Saponin-based vaccine adjuvant

Abstract

Several MS and natural saponin-based vaccine adjuvant candidates have been prepared. MS derivatives were prepared by incorporating terminal functionalized side chains onto the C3 glucuronic acid unit of natural saponins MS I and II through amide formation, while QS analogs were prepared via multi-step organic synthesis. These unnatural saponins exhibit significantly different immunostimulatory activity profiles, suggesting that the structures of the side chain, triterpenoid core, and oligosaccharide domains combine to consolidate each saponin's distinctive immune response enhancement.

Description

Background Art

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 820,477, filed on March 19, 2019, entitled "Saponin - Based Vaccine Adjuvants", the entire content of which is incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Contract R01 GM120159 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] A vaccine adjuvant is a substance used in combination with a vaccine to enhance the host's immune response to specific antigens introduced by the vaccine (Brunner et al., (2010) Immunol. Lett. 128:29 - 35, Kensil et al., (2004) Frontiers Biosci. 9:2972 - 2988, Leroux - Roels G. (2010) Vaccine 28(Suppl 3):C25 - 36, Sharp & Lavelle (2012) Development Therapeutic Agents Handbook John Wiley & Sons, Inc; pp. 533 - 546, Wang W. (2011) World J. Vaccines 1:33 - 78, Weeratna & McCluskie (2011) Recent Advan. Vaccine Adjuvants.pp. 303 - 322; Cox & Coulter (1997) Vaccine 15:248 - 256, Klebanoff et al., (2010) Immunol. Rev. 239:27 - 44, Plotkin SA. (2005) Nat. Med. 11:S5 - S11, Rappuoli & Aderem (2011) Nature 473:463 - 469, Kensil et al., (2005) Vaccine Adjuvants: Immunological and Clinical Principles Humana Press Inc.pp. 221 - 234).

[0004] Vaccine adjuvants also modulate the immune system to a desirable response against certain pathogens. For example, QS-21, which is a mixture of two isomers, is an FDA-approved adjuvant known for its ability to enhance a balanced Th1 / Th2 response in antigen-specific CTL production, which is beneficial for vaccines against intracellular pathogens and cancer (Ragupathi et al., (2011) Expert Rev. Vaccines 10:463-470, Deng et al., (2008) Angew Chem. Int. 47:6395-6398, Kensil CR. (1996) Critical Revs. Therap. Drug Carrier Systs. 13:1-55, Kensil et al., (1991) J. Immun. 146:431-437). It has the potential for a wide range of clinical applications and thus has high demand (Kensil et al., (1991) J. Immun. 146:431-437). The supply of QS-21 is very limited. The natural product is isolated from the bark of Quillaja saponaria Molina (QS), an evergreen tree native to the warm central part of Chile. However, even under current demand, ecological and economic consequences are arising from the overcollection of natural sources ( Ragupathi et al.,(2011)Expert Rev.Vaccines 10:463-470、Martin et al.,(1999)Econ.Bot.53:302-311 ). Furthermore, the abundance of QS-21 in QS bark extracts is low and its isolation is laborious (Kensil et al., (1991) J. Immun. 146:431-437, Ragupathi et al., (2010) Vaccine 28:4260-4267, Wang et al., (2005) J. Am. Chem. Soc. 127:3256-3257). QS-21 also has problems of chemical instability due to two hydrolytically labile ester moieties that complicate its formulation, and the toxicity that limits its dosage also prevents reaching full efficacy. QS-21 analogs bearing a simple dodecyl side chain or a side chain with a terminal carboxyl group have been shown to have different adjuvant activities (Adams et al., (2010) J. Am. Chem. Soc. 132:1939-1945, Chea et al., (2012) J. Am. Chem. Soc. 134:13448-13457).

[0005] Derivatization of Momordica saponins (MS) I(2) and II(3) has been shown to be a potentially viable way to achieve a practical alternative to the desired QS-21 (Wang et al., (2019) J. Med. Chem. 62:9976 - 9982). MS I and II are isolated from the seeds of the perennial Momordica cochinchinensis Spreng (MC) that grows in China and Southeast Asia ( Iwamoto et al.,(1985)Chem.Pharm.Bull.33:464-478、Tang et al.(2011)Handbook Chinese Medicinal Plants,2:768 ). The seeds are widely available and inexpensive. Incorporation of an aliphatic dodecyl chain into MS I with C3 glucuronic acid led to the derivative VSA-1(4a) having an adjuvant activity profile significantly different from that of the native precursor, particularly by enhancing the antigen-specific IgG2a response 。 Two of the MS derivatives 4a and another MS II derivative 5a, although only the structure of the triterpenoid core of 4a differs from that of the chiral acid core of 5a at C16, 5a and its native saponin precursor 3 did not have such a significant change in the IgG1 and IgG2a responses.

Summary of the Invention

Problems to be Solved by the Invention

[0006]

Means for Solving the Problems

[0007] One aspect of the disclosure is a modified saponin having the following formula:

Chemical Formula

[0008] In some embodiments of the present aspect of the disclosure, the carrier can be selected from the group consisting of polyamine polymers, polyethylene glycol amines, poly(ethylene imine), nanocarbons, and amino-containing biomolecules.

[0009] In some embodiments of the present aspect of the disclosure, the modified saponin can have the following formula I:

Chemical formula

[0010] ​In some embodiments of the present aspect of the disclosure, R3 is H and can be. In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain fatty acid having the structure HOOC-(CH2) 6-20 -.

[0011] In some embodiments of the present aspect of the disclosure, R3 can be an alkoxy group having the structure H3C-(CH2) 6-20 -O-CH2.

[0012] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0013] In some embodiments of the present aspect of the disclosure, R4 can be a long-chain alkyl terminated with a functional group selected from the group consisting of an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0014] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)- can be , where R4 can be a long-chain alkyl R6O(CH2) 6-20 -, and R6 can be selected from saccharide units selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.

[0015] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with monophosphoryl lipid A (MPL).

[0016] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with dipalmitoyl-S-glyceril cysteine (Pam2Cys) or tripalmitoyl-S-glyceril cysteine (Pam3Cys).

[0017] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with a muramyl dipeptide unit.

[0018] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with an α-Galcer unit.

[0019] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with an MS I unit.

[0020] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with an MS II unit.

[0021] In some embodiments of the present aspect of the disclosure, the modified saponin can be selected from the group consisting of Formulas A to E below.

Chemical formula

[0022] Another aspect of the disclosure is a pharmaceutical composition comprising a modified saponin having the following formula,

Chemical formula

[0023] ​In some embodiments of the present aspect of the disclosure, the carrier can be selected from the group consisting of polyamine polymers, polyethylene glycol amines, poly(ethylene imine), nanocarbons, and amino-containing biomolecules.

[0024] In some embodiments of the present aspect of the disclosure, the modified saponin can have the following formula I:

Chemical formula

[0025] In some embodiments of the present aspect of the disclosure, R3 is H yes obtained of.

[0026] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)- and obtained , R4 is a long-chain fatty acid having the structure HOOC-(CH2) 6-20 -.​obtained It is.

[0027] In some embodiments of the present aspect of the disclosure, R3 may be an alkoxy group having the structure H3C-(CH2) 6-20 -O-.

[0028] In some embodiments of the present aspect of the disclosure, R3 may be R4-NH-C(O)-, where R4 may be a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0029] In some embodiments of the present aspect of the disclosure, R4 may be a long-chain alkyl terminated with a functional group selected from the group consisting of an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0030] In some embodiments of the present aspect of the disclosure, R3 may be an alkoxy group having the structure H3C-(CH2) 6-20 -O-CH2-.

[0031] In some embodiments of the present aspect of the disclosure, R3 may be R4-NH-C(O)-, where R4 is a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0032] In some embodiments of the present aspect of the disclosure, R4 may be a long-chain alkyl terminated with a functional group selected from the group consisting of an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0033] In some embodiments of the present aspect of the disclosure, R3 may be R4-NH-C(O)-, where R4 may be a long-chain alkyl R6O(CH2) 6-20 -, and R6 may be selected from saccharide units selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.

[0034] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with monophosphoryl lipid A (MPL).

[0035] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)- obtained and R4 can be a long-chain alkyl terminated with dipalmitoyl-S-glycerylcysteine (Pam2Cys) or tripalmitoyl-S-glycerylcysteine (Pam3Cys).

[0036] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with muramyl dipeptide units.

[0037] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with α-Galcer units.

[0038] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with MS I units.

[0039] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with MS II units.

[0040] In some embodiments of the present aspect of the disclosure, the modified saponin can be selected from the group consisting of Formulas A to E below.

Chemical Formula

[0041] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition can further include at least one immunogen.

[0042] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0043] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may be formulated for administration to an animal or human subject.

[0044] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may further comprise at least one cancer therapeutic agent and a pharmaceutically acceptable carrier, and at least one chemotherapeutic agent and a saponin derivative are admixed in a pharmaceutically acceptable formulation or covalently bonded to each other.

[0045] Yet another aspect of the disclosure encompasses embodiments of a method for increasing the immunogenicity of an immunogen when administered to an animal or human subject, the method comprising at least the step of administering to the subject a vaccine comprising a pharmaceutical composition according to the present disclosure.

[0046] Yet another aspect of the disclosure encompasses embodiments of a synthetic route for synthesizing a saponin derivative, the synthetic route comprising coupling a natural saponin with a functionalized side chain molecule, the functionalized side chain comprising an amino group or a hydroxyl group.

[0047] In some embodiments of the present aspect of the disclosure, the natural saponin can be obtained from Momordica cochinchinensis Spreng.

[0048] In some embodiments of the present aspect of the disclosure, the natural saponin can be coupled to the functionalized side chain molecule via an amide formation reaction or an ester formation reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Further aspects of the present disclosure will be more readily understood by considering the detailed description of its various embodiments set forth below in conjunction with the accompanying drawings.

[0050]

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Mode for Carrying Out the Invention

[0051] The present disclosure is not limited to the specific embodiments described and can therefore vary. The technical terms used herein function only to describe specific embodiments and are not intended to limit, as the scope of the present disclosure is limited only by the appended claims.

[0052] When a range of values is provided, unless otherwise clearly indicated by context, each intermediate value between the upper and lower limits of that range to the tenth of the unit of the lower limit, and any other stated value or intermediate value of the stated range, is included within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are included within the disclosure in accordance with any specifically excluded limits of the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0053] Embodiments of the present disclosure will, unless otherwise indicated, employ techniques within the scope of the art, such as those in medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like. Such techniques are well explained in the literature.

[0054] The following examples are described to provide a complete disclosure and description to those skilled in the art of how to carry out the methods and how to use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are by weight, temperature is in °C, and pressure is at approximately atmospheric pressure. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0055] Before the embodiments of the present disclosure are described in detail, it should be understood that, unless otherwise indicated, the present disclosure is not limited to specific materials, reagents, reaction materials, manufacturing processes, dimensions, frequency ranges, applications, etc., and can therefore vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Also, where logically possible, it is also possible in the present disclosure to perform steps in a different order. Embodiments of the present disclosure can be applied to additional embodiments with measurements beyond those described in the examples herein, which are not intended to be limiting. Furthermore, embodiments of the present disclosure can be combined with or integrated with other measurement techniques beyond those described in the examples herein, which are not intended to be limiting.

[0056] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and the following claims, reference will be made to several terms that are defined to have the following meanings, unless the contrary intention is apparent.

