the present invention, characterized in that the saponin derivative has a molecular weight of 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 derivative corresponds to the molecular weight of the saponin derivative excluding the aglycone core and one (for monodesmoside saponins) or two (for bisdesmoside saponins) glycosylation chains. Those skilled in the art will understand that if the saponin derivative has a lower molecular weight than its corresponding underivativeized saponin (for example, as in the case of SO1861-Ac-OH, which corresponds to SO1861 derivatized by deacetylation), then saponin derivatization does not result in any increase in molecular weight, and therefore saponin derivatization conforms to the requirement of Embodiment D14 that the molecular weight be less than 400 g / mol, preferably less than 300 g / mol, and more preferably less than 270 g / mol.

[0102] As those skilled in the art will understand, embodiments D1-D14 may be combined with each other and with other embodiments described herein. For example, embodiments of the present invention provide the following combinations of embodiments D1-D14: · D12, and one or more of D1-D11, D13; · D13, and one or more of D1-D12; · D12, D13, and one or more of D1-D11; · D1, D2, D10, and D12; · D1, D3, D7, D9, and preferably D13; or · D3, D9, D12, and D13.

[0103] Those skilled in the art will understand that these combinations of embodiments D1-D14 can again be combined with other embodiments of the present invention, for example, and preferably embodiment D14.

[0104] Particularly preferred embodiments correspond to embodiments D3, D9, D12, and one or both of D13 and D14. In other words, particularly preferred embodiments include a single saponin derivative having a molecular weight of less than 2500 g / mol, preferably less than 2300 g / mol, more preferably less than 2150 g / mol, and wherein the saponin derivative is • Saponins, particularly not SO1861, in which the aglycone core structure contains an aldehyde group that has been derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol, and in which, preferably, the saponin is not otherwise derivatized; and • It is not an activated ester, which is a reaction product of SO1861 and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-ylmethylene]N-methylmethaneaminium hexafluorophosphate N-oxide (HATU). This invention relates to a saponin derivative.

[0105] A second aspect of the present invention relates to a first pharmaceutical composition comprising the saponin derivative of the present invention and optionally a pharmaceutically acceptable excipient and / or diluent.

[0106] One embodiment comprises a saponin derivative of the present invention, preferably a pharmaceutically acceptable diluent, and further: • A pharmaceutically acceptable salt, preferably a pharmaceutically acceptable inorganic salt, such as ammonium, calcium, copper, iron, magnesium, manganese, potassium, sodium, strontium, or zinc salt, preferably NaCl; and / or • Pharmacologically acceptable buffering systems, e.g., buffering systems containing phosphates, borates, citrates, carbonates, histidines, lactates, tromethamines, glucons, aspartates, glutamates, tartrates, succinates, malates, fumarates, acetates, and / or ketoglutarates. The first pharmaceutical composition comprises the above.

[0107] One embodiment is a first pharmaceutical composition of the present invention comprising a saponin derivative of the present invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is 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.

[0108] One embodiment is a first pharmaceutical composition of the present invention comprising a saponin derivative of the present invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is liquid at a temperature of 25°C and the concentration of the saponin derivative is 10 -12 From 1 mol / l, preferably within the range of 10 -9 From within the range of 0.1 mol / l, more preferably 10 -6 It is within the range of 0.1 mol / l.

[0109] Typically, such first pharmaceutical composition is suitable for use in combination with, for example, ADC or AOC. For example, the first pharmaceutical composition is administered to a patient requiring ADC or AOC therapy, either before the administration of ADC or AOC, together with ADC or AOC, or immediately after the administration of ADC or AOC. For example, the first pharmaceutical composition is mixed with a pharmaceutical composition containing ADC or AOC, and an appropriate dose of the resulting mixture is administered to the patient requiring ADC or AOC therapy. According to the present invention, the saponin derivative contained in the first pharmaceutical composition enhances the efficacy and potency of the effector molecules contained in such ADC or AOC when the saponin derivative and ADC or AOC coexist inside target cells such as tumor cells. Under the influence of the saponin derivative, the effector molecules are released into the cytosol of the target cells to a greater extent compared to when the same cells are contacted with the same dose of ADC or AOC in the absence of the saponin derivative. Therefore, when the effector molecule coexists with the saponin derivative of the first pharmaceutical composition inside 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 the absence of the saponin derivative inside the cell to which the ADC or AOC containing the effector molecule is delivered.

[0110] One embodiment is a first pharmaceutical composition of the present invention, wherein the saponin derivative is the saponin derivative represented by molecule 2:

[0111] [ka]

[0112] Or a SO1861 derivative including single derivatization, where the single derivatization is a conversion by reaction of the carboxyl group of the glucuronic acid moiety of SO1861 with 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), or the saponin derivative is a saponin derivative represented by molecule 3:

[0113]

Chemical formula

[0114] The third aspect of the present invention is · The first pharmaceutical composition of the present invention; and · 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, any one or more of which are included, and optionally, a second pharmaceutical composition containing a pharmaceutically acceptable excipient and / or diluent, relates to a pharmaceutical combination agent containing the above.

[0115] The fourth aspect of the present invention relates to a third pharmaceutical composition containing the saponin derivative of the present invention and further including 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 containing a pharmaceutically acceptable excipient and / or diluent.

[0116] One embodiment is the pharmaceutical combination agent 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 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 a toxin such as saporin and dianthine, and wherein the oligonucleotide is, for example, siRNA, BNA, apolipoprotein B, or HSP27 gene silencing.

[0117] One embodiment is the aforementioned pharmaceutical combination agent or the third pharmaceutical composition of the present invention, wherein the saponin derivative is: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypsoside The saponin derivative is selected from the group consisting of derivatives of A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, their stereoisomers, and combinations thereof. Preferably, the saponin derivative is selected from the group consisting of SO1861 derivatives, GE1741 derivatives, SA1641 derivatives, QS-21 derivatives, and combinations thereof. More preferably, the saponin derivative is an SO1861 derivative or a QS-21 derivative. Even more preferably, the saponin derivative is an SO1861 derivative, and even more preferably, the saponin derivative is represented by molecule 2 or molecule 3.

[0118] One embodiment is a third pharmaceutical composition of the present invention comprising a saponin derivative of the present invention, preferably a pharmaceutically acceptable diluent, and further comprising: a pharmaceutically acceptable salt, preferably a pharmaceutically acceptable inorganic salt, such as ammonium, calcium, copper, iron, magnesium, manganese, potassium, sodium, strontium, or zinc salt, preferably NaCl; and / or a pharmaceutically acceptable buffer system, such as a phosphate, borate, citrate, carbonate, histidine, lactate, tromethamine, gluconate, aspartate, glutamate, tartrate, succinate, malate, fumarate, acetate, and / or a ketoglutarate-containing buffer system. The pharmaceutical composition contains the following:

[0119] One embodiment is a third pharmaceutical composition of the present invention comprising a saponin derivative of the present invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is 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.

[0120] One embodiment is a third pharmaceutical composition of the present invention comprising a saponin derivative of the present invention and a pharmaceutically acceptable diluent, preferably water, wherein the composition is liquid at a temperature of 25°C, and the concentration of the saponin derivative is 10 -12 From 1 mol / l, preferably within the range of 10 -9 From within the range of 0.1 mol / l, more preferably 10 -6 It is within the range of 0.1 mol / l.

[0121] A fifth aspect of the present invention relates to the first pharmaceutical composition of the present invention, the pharmaceutical combination of the present invention, or the third pharmaceutical composition of the present invention for use as a pharmaceutical. In preferred embodiments, a first pharmaceutical composition for pharmaceutical use is provided, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU; the pharmaceutical combination of the present invention comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU; or a third pharmaceutical composition of the present invention is provided, wherein the saponin derivative comprises, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU.

[0122] In another embodiment of the present invention, the saponin derivatives described herein, preferably SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU, are provided for use as pharmaceuticals.

[0123] A sixth aspect of the present invention relates to the first pharmaceutical composition, the pharmaceutical combination, or the third pharmaceutical composition of the present invention for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases. In a preferred embodiment, the first pharmaceutical composition comprises, and preferably consists of, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases. A pharmaceutical composition is provided; the pharmaceutical combination of the present invention comprising, preferably, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU, wherein the saponin derivative comprises SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU, wherein the third pharmaceutical composition of the present invention is provided.

[0124] A seventh aspect of the present invention is an in vitro or ex vivo method for transferring a molecule from the outside of a cell to the inside of the cell, preferably into the cytosol of the cell: a) The process of providing cells; b) A step of providing a molecule to be transferred from the outside of the cell to the inside of the cell provided in step a); c) A step of providing the saponin derivative of the present invention; d) The method comprises the step of contacting the cells of step a) with the molecules of step b) and the saponin derivative of step c) in vitro or ex vivo, thereby establishing the transfer of the molecules from outside the cells to inside the cells.

[0125] One embodiment is a method of the present invention, wherein the cells are human cells, e.g., T-cells, NK-cells, tumor cells, and / or therein, the molecule in 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 the drug is, for example, a toxin, and therein, the oligonucleotide is, for example, siRNA, BNA, and / or the saponin derivative is: SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillaja saponin, Saponinum album, QS-18, Quil-A, Gyp1, Gypsoside The saponin derivative is selected from the group consisting of derivatives of A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, SO1862, SO1904, their stereoisomers, and combinations thereof, preferably the saponin derivative is selected from the group consisting of SO1861 derivatives, GE1741 derivatives, SA1641 derivatives, QS-21 derivatives, and combinations thereof, more preferably the saponin derivative is an SO1861 derivative or a QS-21 derivative, most preferably the saponin derivative is an SO1861 derivative; or in this case, the saponin derivative is an SO1861 derivative comprising a single derivatization, where the single derivatization is an amine of the carboxyl group of the glucuronic acid portion of SO1861. This conversion to an amide is via a reaction with, for example, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) by bonding it to the carboxyl group of the glucuronic acid of SO1861, or (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) by bonding it to the carboxyl group of the glucuronic acid portion of SO1861, or in which the saponin derivative is derivatized by conversion to a hydrazone bond via a reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), at position C shown in the chiral acid aglycone core structure. 23 It contains an aldehyde group; it is an SO1861 derivative represented by molecule 2:

[0126] [ka]

[0127] Alternatively, in this case, the saponin derivative is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH) at position C of the chiral acid aglycone core structure. 23 The SO1861 derivative in which an aldehyde group is contained, wherein the maleimide group of EMCH is derivatized with mercaptoethanol, thereby forming a thioether bond; the SO1861 derivative represented by molecule 3:

[0128] [ka]

[0129] Alternatively, the saponin derivative is not a SO1861 derivative including single derivatization, where single derivatization is the conversion of the carboxyl group of the glucuronic acid moiety of SO1861 by the reaction of 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, or in this case, the saponin derivative is derivatized by conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), at position C shown in the chiral acid aglycone core structure. 23 It contains an aldehyde group; it is an SO1861 derivative represented by molecule 2:

[0130] [ka]

[0131] Alternatively, here, the saponin derivative is a derivative, and in this, i. The saponin derivative comprises an aglycone core structure, where the aglycone core structure is - Reduction to alcohol; - This involves conversion to a hydrazone bond via a reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), where the maleimide group of BMPH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; or - This involves conversion to a hydrazone bond via reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), where the maleimide group of KMUH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; It contains an aldehyde group that has been derivatized by; ii. The first sugar chain contains a carboxyl group of the glucuronic acid moiety, which is derivatized by conversion to an amide bond via reaction with a carboxyl group, preferably 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM); and iii. The second sugar chain contains an acetoxy group (Me(CO)O-) which is derivatized by conversion to a hydroxyl group (HO-) by deacetylation; or iv. The saponin derivative is a derivative comprising any two or three combinations of derivatizations i, ii, and iii, preferably any two combinations of derivatizations i, ii, and iii; Preferably, the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure contains an aldehyde group that is derivatized by conversion to a hydrazone bond via reaction with EMCH, wherein the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; or, wherein the saponin derivative comprises an aglycone core structure, wherein the aglycone core structure contains an aldehyde group, and wherein the first sugar chain is a carboxyl group, preferably N-(2-aminoethyl) The saponin derivative contains a carboxyl group of the glucuronic acid moiety, which is derivatized by conversion to an amide bond via reaction with reimide (AEM); or, in the case where the saponin derivative is SO1861, at least one of the glucuronic acid and acetoxy group (Me(CO)O-) is also derivatized, and the saponin is SO1 A derivative that is 861 and is conditional on the fact that if the carboxyl group of the glucuronic acid moiety of SO1861 is derivatized by the reaction of 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, then at least one of the aldehyde group and the acetoxy group (Me(CO)O-) is also derivatized; or, herein, the saponin is the aglycone core structure of the saponin derivative. The method provides a derivative that is conditional on the following: if the aldehyde group in the derivative is derivatized via a reaction with EMCH and the saponin is SO1861, then at least one of the glucuronic acid and acetoxy group (Me(CO)O-) is also derivatized; and if the saponin is SO1861 and the carboxyl group of the glucuronic acid portion of SO1861 is derivatized by the bonded HATU, then at least one of the aldehyde group and acetoxy group (Me(CO)O-) is also derivatized.

