Pentavalent vaccine against Neisseria meningitidis containing synthetic MenA antigen

Abstract

The present inventors have identified a combination vaccine for immunization against bacterial meningitis caused by multiple pathogens.

Description

[Technical Field]

[0001] All documents cited herein are incorporated herein in their entirety by reference.

[0002] The present invention relates to immunization against bacterial meningitis, and more particularly to combination vaccines for immunization against bacterial meningitis caused by multiple pathogens. [Background technology]

[0003] Neisseria meningitidis is the leading cause of bacterial meningitis and sepsis worldwide, and can lead to outbreaks and epidemics of invasive meningococcal disease. Invasive meningococcal disease occurs globally. Incidence varies by region of the world, but infants, children, and adolescents are most vulnerable to developing invasive disease. The disease progresses rapidly and often leads to a catastrophic outcome. Based on the antigenic differences of their capsular polysaccharides, 12 serotypes of Neisseria meningitidis have been identified. Virtually all disease-associated isolates are encapsulated, and serotypes A, B, C, W, X, and Y are involved in more than 90% of invasive meningococcal infections worldwide. The distribution of these serotypes is geographically and temporally volatile.

[0004] Type B meningitis is a serious and often fatal disease that primarily affects infants and young adults. It is easily misdiagnosed, can result in death within 24 hours of symptom onset, and can cause severe, lifelong disabilities even with treatment.

[0005] Currently, two vaccines designed to induce immunity against serogroup B meningococcus are approved: GSK's BEXSERO and Pfizer's TRUMENBA.

[0006] BEXSERO (also known as C4MenB) is a preparation of outer membrane vesicles (OMVs) from the epidemic strain of Neisseria meningitidis group B NZ98 / 254, containing five meningococcal antigens: Neisserial heparin-binding protein A (NHBA), factor H-binding protein (fHbp) variant 1.1, Neisserial adhesion protein A (NadA), and accessory proteins GNA1030 and GNA2091. Four of these antigens exist as fusion proteins (NHBA-GNA1030 fusion protein and GNA2091-fHbp fusion protein). BEXSERO® is documented in the literature (see, for example, Bai et al. (2011) Expert Opin. Biol. Ther. 11: 969-85, Su & Snape (2011) Expert Rev. Vaccines 10: 575-88).

[0007] TRUMENBA® contains two types of lipid-modified MenB fHbp antigens (v1.55 and v3.45) adsorbed onto aluminum phosphate.

[0008] fHbp (interchangeably known in this art as genome-derived Neisseria antigen (GNA) 1870, LP2086, and protein "741") binds to human factor H (hfH), which is a large (180 kDa) multidomain soluble glycoprotein consisting of 20 complement regulatory protein (CCP) modules linked by short linker sequences. hfH circulates in human plasma and regulates an alternative pathway of the complement system. The functional binding of fHbp to hfH depends primarily on hfH's CCP modules (or domains) 6-7, enhancing the bacteria's ability to resist complement-mediated death. Therefore, fHbp expression enables ex vivo survival in human blood and serum.

[0009] Since various fHbp classification methods have been proposed, a dedicated database with a unified fHbp nomenclature (HyperText Transfer Protocol (http: / / neisseria.org / nm / typing / fhbp, also known as HyperText Transfer Protocol (https: / / pubmlst.org / neisseria / fHbp / ))) is available for assigning new subspecies.

[0010] fHbp is classified into three (major) variants 1, 2, and 3, which are further divided into subspecies / variants fHbp-1.x, fHbp-2.x, and fHbp-3.x, where x indicates a subspecies / variant of a particular peptide. In different nomenclature, subspecies / variants are grouped into subfamily A (corresponding to variants 2 and 3) and subfamily B (corresponding to variant 1) based on sequence diversity.