[0057] Each application and patent cited herein, and each document or reference cited in each application and patent (including during prosecution of each issued patent; "application cited documents"), and each PCT and foreign application or patent corresponding to and / or claiming priority from any of these applications and patents, and each document cited or referenced in each application cited document are hereby expressly incorporated by reference into this specification. Further, the documents or references cited herein, the list of reference documents preceding the claims, or the specification itself, and each of these documents or references ("reference documents cited herein"), and each document or reference cited in each reference document cited herein (including any manufacturer's specification, instruction, etc.) are hereby expressly incorporated by reference into this specification.

[0058] Before describing various embodiments, the following definitions are provided and should be used unless otherwise indicated.

[0059] Definitions As used herein, the term "acyl", alone or in combination, refers to a carbonyl or thiocarbonyl group bonded to a radical selected from, for example, hydride, alkyl (e.g., haloalkyl), alkenyl, alkynyl, alkoxy (including "acyloxy" such as acetyloxy, butyryloxy, iso-butyryloxy, phenylacetyloxy, benzoyloxy, p-methoxybenzoyloxy, and substituted acyloxy such as alkoxyalkyl and haloalkoxy), aryl, halo, heterocyclyl, heteroaryl, sulfonyl (e.g., allylsulfinylalkyl), sulfonyl (e.g., alkylsulfonylalkyl), cycloalkyl, cycloalkenyl, thioalkyl, thioaryl, amino (e.g., alkylamino or dialkylamino), and aralkoxy. Exemplary examples of "acyl" radicals are formyl, acetyl, 2-chloroacetyl, 2-bromoacetyl, benzoyl, trifluoroacetyl, phthaloyl, malonyl, nicotinyl, etc. As used herein, the term "acyl" refers to the group -C(O)R 26 wherein R 26 is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, and heteroarylalkyl. Examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, beozylcarbonyl, etc.

[0060] As used herein, the term "adjuvant molecule" refers to a surface protein capable of inducing an immune response in a host. In certain embodiments, the adjuvant molecule is an adjuvant molecule in a "membrane-anchored form", which indicates that the adjuvant molecule has been engineered to include a signal peptide (SP) and a membrane-anchoring sequence in order to direct protein trafficking and membrane orientation. Thus, in embodiments, an adjuvant molecule in a membrane-anchored form is a recombinant protein that includes a portion of a protein fused to an SP and a membrane-anchoring sequence.

[0061] As used herein, the terms "administering" and "administration" refer to introducing a composition of the present disclosure (e.g., a vaccine, an adjuvant, or an immunogenic composition) to a subject. A preferred route of administration of the vaccine composition is intravenous.

[0062] As used herein, the term "alkoxyl" or "alkoxyalkyl" refers to an alkyl-O-group where the alkyl is as described above. The term "alkoxyl" as used herein refers to, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl, and includes linear, branched or cyclic, saturated or unsaturated oxo-hydrocarbon chains containing C 1-20 that may be referred to.

[0063] As used herein, the term "alkyl," alone or within any of the other terms such as "thioalkyl" and "arylalkyl," means a monovalent saturated hydrocarbon radical that can be straight-chain (i.e., linear) or branched-chain. Alkyl radicals for use in the present disclosure generally contain from about 1 to 20 carbon atoms, particularly about 1 to 10, 1 to 8, or 1 to 7 carbon atoms, more specifically about 1 to 6, or 3 to 6 carbon atoms. Exemplary alkyl radicals include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl, tert-pentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, n-dodecyl, n-tetradecyl, pentadecyl, n-hexadecyl, heptadecyl, n-octadecyl, nonadecyl, eicosyl, docosyl, n-tetracosyl, etc., along with the following branched variations. In certain disclosed embodiments, the alkyl radical is a C1-C6 lower alkyl selected from the group consisting of or including methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, tributyl, sec-butyl, tert-butyl, tert-pentyl, and n-hexyl. The alkyl radical can optionally be substituted with substituents as defined herein at positions that do not significantly interfere with the preparation of the compounds of the present disclosure and do not significantly reduce the effectiveness of the compounds. In certain disclosed embodiments, the alkyl radical is substituted with 1 to 5 substituents including halo, lower alkoxy, lower aliphatic, substituted lower aliphatic, hydroxy, cyano, nitro, thio, amino, keto, aldehyde, ester, amide, substituted amino, carboxyl, sulfonyl, sulfuryl, sulfenyl, sulfate, sulfoxide, substituted carboxyl, halogenated lower alkyl (e.g., CF3), halogenated lower alkoxy, hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, lower alkylcarbonylamino, alicyclic-substituted cycloaliphatic, or aryl (e.g., phenylmethylbenzyl), heteroaryl (e.g., pyridyl), and heterocyclic (e.g., piperidinyl, morpholinyl).The substituents on the alkyl group can themselves be substituted.

[0064] As used herein, the term "antibody" refers to preparations of polyclonal and monoclonal antibodies, as well as hybrid antibodies, modified antibodies, F(ab’)2 fragments, F(ab) fragments, Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof that exhibit the immunological binding properties of the parent antibody molecule.

[0065] As used herein, the term "antibody" further refers to immunoglobulins that specifically bind to the specific spatial and polar organization of another molecule and are thereby defined as complementary. Antibodies can be monoclonal antibodies, polyclonal antibodies, or recombinant antibodies and can be prepared by techniques well known in the art such as immunization of a host and collection of serum (polyclonal), or by preparing continuous hybrid cell lines and collecting the secreted protein (monoclonal), or by cloning and expressing a nucleotide sequence or a mutated version thereof that encodes at least the amino acid sequence necessary for the specific binding of a natural antibody. Antibodies can include intact immunoglobulins or fragments thereof, and such immunoglobulins include various classes and isotypes such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b, and IgG3, IgM, IgY. Examples of such fragments include Fab, Fv, and F(ab’)2, Fab’, scFv, etc. In addition, aggregates, polymers, and conjugates of immunoglobulins or their fragments can be used where appropriate as long as the binding affinity for a particular molecule is maintained.

[0066] As used herein, the term "antigen" refers to a molecule having one or more epitopes that stimulate a host immune system to generate a secretory, humoral, and / or cellular antigen-specific response, or a DNA molecule capable of producing such an antigen in a vertebrate. This term is also used interchangeably with "immunogen". For example, a particular antigen can be a complete protein, a portion of a protein, a peptide, a fusion protein, a glycosylated protein, and combinations thereof. For use with the compositions of the present disclosure, one or more PvDBPII antigens (native proteins or protein fragments) may be provided directly or as part of a recombinant nucleic acid expression system to provide an antigenic PvDBPII product that elicits a host immune response.

[0067] As used herein, the term "antigenic component" refers to a component derived from an organism that is capable of stimulating an immune response in animals, preferably mammals including mice and humans. An antigenic component can be an immunogenic substance. Antigenic components can include components smaller than cells, including organelles, membranes, proteins, lipids, glycoproteins, and other components derived from an organism. An antigenic component can be derived from an entire organism, such as an entire parasite, or a part of an organism, such as a cell or tissue of an organism. Also, a subset of proteins can be purified and recombined, for example, by size fractionation or affinity purification.

[0068] As used herein, the terms "sugar" and "saccharide" refer to polyhydroxyaldehydes, polyhydroxyketones, and their derivatives. Examples of this term include monosaccharides such as erythrose, arabinose, allose, altrose, glucose, mannose, threose, xylose, gulose, idose, galactose, talose, aldhexose, fructose, ketohexose, ribose, and aldopentose. This term also includes carbohydrates composed of monosaccharide units, including disaccharides, oligosaccharides, or polysaccharides. Examples of disaccharides are sucrose, lactose, and maltose. Oligosaccharides generally contain 3 to 9 of their monosaccharides, and polysaccharides contain more than 10 monosaccharide units. Sugars can be members of the D or L series, and include amino sugars, deoxy sugars, and their uronic acid derivatives. In embodiments of the disclosure where the carbohydrate is a hexose, the hexose is glucose, galactose, or mannose, or a substituted hexose sugar residue such as a hexosamine, galactosamine, glucosamine, particularly D-glucosamine (2-amino-2-deoxy-D-glucose) or D-galactosamine (2-amino-2-deoxy-D-galactose). Exemplary pentose sugars include arabinose, fucose, and ribose. Sugar residues from 1,1-linkage, 1,2-linkage, 1,3-linkage, 1,4-linkage, 1,5-linkage, or 1,6-linkage can be linked to the disclosed compounds. The linkage can be through an oxygen atom of the disclosed compounds. The oxygen atom can be replaced one or more times by a --CH2-- or --S-- group.

[0069] As used herein, the term "carboxyl" alone or in combination refers to -C(O)OR 25 - or -C(-O)OR 25 wherein R 25is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, amino, thiol, aryl, heteroaryl, thioalkyl, thioaryl, thioalkoxy, heteroaryl, or heterocyclic, and these may be optionally substituted. In an aspect of the disclosure, the carboxyl group is in an esterified form and may contain a lower alkyl group as the esterifying group. In a particular aspect of the disclosure, -C(O)OR 25It provides esters or amino acid derivatives. The esterified form is also sometimes referred to herein as "carboxylic acid ester" specifically. In the disclosed embodiments, "carboxyl" can be substituted, and in particular, can be substituted with allyl optionally substituted with one or more of amino, amine, halo, alkylamino, aryl, carboxyl, or heterocyclic. Examples of carboxyl groups include tert. alkoxycarbonyl such as methoxycarbonyl, butoxycarbonyl, tert. butoxycarbonyl, etc., and benzyloxycarbonyl, methoxybenzyloxycarbonyl, diphenylmethoxycarbonyl, 2-bromoethoxycarbonyl, 2-iodoethoxycarbonyl tert. butylcarboxyl, 4-nitrobenzyloxycarbonyl, diphenylmethoxy-carbonyl, benzhydroxycarbonyl, di-(4-methoxyphenyl-methoxycarbonyl, 2-bromoethoxycarbonyl, 2-iodoethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, or 2-triphenylsilylethoxycarbonyl, etc., which are arylmethyoxycarbonyls having one or two aryl radicals. The additional carboxyl groups in the esterified form are silyloxycarbonyl groups including organosilyloxycarbonyl. The silicon substituents in such compounds can be substituted with lower alkyl (e.g., methyl), alkoxy (e.g., methoxy), and / or halo (e.g., chlorine). Examples of silicon substituents include trimethylsilyi and dimethyl tert. butylsilyl. In the disclosed embodiments, the carboxyl group can be alkoxycarbonyl, especially methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl, sir heptyloxy carbonyl, especially methoxycarbonyl or ethoxycarbonyl.

[0070] It should be noted that there are some possible inaccuracies in the original text, such as "sir heptyloxy" which might be a misspelling. This translation is based on the best understanding of the provided text.As used herein, the term "composition" refers to a product containing a specified amount of a specified ingredient, as well as any product directly or indirectly resulting from a combination of specified amounts of specified ingredients. Such terms in relation to pharmaceutical compositions include products containing the active ingredient(s) and the inactive ingredient(s) constituting the carrier, as well as any product directly or indirectly resulting from a combination, complex formation, or aggregation of any two or more of the ingredients, or from the dissociation of one or more of the ingredients, or from one or more other types of reactions or interactions of the ingredients. Thus, the pharmaceutical compositions of the present disclosure include any composition prepared by admixing a compound of the present disclosure with a pharmaceutically acceptable carrier.