[0132] In certain embodiments, in vitro or ex vivo methods are provided for transferring a molecule from the outside of a cell to the inside of the cell, preferably into the cytosol of the cell as described herein, wherein the saponin derivative comprises, and preferably consists of, SO1861-Ald-EMCH, SO1861-Ald-EMCH-mercaptoethanol, SO1861-L-N3, or SO1861-Glu-HATU.

[0133] While the present invention has been described in relation to several embodiments, it is intended that those skilled in the art will be able to see, upon reading the specification and examining the drawings, alternatives, modifications, substitutes, and equivalents thereto. The present invention is not limited in any way to the exemplary embodiments. Modifications may be made without moving beyond the scope defined by the appended claims.

[0134] The present invention has been described with reference to several exemplary embodiments. Modifications are possible and fall within the scope of protection as defined in the appended claims. The present invention is further illustrated by the following embodiments, which should not be construed as limiting the invention in any way.

[0135] [Table 2]

[0136] [Table 3]

[0137] [Table 4]

[0138] [Table 5]

[0139] Examples and exemplary embodiment materials: SO1861, SO1832, SO1862 (isomer), and SO1904 were isolated and purified by Analyticon Discovery GmbH from raw plant extracts obtained from Saponaria officinalis L. QS21 (pure), QS18 (fraction), QS17 (fraction), QS7 (fraction), and QS21 (fraction) were purchased from Desert King International, Sandiego, while trastuzumab (Tras, Herceptin®, Roche) and cetuximab (Cet, Erbitux®, Merck KGaA) were purchased from a pharmacy. EGF dianthine was obtained from Escherichia coli (E. coli).The cetuximab-saponin conjugate was manufactured and purchased from Advanced Targeting System (San Diego, CA). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%, Sigma-Aldrich), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB, Elman's reagent, 99%, Sigma-Aldrich), Zeba® spin desalting column (2 mL, Thermo-Fisher), NuPAGE® 4-12% Bis-Tris Protein Gels (Thermo-Fisher), NuPAGE® MES SDS Running Buffer (Thermo-Fisher), Novex® Sharp Pre-stained Protein Standard (Thermo-Fisher), PageBlue® Protein Staining Solution (Thermo-Fisher), Pierce® BCA Protein Assay Kit Kit) (Thermo-Fisher), N-ethylmaleimide (NEM, 98%, Sigma-Aldrich), 1,4-dithiothreitol (DTT, 98%, Sigma-Aldrich), Sephadex G25 (GE Healthcare), Sephadex G50M (GE Healthcare), Superdex 200P (GE Healthcare), isopropyl alcohol (IPA, 99.6%, VWR), tris(hydroxymethyl)aminomethane (Tris, 99%, Sigma-Aldrich), tris(hydroxymethyl)aminomethane hydrochloride (Tris.HCl (Sigma-Aldrich), L-histidine (99%, Sigma-Aldrich), D-(+)-trehalose dihydrate (99%, Sigma-Aldrich), polyethylene glycol sorbitan monolaurate (TWEEN20, Sigma-Aldrich), Dulbecco's phosphate-buffered saline (DPBS, Thermo-Fisher), guanidine hydrochloride (99%, Sigma-Aldrich), ethylenediaminetetraacetate disodium dihydrate (EDTA-Na2, 99%, Sigma-Aldrich), 0.2 μm and 0.45 μm sterile filters (Sartorius), succinimimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Thermo-Fisher), Vivaspin T4 and T15 concentrators (Sartorius), Superdex 200 PG (GE. Healthcare), Tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP, Thermo-Fisher), [O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium-hexafluorophosphate] (HATU, 97%, Sigma-Aldrich), Dimethyl sulfoxide (DMSO, 99%, Sigma-Aldrich), N-(2-aminoethyl)maleimide trifluoroacetate (AEM, 98%, Sigma-Aldrich), L-cysteine ​​(98.5%, Sigma-Aldrich), Deionized water (DI) is Ultrapure Lab Water Newly extracted nickel-nitrilotriacetate agarose (Ni-NTA agarose, Protino), glycine (99.5%, VWR), 5,5-dithiobis(2-nitrobenzoic acid (Ermann reagent, DTNB, 98%, Sigma-Aldrich), S-acetyl mercaptosuccinate anhydride fluorescein (SAMSA reagent, Invitrogen), sodium bicarbonate (99.7%, Sigma-Aldrich), sodium carbonate (99.9%, Sigma-Aldrich), and a PD MiniTrap desalting column using Sephadex G-25 resin (GE Healthcare). PD10 G25 desalting columns (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 columns (Phenomenex), Vivacell ultrafiltration units 10 and 30kDa MWCO (Sartorius), Nalgene Rapid-Flow filters (Thermo-Fisher).

[0140] abbreviation AEM: N-(2-aminoethyl)maleimidotrifluoroacetate AMPD: 2-amino-2-methyl-1,3-propanediol BOP: (benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide EMCH.TFA: N-(ε-maleimidocaproic acid) hydrazide, trifluoroacetate HATU: 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate Min: minutes NMM: 4-methylmorpholine r, t: retention time TCEP: Tris(2-carboxyethyl)phosphine hydrochloride Temp: Temperature TFA: Trifluoroacetic acid

[0141] Analysis method LC-MS method 1 Apparatus: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320nm; SQD: ACQ-SQD2 ESI, mass range neg or neg / pos 1500-2400 or 2000-3000 depending on the molecular weight of the product; ELSD: gas pressure 40 psi, drift tube temperature: 50℃; column: Acquity C18, 50x2.1mm, 1.7μm, temperature: 60℃, flow rate: 0.6 mL / min. Gradient according to the polarity of the product: At0=2%A, t5.0min=50%A, t6.0min=98%A Bt0=2%A, t5.0min=98%A, t6.0min=98%A Post-processing time: 1.0 min; Eluent A: Acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0142] LC-MS method 2, 2 Apparatus: Waters IClass; Bin.Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320nm, SQD: ACQ-SQD2 ESI, mass range depending on the molecular weight of the product; pos / neg 100-800 or neg 2000-3000; ELSD: Gas pressure 40 psi, drift tube temperature: 50°C; Column: Waters XSelect (trademark) CSH C18, 50 x 2.1 mm, 2.5 μm, temperature: 25°C, flow rate: 0.5 mL / min, gradient: t0 min = 5% A, t2.0 min = 98% A, t2.7 min = 98% A, post time: 0.3 min; Eluent A: Acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0143] LC-MS method 3 Instrument: Waters IClass; Bin. Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320nm, SQD: ACQ-SQD2 ESI, mass range depending on product molecular weight pos / neg 105-800, 500-1200 or 1500-2500; ELSD: Gas pressure 40 psi, drift tube temperature: 50°C; Column: Waters XSelect (trademark) CSH C18, 50 x 2.1 mm, 2.5 μm, temperature: 40°C, flow rate: 0.5 mL / min, gradient: t0 min = 5% A, t2.0 min = 98% A, t2.7 min = 98% A, post time: 0.3 min; Eluent A: 0.1% formic acid in acetonitrile; Eluent B: 0.1% formic acid in water.

[0144] LC-MS method 4 Apparatus: Waters IClass; Bin.Pump: UPIBSM, SM: UPISMFTN with SO; UPCMA, PDA: UPPDATC, 210-320nm, SQD: ACQ-SQD2 ESI, mass range depending on the molecular weight of the product; pos / neg 100-800 or neg 2000-3000; ELSD: Gas pressure 40 psi, drift tube temperature: 50°C; Column: Waters Acquity Shield RP18, 50 x 2.1 mm, 1.7 μm, temperature: 25°C, flow rate: 0.5 mL / min, gradient: t0 min = 5% A, t2.0 min = 98% A, t2.7 min = 98% A, post time: 0.3 min; Eluent A: Acetonitrile; Eluent B: 10 mM ammonium bicarbonate in water (pH=9.5).

[0145] Preparative method Preparative MP-LC method 1 Instrument type: Revelleris™ prep MPLC; Column: Waters XSelect™ CSH C18 (145x25mm, 10μm); Flow rate: 40mL / min; Column temperature: Room temperature; Eluent A: 10mM ammonium bicarbonate in water (pH=9.0); Eluent B: 99% acetonitrile + 1% 10mM ammonium bicarbonate in water; Gradient: A t0min=5%B, t1min=5%B, t2min=10%B, t17min=50%B, t18min=100%B, t23min=100%B B t0min=5%B, t1min=5%B, t2min=20%B, t17min=60%B, t18min=100%B, t23min=100%B; Detection UV: 210, 235, 254 nm and ELSD.

[0146] Preparative MP-LC method 2 Instrument type: Revelleris™ prep MPLC; Column: Phenomenex LUNA C18(3) (150x25mm, 10μm); Flow rate: 40mL / min; Column temperature: Room temperature; Eluent A: 0.1% (v / v) formic acid in water; Eluent B: 0.1% (v / v) formic acid in acetonitrile; Gradient: A t0min=5%B, t1min=5%B, t2min=20%B, t17min=60%B, t18min=100%B, t23min=100%B B t0min=2%B, t1min=2%B, t2min=2%B, t17min=30%B, t18min=100%B, t23min=100%B C t0min=5%B, t1min=5%B, t2min=10%B, t17min=50%B, t18min=100%B, t23min=100%B D t0min=5%B, t1min=5%B, t2min=5%B, t17min=40%B, t18min=100%B, t23min=100%B; Detection UV: 210, 235, 254 nm and ELSD.

[0147] Preparative LC-MS method 3 MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XSelect (trademark) CSH (C18, 150x19mm, 10μm); Flow rate: 25ml / min; Column temperature: Room temperature; Eluent A: 100% acetonitrile; Eluent B: 10mM ammonium bicarbonate in water, pH=9.0; Gradient: A t0=20%A, t2.5min=20%A, t11min=60%A, t13min=100%A, t17min=100%A, B t0=5%A, t2.5min=5%A, t11min=40%A, t13min=100%A, t17min=100%A; Detection: DAD (210nm) Detection: MSD (ESI pos / neg) Mass range: 100-800; Fractionation collection based on DAD.