[0011] BEXSERO is expected to provide broad coverage against MenB strains circulating worldwide (Medini D et al., Vaccine 2015; 33: 2629-2636; Vogel U et al. Lancet Infect. Dis. 2013; 13: 416-425; Knzova et al., Epidemiol. Mikrobiol. Imunol. 2014; 63: 103-106; Tzanakaki G et al. BMC Microbiol. 2014; 14: 111; Wasko I et al. Vaccine 2016; 34: 510-515; 6. Simoees MJ et al. PLoS ONE 12 (5): e0176177; and Parikh SR et al. Lancet Infect. Dis. 2017; (17:754-62). Furthermore, after BEXSERO was introduced into the UK's National Infant Immunization Program in September 2015, 10-month data showed 83% vaccine efficacy against all MenB strains after two doses (Parikh SR et al., Lancet 2016; 388: 2775-82).

[0012] However, bactericidal activity is variant-specific, and antibodies produced against one variant do not necessarily provide cross-protection against other variants, although some cross-reactivity has been reported between fHbp v2 and v3 (Masignani V et al., J. Exp. Med. 2003; 197: 789-799). Antibodies produced against the subspecies / variant fHbpv1.1 included in the BEXSERO vaccine show high cross-reactivity with fHbp v1 and low cross-reactivity with fHbp v2 and v3 (Brunelli B et al., Vaccine 2011;29:1072-1081).

[0013] Therefore, despite the efficacy of approved serogroup B meningococcal vaccines such as BEXSERO, there remains a need to develop a vaccine that offers broad coverage of MenB strains and improved immunogenicity without compromising the advantages of, for example, BEXSERO.

[0014] WO2020 / 030782 describes how the strain coverage and immunogenicity of MenB vaccines can be improved by including additional fHbp variants in the immunogenic composition along with the BEXSERO antigen. In particular, WO2020 / 030782 discloses immunogenic compositions comprising fHbp fusion proteins containing modified fHbp v2, v3, and v1.13 or v1.15 polypeptides.

[0015] Vaccine approaches to immunize against serogroups A, C, W, and Y have tended to focus on Neisseria meningitidis capsular polysaccharides (CPSs). Generally, CPSs are T cell-independent antigens, meaning they can evoke an immune response without T cell involvement. This response lacks several key characteristics that characterize T cell-dependent immune responses, such as immunological memory, IgM-to-IgG class switching, and affinity maturation. However, when a portion of the polysaccharide is linked to a carrier protein, it triggers a cellular immune response that produces a memory effect, protecting infants. Such polysaccharides linked to carrier proteins are often referred to as complex carbohydrates and are particularly valuable as vaccines. In this regard, particularly effective vaccines (complex carbohydrate vaccines) can be made by covalently linking the sugar to a carrier protein, either via a linker portion (or spacer) or even by direct coupling of the sugar to a selected carrier protein. In any case, complex carbohydrates can induce a T-cell-dependent immune response with memory and effect even in infants, but non-complex CPS generally do not provide either a memory effect in adults or a substantial immunogenic effect in infants.

[0016] Current serogroup C vaccines (MENJUGATE [Costantino et al. (1992) Vaccine 10: 691-698, Jones (2001) Curr. Opin. Investig. Drugs 2: 47-49], MENINGITEC, and NEISVAC-C) contain conjugated sugars. MENJUGATE and MENINGITEC are CRM 197 One has an oligosaccharide antigen bound to a carrier, while NEISVAC-C uses a complete polysaccharide (de-acetylated) bound to a tetanus toxoid carrier.

[0017] The vaccine products marketed under the trade names MENVEO, MENACTRA, and NIMENRIX all contain conjugate capsule sugar antigens for serogroups Y, W135, C, and A, respectively.

[0018] MENVEO (generic name: Meningococcal (groups A, C, Y, and W-135) oligosaccharide diphtheria CRM197 conjugate vaccine) contains each of the A, C, W135, and Y antigens of CRM197. 197 It is coupled to the carrier.