[0071] When the compounds of the present disclosure are used simultaneously with one or more other drugs, pharmaceutical compositions containing such other drugs in addition to the compounds of the present disclosure are contemplated. Thus, the pharmaceutical compositions of the present disclosure include those that also contain one or more other active ingredients in addition to the compounds of the present disclosure. The weight ratio of the compound of the present disclosure to the second active ingredient may vary and will depend on the effective dosage of each ingredient. Generally, the effective dosage of each will be used. Thus, for example, without intending to be limiting, when the compounds of the present disclosure are combined with another agent, the weight ratio of the compound of the present disclosure to the other agent will generally be in the range of about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. The combination of the compounds of the present disclosure with other active ingredients will also generally be within the aforementioned range, but in each case, the effective dosage of each active ingredient should be used. In such combinations, the compounds of the present disclosure and the other active agents may be administered separately or together. In addition, the administration of one agent may be before, simultaneous with, or after the administration of the other agent(s).

[0072] The disclosed composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository using conventional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Various delivery systems are known and, for example, encapsulation into liposomes, microparticles, microcapsules, etc. can be used to administer the compositions of the present disclosure.

[0073] The disclosed therapeutic composition can include a carrier such as one or more of a polymer, carbohydrate, peptide, or derivatives thereof, which can be covalently bonded directly or indirectly to the compound. The carrier can be substituted with substituents described herein including, but not limited to, one or more alkyl, amino, nitro, halogen, thiol, thioalkyl, sulfate, sulfonyl, sulfinyl, sulfoxide, hydroxyl groups. In an aspect of the disclosure, the carrier is an amino acid including alanine, glycine, proline, methionine, serine, threonine, asparagine, alanyl-alanine, prolyl-methionyl, or glycyl-glycyl. The carrier can also include a molecule that targets the compound of the present disclosure to a particular tissue or organ.

[0074] The disclosed compounds can be prepared using reactions and methods that are generally known to those skilled in the art in view of the knowledge of the compounds and the disclosure of this application, including the examples. The reactions are carried out in solvents that are appropriate for the reagents and materials used and suitable for the reactions to be accomplished. It will be understood by those skilled in organic synthesis that the functionality present in the compounds should be consistent with the proposed reaction steps. This may require modification of the order of the synthetic steps or selection of a particular process scheme over another process scheme in order to obtain the desired compounds of the disclosure. It will also be recognized that another major consideration in the development of synthetic routes is the selection of protecting groups used for the protection of reactive functional groups present in the compounds described in this disclosure. An authoritative treatise that describes many alternatives for those skilled in the art is Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991).

[0075] The disclosed compounds can be formulated into pharmaceutical compositions for administration to a subject by suitable methods known in the art. The pharmaceutical compositions of the present disclosure, or fractions thereof, are selected based on the intended mode of administration and include suitable pharmaceutically acceptable carriers, excipients, and vehicles consistent with conventional pharmaceutical practice. Suitable pharmaceutical carriers, excipients, and vehicles are described in the standard text Remington: The Science and Practice of Pharmacy (21st Edition, 2005, University of the Sciences in Philadelphia (Editor), Mack Publishing Company), and The United States Pharmacopeia: National Formulary (USP 24 NF19), published in 1999. As an example of oral administration in the form of capsules or tablets, the active ingredient can be combined with an orally non-toxic pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, methylcellulose, magnesium stearate, glucose, calcium sulfate, dicalcium phosphate, mannitol, sorbitol, etc. In the case of oral administration in liquid form, the active ingredient can be combined with any orally non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Suitable binders (e.g., gelatin, starch, corn sweeteners, natural sugars including glucose, natural and synthetic gums, and waxes), lubricants (e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride), disintegrants (e.g., starch, methylcellulose, agar, bentonite, and xanthan gum), flavoring agents, and coloring agents can also be combined with the compositions or their components. The compositions described herein may further include wetting agents or emulsifying agents, or pH buffering agents.

[0076] As used herein, the term "immunogenic composition" is one that, when injected into a host, elicits specific antibody production or cellular immunity.

[0077] The immunogenic compositions and / or vaccines of the present disclosure can be formulated by any of the methods known in the art. They can typically be prepared as injectable formulations, either as liquid solutions or suspensions, or as formulations for nasal administration. Prior to injection or other administration, a suitable solid form may be prepared in a solution in a liquid or a suspension in a liquid. The preparations may also, for example, be emulsified or be proteins / peptides encapsulated in liposomes.

[0078] The active immunogenic ingredient is often mixed with an excipient or carrier that is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include, but are not limited to, water, saline, dextrose, glycerol, ethanol, etc., and combinations thereof. The concentration of the immunogenic polypeptide in an injectable aerosol formulation or a nasal formulation is usually in the range of about 0.2 to 5 mg / ml. Similar doses can be administered to other mucosal surfaces.

[0079] In addition, if desired, the vaccine may contain small amounts of auxiliary substances such as wetting or emulsifying agents, pH buffers, and / or other agents that enhance the effectiveness of the vaccine. Examples of agents that may be effective include, but are not limited to, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1’-2’-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, also known as MTP-PE), and RIBI containing three components extracted from bacteria in a 2% squalene / Tween80 emulsion: monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS). The effectiveness of the auxiliary substance can be determined by measuring the amount of antibodies (especially IgG, IgM, or IgA) directed against the immunogen resulting from administration of the immunogen in the vaccine containing the adjuvant in question. Additional formulations and modes of administration may also be used.

[0080] The immunogenic compositions and / or vaccines of the present disclosure can be administered in a manner compatible with a dosage formulation in a manner known in the art, and in such amounts and in such a manner as to be prophylactically and / or therapeutically effective. Generally, it is within the range of about 1 to 1,000 micrograms of viral surface envelope glycoprotein per dose and / or adjuvant molecule per dose, and more generally within the range of about 5 to 500 micrograms of glycoprotein per dose and / or adjuvant molecule per dose. The amount administered depends on the nature of the antigen and / or adjuvant molecule, the subject being treated, the ability of the host immune system to synthesize antibodies, and the degree of protection desired. The exact amount of active ingredient that needs to be administered can depend on the judgment of a physician or veterinarian and can be specific to each individual, but such determinations are within the scope of the skills of such a professional.

[0081] The vaccine or immunogenic composition can be administered as a single dose, a two-dose schedule, for example, at intervals of 2 to 8 weeks, or a multiple-dose schedule. The multiple-dose schedule can include that the primary step of vaccination can include 1 to 10 or more separate doses, followed by other doses (for example, the second dose at 1 to 4 months and, optionally, subsequent doses (if any) several months later) administered at subsequent time intervals necessary to maintain and / or enhance the immune response.

[0082] As used herein, the term "immunogenic fragment" refers to a fragment of an immunogen that contains one or more epitopes and, thus, can modulate an immune response or act as an adjuvant to an antigen administered concomitantly. Such fragments can be identified using any number of epitope mapping techniques well known in the art (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Morris, G.E., Ed., 1996) Humana Press, Totowa, NJ).

[0083] The immunogenic fragment can be at least about 2 amino acids in length, more preferably about 5 amino acids in length, and most preferably at least about 10 to about 15 amino acids in length. There is no critical upper limit to the length of the fragment that can include substantially the full-length of the protein sequence or even a fusion protein containing two or more epitopes.

[0084] As used herein, the term "immunoglobulin" refers to a class of proteins that exhibit antibody activity and bind to other molecules (e.g., antigens and certain cell surface receptors) with a high degree of specificity. Immunoglobulins can be classified into five classes: IgM, IgG, IgA, IgD, and IgE. IgG is the most abundant class of antibodies in the body and is envisioned to have a twisted "Y" shaped structure. Except for IgM, immunoglobulins are composed of four peptide chains linked by intra-chain and inter-chain disulfide bonds. IgG is composed of two polypeptide heavy chains (H chains) and two polypeptide light chains (L chains) coupled by non-covalent disulfide bonds.

[0085] As used herein, the term "immunological response" refers to the development of a humoral and / or cellular immune response in a subject to an antigen present in a composition of interest. For the purposes of the present disclosure, a "humoral immune response" refers to an immune response mediated by antibody molecules, and a "cellular immune response" is an immune response mediated by T lymphocytes and / or other white blood cells.

[0086] One aspect of cellular immunity involves an antigen-specific response by cytotoxic T cells (“CTLs”). CTLs associate with and are presented by proteins encoded by the major histocompatibility complex (MHC), and have specificity for peptide antigens expressed on the surface of cells. CTLs serve to induce and facilitate the destruction of intracellular microorganisms or the lysis of cells infected with such microorganisms. Another aspect of cellular immunity involves an antigen-specific response by helper T cells. Helper T cells act to stimulate the functions of non-specific effector cells and to focus the activity of non-specific effector cells on cells that associate with MHC molecules on their surface and present peptide antigens. “Cellular immune response” also refers to the production of cytokines, chemokines, and other such molecules produced by activated T cells and / or other leukocyte cells, including those derived from CD4+ and CD8+ T cells. Thus, an immunological response can include one or more of the following effects: production of antibodies by B cells, and / or activation of suppressor T cells and / or γδ T cells specifically directed against one or more antigens present in the composition or vaccine of interest. These responses can function to neutralize infectivity and / or provide protection to the immunized host by mediating antibody complement or antibody-dependent cellular cytotoxicity (ADCC). Such responses can be determined using standard immunoassays and neutralization assays well known in the art.

[0087] As used herein, the term “immunogenic amount” refers to an amount capable of inducing the production of antibodies directed against a virus in a host to which the vaccine has been administered.

[0088] As used herein, the term "immunogenic carrier" refers to a composition that improves the immunogenicity of virosomes from any of the viruses discussed herein. Such carriers include, but are not limited to, proteins and polysaccharides, and microspheres formulated using biodegradable polymers such as, for example, DL-lactide-coglycolide, liposomes, and bacterial cells and membranes. Protein carriers can be conjugated to proteases or peptides derived therefrom to form fusion proteins by recombinant or synthetic techniques or by chemical coupling. Useful carriers and methods of coupling such carriers to polypeptide antigens are known in the art.

[0089] As used herein, the term "immunopotentiator" is intended to mean a substance that, when mixed with an immunogen, induces a higher immune response than the immunogen alone. For example, an immunopotentiator can improve immunogenicity and provide an excellent immune response. Immunopotentiators can act, for example, by improving the expression of co-stimulators on macrophages and other antigen-presenting cells.

[0090] As used herein, the terms "subject", "individual", or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals such as mammals. Mammals include, but are not limited to, any member of the class Mammalia. Mammals as subjects or patients in the present disclosure can be from the families of primates, carnivores, proboscideans, perissodactyls, artiodactyls, rodents, and lagomorphs. In certain embodiments, the mammal is a human. In other embodiments, animals can be treated, and the animals can be vertebrates including both birds and mammals. In aspects of the disclosure, the term includes food or pet livestock including horses, cows, sheep, poultry, fish, pigs, dogs, cats, as well as zoo animals, goats, apes (e.g., gorillas or chimpanzees), and rodents such as rats and mice.

[0091] As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle for use in animals and more specifically in humans, which is approved by the regulatory agencies of the Federal or State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias, when the disclosed probes are administered together. Such pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. Pharmaceutical carriers can be, for example, physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. When administered to a patient, the probe and the pharmaceutically acceptable carrier can be sterilized. When the probe is administered intravenously, water is a useful carrier. Aqueous solutions of physiological saline, as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, etc. The composition can also contain, if desired, small amounts of wetting or emulsifying agents, or pH buffering agents. The composition can advantageously be in the form of a solution, an emulsion, a sustained release formulation, or any other form suitable for use.

[0092] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio.