[0148] Preparative LC-MS method 4 MS instrument type: Agilent Technologies G6130B Quadrupole; HPLC instrument type: Agilent Technologies 1290 preparative LC; Column: Waters XBridge Protein (C4, 150x19mm, 10μm); Flow rate: 25ml / min; Column temperature: Room temperature; Eluent A: 100% acetonitrile; Eluent B: 10mM ammonium bicarbonate in water, pH=9.0; Gradient: A t0=2%A, t2.5min=2%A, t11min=30%A, t13min=100%A, t17min=100%A B t0=10%A, t2.5min=10%A, t11min=50%A, t13min=100%A, t17min=100%A C t0=5%A, t2.5min=5%A, t11min=40%A, t13min=100%A, t17min=100%A Detection: DAD (210nm); Detection: MSD (ESI pos / neg) Mass range: 100-800; Fractionation based on DAD

[0149] Flash chromatography Grace Reveleris X2(registered trademark) C-815Flash; Solvent delivery system: 3-piston pump with autopriming, 4 independent channels with up to 4 solvents in a single run, auto-switch line in case of solvent depletion; Maximum pump flow rate 250 mL / min; Maximum pressure 50 bar (725 psi); Detection: UV 200-400 nm, combination of up to 4 UV signals and full UV range scan, ELSD; Column size: 4-330 g on the instrument, Luer type, 750 g to 3000 g, with optional holder.

[0150] Example 1: Synthesis of saponin derivatives The following modified SO1861 saponins, i.e., saponin derivatives, were synthesized from naturally occurring SO1861 as summarized in Table A2:

[0151] [Table 6]

[0152] [Table 7]

[0153] [Table 8]

[0154] [Table 9]

[0155] [Table 10]

[0156] For details on the synthesis of the following SO1861 derivatives, please refer to Table A2 and the drawings.

[0157] Synthesis of SO1861-Ald-EMCH (molecule 2); see Figures 60 and 61A. SO1861 (59 mg, 31.7 μmol) and EMCH (301 mg, 888 μmol) derived from Saponaria officinalis were placed in a round-bottom flask equipped with a stirrer and dissolved in 13 mL of methanol. TFA (400 μL, cat.) was added to the solution, and the reaction mixture was stirred at 800 rpm for 3 hours at room temperature on an RCT B magnetic stirrer (IKA Labortechnik). After 3 hours of stirring, the mixture was diluted with either MilliQ water or PBS and dialyzed thoroughly for 24 hours in either MilliQ water or PBS using a regenerated cellulose membrane tube (Spectra / Por7) with MWCO 1 kDa. After dialyzing, the solution was lyophilized to obtain a white powder. Yield 62.4 mg (95%). The dried aliquots were further processed. 1These were used for characterization by 1H NMR and MALDI-TOF-MS.

[0158] 1 ¹H NMR (400 MHz, methanol-D4) (SO1861): δ = 0.50-5.50 (m, saponin triterpenoid and sugar backbone proton), 9.43 (1H, s, saponin aldehyde proton, H a ). 1 ¹H NMR (400 MHz, methanol-D4) (SO1861-Ald-EMCH, PBS workup): δ = 0.50-5.50 (m, saponin triterpenoid and sugar backbone proton), 6.79 (2H, s, maleimide proton, H c ),7.62-7.68(1H,m,hydrazone proton,H b ). MALDI-TOF-MS (RP mode): m / z 2124 Da ([M+K] + ,saponin-EMCH),m / z 2109 Da([M+K] + ,SO1861-ALD-EMCH),m / z 2094 Da([M+Na] + (SO1861-ALD-EMCH). See Figure 61A. MALDI-TOF-MS(RN mode): m / z 2275 Da([MH] - ,Saponin EMCH conjugate), 2244 Da([MH] - ,Saponin EMCH conjugate),2222 Da([MH] - ,Saponin EMCH conjugate), 2178 Da([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] - ,SO1861-ALD-EMCH),2038 Da([MH] -,SO1832-EMCH),1936 Da([MH] - ,SO1730-EMCH),1861 Da([MH] - SO1861). SO1861-ALD-EMCH is a molecule with two molecules (chemical formula: C 93 H 143 N3O 48 , expressed by precise mass: 2069.88):

[0159] [ka]

[0160] To examine the pH-dependent hydrolysis of the hydrazone bond, SO1861-Ald-EMCH was dissolved in HCl solution at pH 3, and MALDI-TOF-MS spectra were recorded at two different time points (Figure 62). As shown in Figures 62A and 62B, a clear decreasing trend in the peak corresponding to SO1861-Ald-EMCH at m / z 2070 is observed in Figure 61B. Since SO1861 is produced during hydrolysis, an increase in the peak at m / z 1861Da was recorded, accompanied by a decreasing trend at m / z 2070Da. These results indicate that the hydrazone bond is susceptible to hydrolysis and is cleaved even when bound to SO1861.

[0161] Synthesis of SO1861-Ald-EMCH-mercaptoethanol (molecule 3; block SO1861-Ald-EMCH); see Figures 60 and 61B. The maleimide group of SO1861-Ald-EMCH undergoes rapid and specific Michael addition with thiols when performed in the pH range of 6.5-7.5. To SO1861-Ald-EMCH (0.1 mg, 48 nmol), 200 μL of mercaptoethanol (18 mg, 230 μmol) was added, and the solution was shaken at 800 rpm for 1 hour at room temperature on a ThermoMixer C (Eppendorf). After 1 hour of shaking, the solution was diluted with methanol and dialyzed thoroughly with methanol for 4 hours using a MWCO 1 kDa regenerated cellulose membrane tube (Spactra / Por7). After dialyzing, SO1861-Ald-EMCH-mercaptoethanol was given (molecules 3), and aliquots were taken and analyzed by MALDI-TOF-MS.

[0162] MALDI-TOF-MS (RP mode): m / z 2193 Da ([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 Figure 61B. SO1861-Ald-EMCH-mercaptoethanol is a molecule with 3 molecules (chemical formula: C 95 H 149 N3O 49 S, represented by precise mass: 2147.90:

[0163] [ka]

[0164] Synthesis of SO1861-Glu-AMPD (molecule 3A); see Figure 1. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight. Next, the product was separated by LC-MS. 3 The compound was re-purified using [method / tool ​​name]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (20.2 mg, 67%) as a white, fluffy solid. Purity based on LC-MS = 93% (Chemical formula: C 87 H 139 NO 47 , exact mass:1949,85)LRMS(m / z):1949[M-1] 1- LC-MS rt(min):2.45 1B

[0165] Synthesis of SO1861-Ald-OH (molecule 6); see Figure 2. 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 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was diluted with water (0.50 mL) and preparative MP-LC was performed. 2 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (15.9 mg, 79%) as a white, fluffy solid. Purity was 97% based on LC-MS (chemical formula: C 83 H 132 O 46 , exact mass:1864,80)LRMS(m / z):1865[M-1] 1- (See Figures 15 and 16) LC-MS rt(min): 1.95 1B

[0166] Synthesis of SO1861-Ac-OH (molecule 8); see Figure 3. SO1861 (9.30 mg, 4.99 μmol) was mixed with a solution of sodium hydroxide (2.00 mg, 0.050 mmol) in water (0.25 mL) and methanol (0.25 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and analyzed by preparative MP-LC. 2was subjected to. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to obtain the title compound (8.86 mg, 97%) as a white fluffy solid. Purity based on LC-MS = 97% (chemical formula: C 81 H 128 O 45 , exact mass: 1820,77) LRMS (m / z): 1820 [M - 1] 1- LC-MS r.t. (min): 1.83 1B

[0167] Synthesis of SO1861-(Ald-OH)-(Glu-AMPD) (Molecule 9); see Figure 4 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC [[ID=...]] 2 was subjected to. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to obtain 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 , exact mass: 1951,87) LRMS (m / z): 1952 [M - 1] [[ID=2...]] 1- (see Figure 17) LC-MS r.t. (min): 2.45 1B

[0168] Synthesis of SO1861-(Ald-OH)-(Ac-OH) (Molecule 10); see Figure 5 To SO1861-Ald-OH (26.8 mg, 0.014 mmol) was added a solution of sodium hydroxide (5.74 mg, 0.144 mmol) in water (0.50 mL) and methanol (0.50 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was subjected to preparative MP-LC 2was subjected to. The fractions corresponding to the product were immediately pooled together, 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 , exact mass: 1822.79) LRMS (m / z): 1822 [M - 1] 1- LC-MS r.t. (min): 1.81 1B

[0169] Synthesis of SO1861-(Ac-OH)(Glu-AMPD) (Molecule 11); see Figure 6 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 minute and left standing at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC 2 was subjected to. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight. Next, the product was re-purified using the first preparative MP-LC 2 and then preparative MP-LC 3 The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to give the title compound (9.47 mg, 63%) as a white fluffy solid. Purity based on LC-MS = 98% (chemical formula: C 85 H 137 NO 46 , exact mass: 1907.84) LRMS (m / z): 1908 [M - 1] 1- LC-MS r.t. (min): 2.31 1B

[0170] Synthesis of SO1861-(Ald-OH)-(Ac-OH)(Glu-AMPD) (Molecule 12); see Figure 7 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2 The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight. The product was then subjected to further preparative MP-LC. 2 The product was purified using [method / method]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (7.21 mg, 80%) as a white, fluffy solid. Purity based on LC-MS = 97.8% (Chemical formula: C 85 H 139 NO 46 , exact mass: 1909,86) LRMS (m / z): 1910 [M-1] 1- LC-MS rt(min):2.21 1B

[0171] Synthesis of SO1861-(Ald-EMCH)-(Glu-AMPD) (molecule 14); see Figure 8. SO1861-Glu-AMPD (10.6 mg, 5.43 μmol) and EMCH.TFA (9.22 mg, 0.027 mmol) were dissolved in methanol (super-anhydrous, 0.50 mL). Next, TFA (1.66 μL, 0.022 mmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight. The product was then subjected to preparative MP-LC. 3 The product was re-purified using [method / method]. The fractions corresponding to the product were immediately pooled together. The resulting solution was neutralized with formic acid, frozen, and freeze-dried overnight to obtain 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, exact mass: 2156,95). LRMS (m / z): 2156 [M-1] 1- LC-MS rt(min):2.64 1B

[0172] Synthesis of SO1861-(Ald-EMCH)-(Ac-OH) (molecule 15); see Figure 9. SO1861-Ac-OH (9.05 mg, 4.97 μmol) and EMCH.TFA (8.43 mg, 0.025 mmol) were dissolved in methanol (super-anhydrous, 0.50 mL). Next, TFA (1.52 μL, 0.022 mmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and analyzed by preparative MP-LC. 1 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain 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 , exact mass:2027,87). LRMS(m / z):2028[M-1] 1- LC-MS rt(min):1.96 1B

[0173] Synthesis of SO1861-(Ald-EMCH)-(Ac-OH)-(Glu-AMPD) (molecule 16); see Figure 10. SO1861-(Ac-OH)-(Glu-AMPD) (6.00 mg, 3.14 μmol) and EMCH.TFA (5.33 mg, 0.016 mmol) were dissolved in methanol (super-anhydrous, 0.50 mL). Next, TFA (0.96 μL, 0.013 mmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and analyzed by preparative MP-LC. 1 The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight. The product was then subjected to preparative MP-LC. 3The solution was re-purified using [method / method]. The fractions corresponding to the products were immediately pooled together. The resulting solution was neutralized with formic acid, frozen, and freeze-dried overnight to obtain 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 , exact mass: 2114,94) LRMS (m / z): 2115 [M-1] 1- LC-MS rt(min):2.55 1B

[0174] Synthesis of SO1861-Glu-AEM (molecule 18); see Figure 11. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was preparatively divided by MP-LC. 2 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (7.82 mg, 71%) as a white, fluffy solid. Purity based on LC-MS = 95% (Chemical formula: C 89 H 136 N2O 47 , exact mass:1984,83). LRMS (m / z): 1985 [M-1] 1- LC-MS rt(min):2.62 1B