[0019] In MENACTRA (generic name: meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccine), the A, C, W135, and Y antigens are bound to the diphtheria toxoid carrier.

[0020] In NIMENRIX (generic name: meningococcal polysaccharide group A, C, W-135 and Y conjugate vaccine), each of the A, C, W135, and Y antigens is conjugated to a tetanus toxoid carrier.

[0021] Among the meningococcal (N. meningitidis) capsular polysaccharides, the meningococcal (N. meningitidis) serotype A capsular polysaccharide (MenA CPS) is known to be affected by inherent chemical instability in water (see, e.g., Frasch et al., Adv. Biotechnol. Processes, 1990, 12, 123-145). As a result of this instability, serogroup A antigens are supplied in solid lyophilized form. Therefore, vaccines containing serogroup A antigens, such as MENVEO, must currently be supplied in two vials that are combined (reconstituted) before administration. The MenCYW-135 component of the conjugate vaccine is supplied as a liquid, which is used to reconstitute the MenA lyophilized conjugate vaccine component to form a complete vaccine product at the time of administration. However, supplying vaccine products in this manner is inconvenient, and a single, completely liquid formulation is considered most advantageous.

[0022] Furthermore, it is considered advantageous to provide a fully liquid pentavalent vaccine composition that provides immunoprotection against infection by each of the meningococcal (N. meningitidis) serogroups A, B, C, W135, and Y.

[0023] MenA CPS is composed of (1→6) linked 2-acetamido-2-deoxy-α-D-mannopyranosylphosphate repeating units, and the hydrolytic instability of MenA polysaccharide is mainly due to hydrolysis of phosphodiester linkages promoted by the ring oxygen and N-acetamide. In fact, both the ring oxygen and the N-acetyl group (NHAc) destabilize the phosphodiester glycoside linkage, and it has been recognized that the axial position of NHAc also contributes to this mechanism, as shown in scheme A below (Berti et al., Vaccine, 2012, 30, 6409-6415). [ka]

[0024] The availability of hydrolysis-resistant MenA polysaccharide mimics would be highly attractive for developing more stable conjugate vaccines. Stabilization of CPS can be achieved in various ways, and MenA CPS analogs in which the ring oxygen is replaced with a methylene group have been reported in the prior art. In particular, as shown in Scheme B, replacing the oxygen in the ring with carbon prevents the destabilization described in Scheme A. [ka]

[0025] Toma et al., Org. Biomol. Chem., 2009, 7, 3734-3740, describes the preparation of O-(2-acetamido-2-deoxy-5a-carba-alpha-D-mannopyranosyl) phosphate, a monomer in which the pyranose oxygen in the repeating unit of MenA CPS is replaced with a methylene group. This document only refers to the chemical synthesis of the monomer itself.

[0026] Gao et al. (Org. Biomol. Chem. 2012, 10(33), 6673, and ACS Chem. Biol. 2013, 8(11), 2561) and Ramella D. et al. (Eur J. Org. Chem, 2014, 5915-5924) describe the stabilization of glycosyl 1-O-phosphate by using carba sugars in which a methylene group replaces the pyranose oxygen atom. They also report the conjugation of synthetic carba trimers to protein carriers, but do not further investigate the behavior of carba analogs with higher degrees of polymerization. There is also no mention of carba analogs with specific levels of acetylation and / or specific acetylation patterns. Furthermore, the trimers examined are unlikely to inhibit the binding of anti-MenA CPS antibodies, indicating that the described derivatives are relatively weak conjugate antigens. For natural MenA polysaccharides, the recommended degree of acetylation is 75–90%, and WHO guidelines suggest that MenA vaccines should have at least 61.5% O-acetylation. However, the authors further disclose that acetylation significantly alters the hydrophobicity of O-acetylated conjugate polysaccharides. Therefore, considering structural conformational changes and conformational differences with natural polysaccharides, predicting the optimal level and pattern of acetylation for carba analogs is not trivial. This has been confirmed in recent in silico studies that have shown conformational differences between carba analogs and natural polysaccharides in increasing oligomer length (Carbohydrate Research, 486 (2019) 107838). [Overview of the Initiative]