[0093] As used herein, the term "vaccine" refers to an immunogenic amount of one or more virosomes, fragments thereof, or subunits thereof. Such vaccines can include them in combination with another protein or other immunogen, such as one or more viral surface envelope glycoproteins and portions thereof, as well as adjuvant molecules and portions thereof on the surface of the virosome, or one or more additional viral components that naturally associate with viral particles or epitope peptides derived therefrom.

[0094] In 1974, Dalsgaard et al. ("Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254) first described that a saponin preparation isolated from the South American tree Quillaja saponaria Molina has adjuvant activity. The purified fragment of Quil A has been isolated by HPLC and this fragment retains adjuvant activity without the toxicity associated with Quil A (EP 0 362278), for example QS7 and QS21 (also known as QA7 and QA21). QS-21 is a natural saponin derived from the bark of Quillaja sapanaria Molina that induces CD8+ cytotoxic T cells (CTLs), Th1 cells, and a predominant IgG2a antibody response.

[0095] The disclosed saponin can be used at a level of 1 to 100 μg per human dose of the adjuvant composition, about 50 μg, for example 40 - 60 μg, preferably 45 - 55 μg, or 49 - 51 μg, or 50 μg. In some embodiments, the human dose of the adjuvant composition can contain QS21 at a level of about 25 μg, for example 20 - 30 μg, preferably 21 - 29 μg, or 22 - 28 μg, or 28 - 27 μg, or 24 - 26 μg, or 25 μg.

[0096] When the adjuvant is combined with the liquid form of the antigenic composition, the adjuvant composition is of a volume suitable for human dosage, which is approximately half of the intended final dosage of the human dosage. For example, for an intended final human dosage of 1 μl, the volume of the adjuvant is 500 μl, or for an intended final human dosage of 0.5 ml, the volume is 250 μl. When combined with the antigen composition, the adjuvant composition is diluted to provide the final human dosage of the vaccine. The final volume of such a dosage will of course vary depending on the initial volume of the adjuvant composition and the volume of the antigen composition added to the adjuvant composition. In an alternative embodiment, the aqueous adjuvant is used to reconstitute the lyophilized antigen composition. In this embodiment, the volume suitable for the human dosage of the adjuvant composition is approximately equal to the final volume of the human dosage. The liquid adjuvant composition is added to the vial containing the lyophilized antigen composition and is used to reconstitute the lyophilized antigen composition.

[0097] Abbreviations IgG, immunoglobulin G; Th, T helper cell; CTL, cytotoxic T lymphocyte; rha, rhamnose; xyl, xylose; OVA, ovalbumin; NMM, N-methylmorpholine; HOBt, hydroxybenzotriazole; EDC HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DCM, dichloromethane; MeCN, acetonitrile; THF, tetrahydrofuran; rHagB, recombinant hemagglutinin B; s.c., subcutaneous; ESI-TOF, electrospray ionization time-of-flight mass spectrometer; ELISA, enzyme-linked immunosorbent assay;

[0098] Discussion The advantages of new structurally defined saponin adjuvants from natural sources other than the bark of Quillaja saponaria Molina are multiple. First, the plant Momordica cochinchinensis Spreng is a perennial vine that is easy to grow, avoiding the drawback of limited supply of QS saponins. Second, the abundance of saponins in MS seeds is high (Figure 1), and their isolation is more efficient than that of QS saponins and thus cost-effective. M. cochinchinensis Spreng grows mainly in China and Southeast Asia. The seeds (Mubiezi) have been used in China for over 1000 years as a traditional Chinese medicine for the treatment of ulcers, mastitis, eruptions, hemorrhoid oozing, hemorrhoids, eczema, and neurodermatitis. In recent years, extracts from M. cochinchinensis seeds have been evaluated for their adjuvant effect and safety in an experimental swine vaccine against foot-and-mouth disease (FMD). MS saponins showed a synergistic effect with oil emulsions in enhancing antigen-specific IgG in guinea pigs. However, in comparison of adjuvant activity against other adjuvants, namely Freund's adjuvant, Quil A (QA), and propolis, in chickens immunized with antigen F4 fimbriae, MS saponins showed lower ability than Freund's adjuvant and QA in enhancing IgG responses in both serum and egg yolk. These results are consistent with the observation that deacylated QS-17 / 18, which does not contain a fatty side chain, showed only humoral immunity.

[0099] Two natural MS saponins from Momordica cochinchinensis (Lour.). Seeds of Spreng were isolated by using the procedure published as outlined in Figure 11. Two MS derivatives, namely 5 and 6 from natural saponins 3 and 4, respectively, were then synthesized by using a normal one-step amide formation reaction (Figure 3).

[0100] Using pure natural saponins 3 and 4, and their available derivatives 5 and 6 (Figure 7), their ability to enhance the antibody response against chicken egg ovalbumin (OVA) was evaluated. The known saponin adjuvant GPI-0100 was used as a positive control. Thus, groups of female BALB / c mice (8 - 10 weeks old, 6 mice per group) were immunized by the subcutaneous (s.c.) route on days 0, 14, and 28 with OVA alone (20 μg), or OVA containing GPI-0100, or OVA containing 100 μg doses of saponins 3 - 6. The mice were weighed and serum samples were collected before each immunization and 6 weeks after the first immunization. As shown in Figures 6A - 6C, 9A - 9C, and Table 1, the levels of serum IgG antibody activity against OVA were determined by enzyme-linked immunosorbent assay (ELISA).

[0101] Serum IgG responses were detected in all groups by 2 weeks after the first immunization. In mice that received OVA + adjuvant after the second immunization, a marked increase was seen in the level of IgG anti-OVA antibody activity, and the magnitude of the response continued to increase after the third immunization (Table 1). GPI-0100, as a positive control, was significantly higher than that seen with antigen alone at 2 weeks (P < 0.05), 4 weeks (P < 0.001), and 6 weeks (P < 0.001), enhancing the IgG response to OVA. Similar to GPI-0100, adjuvant VSA-1 (5, a derivative of native MS I(3)) showed a significantly higher anti-OVA IgG response than mice that received antigen alone at 2 weeks (P < 0.01), 4 weeks (P < 0.001), and 6 weeks (P < 0.01), as well as mice that received OVA + 3 at 2 weeks (P < 0.001), 4 weeks (P < 0.001), and 6 weeks (P < 0.01). Saponin 3 did not show a significant difference in the IgG response compared to the OVA group. Saponin 4 showed a significant difference in the IgG response compared to the OVA group at 2 weeks (P < 0.01) and 4 weeks (P < 0.001), but not at 6 weeks. Adjuvant 6 (a derivative of native MS II(4)) showed a significantly higher anti-OVA IgG response than the OVA group at 4 weeks (P < 0.001) and 6 weeks (P < 0.05), but did not show a significant difference from its parent compound 4 until 6 weeks (P < 0.05). No signs of toxicity based on body weight monitoring were observed in any of the mice.

[0102] Next, the IgG subclass antibody responses induced by different adjuvants were analyzed. All adjuvants showed significantly higher IgG1 responses at week 2 and week 4 than the mice in the OVA group (Table 1). However, at week 6, only GPI-0100 (P<0.001), 5 (P<0.05), and 6 (P<0.01) showed significantly higher IgG1 responses than the OVA group. In the IgG2a evaluation (Figure 6B), there was no significant difference between groups at week 2, but GPI-0100 (P<0.001) and VSA-1(5) (P<0.001) induced significantly higher IgG2a than the OVA group at week 4. At week 6, GPI-0100 (P<0.05) and VSA-1(5) (P<0.01) still showed significantly higher IgG2a titers than those seen in mice using OVA alone or adjuvants 3 (P<0.01), 4 (P<0.01), or 6 (P<0.01), but there was no significant difference between the groups of GPI-0100 and VSA-1.

[0103] Analysis of the IgG2a / IgG1 ratio of the anti-OVA response at week 6 revealed that adjuvant 5 had a significantly higher ratio (P<0.01) than OVA alone or OVA containing 3, 4, or 6, but there was no significant difference from GPI-0100 (Figure 8). The minor IgG2a responses from the groups without adjuvant or containing adjuvants 3, 4, or 6 suggest that a Th2-biased immune response was selectively induced in these groups. Since GPI-0100 is known for its ability to enhance a mixed Th1 / Th2 response along with CTL production, the similar IgG2a / IgG1 distribution between GPI-0100 (0.194, Table 1, entry 2)) and adjuvant 5 (0.312, Table 1, entry 5) suggests that these two adjuvants may have similar activity profiles.

Table 1

[0104] For comparison, as shown in FIGS. 8A-8C, adjuvants 3-6 were also evaluated for enhancing the immune response to rHagB, a recombinant non-fimbrial adhesin hemagglutinin B derived from Porphyromonas gingivalis. The antigen is an etiological factor of periodontal disease, and the efficacy of rHagB in inducing a protective immune response against P. gingivalis-induced alveolar bone loss has been demonstrated in an experimental animal model. By using the same procedure, the results with the rHagB antigen (at a dose of 35 μg) were similar to those with the OVA antigen, and the IgG, IgG1, and IgG2a data are summarized in FIGS. 8A-8C.

[0105] The antigen rHagB stimulated a strong humoral immune response. The IgG and IgG1 titers in the group without adjuvant were lower than those in the groups with adjuvant at 2 and 4 weeks, but the difference was minimal at 6 weeks (except for IgG in the group using 5 (P<0.05)) (FIGS. 8A and 8B, respectively). For IgG2a (FIG. 8C), mice using GPI-0100 or 5 showed significantly higher activity than the group using rHagB alone at 2 weeks (P<0.001), 4 weeks (P<0.001), and 6 weeks (P<0.05), which is consistent with what has been observed with the OVA antigen. Also, mice using 6 showed significantly higher IgG2a titers than the rHagB control group at 2 weeks (P<0.05) and 4 weeks (P<0.01), but not at 6 weeks.

[0106] These results showed that a new derivative (5) obtained by incorporating a simple and chemically stable fatty side chain into natural Momordica saponin I (3) not only retains and improves the IgG1 immunity of 3, but also regulates its adjuvant activity profile by inducing a significant IgG2a immune response. However, when the same strategy was applied to Momordica saponin II (4), no significant increase in IgG2a was observed. Without wishing to be bound by any one theory, considering that the only structural difference between the two MS derivatives is the triterpenoid core, i.e., gypsogenin (R1 = H) versus kierraic acid (R1 = OH), it seems that the structure of the triterpenoid core, rather than the hydrophilic-lipophilic balance of the saponin structure, plays an important role in influencing the adjuvant activity of derivatives 5 and 6.

[0107] The acute toxicity of adjuvant VSA-1 (5) was evaluated by using the same procedure as for GPI-0100. Thus, 10-week-old female BALB / c mice were given an s.c. injection of the indicated doses of the adjuvant in 0.1 mL of PBS behind the neck.

Table 2

[0108] All mice in the groups treated with 5 (5000 μg) and Quil-A died within 5 days after injection. All surviving mice appeared to have a healthy coat and to be behaving normally. None of the surviving mice appeared lethargic by day 7, and no lesion formation was observed in any of the mice. The data in Table 2 showed that the acute toxicity of 5 was similar to that of GPI-0100 but much lower than that of Quil A.