[0175] Synthesis of SO1861-(Glu-AEM)-(Ac-OH) (molecule 19); see Figure 12. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain 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 , exact mass:1942,82). LRMS(m / z):1944[M-1] 1- LC-MS rt(min):2.47 1B

[0176] Synthesis of SO1861-(Glu-AEM)-(Ald-OH) (molecule 20); see Figure 13. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain 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 47 , exact mass:1986,85). LRMS (m / z): 1987 [M-1] 1- LC-MS rt(min):2.62 1B

[0177] Synthesis of SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) (molecule 21); see Figure 14. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2 The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain 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 , exact mass: 1944,84). LRMS(m / z):1944[M-1] 1- LC-MS rt(min):2.46 1B

[0178] Synthesis of SO1861-L-N3 (molecule 23); see Figure 36. Chemical formula:C 94 H 151 N5O 50 , exact mass: 2149,94

[0179] Synthesis of SO1861-L-NHS (molecule 25); see Figure 37. 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 minute and allowed to stand at room temperature. After 30 minutes, the reaction mixture was added dropwise to diethyl ether (40 mL). After centrifugation (7800 RPM, 5 min), the supernatant was decanted, and the pellet was resuspended in diethyl ether (20 mL) and centrifuged again. After decanting the supernatant, the residue was dissolved in water / acetonitrile (3:1, v / v, 3 mL), the resulting solution was immediately frozen, and lyophilized overnight to obtain the title compound (8.81 mg, 96%) as a white, fluffy solid. Purity based on LC-MS = 84%. Contains 14% hydrolyzed NHS (chemical formula: C 117 H 169 N7O 55 , exact mass: 2552,06). LRMS (m / z): 2551 [M-1] 1- LC-MS rt(min):2.76 / 2.78 2 (Dual peak due to isomers)

[0180] Synthesis of SO1861-Glu-HATU (molecule 26); see Figure 59. To generate SO1861-Glu-HATU, the carboxyl group of SO1861 is activated by a reagent used in peptide coupling chemistry to produce active esters, 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.

[0181] The following modified QS-21 saponins, i.e., saponin derivatives, were synthesized from naturally occurring QS-21:

[0182] [Table 11]

[0183] [Table 12]

[0184] Synthesis of QS21-Ald-OH (molecule 27); see Figure 38. QS21 (9.41 mg, 4.73 μmol) was dissolved in methanol (0.50 mL). Next, sodium borohydride (1.79 mg, 0.047 mmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 30 minutes, 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 obtain the title compound (4.68 mg, 50%) as a white, fluffy solid. Purity was 99% based on LC-MS (accurate mass: 1990, 4 isomers: Api / Xyl(2:1)). LRMS (m / z): 1990 [M-1] 1- LC-MS rt(min):1.25 / 2.31 1B (Dual peak, 17 / 83 UV-area, attributed to the mixture QS21)

[0185] Synthesis of QS21-Glu-AEM (molecule 28); see Figure 39. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC2A. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to obtain the title compound (1.80 mg, 70%) as a white, fluffy solid. Purity was 92% based on LC-MS (accurate mass: 2110, 4 isomers: Api / Xyl(2:1)). LRMS (m / z): 2110 [M-1] 1- LC-MS rt(min):2.84 / 2.93 1B(Dual peak, attributed to QS21, a 10 / 90 UV-area % mixture)

[0186] Synthesis of QS21-(Ald-OH)-(Glu-AEM) (molecule 29); see Figure 40A. 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was subjected to preparative MP-LC2A. The fractions corresponding to the product were immediately pooled together, frozen, and lyophilized overnight to obtain the title compound (1.46 mg, 72%) as a white, fluffy solid. Purity was 92% based on LC-MS (accurate mass: 2112, 4 isomers: Api / Xyl(2:1)). LRMS (m / z): 2112 [M-1] 1- LC-MS rt(min):2.83 / 2.92 1B (Dual peak, 7 / 93 UV-area % due to QS21 mixture)

[0187] Synthesis of QS21-Ald-EMCH (Figure 40B, Molecule 30) QS21 (4.82 mg, 2.42 μmol) and EMCH.TFA (4.11 mg, 0.012 mmol) were dissolved in methanol (super-anhydrous, 0.25 mL). The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and subjected to preparative MP-LC. 2A The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight. The product was then subjected to preparative MP-LC. 2A The compound was re-purified using [method / tool ​​name]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.78 mg, 52%) as a white, fluffy solid. Purity was 96% based on LC-MS. LRMS (m / z): 2196 [M-1] 1- LC-MS rt(min):2.44 1B(Multiple peaks caused by the mixture QS21)

[0188] Synthesis of QS21-Glu-AMPD (Figure 40C; Molecule 31) 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2A The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (3.76 mg, 74%) as a white, fluffy solid. Purity was 94% based on LC-MS. LRMS (m / z): 2076 [M-1] 1- LC-MS rt(min):2.78 1B (Multiple peaks caused by the mixture QS21)

[0189] Synthesis of QS21-(Ald-EMCH)-(Glu-AMPD) (Figure 40D; Molecular 32) QS21-Glu (2.47 mg, 1.19 μmol) and EMCH.TFA (2.02 mg, 5.95 μmol) were dissolved in methanol (super-anhydrous, 100 μL). Next, TFA (0.36 μL, 4.76 μmol) was added. The reaction mixture was shaken for 1 minute and allowed to stand at room temperature. After 2 hours, the reaction mixture was separated and analyzed by preparative MP-LC. 2A The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.25 mg, 83%) as a white, fluffy solid. Purity was 95% based on LC-MS. LRMS (m / z): 2283 [M-1] 1- LC-MS rt(min):2.88 1B (Multiple peaks caused by the mixture QS21)

[0190] Synthesis of QS21-(Ald-OH)-(Glu-AMPD) (Figure 40E; Molecule 33) 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 minute and allowed to stand at room temperature. After 1 hour, the reaction mixture was separated by preparative MP-LC. 2A The mixture was subjected to [a specific procedure]. The fractions corresponding to the product were immediately pooled together, frozen, and freeze-dried overnight to obtain the title compound (2.16 mg, 42%) as a white, fluffy solid. Purity was 96% based on LC-MS. LRMS (m / z): 2077 [M-1] 1- LC-MS rt(min):2.77 1B (Multiple peaks caused by the mixture QS21)

[0191] Example 2: Activity of saponin derivatives - Preliminary experiment The saponin modifications described herein were found not to substantially interfere with the saponin's ability to promote endosomal escape (modified saponin or saponin released from conjugate inside the endosome). The experimental results are summarized in Table Ex2 below.

[0192] Chemically modified saponin SO1861 showed reactivity in cell-based bioassays, measured by relative cell viability as a reading. HeLa cells were incubated for 72 hours in the following configuration, and cell viability was evaluated before and after the 72-hour incubation. In the experiment, cells were exposed to a 1.5 pM dianthin-EGF conjugate. The negative control was cells incubated with a buffer vehicle and 10 micrograms / ml saponin without dianthin-EGF. Cell viability was set to 100% for controls where both saponin and EGF-dianthin were excluded. The positive control was 10 micrograms / ml unmodified saponin SO1861 + dianthin-EGF. Cell viability after 72 hours was essentially 0%. For chemically modified saponin variants, 10 micrograms / ml saponin was tested in combination with 1.5 pM dianthin-EGF. SO1861-Ald-EMCH reduced cell viability at 10 micrograms / ml.

[0193] These data demonstrate that saponins can be modified at free aldehyde groups or free carbonyl groups without losing their endosomal escape-promoting activity.

[0194] [Table 13]

[0195] Example 3: Activity of Saponin Derivatives - Detailed Study Various saponins (e.g., SO1861, QS-21) were co-administered to cells as "free" non-conjugate molecules in combination with ligand-toxin fusions (e.g., EGF-dianthine) or antibody-protein toxin conjugates, resulting in enhanced cytotoxic activity of target-expressing cells.

[0196] 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 various intramolecular positions (single, double, or triple modification), thereby providing a series of saponin derivatives outlined in Tables A2 and A3. The saponin derivatives were examined for: 1) endosomal escape activity of ligand toxin (modified SO1861 / QS21 titration + 5 pM EGF dianthin) in EGFR-expressing cells (HeLa and A431); 2) intrinsic cytotoxicity to HeLa and A431 (modified SO1861 / QS21 titration); and 3) human erythrocyte hemolytic activity (modified SO1861 / QS21 titration on human erythrocytes).

[0197] To measure endosomal escape-promoting activity, modified SO1861 was titrated in EGFR-expressing cells (HeLa and A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantine (see Figures 18A-B and 19A-B). Furthermore, endosomal escape-promoting activity was also measured for saponin derivatives titrated in the presence of a non-effective constant concentration of 5 pM EGF-diantine (see Figures 23A-B for comparisons between SO1861 and SO1861-Ald-EMCH and blocked SO1861-Ald-EMCH, and Figures 24A-B for comparisons between SO1861 and SO1861-(Ald-EMCH)-(Ac-OH), SO1861-(Ald-EMCH)-(Glu-AMPD), and SO1861-(Ald-EMCH)-(Ac-OH)-(Glu-AMPD)).This is because modified saponins with a single modification have the following concentrations compared to SO1861: SO1861-Ald-OH: IC50 = 600 nM in HeLa and A431: IC50 = 600 nM; SO1861-Glu-AMPD: IC50 = 600 nM in HeLa and A431: IC50 = 600 nM; SO1861-Ac-OH: IC50 = 1000 nM in HeLa and A431: IC50 = 600 nM. The activity of SO1861-Glu-AEM was 800nM, with IC50=1500nM and A431:IC50=2000nM on HeLa, and SO1861-Ald-EMCH was 2000nM and A431:IC50=2000nM on HeLa. Furthermore, the activity of the double modified form was 800nM, with the following IC50 values: SO1861-(Ac-OH)-(Glu-AMPD) was 800nM on HeLa. =3000nM and A431:IC50=3000nM, for SO1861-(Ald-OH)-(Glu-AMPD), in HeLa:IC50=4000nM and A431:IC50=4000nM, for SO1861-(Ald-OH)(Ac-OH), in HeLa:IC50=4000nM and A431:IC50=5000nM, for SO1861-(Glu-AEM)-(Ac-OH), HeL This indicates that activity was observed with a:IC50=8000nM and A431:IC50=4000nM, with SO1861-(Ald-EMCH)-(Ac-OH) with HeLa:IC50=8000nM and A431:IC50=10,000nM, and with SO1861-(Glu-AEM)-(Ald-OH) with HeLa:IC50=40,000nM and A431:IC50=20,000nM. The triple-modified compounds tested showed no activity at these concentrations. In the unmodified SO1861 control, the following IC50 values ​​were observed: HeLa:IC50=100nM and A431:IC50=200nM. The endosomal escape enhancement of modified QS21 was measured by titrating EGFR-expressing cells with saponin derivatives in the presence of a non-effective constant concentration of 5 pM EGF-dianthine (Figures 30A and 30B).This indicates that the following modified QS21 showed activity compared to unmodified QS21 at the following concentrations: QS21 at IC50=200nM in HeLa and A431; QS21-Ald-OH at IC50=600nM in HeLa and A431; QS21-Glu-AEM at IC50=600nM in HeLa and A431; and QS21-(Ald-OH)-(Glu-AEM) at IC50=1500nM in HeLa and A431 at IC50=3000nM. In conclusion, both unmodified SO1861 and QS21 were effective in HeLa and A431 cells at IC50=200nM. Single SO1861 / QS21 modifiers (SO1861-Ald-EMCH, blocked SO1861-Ald-EMCH, SO1861-Glu-AMPD, SO1861-Ald-OH, SO1861-Ac-OH, SO1861-Glu-AEM, QS21-Ald-OH, QS21-Glu-AEM) showed activity in HeLa or A431 cells at IC50 = 600 nM–2000 nM (Tables A5 and A6), while double SO1861 / QS21 modifiers showed activity in HeLa or A431 cells at IC50 = 1500 nM–40,000 nM (Tables A5 and A6). No activity was observed for the triple SO1861 modifier up to 20,000 nM.