[0027] Therefore, there is a need to identify carbaMenA analog polysaccharide derivatives that have good stability, exhibit a good immunogenicity profile, can be obtained following a reliable and simple synthetic approach, and are suitable for inclusion in a complete liquid pentavalent vaccine composition that provides immunoprotection against infection by each of the meningococcal (N. meningitidis) serogroups A, B, C, W135, and Y.

[0028] A first aspect of the present invention is an aqueous immunogenic composition that, after administration to a subject, can induce a bactericidal immune response against serogroups A, B, C, W135 and Y of Neisseria meningitidis, wherein the composition i. Conjugate serogroup A antigen, ii. Conjugate serogroup C antigen, iii. Conjugate serogroup W135 antigen, iv. Conjugate serogroup Y antigen; and v. One or more polypeptide antigens from serogroup B Includes, The present invention provides an aqueous immunogenic composition in which (ii), (iii), and (iv) are capsular sugar antigens and (i) is a synthetic analog of serogroup A capsular sugar.

[0029] In a preferred embodiment of the first aspect, the conjugated serogroup A antigen is an oligomeric conjugate comprising an oligomer of the following formula (Ia) or (Ib). [ka] During the ceremony, In the oligomer, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and can be the same or different in each repeating unit; R yis H or -C(O)CH3 and can be the same or different in each repeating unit; R x or R y at least one of which is -C(O)CH3 in at least one repeating unit; Az is an aza substituent selected from the group consisting of -NH(CO)R 1 , -N(R 1 )2 and -N3, and R 1 is independently selected from the group consisting of H, linear or branched C1-C6-alkyl, and linear or branched C1-C6-haloalkyl; Z is (i) a protecting group, (ii) a functional linker for conjugation to a protein, or (iii) linear or branched C1-C6 alkyl, optionally substituted phenyl, -C(O)Y, or linear or branched C1-C6-alkyl-X where Y is H, linear or branched C1-C6-alkyl, or a protecting group, X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -S-C≡N.

[0030] In a preferred embodiment of the first aspect, the conjugate serum group A antigen is an oligomer conjugate of the following formula (IIa) or (IIb).

Chemical formula

[0031] A second aspect of the present invention provides a method for inducing an immune response in a mammal, comprising administering an immunogenic composition according to the first aspect.

[0032] A third aspect of the present invention provides an immunogenic composition according to the first aspect for medical use.

[0033] A fourth aspect of the present invention provides an immunogenic composition according to the first aspect for use as a vaccine.

[0034] A fifth aspect of the present invention provides an immunogenic composition according to the first aspect for use in a method for inducing an immune response in mammals.

[0035] A sixth aspect of the present invention provides an immunogenic composition according to the first aspect for use in immunizing mammals against Neisseria meningitidis infection. [Brief explanation of the drawing]