[0109] Momordica saponins I and II have been derivatized by coupling them with dodecylamine at the C3 glucuronic acid site. The resulting derivatives exhibit an immunostimulatory activity profile that is significantly different from that of their natural parent saponins. In particular, the derivative of Momordica saponin I(3), adjuvant VSA-1(5), induces a significantly higher IgG2a response than the corresponding natural product. The production of its IgG1 and IgG2a is similar to that of GPI-0100, suggesting a potential mixed Th1 / Th2 immune response to specific antigens, which is different from the Th2 immunity induced by natural saponins. Toxicity evaluation shows that VSA-1(5) has a toxicity profile similar to that of GPI-0100 and is much less toxic than the widely used natural saponin mixture Quil A. These results demonstrate that derivatizing Momordica saponins is a feasible way to readily access structurally defined saponin immunostimulants with low toxicity against mixed Th1 / Th2 immune responses. Considering that MS saponins are readily available and easy to isolate, it would be useful for large-scale preparation of MS derivatives for potential preclinical studies and clinical applications.

[0110] Two MS I(2) derivatives 4b and 4c (Scheme 1, Figure 16) were prepared. The side chain of 4b has a terminal ester group, and 4c has the same side chain as QS-21 analog 7. Similarly, the corresponding two MS II(3) derivatives 5b and 5c were also prepared. By using the published procedures, two natural MS saponins (2 and 3) were isolated from commercially available and inexpensive MC seeds. Then, derivatives 4b and 5b were synthesized from their respective natural saponins and side chains by using the usual amide formation method. Hydrogenolysis of 4b and 5b gave 4c and 5c in high yields, respectively. Under the hydrogenolysis conditions, the C12 alkene moiety in the chiral acid core remained intact.

[0111] Next, their ability to enhance the antibody response against chicken ovalbumin (OVA) was evaluated. The known saponin adjuvant GPI-0100 was one of the positive controls. As another positive control, the MS derivative VSA-1(4a) reported in recent years was also used. Its high IgG1 / IgG2a production and low toxicity are similar to those of GPI-0100. Therefore, female BALB / c mouse groups (8 - 10 weeks old, 6 mice per group) were immunized via the subcutaneous route (s.c.) on days 0, 14, and 28 with OVA alone (20 μg), OVA containing GPI-0100, or OVA containing 100 μg of the MS saponin derivative. The mice were weighed, and serum samples were collected before each immunization and 2 weeks after the last immunization. The level of serum antibody activity against OVA was determined by enzyme-linked immunosorbent assay (ELISA).

[0112] Mice in all groups had a serum anti-OVA IgG response by 2 weeks after the first immunization, and the levels of IgG titers continued to increase at 4 and 6 weeks (Figure 9A). At 4 and 6 weeks, all adjuvant groups showed significantly higher IgG activity than the OVA control group without adjuvant. The same trend was observed in anti-OVA IgG1 activity (Figure 9B), but at 6 weeks, the OVA + 5c group showed no statistical difference from the OVA control group. Regarding IgG2a activity (Figure 9C), the OVA control group maintained the same activity over time, while the other groups with adjuvant (excluding the OVA + 5c group) showed a stable increase in IgG2a titers at 4 and 6 weeks. The adjuvant VSA-2(5b) significantly enhanced IgG2a activity at 6 weeks compared to the OVA group (P < 0.01), OVA + 4b (P < 0.05), and OVA + 5c group (P < 0.01), but showed no significant difference from the positive control group and the OVA + 4c group.

[0113] Considering the structural similarity between MS I / II (Figure 1) and deacylated QS-17 / 18 (8) (Scheme 2, Figure 17), QS-17 / 18 derivatives similar to 5b / 5c were synthesized to see how the slight differences in the C3 and C28 oligosaccharide domains affect adjuvanticity using the same side chains. QS-17 / 18 derivative 9a was previously synthesized and evaluated, and new derivatives 9b and 9c were prepared by using the same synthetic route. Thus, first, the fully protected intermediate 10 was subjected to debenzylation to remove all benzyl protecting groups. The carboxyl group of the C3 glucuronic acid was exposed. Under hydrogenolysis conditions, all triethylsilyl groups were also removed in the same way. The side chains were then installed by subsequent amide bond formation reactions, similar to the synthesis of 4b and 5b. After removing the two acetyl groups at the C28 position on the fucosyl unit under basic conditions, adjuvant candidate 9b was obtained. Debenzylation of 9b gave rise to adjuvant candidate 9c.

[0114] The abilities of 9b and 9c to enhance antigen-specific antibody activity were compared with those of 5b and 5c. The rHagB antigen (recombinant and non-fimbrial adhesin hemagglutinin B from Porphyromonas gingivalis) was used. Previous studies have shown that it is effective in inducing protective immunity against P. gingivalis-induced alveolar bone loss. Female BALB / c mouse groups (8 - 10 weeks old, 6 mice per group) were immunized subcutaneously (s.c.) on days 0, 14, and 28 with rHagB alone (35 μg), rHagB containing GPI-0100, or rHagB containing 100 μg of the saponin adjuvant. The same immunization and ELISA evaluation procedures as for the OVA antigen were used.

[0115] Mice in all groups showed serum anti-rHagB IgG, IgG1, and IgG2a responses by 2 weeks after the first immunization, and the levels of antibody titers continued to increase at 4 and 6 weeks (Figures 10A - 10C). At 2 and 4 weeks, all groups showed significantly higher IgG titers than the adjuvant-free rHagB control group, but at 6 weeks, only the GPI-0100 group showed a statistical difference from the rHagB group. The IgG1 response had a similar trend as seen in IgG (Figure 10B), but at 6 weeks, the OVA + 9a and OVA + 9b groups showed a statistical difference from the rHagB group. Regarding the IgG2a response, all groups showed significantly higher activity than the rHagB control group at 2, 4, and 6 weeks (Figure 10C).

[0116] VSA-2(5b) enhanced IgG2a higher than the positive control GPI-0100 at 4 weeks (P < 0.05) and 6 weeks (P < 0.05). VSA-2(5b) also showed higher IgG2a activity than the QS-17 / 18 derivative 9b at 2 weeks (P < 0.01), 4 weeks (P < 0.01), and 6 weeks (P < 0.01), and than 9c at 2 weeks (P < 0.05), 4 weeks (P < 0.01), and 6 weeks (P < 0.01). Different from the OVA antigen (Figure 10C), VSA-2(5b) did not show a significant difference from 5c in enhancing the IgG2a response.

[0117] Since the production of IgG1 or IgG2a in mice is enhanced by their respective Th2 or Th1 cytokines, their relative amounts can be used as a provisional indicator of the involvement of Th2 and Th1 immunity enhanced by an adjuvant. For the rHagB antigen, VSA-2(5b) showed a significantly higher IgG2a / IgG1 ratio than other groups (except 5c) at 2, 4, and 6 weeks (Figure 18). These results suggest that 5b (and 5c) may be able to enhance Th1 immunity more than GPI-0100 can, which would be beneficial when strong Th1 immunity is desired.

[0118] The immunological evaluation of semi-synthetic MS derivatives and synthetic QS analogues revealed that with different protein antigens (i.e., OVA or rHagB), VSA-2(5b) showed an IgG2a / IgG1 ratio equal to or higher than that of GPI-0100 with similar overall IgG production. With the OVA antigen, MS derivative 4a significantly improved IgG2a production compared to 5a. Figures 4a and 5b also showed similar IgG2a production (no significant difference, Figure 10C). The only difference between 5a and 5b is their side chains, suggesting that the side chain structure affects the antibody activity profile of the induced immunological response. Furthermore, derivatives 5b and 5c with different side chains also showed different antibody activities when combined with the OVA antigen but similar activities when combined with the rHagB antigen. Saponins 4b, 5b, and 9b with different core structures having the same side chain showed different antibody-inducing stimulatory activities. Two saponins, 4b and 5b, differ only in their respective triterpenoid cores, and 5b has an excess C16 OH (kila acid core) compared to the diosgenin core of 4b. Saponins 5b and 9b have the same triterpenoid core (i.e., kila acid) and side chain but differ slightly in their C3 and C28 oligosaccharide domains. All these saponins have a similar hydrophilic-lipophilic balance (HLB) and they showed similar overall IgG activities. However, their ability to enhance IgG2a production was significantly different, indicating that the specific structure of the saponins, i.e., the structural details of the side chain, triterpenoid core, and oligosaccharide domain, affects the details of the immune response.

[0119] Several MS- and QS-saponin-based vaccine adjuvant candidates have been prepared. The MS derivatives were prepared by incorporating terminal functionalized side chains into the C3 glucuronic acid units of natural saponins MS I and II through amide formation reactions, and the QS analogs were prepared via multi-step organic syntheses. These unnatural saponins exhibit significantly different immunostimulant activity profiles, suggesting that the structures of the side chains, triterpenoid core, and oligosaccharide domain work together to converge the characteristic immune response enhancement of each saponin.

[0120] Among the various adjuvant candidates, VSA-2(5b), a derivative of MS II, consistently improved IgG2a production when delivered with either OVA or rHagB antigen. For the antigen rHagB, it induced a significantly higher IgG2a response than the well-studied semi-synthetic saponin adjuvant derived from QS saponin and known as the positive control GPI-0100, which is known for its ability to induce balanced Th1 / Th2 immunity. The results of the present disclosure demonstrate that Momordica saponins are a viable natural source of saponins useful for the preparation of unnatural saponin adjuvants with different adjuvant activities through simple chemical derivatization, and it can be seen that VSA-2(5b), as a useful MS-based immunostimulant (in addition to the known VSA-1(4a)), is particularly useful in its unique ability to enhance the IgG2a response.

[0121] Accordingly, the compounds and pharmaceutical compositions of the present disclosure can be used in combination with one or more other therapeutic agents for treating viral infections and other diseases. For example, the compounds of the present disclosure and the pharmaceutical compositions provided herein can be used in combination with other antiviral agents for treating viral infections.

[0122] One aspect of the disclosure is a modified saponin having the following formula:

Chemical formula

[0123] In some embodiments of the present aspect of the disclosure, the carrier can be selected from the group consisting of polyamine polymers, polyethylene glycol amines, poly(ethylene imine), nanocarbons, and amino-containing biomolecules.

[0124] In some embodiments of the present aspect of the disclosure, the modified saponin can have the following formula I:

Chemical formula

[0125] In some embodiments of the present aspect of the disclosure, R3 can be H as follows.

[0126] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain fatty acid having the structure HOOC-(CH2) 6-20 -.

[0127] In some embodiments of the present aspect of the disclosure, R3 can be an alkoxy group having the structure H3C-(CH2) 6-20 -O-CH2.

[0128] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0129] In some embodiments of the present aspect of the disclosure, R4 can be a long-chain alkyl terminated with a functional group selected from the group consisting of an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0130] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)- obtained , where R4 can be a long-chain alkyl R6O(CH2) 6-20 -, and R6 can be selected from saccharide units selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.

[0131] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with monophosphoryl lipid A (MPL).

[0132] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with dipalmitoyl-S-glycerylcysteine (Pam2Cys) or tripalmitoyl-S-glycerylcysteine (Pam3Cys).

[0133] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with muramyl dipeptide units.

[0134] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with α-Galcer units.

[0135] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with MS I units.

[0136] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with MS II units.

[0137] In some embodiments of the present aspect of the disclosure, the modified saponin can be selected from the group consisting of Formulas A to E below.

Chemical formula

[0138] Another aspect of the disclosure is a pharmaceutical composition comprising a modified saponin having the following formula:

Chemical formula

[0139] In some embodiments of this aspect of the disclosure, the carrier can be selected from the group consisting of polyamine polymers, polyethylene glycol amines, poly(ethylene imine), nanocarbons, and amino-containing biomolecules.