[0198] As described above, to measure endosomal escape-promoting activity, SO1861 derivatives, QS21 derivatives, and their underivativeated counterparts were titrated in EGFR-expressing cells (HeLa and A431) in the presence of a non-effective constant concentration of 5 pM EGF-diantine. This indicates that underivativeated SO1861, underivativeated QS21, and the QS21 derivative QS21-Glu-AMPD were effective in HeLa and A431 cells with an IC50 of 200 nM. Single SO1861 / QS21 modified compounds (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-A EM showed activity in HeLa or A431 at IC50 = 600 nM–2000 nM (Tables A5 and A6), while the double SO1861 modifier and the double QS21 modifier showed activity in HeLa or A431 at IC50 = 1500 nM–40,000 nM (Tables A5 and A6). No activity was observed for the triple SO1861 modifier up to 20,000 nM.

[0199] For toxicity testing, modified SO1861 was titrated in HeLa cells (see Figures 20A and 21A) and A431 cells (see Figures 20B and 21B). Figures 25A and 25B illustrate the details of toxicity testing for SO1861, SO1861-Ald-EMCH, and blocked SO1861-Ald-EMCH, while Figures 26A and 26B show details for SO1861, SO1861-(Ald-EMCH)-(Ac-OH), SO1861-(Ald-EMCH)-(Glu-AMPD), and SO1861-(Ald-EMCH)-(Ac-OH)-(Glu-AMPD). This indicates that unmodified SO1861 exhibits the strongest intrinsic toxicity to HeLa cells: IC50 = 2000 nM, while the single-modified SO1861-Ac exhibits toxicity to HeLa cells at IC50 = 10,000 nM. For all other SO1861 derivatives, the intrinsic toxicity (IC50) to HeLa cells was higher than 20,000 nM. In A431 cells, toxicity of unmodified SO1861 was observed at IC50: 1000 nM, while the single-modified SO1861-Ac-OH, SO1861-Ald-OH, SO1861-Glu-AMPD, and SO1861-Ald-EMCH exhibited toxicity at (acceptable) IC50 = 2000 nM, IC50 = 7000 nM, IC50 = 20,000 nM, and IC50 = 30,000 nM. To assess the toxicity of modified QS21, saponin derivatives were titrated in HeLa cells (see Figure 31A) and A431 cells (see Figure 31B). This indicates that QS21 exhibits toxicity in HeLa cells: IC50 = 6000 nM and A431 cells: IC50 = 3000 nM; QS21-Ald-OH exhibits toxicity in HeLa cells: IC50 = 20,000 nM and A431 cells: IC50 = 20,000 nM; QS21-Glu-AEM exhibits toxicity in HeLa cells: IC50 => 100,000 nM and A431 cells: IC50 => 100,000 nM; and QS21-(Ald-OH)-(Glu-AEM) exhibits toxicity in HeLa cells: IC50 => 100,000 nM and A431 cells: IC50 => 100,000 nM. No toxicity was observed up to 100,000 nM with SO1861 or QS21 double modifiers, or with SO1861 triple modifiers.As described above, unmodified or modified SO1861 or QS21 were titrated in HeLa and A431 cells. This shows that unmodified SO1861 showed toxicity with IC50=1000nM (HeLa) and IC50=2000nM (A431), while QS21 showed toxicity with IC50=6000nM (HeLa) and IC50=3000nM (A431), and QS21-Glu-AMPD showed toxicity with IC50=9000nM (HeLa) and IC50=5000nM (A431) (Tables A5 and A6). Single SO1861 modifiers and in HeLa cells Regarding the single QS21 modifiers, SO1861-Ald-EMCH, SO1861-Ald-EMCH (block), SO1861-(Ald-OH), SO1861-Glu-AEM, QS21-Ald-EMCH, and QS21-Glu-AEM showed no toxicity up to 100,000 nM, whereas SO1861-Glu-AMPD, SO1861-Ac-OH, and QS21-(Ald-OH) had IC50s of 20,000 nM and 10,000 nM, respectively. Toxicity was observed at 00 nM and IC50 = 20,000 nM (Tables A5 and A6). In A431 cells, SO1861-Glu-AEM and QS21-Glu-AEM showed no toxicity up to 100,000 nM, whereas toxicity was observed for SO1861-Ald-EMCH (IC50 = 30,000 nM), SO1861-Ald-EMCH (blocked) (IC50 = 30,000 nM), SO1861-(Ald-OH) (IC50 = 7000 nM), and SO1 Toxicity was observed for 861-Ac-OH (IC50=2000nM), SO1861-Glu-AMPD (IC50=20,000nM), QS21-Ald-EMCH (IC50=30,000nM), and QS21-(Ald-OH) (IC50=20,000nM) (Tables A5 and A6). No toxicity was observed up to 100,000 nM for the SO1861 double modifier, the QS21 double modifier, or the SO1861 triple modifier (Tables A5 and A6).

[0200] In addition, the hemolytic activity of unmodified and modified SO1861 was measured by a human erythrocyte hemolysis assay (see Figures 22, 27, 28, and 32). This indicates that unmodified SO1861 showed activity with an IC50 of 8000 nM, and unmodified QS21 showed activity with an IC50 of 3000 nM. While the single modified compound SO1861-Ald-EMCH showed no hemolytic activity up to 1,000,000 nM, hemolytic activity in human red blood cells was observed for SO1861-Ald-EMCH (blocked) (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). Regarding the SO1861 and QS21 double modifiers, 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), whereas lysis activity was not observed for the latter. Hemolysis was observed with 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 up to 100,000 nM for any of the triple-modified SO1861 compounds. In the hemolysis assay, hemolytic activity was observed with unmodified SO1861 at IC50=8000 nM, with unmodified QS21 at IC50=3000 nM, and with modified QS21-Glu-AMPD at IC50=3000 nM.The single modified compound SO1861-Ald-EMCH showed no hemolytic activity up to 1,000,000 nM, whereas the hemolytic activity of human red blood cells was as follows: SO1861-Ald-EMCH (block) (IC50=300,000 nM), SO1861-(Ald-OH) (IC50=30,000 nM), SO1861-Ac-OH (IC50=20,000 nM), S This was observed with O1861-Glu-AMPD (IC50=20,000nM), SO1861-Glu-AEM (IC50=30,000nM), QS21-Ald-EMCH (IC50=30,000nM), QS21(Ald-OH) (IC50=20,000nM), and QS21-Glu-AEM (IC50=10,000nM) (Tables A5 and A6). For the SO1861 double modifier or QS21 double modifier, no hemolytic activity (up to 1,000,000 nM) was observed 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), whereas hemolytic activity was observed for SO1861-(Ald-OH)-(Glu-AEM). Hemolytic activity was observed for SO1861-(Ald-OH)-(Ac-OH)(IC50=100,000nM), SO1861-(Ac-OH)-(Glu-AMPD)(IC50=200,000nM), SO1861-(Ac-OH)-(Glu-AMPD)(IC50=140,000nM), SO1861-(Ald-OH)-(Glu-AEM)(IC50=100,000nM), and QS21-(Ac-OH)-(Glu-AMPD)(IC50=40,000nM) (Tables A5 and A6). No hemolytic activity was observed up to 100,000 nM in the SO1861 triple-modified compounds (Tables A5 and A6).

[0201] We examined the endosomal escape-promoting activity (saponin + 5pM cetuximab-saponin titration in HeLa and A431 cells, see Figures 33A-B), toxicity (saponin titration in HeLa and A431 cells, see Figures 34A-B), and hemolytic activity (saponin titration in human erythrocytes, see Figures 32 and 35) of various QS saponin fractions. This showed that QS21 (fraction), QS17 (fraction), and QS18 (fraction) were active at 200 nM in HeLa and A431 cells, while QS7 (fraction) was active at IC50 = 6000 nM (HeLa) and 10,000 nM (A431). Through toxicity measurements, QS21 (fraction), QS17 (fraction), and QS18 (fraction) were observed to be toxic in HeLa cells and A431 cells with an IC50 of 10,000 nM, while QS7 (fraction) showed toxicity with an IC50 of 20,000 nM (Figure 34). Next, a hemolysis assay was performed, which showed hemolytic activity for QS21 (fraction) at an IC50 of 3,000 nM, and for QS17 (fraction) and QS18 (fraction) at an IC50 of 5,000 nM, while no hemolytic activity was detected in QS7 (fraction) up to 20,000 nM (Figure 35).

[0202] The hemolytic activity of various SO saponins (SO1862 (isomers), SO1832, SO1904) and antibody-SO1861 conjugates (cetuximab-SO1861(DAR4), trastuzumab-SO1861(DAR4)) was examined. This showed that the hemolytic activity of SO1862 (isomers, SO1832, SO1904) was comparable to that of SO1861 (IC50 = 10,000 nM) (Figure 29). Cetuximab-SO1861(DAR4) showed no hemolytic activity up to 60,000 nM, while trastuzumab-SO1861(DAR4) showed initial hemolytic activity after 60,000 nM (IC50 = 200,000 nM) (Figure 29).

[0203] When comparing the cytotoxicity, hemolytic activity, and endosomal escape-promoting activity of SO1861, SO1861-Ald-EMCH, and SO1861-Ald-EMCH-mercaptoethanol (blocked SO1861-Ald-EMCH), the latter two were similarly or essentially equally cytotoxic, hemolytic, and active in endosomal escape-promoting bioassays, and were less cytotoxic and hemolytic than SO1861. See also Tables A5 and A6.

[0204] Cell viability assay Cell viability was assessed using the MTS assay according to the manufacturer's instructions (CellTiter96® AQueous One Solution Cell Proliferation Assay, Promega). The MTS solution was diluted 20x in phenol red-free DMEM (PAN-Biotech GmbH) supplemented with 10% FBS (PAN-Biotech GmbH). Cells were washed once with 200 μL of PBS per well, and then 100 μL of diluted MTS solution was added per well. Plates were incubated at 37°C for approximately 20–30 minutes. Subsequently, optical density at 492 nm was measured using a Thermo Scientific Multiskan FC plate reader (Thermo Scientific). For quantification, the background signal of the "medium only" well was subtracted from all other wells, and then the background-corrected signal of the untreated well was divided by the corrected background signal of the treated well to calculate the untreated / treated cell ratio.

[0205] FACS analysis Cells were seeded in DMEM (PAN-Biotech GmbH) supplemented with 10% fetal bovine serum (PAN-Biotech GmbH) and 1% penicillin / streptomycin (PAN-Biotech GmbH) at 500,000c / plate in 10cm dishes and incubated for 48 hours until a concentration of 90% was achieved (5% CO2, 37°C). Next, the cells were treated with trypsin (TrypIE Express, Gibco Thermo Scientific) to isolate single cells. 0.75 x 10 6 The cells were transferred to a 15 mL Falcon tube and centrifuged (1,400 rpm, 3 min). The supernatant was discarded, while the cell pellet was left submerged in water. The pellet was lysed by gently tapping the Falcon tube on a vortex shaker, and the cells were hydrated in 4 mL of cold PBS (Mg 2+ and Ca 2+ The cells were washed with free, 2% FBS. After washing, the cells were immersed in 3 mL of cold PBS (Mg 2+ and Ca 2+ Resuspend the cells in free PBS (2% FBS) and divide equally into three round-bottom FACS tubes (1 mL / tube). Centrifuge the cells again and rinse in 200 μL of cold PBS (Mg 2+ and Ca 2+ 200 μL of antibody solution (free, 2% FBS) or 195 μL of cold PBS (Mg 2+ and Ca 2+ The cells were resuspended in 5 μL of antibody in free PBS (2% FBS). APC mouse IgG1, κ isotype Ctrl FC (#400122, Biolegend) was used as the isotype control, and APC anti-human EGFR (#352906, Biolegend) was used. The samples were incubated on a tube roller mixer at 4°C for 30 minutes. The cells were then chilled in cold PBS (Mg 2+ and Ca 2+Cells were washed three times with free PBS (2% FBS) and fixed at room temperature for 20 minutes using a 2% PFA solution in PBS. The cells were washed twice with cold PBS and resuspended in 250–350 μL of cold PBS for FACS analysis. Samples were analyzed using the BD FACSCanto II flow cytometry system (BD Biosciences) and FlowJo software. The results of the FACS analysis are summarized in Table A4.

[0206] [Table 14]

[0207] Hemolysis assay Red blood cells (RBCs) were separated from the buffy coat using a Ficoll gradient. The resulting RBC pellet (~4-5 ml) was mixed with 50 ml of DPBS (Ca 2+ / Mg 2+ The cells were washed twice with PAN-Biotech GmbH. The cells were pelleted by centrifugation at 800xg for 10 minutes at room temperature. RBCs were counted and, based on the total cell count, DPBS (Ca 2+ / Mg 2+ (None) The solution was resuspended at 500,000,000 c / ml.

[0208] Saponin dilution, DPBS (Ca 2+ / Mg 2+ The solution was prepared at a final intensity of 1.11x in PAN-Biotech GmbH. For the positive lysis control, a 0.02% Triton-X100 solution was used in DPBS. + / +The following preparations were made. 135 μl of all compound solutions were dispensed into each well of a 96-well V-bottom plate. 15 μl of RBC suspension was added and mixed briefly (10 seconds - 600 rpm). The plate was incubated at room temperature for 30 minutes with gentle agitation. The plate was then rotated at 800xg for 10 minutes to pelletize the RBCs, and 100-120 μl of the supernatant was transferred to a standard 96wp (96-well plate). Subsequently, the OD at 405 nm was measured using a Thermo Scientific Muliskan FC plate reader (Thermo Scientific). Quantification was performed using DPBS. + / + The percentage of hemolysis compared to 0.02% Triton-X100 was calculated by subtracting the background signal of the "only" well from all other wells, and then dividing the corrected background signal of the treated well by the corrected background signal of the 0.02% Triton-X100 well (x100).

[0209] [Table 15]

[0210] [Table 16]

[0211] Example 4: Critical micelle concentration (CMC) of saponin derivatives material and method The critical micelle concentrations (CMCs) of saponin derivatives derived from Saponaria officinalis (SO) (Table A7) and Quillaya Saponaria (QS) (Tables A8 and A9) were determined using a fluorimetric method developed by DeVendittis et al. The following measurements were taken using the method described in "Analytical Biochemistry" (Fluorescence Measurement Method for Estimating the Critical Micelle Concentration of Surfactants), August 1981, Vol. 115, No. 2, pp. 278-286):

[0212] Purified water (MQ) or PBS (Dulbecco's PBS) + / + The emission spectra of 8-anilinonaphthalene-1-sulfonic acid (ANS) in either of the following locations were measured at dry weight saponin concentrations ranging from 1 to 1400 μM to cover the range below and above the CMC. Above the CMC, the fluorescence yield of ANS increases and the wavelength of maximum emission decreases due to the positioning of the fluorescent dye within the micelle. Fluorescence yields were recorded with a Fluoroskan Ascent FL (Thermo Scientific) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. 6 μg of ANS at a concentration of 75.86 μM was used per sample and measurement.

[0213] result SO1861 Saponin Chemical modifications to the functional groups aldehyde (Ald) and glucuronic acid (Glu), and the removal of the acetyl group (Ac), strongly affected the micelle properties of individual saponins. As shown in Figure 42, single modifications to individual functional groups on SO1861 saponins significantly affected the micelle formation ability, represented by the slope of the relative fluorescence value of the resulting ANS. Modifications to glucuronic acid (SO1861-Glu-AMPD, SO1861-Glu-AEM) clearly resulted in steeper slopes (Figure 42), and lower CMCs were obtained, such as 185 μM for natural SO1861. Similar observations were obtained for the block SO1861-Ald-EMCH sample. However, modifications with aldehyde and acetyl groups (SO1861-Ald-OH, SO1861-Ald-EMCH, SO1861-Ac-OH) resulted in a significantly flatter slope (Figure 42) and higher CMC values ​​than those for natural SO1861. The SO1861-Ald-EMCH sample was particularly interesting because the resulting slope was almost flat and no CMC could be measured even at concentrations up to 800 μM.

[0214] Similar observations regarding the modification site have been obtained for double modification (Figure 43) and triple modification (Figure 44) of SO1861 saponin. Modifications to the 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 a steeper ANS fluorescence yield slope and therefore lower CMC values, while modifications to the aldehyde and acetyl positions (SO1861-(Ald-OH)-(Ac-OH), SO1861-(Ald-EMCH)-(Ac-OH)) resulted in a flatter ANS fluorescence yield slope and therefore increased CMC values ​​compared to natural SO1861 (Table A7).

[0215] When comparing the triple-modified saponins SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) and SO1861-(Ald-OH)-(Ac-OH)-(Glu-AMPD), the modification of glucuronic acid in SO1861-(Glu-AEM)-(Ald-OH)-(Ac-OH) resulted in a flatter slope of individual ANS fluorescence yields, whereas the modification of glucuronic acid in SO1861-(Ald-OH)-(Ac-OH)-(Glu-AMPD) resulted in a steeper slope of individual ANS fluorescence yields compared to natural SO1861 (Figure 44). These results demonstrate the importance of modifications at the aldehyde and / or acetoxy position when CMC is considered, as the aldehyde and / or acetoxy modifications can increase the CMC, at least partially mitigating the negative effects of the aldehyde and / or acetoxy modifications, even in Glu-modified derivatives (which have a lower CMC than free saponins).

[0216] [Table 17]

[0217] QS saponin Saponins derived from Quillaja saponaria (QS), QS7, QS17, QS18, QS21 CMC values ​​were measured for Frac and QS21 SP, and are shown in Table A8. As shown in Figure 45, the slope of the ANS fluorescence yield for each QS saponin is consistent with the derived CMC values. The obtained CMC values ​​show a decreasing trend, starting with the highest CMC value of 49 μM for QS21 SP, and decreasing towards QS-17, QS-18, and QS-21 Frac, all showing similar CMC values ​​around 70 μM. Finally, a CMC value of 230 μM was obtained for QS-7.

[0218] 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).

[0219] For single-modification QS21 saponins QS21-Ald-EMCH (molecule 30; Figure 40B), QS21-Glu-AEM, QS21-(Ald-OH), and QS21-Glu-AMPD (Figures 47A, 47B), only the AMPD modification to the glucuronic acid (QS21-Glu-AMPD, Figure 47B) resulted in a steeper ANS fluorescence yield slope compared to natural QS21, which produced a lower CMC value of 40 μM (Table A9). All other single-modification QS21, both at the glucuronic acid (QS21-Glu-AEM) and aldehyde positions (QS21-(Ald-OH), QS21-Ald-EMCH), resulted in a flatter ANS fluorescence yield slope than natural QS21 (Figure 47B).

[0220] Similar to the findings regarding the double modification of Saponaria officinalis saponin SO1861, the AldGlu modification of QS21 saponin (QS21-(Ald-OH)-(Glu-AMPD), Figure 40E, Molecule 33, Figure 47C) also resulted in a steeper slope of ANS fluorescence yield compared to natural QS21, which yielded a lower CMC value of 39 μM (Table A9). All other QS21 double modifications at both the glucuronic acid and aldehyde positions (QS21-(Ald-OH)-(Glu-AEM), QS21-Ald-EMCH-(Glu-AEM), Figure 47C) resulted in flatter ANS fluorescence yields than natural QS21.

[0221] [Table 18]

[0222] [Table 19]

[0223] Example 5: Endosome escape-promoting activity of SO1861 and SO1861-Ald-EMCH SO1861 and SO1861-Ald-EMCH (also referred to as SO1861-EMCH, e.g., Figures 48-58) were examined for their ability to promote the endosomal escape of targeted proteotoxins. For this purpose, SO1861 or SO1861-Ald-EMCH was titrated against constant concentrations of 10 pM cetuximab-saporin (cetuximab conjugated to the proteotoxin and saporin at DAR4) in EGFR-expressing cells (A431). This was because SO1861 (IC50=800 nM) and SO1861-Ald-EMCH (IC50=2000 nM) were found to be 10 pM The combination of cetuximab and saporin induced effective cytotoxicity in A431 cells, whereas SO1861 or SO1861-Ald-EMCH alone showed no cytotoxic activity (Figure 48).

[0224] Next, cetuximab-diantine or cetuximab-saporin were titrated against various constant concentrations of SO1861 or SO1861-Ald-EMCH. This demonstrated efficient cell killing by low pM concentrations of cetuximab-diantine (IC50=1 pM, Figure 49) or cetuximab-saporin (IC50=0.5 pM, Figure 50) in the presence of 4000 nM SO1861-Ald-EMCH, 4829 nM SO1861-Ald-EMCH, or 1500 nM SO1861. This cytotoxic effect was observed to be... It was not observed with 300 nM SO1861 or 300 nM SO1861-Ald-EMCH (Figures 49 and 50).

[0225] Next, SO1861 or SO1861-Ald-EMCH was titrated against a constant concentration of 10 pM EGF dianthin (EGFR-targeted fusion protein toxin) in EGFR-expressing cells (A431). This indicates that SO1861 (IC50 = 800 nM) and SO1861-Ald-EMCH (IC50 = 2000 nM), in combination with 10 pM EGF dianthin, induced efficient cell killing of A431 cells, while SO1861 or SO1861-Ald-EMCH alone showed no cell-killing activity (Figure 51).

[0226] Next, EGF dianthin was titrated against various constant concentrations of SO1861 or SO1861-Ald-EMCH. This demonstrated efficient cell killing by low pM concentrations of EGF dianthin (IC50 = 0, 1 pM, Figure 52) in the presence of 4829 nM SO1861-Ald-EMCH or 1500 nM SO1861. This cell-killing effect was not observed with 10 nM SO1861 or 300 nM SO1861 (Figure 52).

[0227] Next, trastuzumab-diantin or trastuzumab-saporin (trastuzumab conjugated to the protein toxin saporin at DAR4) was titrated against HER2-expressing cells (SK-BR-3) at constant concentrations of 1500 nM SO1861 or 4000 nM SO1861-Ald-EMCH. Low pM concentrations of trastuzumab-dianthine (IC50=0,1 pM) or trastuzumab-saporin (IC50=0,1 pM) demonstrated efficient cell killing in the presence of SO1861 or 4000 nM SO1861-Ald-EMCH (Figure 53).

[0228] All of these results, outlined in Figures 48-53, demonstrate that SO1861-Ald-EMCH efficiently facilitates endosomal escape and cytoplasmic delivery of targeted protein toxins, thereby significantly reducing the effective concentration of targeted protein toxins from the nM range to the pM range.