[0140] In some embodiments of this aspect of the disclosure, the modified saponin can have the following formula I [Chemical formula] wherein q1 can be H or OH, q2 and q3 can each independently be selected from CHO, CH3, CH2OH, H, or a component of an acetal group, R3 can be H, a methyl group, a carboxyl group (R excluding H-O-C(O)- 4 -O-C(O)-) , R4-NR5-C(O)- 、 R4-O- and R 4 -O-CH 2 - and can be selected from the group consisting of, R4 and R5 can each independently be H、 the structure R6(CH2) 1-20 - or R6[(CH2) 1-20 O 0-1 (CH2) 0-20 1-20 and can be a straight chain having, R6 can be H, OH, COO(CH2) 0-6 H, COOBn, C(O)NR7Bn, NR7Bn, OBn, a saccharide unit, Momordica saponin I or II, muramyl dipeptide, monophosphoryl lipid A (MPL) unit, α-Galcer unit, dipalmitoyl-S-glycerylcysteine (PamCys) unit, or a functional group of a carrier, and R7 can be H or an alkyl group.​

[0141] In some embodiments of the present aspect of the disclosure, R3 is H is obtainable such.

[0142] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)- obtained and R4 is a long-chain fatty acid having the structure HOOC-(CH2) 6-20 -. obtainable such.

[0143] In some embodiments of the present aspect of the disclosure, R3 can be an alkoxy group having the structure H3C-(CH2) 6-20 -O-.

[0144] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0145] In some embodiments of the present aspect of the disclosure, R4 can be a long-chain alkyl terminated with a functional group selected from an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0146] In some embodiments of the present aspect of the disclosure, R3 can be an alkoxy group having the structure H3C-(CH2) 6-20 -O-CH2-.

[0147] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 is a long-chain alcohol having the structure HO-(CH2) 6-20 -.

[0148] In some embodiments of the present aspect of the disclosure, R4 can be a long-chain alkyl terminated with a functional group selected from an ester group, an ether group, an amino group, a cyano group, a carbonyl group, an azide group, and an aromatic group.

[0149] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with R6O(CH2) 6-20 -, and R6 can be selected from saccharide units selected from the group consisting of monosaccharides, disaccharides, and trisaccharides.

[0150] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with monophosphoryl lipid A (MPL).

[0151] In some embodiments of the present aspect of the disclosure, R3 is R4-NH-C(O)-, and R4 can be a long-chain alkyl terminated with dipalmitoyl-S-glycerylcysteine (Pam2Cys) or tripalmitoyl-S-glycerylcysteine (Pam3Cys).

[0152] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with muramyl dipeptide units.

[0153] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with α-Galcer units.

[0154] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with MS I units.

[0155] In some embodiments of the present aspect of the disclosure, R3 can be R4-NH-C(O)-, where R4 can be a long-chain alkyl terminated with MS II units.

[0156] In some embodiments of the present aspect of the disclosure, the modified saponin can be selected from the group consisting of Formulas A to E below.

Chemical formula

[0157] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may further comprise at least one immunogen.

[0158] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0159] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may be formulated for administration to an animal or human subject.

[0160] In some embodiments of the present aspect of the disclosure, the pharmaceutical composition may further comprise at least one cancer therapeutic agent and a pharmaceutically acceptable carrier, and at least one chemotherapeutic agent and a saponin derivative are admixed in a pharmaceutically acceptable formulation or covalently bonded to each other.

[0161] Yet another aspect of the disclosure encompasses embodiments of a method for increasing the immunogenicity of an immunogen when administered to an animal or human subject, the method comprising at least the step of administering to the subject a vaccine comprising a pharmaceutical composition according to the present disclosure.

[0162] Yet another aspect of the disclosure encompasses embodiments of a synthetic route for synthesizing a saponin derivative, the synthetic route comprising coupling a natural saponin with a functionalized side chain molecule, the functionalized side chain comprising an amino group or a hydroxyl group.

[0163] In some embodiments of the present aspect of the disclosure, the natural saponin can be obtained from Momordica cochinchinensis Spreng.

[0164] In some embodiments of the present aspect of the disclosure, the natural saponin can be coupled to the functionalized side chain molecule via an amide formation reaction or an ester formation reaction.

[0165] Embodiments of the present disclosure are described in connection with examples, as well as corresponding text and drawings, but there is no intention to limit the disclosure to these described embodiments. Rather, it is intended to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.

Example

[0166] Example 1 Summary The organic solution was concentrated by rotary evaporation at about 12 Torr. Flash column chromatography was performed using 230 - 400 mesh silica gel. Thin layer chromatography was performed using a glass plate pre-coated to a depth of 0.25 mm with 230 - 400 mesh silica gel impregnated with a fluorescent indicator (254 nm). Infrared (IR) data is presented as absorption frequency (cm -1 ). Proton and carbon-13 nuclear magnetic resonance ( 1 1H NMR or 13 13C NMR) spectra were recorded on 400, 700, and 850 MHz NMR spectrometers, and chemical shifts are expressed in parts per million (δ scale) downfield from tetramethylsilane, referenced to residual protium in the NMR solvent (CHCl3: δ = 7.26). The data is presented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and / or multiple resonances, AB = AB quartet), coupling constant in Hertz (Hz), and integration. Anhydrous solvents were used without distillation. Solvents for workup and column chromatography were obtained from vendors and used without further purification. The purity of the products for immunological studies was determined by a combination of HPLC and 1 1H NMR, and the measured value was >95%.

[0167] Example 2 The precursor of the disclosed adjuvant was isolated from the seed saponins of Momordica cochinchinensis SPRENG. (Cucurbitaceae), which is a more accessible source of adjuvants and adjuvant precursors than adjuvants of other saponin series.

[0168] According to the flowchart shown in FIG. 11, the natural products MS-I, MS-II, and MS-C isolated from the seed saponins of Momordica cochinchinensis Spreng. (Cucurbitaceae) are shown in FIGS. 12A to 12C, respectively.

[0169] Example 3 Side chain options: Using standard amide formation procedures (e.g., as shown in Figure 5), side chains can be incorporated to produce various saponin derivatives. Preliminary studies have revealed that the structure of the incorporated side chain has a significant impact on adjuvant activity in terms of the magnitude and nature of the stimulated immune response. Thus, analogs with different side chains can be synthesized. QS-21 analogs incorporating side chains terminated with polar functional groups significantly improve the adjuvant activity of the derivatives. The side chains have terminal carboxyl groups. Previous studies have shown that terminal carboxyl groups can change the IgG subclass distribution and result in more IgG2a production, which may be related to enhanced Th1 immunity. Side chains b and c can fine-tune the balance between the hydrophilicity and hydrophobicity of the whole molecule, which may be related to adjuvant activity. Side chains d - f could provide similar insights as side chains a - c. Previous SAR studies of QS-21 analogs have also shown that side chain h with a terminal sugar unit also significantly improves the adjuvant activity of the analogs. Side chain g is a simplified version of side chain h. Side chain i has a terminal aldehyde moiety. SAR studies have shown that the carbonyl group on the chiral acid core of natural QS-21 is essential for the exceptional adjuvant activity of QS-21. It has been suggested that the carbonyl group can react with an amino group on the T cell surface receptor to form an imine. This Schiff base formation may presumably provide a co-stimulatory signal and result in T cell activation and Th1 immunity. Incorporation of an additional aldehyde moiety can improve the Schiff base-induced interaction between the adjuvant molecule and the T cell surface receptor, and thus improve Th1 immunity. Side chain j has a terminal tukaresol moiety bearing an aromatic carbonyl group. Tukaresol has been studied as an adjuvant and improves antigen-specific humoral and cellular immune responses. Side chains k and l (each having two cis / trans isomers), which are derived from natural saponins such as escin and diosphyllasaponin, can result in an improvement in cell-mediated immunity by recombining them with natural saponins that already show high humoral immunity.

[0170] QS-MPL combination adjuvant, MPL side chain 23d: The terminal group can also be derived from established adjuvant moieties. In animal models, those of QS-21 and its variants can act synergistically with other adjuvants such as monophosphoryl lipid A (MPL, a TLR4 agonist) (Ashtekar et al., (2012) PloS one. 7: e50460). MPL is known for TLR4 activation and significantly enhances Th1-type cellular and humoral immune responses. Typically, it enhances serum Ab titers 10 - 20-fold compared to vaccine alone. In human vaccine trials, MPL has been shown to have a safety profile similar to that of alum (Wang et al., (2016) J. Org. Chem. 81: 9560 - 9566). Therefore, MPL side chain 23d can be incorporated into saponin derivatives to produce the corresponding QS-MPL single-molecule combination adjuvant.

[0171] QS-Pam2Cys combination adjuvant, Pam2Cys side chain 23e: Pam2Cys and Pam3Cys, synthetic analogs of bacterial lipopeptides, are two TLR 2 agonists used as vaccine adjuvants in preclinical studies. These lipid adjuvants enhance both humoral and cell-mediated responses, but they are not very effective in enhancing CTL responses. They have been shown to be effective in epitope-based vaccines and do not exhibit the harmful side effects commonly associated with many other adjuvant formulations. Chemical incorporation of Pam3Cys into a fully synthetic carbohydrate-based cancer vaccine has shown results demonstrating that chemically connecting the TLR 2 agonist is achievable for enhancing the immune response. The synthesis of appropriately protected Pam2Cys moieties is a common practice and is known in the literature.

[0172] Example 4 4b (14.4 mg, 78%) of 11H NMR (600 MHz, CD3OD) (Characteristic protons) δ 9.49 (s, 1H), 7.41 - 7.37 (m, 4H), 7.35 (m, 1H), 5.36 (d, J = 1.5 Hz, 1H), 5.34 (d, J = 8.2 Hz, 1H), 5.29 (t, J = 3.3 Hz, 1H), 5.15 (s, 2H), 5.05 (d, J = 1.5 Hz, 1H), 4.67 (d, J = 7.9 Hz, 1H), 4.60 (d, J = 7.8 Hz, 1H), 4.52 (d, J = 7.6 Hz, 1H), 4.51 (d, J = 6.9 Hz, 1H), 4.48 (d, J = 7.2 Hz, 1H), 4.29 (t, J = 2.4 Hz, 1H), 4.04 (dd, J = 3.2, 1.9 Hz, 1H), 4.01 (dd, J = 11.4, 5.3 Hz, 1H), 3.16 (t, J = 10.9 Hz, 1H), 3.07 (dd, J = 9.1, 8.0 Hz, 1H), 2.83 (dd, J = 12.9, 3.6 Hz, 1H), 2.40 (t, J = 7.4 Hz, 2H), 2.07 (td, J = 12.8, 2.3 Hz, 1H), 1.02 (s, 3H), 0.94 (s, 3H), 0.93 (s, 3H), 0.83 (s, 3H); 13 13C NMR (150.9 MHz, CD3OD) δ 209.3, 176.5, 173.8, 169.9, 169.8, 143.6, 136.4, 128.2, 127.9, 127.8, 121.8, 104.6, 103.9, 103.7, 102.8, 102.5, 101.8, 100.0, 94.0, 87.3, 84.4, 84.1, 81.5, 77.7, 77.6, 76.6, 76.1, 76.0, 75.4, 74.8, 74.5, 74.0, 73.6, 73.0, 72.8, 72.4, 72.2, 71.6, 71.3, 70.8, 70.7, 70.5, 70.1, 69.9, 69.6, 69.2, 69.1, 68.1, 67.4, 65.8, 65.7, 65.6, 60.8, 54.9, 46.6, 46.0, 41.8, 41.6, 39.6, 38.9, 38.7, 38.0, 35.7, 33.7, 33.5, 32.2, 32.1, 31.5, 30.1, 29.2, 29.1, 29.0, 28.9, 28.8, 27.5, 26.5, 24.8, 24.7, 24.4, 23.2, 22.8, 22.6, 20.2, 17.1, 16.5, 16.4, 15.1, 15.0, 9.5; HRMS (ESI-TOF) m / z: C 94H 148 NO 41 of [M+H] + Calculated value 1946.9527, measured value 1946.9496.