[0229] SO1861-Ald-EMCH was tested for its ability to promote endosomal escape of antisense oligonucleotides (BNA, cross-linked nucleic acids) against HSP27 mRNA. For this purpose, SO1861-Ald-EMCH was titrated against constant concentrations of 100 nM HSP27BNA, 100 nM cetuximab-HSP27BNA (cetuximab conjugated to HSP27BNA at DAR4), or 100 nM trastuzumab-HSP27BNA (trastuzumab conjugated to HSP27BNA at DAR4) in EGFR / HER2-expressing cells (A431). This indicates that SO1861-Ald-EMCH (IC50 = 700 nM) was found to promote endosomal escape of antisense oligonucleotides (BNA, cross-linked nucleic acids) against HSP27 mRNA. The combination of 100nM HSP27BNA, 100nM cetuximab-HSP27BNA (Figure 54), or 100nM trastuzumab-HSP27BNA demonstrated (or did not demonstrate) efficient induction of HSP27 gene silencing cells. SO1861-Ald-EMCH alone did not show any HSP27 gene silencing activity (Figure 54).

[0230] Next, cetuximab-HSP27BNA (DAR1.5 or DAR4) and trastuzumab-HSP27BNA (DAR4.4) were titrated against various constant concentrations of SO1861-Ald-EMCH in EGFR(A431) or HER2(SK-BR-3) expressing cells. This demonstrated efficient HSP27 gene silencing in A431 cells with low nM concentrations of cetuximab-HSP27BNA (IC50 = 0.5 nM, Figure 55) in the presence of 4000 nM SO1861-Ald-EMCH, whereas cetuximab-HSP27BNA alone, or cetuximab-HSP27BNA + 100 nM SO1861-Ald-EMCH, showed no gene silencing activity or only slight activity at very high concentrations (IC50 > 100 nM; Figure 550). In SKBR-3 cells, trastuzumab HSP27BNA (IC50 = 0.5 nM, Figure 56) showed efficient HSP27 gene silencing activity in the presence of 4000 nM SO1861-Ald-EMCH, whereas trastuzumab-HSP27BNA alone, or trastuzumab-HSP27BNA + 100 nM SO1861-Ald-EMCH, showed only slight gene silencing activity (IC50 > 100 nM; Figure 56).

[0231] Next, untargeted HSP27BNA was titrated against constant concentrations of SO1861-Ald-EMCH in various cell lines. This demonstrated efficient HSP27 gene silencing in A431, A2058, and SK-BR-3 cells with low nM concentrations of HSP27BNA (IC50(SK-BR3)=2nM; IC50(A431)=10nM; IC50(A2058)=10nM) in the presence of 4000nM or 4829nM SO1861-Ald-EMCH (Figures 57 and 58), whereas HSP27BNA alone induced gene silencing at much higher concentrations (IC50(SK-BR3)=300nM; IC50(A431)=1000nM; IC50(A2058)>1000nM) (Figures 57 and 58). When comparing the activity of HSP27BNA (with and without SO1861-Ald-EMCH) with that of HSP27LNA (LNA, Loc Nucleic Acid), the inventors observed that the endosomal escape / gene silencing enhancement coefficients were similar, but at higher HSP27LNA concentrations compared to HSP27BNA (Figure 58).

[0232] All of this indicates that SO1861-Ald-EMCH efficiently promotes endosomal escape and cytoplasmic delivery of targeted and untargeted antisense BNA oligos, thereby significantly reducing the effective concentrations of targeted and untargeted antisense oligos from the μM range to the lower nM range.

[0233] material Trastuzumab (Tras, Herceptin®, Roche) and cetuximab (Cet, Erbitux®, Merck KGaA). Diantin-cys was manufactured and purchased from Proteogenix, France, and EGF-diantin was manufactured from Escherichia coli (E. coli) using standard procedures. Cetuximab-saporin and trastuzumab-saporin conjugates were manufactured and purchased from Advanced Targeting Systems (San Diego, CA).

[0234] method Flash chromatography Grace Reveleris X2(registered trademark) C-815 Flash; Solvent delivery system: 3-piston pump with autopriming, 4 independent channels with up to 4 solvents in a single run, auto-switch line for solvent depletion; Maximum pump flow rate 250 mL / min; Maximum pressure 50 bar (725 psi); Detection: UV 200-400 nm, combination of up to 4 UV signals and full UV range scan, ELSD; Column size: 4-330 g on the instrument, Luer type, 750 g to 3000 g, with optional holder.

[0235] HSP27BNA Oligo Sequence Zhang et al. (2011) [Y Zhang, Z Qu, S Kim, V Shi, B HSP27 BNA oligo(5'-GGCacagccagtgGCG-3')([SEQ ID NO:2]) was ordered from Bio-Synthesis Inc. (Lewisville, Texas) with and without a 5'-thiol C6 linker. HSP27 LNA oligo(5'-ggcacagccagtggcg-3')([SEQ ID NO:3]) was ordered from Bio-Synthesis Inc. (Lewisville, Texas).

[0236] RNA isolation and gene expression analysis Cell-derived RNA was isolated and analyzed according to a standard protocol (Biorad). The qPCR primers used are shown in Table A10.

[0237] [Table 20]

[0238] Trastuzumab-saporin and cetuximab-saporin synthesis Custom mAB-superin conjugates are used in Advanced Targeting Manufactured and purchased from Systems (San Diego, CA).

[0239] Trastuzumab-dianthine and cetuximab-dianthine synthesis Dianthine-Cys (17.0 ml, ~9.6 mg) was concentrated by ultrafiltration using a Vivaspin T15 filter tube (3,000 g, 20°C, 10 minutes). The resulting 3.25 ml aliquot was gel filtered using a 10 ml Zeba spin column eluted with TBS pH 7.5.

[0240] Trastuzumab (mAB) or cetuximab (mAB) (0.30 ml, ~10 mg) was diluted to 10 mg / ml with DPBS pH 7.5, desalted through a 5 ml Zeba spin column eluting with DPBS pH 7.5, and normalized to 2.50 mg / ml. Aliquots of mAb were prepared in DMSO with freshly prepared SMCC solution (1.00 mg / ml, 4.20 molar equivalents, 13.9 x 10⁴). -5 The mixture was briefly vortexed and then incubated at 20°C for 60 minutes using roller mixing. The reaction was then carried out by adding aliquots (2.0 mg / ml, 5.0 molar equivalents, 69.5 x 10) of freshly prepared glycine solution in DPBS pH 7.5. -5Quenched by adding mmol. After gel filtration using a 10 ml zeba spin column that elutes at TBS pH 7.5, mAb-SMCC (4.27 mg, 2.80 x 10) was extracted. -5 mmol (1.514 mg / ml) was obtained.

[0241] Diantine-Cys (7.54 mg, 25.3 x 10) -5 A liquot of freshly prepared TCEP solution (1.00 mg / ml, 0.5 molar equivalent, 12.6 x 10) was added to mmol, 2.258 mg / ml in TBS pH 7.5. -5 Dianthine-SH (6.0 mg, 20.2 x 10⁻¹⁴) was added, the mixture was vortexed briefly, and then incubated at 20°C for 60 minutes using roller mixing. Subsequently, gel filtration using a 10 ml Zeba spin column eluted at TBS pH 7.5 was performed, yielding dianthine-SH (6.0 mg, 20.2 x 10⁻¹⁴). -5 mmol, 1.722 mg / ml, dianthine (SH=1:1) was obtained.

[0242] To bulk mAb-SMCC, an aliquot of dianthine-SH (7.20 molar equivalents) was added, the mixture was vortexed briefly, and then incubated overnight at 20°C. After approximately 16 hours, an aliquot of freshly prepared NEM solution (2.50 mg / ml, 5.0 molar equivalents, 10¹ x 10¹⁴) was added to TBS pH 7.5. -5 The reaction was quenched by the addition of mmol. The reaction mixture was filtered to 0.45 μm and then ultrafiltration was performed using a Vivaspin T15 filter tube (3,000 g, 20°C, 15 minutes). The solution was concentrated to <2 ml. The conjugate was purified by gel filtration using a 1.6 x 35 cm Superdex 200PG column eluted with DPBS pH 7.5.

[0243] Antibody-(L-HSP27 BNA) n [On HSP27 BNA disulfide] Trastuzumab-(L-HSP27) via PEG4-SPDP in DAR4 4 Cetuximab-(L-HSP27)4 Synthesizing and PEG4-SPDP-mediated synthesis of cetuximab-(L-HSP27) in DAR2 2 synthesis Trastuzumab and cetuximab will hereafter be referred to as "Ab". Ab was conjugated to HSP27 BNA disulfide via a tetra(ethylene glycol) succinimidyl 3-(2-pyridyldithio)propionate (PEG4-SPDP) linker, which forms an unstable (L) disulfide bond between Ab and HSP27 BNA. The procedure is described exemplarily for trastuzumab-(L-HSP27 BNA)4:

[0244] HSP27 BNA disulfide oligo (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) using roller mixing at 20°C for 30 minutes. Oligo-SH was then purified using a PD10 G25 desalting column eluting into TBS pH 7.5 and used immediately. Oligo-SH was obtained (2.48 mg, 90%, 1.24 mg / ml, SH to oligo ratio = 0.8).

[0245] Trastuzumab (1.5 mg, 10.3 nmol, 2.50 mg / ml) was reacted with an aliquot (6.81 molar equivalents, 70.1 nmol, 39 μg) of a freshly prepared PEG4-SPDP solution in DMSO (1 mg / ml) using roller mixing at 20°C for 60 minutes. The reaction was then quenched with glycine (15.1 μl of a freshly prepared 2 mg / ml solution in TBS pH 7.5), followed by desalting by elution with TBS pH 7.5 using a Zeba desalting column. Aliquots of the resulting Tras-S-PEG4-SPDP were collected and analyzed by UV-Vis analysis. SPDP uptake was measured by releasing pyridyl-2-thione (PDT) using TCEP and then performing UV-Vis analysis at 343 nm (SPDP to Ab ratio: 4). The remaining Tras-(S-PEG4-SPDP)4 was reacted with a newly prepared aliquot of HSP27 oligonucleotide (oligo-SH) (8 molar equivalents, 82.4 nmol, 1.24 mg / ml) and incubated overnight at 20°C using roller mixing. After 17 hours, the conjugate was analyzed by UV-Vis analysis, and the incorporation of HSP27 by pyridyl-2-thione (PDT) substitution was confirmed at 343 nm. The crude conjugate was purified using a 1.6 x 33 cm Sephadex G50 column eluted with DPBS pH 7.5. The resulting trastuzumab-(L-HSP27)4 was obtained as a single fraction. Yield: nd, Purity: 96%, HSP27 BNA to Ab ratio = 4.4

[0246] Example 6 - Endosome escape-promoting activity of saponins Previously, the efficacy of various saponins (SO1861, SA1642) was demonstrated when they were co-administered to cells as "free" non-conjugate molecules in combination with ligand-toxin fusions (e.g., EGF dianthin) or antibody-protein-toxin conjugates, resulting in enhanced cytotoxic activity of target-expressing cells. Here, three different saponin molecules (SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904) isolated from the root extract of Saponaria officinalis were used in HeLa(EGFR +Cells were titrated with and without a non-effective constant concentration of 1.5 pM EGF dianthin. This showed a strong enhancement of cytotoxic activity for all tested saponin variants compared to treatment without EGF dianthin (IC50 = 300 nM; Figure 63A). Next, EGF dianthin was titrated with constant concentrations of saponins (~1000 nM), which showed a strong enhancement of targeted cell killing at low pM concentrations of EGF dianthin, as observed for all saponins used: SO1861, SO1862 (an isomer of SO1861), SO1832, and SO1904 (IC50 = 0.4 pM; Figure 63B). EGF dianthin alone could only induce cell killing at very high concentrations (IC50 = 10,000 pM). This indicates that all of these specific types of saponins possess an intrinsic ability to efficiently induce endosomal escape using only very small amounts of available targeted toxins.