[0173] Example 5 4c (56.0 mg, 98%) of 1 H NMR (600 MHz, CD3OD) (characteristic protons) δ 9.48 (s, 1H), 5.35 - 5.32 (m, 2H), 5.28 (s, 1H), 5.05 (s, 1H), 4.46 (d, J = 7.8 Hz, 1H), 4.60 (d, J = 7.8 Hz, 1H), 4.51 (d, J = 7.5 Hz, 2H), 4.48 (m, 1H), 4.28 (s, 1H), 4.05 (s, 1H), 4.01 (dd, J = 11.3, 5.1 Hz, 1H), 3.16 (t, J = 11.0 Hz, 1H), 3.07 (t, J = 8.5 Hz, 1H), 2.83 (d, J = 10.4 Hz, 1H), 2.31 (t, J = 7.4 Hz, 1H), 2.07 (t, J = 13.2 Hz, 1H), 1.02 (s, 3H), 0.81 (s, 3H); 13 C NMR (150.9 MHz, CD3OD) δ 209.4, 176.5, 176.3, 169.8, 143.5, 121.8, 104.6, 103.9, 103.6, 102.8, 102.5, 101.9, 100.0, 94.0, 87.2, 84.4, 84.2, 81.5, 77.6, 76.6, 76.1, 76.0, 75.4, 74.8, 74.5, 74.0, 73.6, 73.01, 72.98, 72.4, 72.2, 71.6, 71.3, 70.8, 70.6, 70.5, 70.1, 69.9, 69.6, 69.2, 69.1, 68.1, 67.5, 65.7, 65.6, 60.8, 54.9, 46.6, 46.0, 41.8, 41.6, 39.6, 38.8, 38.0, 35.7, 33.6, 32.2, 31.6, 30.2, 29.4, 29.2, 29.14, 29.11, 28.9, 27.5, 26.5, 24.9, 24.7, 24.4, 23.2, 22.8, 22.7, 20.2, 17.1, 16.6, 16.4, 15.2, 15.0, 9.5; HRMS (ESI-TOF) m / z: C 87 H 142 NO 41 of [M+H] +Calculated value 1856.9057, measured value 1856.8998.

[0174] Example 6 of 5b (12.5 mg, 72%) 1 H NMR (700 MHz, CD3OD) (characteristic protons) δ 9.51 (s, 1H), 7.39 - 7.38 (m, 4H), 7.34 (m, 1H), 5.44 (d, J = 1.5 Hz, 1H), 5.34 (t, J = 3.2 Hz, 1H), 5.24 (d, J = 8.3 Hz, 1H), 5.15 (s, 2H), 5.05 (d, J = 1.3 Hz, 1H), 4.75 (d, J = 7.9 Hz, 1H), 4.57 (d, J = 7.8 Hz, 1H), 4.54 (s, 1H), 4.52 - 4.47 (m, 2H), 4.46 (d, J = 7.6 Hz, 1H), 4.26 (t, J = 3.2, 1.8 Hz, 1H), 4.06 - 4.02 (m, 2H), 3.19 (t, J = 10.7 Hz, 1H), 3.15 (dd, J = 9.2, 8.1 Hz, 1H), 2.92 (dd, J = 9.4, 4.2 Hz, 1H), 2.40 (t, J = 7.3 Hz, 2H), 2.31 (t, J = 13.6 Hz, 1H), 1.44 (s, 3H), 1.26 (d, J = 6.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 3H), 1.19 (s, 3H), 1.21 (s, 3H), 1.04 (s, 3H), 0.95 (s, 3H), 0.89 (s, 3H), 0.82 (s, 3H); 1313C NMR (150.9 MHz, CD3OD) δ 209.6, 175.5, 173.8, 169.8, 143.5, 136.4, 128.2, 128.1, 127.9, 127.8, 121.5, 104.7, 103.8, 103.5, 102.8, 102.7, 101.9, 99.3, 94.0, 87.4, 84.6, 84.5, 82.1, 77.3, 76.8, 76.4, 76.0, 75.42, 75.38, 75.1, 74.2, 74.0, 73.6, 73.3, 73.0, 72.4, 72.3, 71.6, 71.51, 71.45, 70.8, 70.7, 70.5, 70.1, 70.0, 69.6, 69.2, 69.1, 68.0, 67.4, 65.8, 65.7, 60.8, 60.6, 54.8, 48.5, 46.6, 41.6, 41.1, 39.7, 38.7, 38.0, 35.7, 35.2, 33.7, 32.8, 32.0, 30.5, 29.9, 29.4, 29.2, 29.1, 29.0, 28.9, 28.7, 26.5, 25.9, 24.7, 23.3, 23.1, 20.0, 17.0, 16.5, 16.4, 15.1, 9.6; HRMS (ESI-TOF) m / z: C 94 H 148 NO 42 of [M + H] + Calculated value 1962.9476, measured value 1962.9436.

[0175] Example 7 5c (11.0 mg, 96%) of 11H NMR (700 MHz, CD3OD) (Characteristic protons) δ 9.51 (s, 1H), 5.44 (d, J = 1.3 Hz, 1H), 5.35 (t, J = 3.4 Hz, 1H), 5.25 (d, J = 8.3 Hz, 1H), 5.05 (s, 1H), 4.75 (d, J = 7.8 Hz, 1H), 4.57 (d, J = 7.9 Hz, 1H), 4.54 (s, 1H), 4.45 - 4.47 (m, 2H), 4.46 (d, J = 7.6 Hz, 1H), 4.25 (s, 1H), 4.06 - 4.00 (m, 2H), 3.19 (t, J = 11.3 Hz, 1H), 3.15 (dd, J = 9.3, 8.1 Hz, 1H), 2.31 (t, J = 13.9 Hz, 1H), 2.29 (t, J = 7.5 Hz, 2H), 1.44 (s, 3H), 1.21 (s, 3H), 1.05 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H), 0.82 (s, 3H); HRMS (ESI-TOF) m / z: C 87 H 142 NO 42 of [M + H] + Calculated value 1872.9006, measured value 1872.9016.

[0176] Example 8 Preparation of 9b and 9c: Hydrogen gas was applied to conjugate 10 (30.0 mg, 7.9 mmol) and 10% Pd / C (6.0 mg) in 1.5 mL of THF / MeOH (2:1) at 55 psi for 16 hours. The suspension was then filtered through a Celite plug, concentrated, and redissolved in 0.6 mL of EtOH / H2O (v / v 5:1). To the solution were added benzyl 11-aminodecanoate hydrochloride (6.4 mg, 20 μmol), N-methylmorpholine (NMM) (13.0 mg, 127 μmol), hydroxybenzotriazole (HOBt) (8.8 mg, 58 μmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC .HCl (11.4 mg, 58 μmol) was added at room temperature. The reaction mixture was stirred for 1 day and then filtered. The filtrate was purified by RP-HPLC using a semi-Prep C18, 250x10 mm, 5 micron column, and an H2O / MeCN gradient (90% - 10% H2O over 30 minutes at a flow rate of 3 mL / min). The desired product had a retention time of 23 minutes. The fractions were concentrated at room temperature on a rotary evaporator to remove MeCN, and then the remaining water was removed by lyophilization to afford the intermediate as a white solid (8.0 mg, 52%) in two steps. The intermediate was dissolved in methanol (0.5 mL) and H2O (0.3 mL) and treated with K2CO3 (20 mg) overnight. The reaction mixture was neutralized with acetic acid and purified by RP-HPLC using a semi-Prep C18, 250x10 mm, 5 micron column, and an H2O / MeCN gradient (90% - 10% H2O over 45 minutes at a flow rate of 3 mL / min). The desired product had a retention time of 23 minutes. The fractions were concentrated at room temperature on a rotary evaporator to remove MeCN, and then the remaining water was removed by lyophilization to afford 9b as a white solid (4.4 mg, 57%). 9c was obtained as a white solid (4.0 mg, 96%) by using the same debenzylation procedure described for 4c / 5c.

[0177] Example 9 of 9b 11H NMR (600 MHz, CD3OD) (Characteristic protons) δ 9.38 (s, 1H), 7.87 (t, J = 5.5 Hz, 1H), 7.27 - 7.24 (m, 4H), 7.22 (m, 1H), 5.22 - 5.19 (m, 2H), 5.17 (d, J = 8.2 Hz, 1H), 5.03 (s, 2H), 4.71 (d, J = 7.7 Hz, 1H), 4.63 (d, J = 7.9 Hz, 1H), 4.48 (d, J = 7.8 Hz, 1H), 4.44 (d, J = 7.7 Hz, 1H), 4.41 (d, J = 7.5 Hz, 1H), 4.38 (s, 1H), 4.34 (d, J = 7.4 Hz, 1H), 4.16 (dd, J = 2.9, 1.9 Hz, 1H), 2.85 (dd, J = 13.9, 4.0 Hz, 1H), 2.28 (t, J = 7.3 Hz, 2H), 2.21 (t, J = 13.3 Hz, 1H), 1.29 (s, 3H), 1.11 (d, J = 6.4 Hz, 3H), 1.07 (s, 3H), 0.90 (s, 3H), 0.84 (s, 3H), 0.78 (s, 3H), 0.66 (s, 3H); 13 13C NMR (176.0 MHz, CD3OD) δ 211.4, 177.1, 175.2, 170.8, 145.0, 137.8, 129.6, 129.3, 129.2, 123.1, 105.9, 105.3, 105.0, 104.8, 104.7, 103.7, 101.7, 95.3, 95.2, 88.3, 87.0, 86.3, 83.0, 78.9, 78.23, 78.20, 78.0, 77.8, 77.7, 77.0, 76.4, 76.3, 75.37, 75.34, 75.26, 75.0, 74.9, 74.6, 73.6, 73.5, 72.7, 71.4, 71.3, 71.1, 71.0, 70.7, 69.9, 68.9, 67.2, 66.6, 62.3, 61.9, 56.3, 42.7, 42.2, 41.1, 40.1, 39.4, 37.1, 36.6, 36.5, 35.1, 33.8, 33.4, 32.1, 31.3, 30.8, 30.6, 30.52, 30.45, 30.3, 30.2, 27.9, 27.3, 26.1, 26.0, 24.8, 24.5, 21.6, 18.7, 17.8, 16.52, 16.45, 11.0; HRMS (ESI-TOF) m / z: C 93 H 145 NO 42 Na of [M + Na] +Calculated value 1970.9139, measured value 1970.9172.