[0247] To extend this study, saponins from other sources were analyzed. Saponin (GE1741) purified from the root extract of Gypsophila elegans M.Bieb was titrated against HeLa cells with and without 1.5 pM EGF dianthine and compared to purified SO1861. GE1741 also promoted EGF dianthine-induced HeLa cell killing, but with slightly lower efficiency compared to SO1861 (GE1741 IC50 = 800 nM, Figure 63C), and also showed higher general toxicity (IC50 = 5,000 nM without EGF dianthine, Figure 63C). In similar tests, mixtures of different partially purified saponins from Quillaja saponaria (QSmix1-3) were co-administered to HeLa cells with 1.5 pM EGF dianthin, and two of the three (QSmix1 and QSmix3) showed activity similar to SO1861 (IC50 QSmix / QSmix3 = 300 nM; Figure 63D). QSmix(2) was less efficient in promoting 1.5 pM EGF dianthin-induced cell killing (IC50 = 2000 nM; Figure 63D), however, no general toxicity was observed. This indicates that certain types of saponins are available in QS extracts and efficiently induce endosomal extrusion of the targeted ligand toxin EGF dianthin. Therefore, the saponins described in this embodiment, such as Quillaja saponaria saponin, GE1741, SO1861, SO1862, SO1832, and SO1904, are particularly attractive saponins for derivatization according to the present invention.

[0248] Example 7: Endosome escape-promoting activity of saponins and saponin derivatives Unstable / acid-sensitive derivatives (Ald-EMCH or SO1861-L-N3 (also referred to as SO1861-N3 and SO1861-azide or SO1861-N3 / azide)) were applied to SO1861 via an aldehyde group to generate SO1861-Ald-EMCH or SO1861-L-N3. To confirm the activity of SO1861-Ald-EMCH, EGFR expression (A431, HeLa) was used. For both non-expressing cells (A2058) and non-expressing cells, this molecule was titrated with and without a fixed non-effective concentration (1.5 pM) of EGF dianthine. In all three cell lines, SO1861 alone showed a strong decrease in cell viability, whereas SO1861-Ald-EMCH as a single compound showed no toxicity up to 25,000 nM (Figure 64A-C). When SO1861-Ald-EMCH was combined with 1.5 pM EGF dianthine, EGFR + In A431 and HeLa cells, a strong target-specific decrease in cell viability was observed (IC50 = 3,000 nM, Figure 64A, B), whereas EGFR - -A2058 cells were completely unaffected (Figure 64C). Similar results were obtained for SO1861-L-N3. 1.5 pM SO1861-L-N3 administered co-administered with EGF-dianthine also showed efficient cytotoxicity against A431 and HeLa cells (IC50 = 3,000 nM), however, without EGF-dianthine, general toxicity was observed above 10,000 nM (Figures 64D, 64E).

[0249] HATU was conjugated to SO1861 via the carboxylic acid group of SO1861 to produce SO1861-(S), also known as SO1861-HATU or SO1861-Glu-HATU. To measure its activity, various concentrations of SO1861-(S) were co-administered with 1.5p MEGF dianthin in EGFR-expressing HeLa cells, and its cytotoxic activity was examined. SO1861-(S) showed similar activity to SO1861, indicating that conjugation to the carboxylic acid group did not affect the endosomal escape-promoting activity of this molecule, similar to what was observed with SO1861-Ald-EMCH (Figure 65).

Claims

1. A saponin-based chemically modified saponin comprising a triterpene aglycone core structure and at least one of a first sugar chain and a second sugar chain bonded to the triterpene aglycone core structure, The aforementioned chemically modified saponin has a chiric acid or gypsogenin aglycone core structure, and in this, The aforementioned chemically modified saponin is molecule 1: 【Chemistry 1】 It corresponds to saponins, which are represented by In this, R is hydrogen in gypsogenin or hydroxyl in chiric acid, and in this, the first sugar chain A １ This is Gal-(1→2)-[Xyl-(1→3)]-GlcA, and the second sugar chain A ２ These are hydrogen, monosaccharides, or linear or branched oligosaccharides. In this case, at least one of the following chemical modifications is present: i. The chemically modified saponin is C ２３ The aglycone core structure contains an aldehyde group that is chemically modified, ii. The first sugar chain A １ However, it contains a carboxyl group of a chemically modified glucuronic acid moiety: or iii. The second sugar chain A ２ However, it contains a chemically modified acetoxy (Me(CO)O-) group; or iv. The chemically modified saponin includes any combination of chemical modifications i, ii, and iii. Chemically modified saponins (excluding SO1861 saponin).

2. The following chemical modifications: i. Position C of chiric acid or gypsogenin ２３ The aldehyde group in - Reduction to alcohol; - This involves conversion to a hydrazone bond via reaction with N-ε-maleimidocaproic acid hydrazide (EMCH), thereby providing saponin-Ald-EMCH, where the maleimide group of EMCH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; - Conversion to a hydrazone bond via reaction with N-[β-maleimidopropionic acid]hydrazide (BMPH), in which the maleimide group of BMPH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; or - This involves conversion to a hydrazone bond via a reaction with N-[κ-maleimidoundecanoic acid]hydrazide (KMUH), in which the maleimide group of KMUH is optionally derivatized by the formation of a thioether bond with mercaptoethanol; Chemically modified by; ii. A １ The carboxyl group of the glucuronic acid portion is chemically modified by conversion to an amide bond via reaction with 2-amino-2-methyl-1,3-propanediol (AMPD) or N-(2-aminoethyl)maleimide (AEM), thereby providing saponin-Glu-AMPD or saponin-Glu-AEM; and iii. A ２ One or more acetoxy groups are chemically modified by conversion to hydroxyl groups (HO-) through deacetylation. A chemically modified saponin according to claim 1, wherein at least one of the following is present.

3. If the second sugar chain is present, Glc-, Gal-, Rha-(1→2)-[Xyl-(1→4)]-Rha-, Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha-, Ara-, Xyl-, Xyl-(1→4)-Rha-(1→2)-[R1-(→4)]-Fuc- where R1 is 4E-methoxycinnamic acid, Xyl-(1→4)-Rha-(1→2)-[R2-(→4)]-Fuc- where R2 is 4Z-methoxycinnamic acid, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc-Fuc-,Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-OAc-Fuc-,Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R3-(→4)]-3-OAc-Fuc- where R3 is 4E-methoxycinnamic acid, Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc-, (Ara- or Xyl-)(1→3)-(Ara- or Xyl-)(1→4)-(Rha- or Fuc-)(1→2)-[4-OAc-(Rha- or Fuc-)(1→4)]-(Rha- or Fuc-), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc-, Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc-, Ara / Xyl-(1→4)-Rha / Fuc-(1→4)-[Glc / Gal-(1→2)]-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R4-(→4)]-Fuc- where R4 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R5-(→4)]-Fuc- where R5 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc-, 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc--Rha-(1→3)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui-(1→4)]-Fuc-, Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc-Qui-(1→4)]-Fuc-, Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc-, Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc-, Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc-, Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-, Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4OAc-Fuc-,Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R6-(→4)]-Fuc- where R6 is 5-O-[5-O-Rha-(1→2)-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R7-(→4)]-Fuc- where R7 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api / Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R8-(→4)]-Fuc- where R8 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R9-(→4)]-Fuc- where R9 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R10-(→4)]-Fuc- where R10 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R11-(→3)]-Fuc- where R11 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid), Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R12-(→3)]-Fuc- where R12 is 5-O-[5-O-Ara / Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoic acid) Glc-(1→3)-[Glc-(1→6)]-Gal-, and Those derivatives, Selected from, A chemically modified saponin according to claim 1 or 2.

4. The aforementioned saponins are NP-017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE Selected from X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2, GE1741, SO1542, SO1584, SO1658, SP1674, SO1832, QS-7, QS-7 api, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, or QS-21 B-xylo, A chemically modified saponin according to any one of claims 1 to 3.

5. The chemically modified saponin according to any one of claims 1 to 4, wherein the chemically modified saponin comprises the aglycone core structure, wherein the aglycone core structure contains an aldehyde group, and wherein the first sugar chain contains the chemically modified carboxyl group.

6. The chemically modified saponin according to any one of claims 1 to 5, provided that the aldehyde group in the aglycone core structure is chemically modified and the saponin is saponin-Ald-EMCH, then at least one glucuronic acid is also chemically modified.

7. A first pharmaceutical composition comprising the chemically modified saponin according to any one of claims 1 to 6, and a pharmaceutically acceptable excipient and / or diluent.

8. - The first pharmaceutical composition described in claim 7, A second pharmaceutical composition comprising any one or more of the following: 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, A combination of pharmaceuticals containing the following ingredients.

9. A third pharmaceutical composition comprising the chemically modified saponin described in any one of claims 1 to 6, and further comprising any one or more of the following: 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. thing.

10. The pharmaceutical combination according to claim 8, wherein the second pharmaceutical composition comprises any one or more of the following: an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate.

11. The antibody-drug conjugate is an IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more VH domains, single domain antibody, V ＨＨ , or a conjugate of an antibody containing V of camelids Ｈ The pharmaceutical combination according to claim 8 or 10, which is a conjugate of an antibody.

12. The third pharmaceutical composition according to claim 9, wherein the third pharmaceutical composition comprises any one or more of the following: an antibody-drug conjugate, a receptor-ligand-drug conjugate, an antibody-oligonucleotide conjugate, or a receptor-ligand-oligonucleotide conjugate.

13. The antibody-drug conjugate is IgG, Fab, scFv, immunoglobulin, immunoglobulin fragment, one or more VH domains, single-domain antibody, V ＨＨ , or camel species V Ｈ A third pharmaceutical composition according to claim 9 or 12, which is a conjugate of an antibody containing the above.

14. A third pharmaceutical composition according to claim 9, 12, or 13 for use as a pharmaceutical.

15. A third pharmaceutical composition according to any one of claims 9 and 12 to 14, for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

16. Use of the third pharmaceutical composition according to any one of claims 9 and 12 to 15 for the manufacture of a pharmaceutical for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

17. A pharmaceutical combination agent according to any one of claims 8 and 10-11, for use as a pharmaceutical.

18. A combination of pharmaceutical agents according to any one of claims 8, 10-11, and 17, for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

19. Use of a pharmaceutical combination according to any one of claims 8, 10-11, and 17-18 for the manufacture of a pharmaceutical for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

20. A first pharmaceutical composition according to any one of claims 7-8, for use as a pharmaceutical.

21. The first pharmaceutical composition according to any one of claims 7-8 and 20, for use in the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

22. Use of the first pharmaceutical composition according to any one of claims 7-8 and 20-21 for the manufacture of a medicament for the treatment or prevention of cancer, infectious diseases, viral infections, hypercholesterolemia, primary hyperoxaluria, hemophilia A, hemophilia B, alpha-1 antitrypsin-related liver disease, acute hepatic porphyria, transthyretin-mediated amyloidosis, or autoimmune diseases.

23. An in vitro or ex vivo method for transferring a molecule from the outside of a cell to the inside of the cell, comprising: a) The process of providing cells; b) A step of providing the molecules to be transferred from the outside of the cells to the inside of the cells provided in step a); c) A step of providing a saponin derivative according to any one of claims 1 to 6; d) A method comprising the step of contacting the cells of step a) with the molecule of step b) and the saponin derivative of step c) in vitro or ex vivo, thereby establishing the transfer of the molecule from outside the cell to inside the cell.

24. The method according to claim 23, wherein the cells are human cells including T-cells, NK-cells, and tumor cells, and / or wherein the saponin derivative is a saponin derivative according to any one of claims 1 to 6, and / or wherein the molecule in 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.

Images