[0178] Example 10 of 9c 1 1H NMR (700 MHz, CD3OD) (characteristic protons) δ 9.50 (s, 1H), 7.95 (t, J = 5.9 Hz, 1H), 5.36 - 5.32 (m, 2H), 5.30 (d, J = 8.2 Hz, 1H), 4.75 (d, J = 7.9 Hz, 1H), 4.60 (d, J = 7.8 Hz, 1H), 4.56 (d, J = 7.7 Hz, 1H), 4.54 (d, J = 7.6 Hz, 1H), 4.50 (s, 1H), 4.46 (d, J = 7.5 Hz, 1H), 4.29 (dd, J = 3.2, 1.7 Hz, 1H), 2.98 (dd, J = 14.1, 3.8 Hz, 1H), 2.33 (t, J = 13.8 Hz, 1H), 2.33 (t, J = 7.4 Hz, 2H), 1.41 (s, 3H), 1.31 (d, J = 6.2 Hz, 3H), 1.24 (d, J = 6.4 Hz, 3H), 1.21 (s, 3H), 1.03 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.79 (s, 3H); HRMS (ESI-TOF) m / z: C 86 H 139 NO 42 Na of [M+Na] + Calculated value 1880.8669, measured value 1880.8645.

[0179] Example 11 Antigens: Chicken ovalbumin (Vac-pova) for in vivo use was purchased from InvivoGen. Recombinant Porphyromonas gingivalis HagB was prepared as previously described (Zhang et al., (2003) Vaccine 21:4459-4471, Zhang et al., (2004) Infect. Immun. 72:637-644, Zhang et al., (2005) Infect. Immun. 73:3990-3998). Briefly, the HagB gene was cloned from P. gingivalis 381 into a pET vector with a lac promoter and a histidine tag and expressed in Escherichia coli JM109. Protein expression was induced after induction with isopropyl-β-D-thiogalactopyranoside (IPTG). rHagB was purified from the soluble fraction of bacterial lysates by using a His-binding resin column according to the manufacturer's instructions (Novagen, Madison, WI). The purity of rHagB was confirmed by silver staining and Western blot analysis using a rabbit anti-rHagB antibody. The concentration of rHagB was estimated by a bicinchoninic acid protein determination assay (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard.

[0180] Example 12 Mice and immunization: BALB / c mice used in this study were from Frederick Cancer Research (Fredrick, MD). To evaluate the adjuvant activity of the MS saponin-based adjuvant, female mouse groups (8-10 weeks old, 6 mice per group) were immunized by the subcutaneous (s.c.) route with OVA (20 μg) or rHagB (35 μg) alone, or with appropriate adjuvants such as antigen plus GPI-0100 (100 μg) or MS adjuvant (100 μg) at days 0, 14, and 28. Before each immunization and 2 weeks after the last immunization, the mice were weighed and blood samples were collected from the lateral tail vein by using a heparinized capillary pipette. Serum was obtained after centrifugation and stored at -20 °C until assay.

[0181] Example 13 Evaluation of antibody response: The levels of specific serum IgG and IgG subclasses against OVA or rHagB in each group were determined by enzyme-linked immunosorbent assay (ELISA). rHagB (1 μg / ml), OVA (0.1 μg / ml), or the optimal amount of goat anti-mouse IgG, IgG1 7, or IgG2a in borate buffered saline (BBS; 100 mM NaCl, 50 mM boric acid, 1.2 mM Na2B4O 、 , pH 8.2) was used to coat Maxisorpmicrotiter plates (NUNC International, Roskilde, DK) overnight at 4°C. The plates were blocked for 2 hours at room temperature with 1% bovine serum albumin (BSA) and 0.02% sodium azide in BBS. Serial two-fold dilutions of serum samples were added in duplicate to the plates. To generate a standard curve, serial dilutions of mouse immunoglobulin reference serum (MP Biomedicals, Solon, OH) were added to two columns of wells of each plate coated with the appropriate anti-mouse IgG or IgG subclass reagent. After incubation (overnight at 4°C) and washing of the plates, horseradish peroxidase-conjugated goat anti-mouse IgG or IgG subclass antibody was added to the appropriate wells. After incubation for 4 hours at room temperature, the plates were washed and developed with o-phenylenediamine substrate containing hydrogen peroxide. Color development was recorded at 490 nm. The concentration of the antibody was determined by interpolation against a standard curve generated using mouse immunoglobulin reference serum and constructed by a computer program based on a four-parameter logistic algorithm (Softmax / Molecular Devices Corp., Menlo Park, CA).

[0182] Example 14 Statistical analysis: Statistical significance in antibody responses was evaluated by t-tests using GraphPad Prism 8 (with non-parametric and Mann-Whitney tests for unpaired two groups). A difference with a P-value < 0.05 was considered significant.

[0183] Example 15 General structure [Chemical formula] wherein q1 is H or OH, q2 and q3 are each independently selected from CHO, CH3, CH2OH, H, or a component of an acetal group, f3 and f4 are each independently OH, or acetyl, or C3 and C4 of a fucosyl unit, and f3 and f4 form a cyclic ketal ring or a cyclic carbonate ester, f5 is H, a methyl group, a a carboxyl group R 4 -O-C(O)- , R4-NR5-C(O)-, R4-O- and R 4 -O-CH 2 - selected from the group consisting of R4 and R5 are each independently a straight chain having the structure R6(CH2) 1-20 - or R6[(CH2) 1-20 O 0-1 (CH2) 0-20 1-20 and R6 is H, OH, COO(CH2) 0-6 H, COOBn, C(O)NR7Bn, NR7Bn, OBn, a saccharide unit, Momordica saponin I or II, muramyl dipeptide, monophosphoryl lipid A (MPL) unit, α-Galcer unit, dipalmitoyl-S-glyceryl cysteine (PamCys) unit, or a functional group of a carrier, R7 is H or an alkyl group, ga 5 is H, a methyl group, a carboxyl R excluding H-O-C(O)- 4​-O-C(O)-, R 4 -NR 5 -C(O)-, R 4 -O-, and R 4 -O-CH 2 - and is selected from the group consisting of, R 4 and R 5 are each independently H, a structure R 6 (CH 2 ) 1-20 - or R 6 [(CH 2 ) 1-20 O 0-1 (CH 2 ) 0-20 ] 1-20 is a straight chain having, R 6 is H, OH, COO(CH 2 ) 0-6 H, COOBn, C(O)NR 7 Bn, NR 7 Bn, OBn, saccharide unit, Momordica saponin I or II, muramyl dipeptide, monophosphoryl lipid A (MPL) unit, α-Galcer unit, dipalmitoyl-S-glyceryl cysteine (PamCys) unit, or a functional group of a carrier, R 7 is H or an alkyl group, r3 is H, a monosaccharide, a disaccharide, or a trisaccharide, x3 is H, a monosaccharide (excluding xylose), or a disaccharide, ga3 is H, a monosaccharide, or a disaccharide.

[0184] Example 16 Synthetic derivatives: These semi-synthetic products were prepared from the derivatization of the carboxyl groups of the glucuronic acid units of the natural products (MS-A, MS-B, and MS-C) (Figures 12A - 12C, respectively).

Chemical formula

[0185] Example 17 Saponin-MPL conjugate

Chemical formula

[0186] Example 18 Saponin-Pam2Cys conjugate [Chemical formula]

[0187] Example 19 Synthetic derivative: [Chemical formula] [Chemical formula] [Chemical formula]

[0188] Example 20 Anti-rHagB antibody formation induced by rHagB containing various saponin adjuvants disclosed in mice: Generation of IgG, Ig1, and Ig2a in female, BALB / c mice (6 mice / group) immunized at 200 μl / mouse: Administration at 100 μl / site, 2 sites / dorsal s.c. / mouse is shown in Figures 13 - 15 respectively.

[0189] Immunized with the following: A, 20 μg of rHagB; B, 20 μg of rHagB + 100 μg of GPI-0100; G, 20 μg of rHagB + 100 μg of MA; H, 20 μg of rHagB + 100 μg of MB; J, 20 μg of rHagB + 100 μg of MA-N; K, 20 μg of rHagB + 100 μg of MA-X; L, 20 μg of rHagB + 100 μg of MA-XBn; M, 20 μg of rHagB + 100 μg of MB-N; N, 20 μg of rHagB + 100 μg of MB-X; O, 20 μg of rHagB + 100 μg of MC-N.

Claims

1. A modified saponin having Formula I: 【Chemical 2】 Wherein, q １ is H or OH, and q ２ and q ３ are each independently selected from CHO, CH ３ CH ２ OH, or H, R ３ is R4-NH-C(O)-, and R ４ is H, a structural R ６ (CH ２ ) １－２０ - or R ６ [(CH ２ ) １－２０ O ０－１ (CH ２ ) ０－２０ １－２０ is a straight chain having R ６ is a functional group of a carrier selected from the group consisting of H, OH, COO(CH ２ ), ０－６ H, COOBn, C(O)NR ７ Bn, NR ７ Bn, OBn, saccharide unit, Momordica saponin I or II, muramyl dipeptide, monophosphoryl lipid A (MPL) unit, α-GalCer unit, dipalmitoyl-S-glycerylcysteine (PamCys) unit, or a polyamine polymer, polyethylene glycol amine, poly(ethyleneimine), nanocarbon, and an amino-containing biomolecule, R ７ is H or an alkyl group.

2. R ４ is a long-chain fatty acid having the structure HOOC-(CH ２ ) ６－２０ -, the modified saponin according to claim 1.

3. R ４ is a long-chain alcohol having the structure HO-(CH ２ ), ６－２０ -; or R ４ is a long-chain alkyl terminated with a functional group selected from an amino group, a cyano group, an azide group, and an aromatic group, the modified saponin according to claim 1.

4. R ４ is long-chain alkyl R ６ O(CH ２ ) ６－２０ -, and R ６ is selected from saccharide units selected from the group consisting of monosaccharides, disaccharides, and trisaccharides, the modified saponin according to claim 1.

5. R ４ The modified saponin according to claim 1, wherein R is a long-chain alkyl terminated with monophosphoryl lipid A (MPL).

6. R ４ is a long-chain alkyl terminated with dipalmitoyl-S-glycerylcysteine (Pam ２ Cys) or tripalmitoyl-S-glycerylcysteine (Pam ３ Cys), the modified saponin according to claim 1.

7. R ４ is a long-chain alkyl terminated with a muramyl dipeptide unit; or R ４ is a long-chain alkyl terminated with an α-GalCer unit; or R ４ is a long-chain alkyl terminated with Momordica saponin I unit; or R ４ The modified saponin according to claim 1, wherein R is a long-chain alkyl terminated with a Momordica saponin II unit.

8. The modified saponin according to Claim 1, wherein the modified saponin is selected from the group consisting of the following Formulas A to E. [Chemical Formula 3]

9. A pharmaceutical composition comprising a modified saponin having Formula I [Chemical Formula 5] Wherein, q １ is H or OH, and q ２ and q ３ are each independently selected from CHO, CH ３ , CH ２ OH, or H, R ３ is R4-NH-C(O)-, R ４ is H, the structure R ６ (CH ２ ) １－２０ - or R ６ [(CH ２ ) １－２０ O ０－１ (CH ２ ) ０－２０ １－２０ is a straight chain having​ R ６ is a functional group of a carrier selected from the group consisting of H, OH, COO(CH ２ ), ０－６ H, COOBn, C(O)NR ７ Bn, NR ７ Bn, OBn, saccharide unit, Momordica saponin I or II, muramyl dipeptide, monophosphoryl lipid A (MPL) unit, α-GalCer unit, dipalmitoyl-S-glyceryl cysteine (PamCys) unit, or a polyamine polymer, polyethylene glycol amine, poly(ethylene imine), nanocarbon, and an amino-containing biomolecule. R ７ is H or an alkyl group.

10. The pharmaceutical composition according to Claim 9, wherein the modified saponin is selected from the group consisting of the following Formulas A to E. [[Chemical Formula 6]]

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