[0036] [Figure 1]Figure 1 shows 1H-NMR monitoring of three reaction steps for the random O-acetylation of carba analog DP8, i.e., formula (Ia) n=8 (i.e., the oligomer is acetylated with one or more Rx and / or Ry, in other words, at least one of Rx and / or Ry is -C(O)Me). [Figure 2] Figure 2 shows the 1H-NMR of the final random O-acetylated carba analog DP8 (i.e., equation (Ia) where n=8), with integrals for determining the acetylation percentage. [Figure 3] Figure 3 shows the 31P NMR spectrum of the final random O-acetylated carba analog DP8 (formula (Ia)). This spectrum shows simultaneous acetylation at the C3+C4 position in approximately 44% of cases, and acetylation at either C3 or C4 in approximately 28% of cases. 27% of the molecule remains unacetylated. [Figure 4] Figure 4 shows the conjugation method for oligomers according to the present invention, including the determination of crude reaction CRM197 and SDS-PAGE characteristics. [Figure 5-1] Figure 5 shows the inhibition of binding of anti-MenA antibodies to CPS. Competitive ELISA using anti-MenA mAbs (Figure 5A) and anti-MenA polyclonal serum (Figure 5B) with non-acetylated carbaMenA oligomers of different lengths as inhibitors and CPS as the coating. (Figure 5C) Competitive SPR of binding between anti-MenA mAbs and immobilized biotinylated CPS. In A and B, MenA CPS and de-OAc CPS were used as positive controls, and β-glucan laminarin was used as a negative control. In C, MenA CPS and its fragments were used as positive controls, and anti-MenC mAbs were used as a negative control when flowed onto the chip. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 6-1]Figures 6A, 6B, 6C, and 6D show the immune response induced by neocomplex carbohydrates. The Figure 6A, 6B, and 6C panels show antibody titers reported as geometric mean (horizontal bars) and 95% CI (vertical bars). The Figure 6D panel shows rSBA titers reported as geometric mean (horizontal bars) and 95% CI (vertical bars). Ranks were compared using the two-sided Mann-Whitney test; n=10. Pre-immunization was a negative control in both types of analysis. Figure 6A) Anti-MenA IgG titers estimated in individual mouse serum after a second boost against natural MenA CPS. p<0.0001 between avDP~15 MenA and carbaDP6 / DP8 conjugate. Figure 6B) Anti-de-Oacetylated MenA IgG titers measured against de-Oacetylated MenA CPS conjugated to HSA. The p-values ​​between avDP~15 and carba DP8 conjugate, and between avDP8.5 and carba DP6 conjugate were p=0.002; between avDP8.5 and carba DP8 conjugate was p=0.003; and between avDP~15 and carba DP6 conjugate was p=0.004. Figure 6C) Estimated anti-MenA IgG titers in individual serums after a second boost using MenA CPS for coating. Comparison between avDP15 and Ac-carba DP8 conjugate was p=0.0011. Figure 6D) Bactericidal titers of human and rabbit serum measured after the third injection in pooled mouse serum and individual mouse serum, respectively. No significant differences were observed when comparing the ranks. Immunosensitization was performed in two series, and data from representative experiments are shown here. *Human and rabbit SBA titers measured after the third injection in pooled serum; **Human and rabbit SBA titers measured after the third injection in pooled serum from responder mice. The Y-axis shows "Anti-Men A CPS IgG (GMT 95% CI)". [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7]Figure 7 shows the ELISA titers measured after three vaccine administrations. Anti-MenA polysaccharide IgG antibodies were evaluated using CRM197 conjugates of random O-acetylated carbamenA analog DP8, compared to CRM197 conjugates of selectively 3-O-acetylated carbamenA DP8 and a natural MenA-CRM197 vaccine as a benchmark (i.e., positive control). [Figure 8-1] Figures 8A and 8B show ELISA titers after two and three doses of the vaccine. The p-value indicates a comparison between the benchmark MenA-CRM197 natural group and other vaccinated groups. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9] Figure 9 shows the SBA titer after three doses of the vaccine. Human complement-mediated bactericidal titer was measured in serum induced with CRM197 conjugates of random O-acetylated carbamenA analog DP8, compared to CRM197 conjugates of selectively 3-O-acetylated carbamenA DP8 and a natural MenA-CRM197 vaccine as a benchmark (i.e., positive control). [Figure 10] Figure 10 shows the SBA titers obtained in rabbits (rSBA) and human complement (hSBA) after two and three doses of the vaccine according to the present invention (DP8-OAc), as well as the SBA titers for a vaccine not according to the present invention (DP6-OAc). [Figure 11] Figure 11 shows the total IgG titers of single and pooled serum measured by HT-ELISA for MenA formulations containing benchmark MenABCWY and solid (lyophilized) MenA components versus corresponding complete liquid formulations containing randomly acetylated carba MenA antigens. [Figure 12] Figure 12 shows the functional antibody response measured by rSBA and SBA for MenA formulations containing benchmark MenABCWY and solid (lyophilized) MenA components versus corresponding complete liquid formulations containing randomly acetylated carba MenA antigens. [Figure 13]Figure 13 shows the SDS-PAGE and Western blot characteristics of the CarbaMenA DP8 and DP10 conjugates. [Figure 14-1] Figure 14 shows the ELISA titers measured after three doses of a vaccine containing random O-acetylated carbamenA analog DP10 combined with BNGCWY, compared to the ABNGCWY vaccine as a benchmark (i.e., positive control). Figure 14A shows anti-MenA polysaccharide IgG antibodies, Figure 14B shows anti-MenC, anti-MenW, and anti-MenY polysaccharide IgG antibodies, and Figure 14C shows anti-NadA, anti-FHbp variant 1.1, anti-NHBA, anti-231.13NB, and anti-OMV protein IgG antibodies. Here, for each antigen, the ABNGCWY vaccine benchmark is shown in the left bar, and the random O-acetylated carbamenA analog DP10 combined with BNGCWY is shown in the right bar. [Figure 14-2] This is a continuation of Figure 14-1. [Figure 14-3] This is a continuation of Figure 14-2. [Figure 15-1]Figure 15 shows the SBA titer after three doses of vaccine. Figure 15A shows the human complement-mediated bactericidal titer measured in serum induced with random O-acetylated carbamenA analog DP10 combined with BNGCWY, compared to the ABNGCWY vaccine as a benchmark (i.e., positive control), using strains 3125 and F8238 MenA. Figure 15B shows the human complement-mediated bactericidal titer measured in serum induced with random O-acetylated carbamenA analog DP10 combined with BNGCWY, compared to the ABNGCWY vaccine as a benchmark (i.e., positive control), using strains C11 (for MenC), 240070 (for MenW), and 860800 (for MenY). Figure 15C shows the human complement-mediated bactericidal titers measured in serum induced with random O-acetylated carbaMenA analog DP10 combined with BNGCWY, compared to the ABNGCWY vaccine as a benchmark (i.e., positive control), using strains 96217 (for NadA), M14459 (for fHbp variant 1.1), M13530 (for NHBA), M08-240104 (for fHbp variant 2), M01-240320 (for fHbp variant 3), M15-240084 and M08-0240264 (for fHbp variant 1.13) and NZ98 / 254. [Figure 15-2] This is a continuation of Figure 15-1. [Figure 15-3] This is a continuation of Figure 15-2. [Modes for carrying out the invention]

[0037] Detailed description of the invention The present invention provides an aqueous immunogenic composition that, after administration to a subject, can induce a bactericidal immune response against Neisseria meningitidis serogroups A, B, C, W135, and Y. Advantageously, the composition is provided as a complete liquid formulation, meaning that each antigenic component can be stably combined in a single aqueous dose without the need for lyophilization. The immunogenic composition is: i. Conjugate serogroup A antigen, ii. Conjugate serogroup C antigen, iii. Conjugate serogroup W135 antigen, iv. Conjugate serogroup Y antigen; and v. One or more polypeptide antigens from serogroup B Includes, (ii), (iii), and (iv) are capsular sugar antigens, and (i) is a synthetic analog of serogroup A capsular sugar. Preferably, the sugar antigen is an oligosaccharide.

[0038] Conjugate serogroup A antigenic component The serogroup A antigenic component of the immunogenic composition of the present invention is a synthetic polysaccharide carba analog (i.e., in which the ring oxygen of the mannosamine unit is replaced with methylene). In a preferred embodiment, the polysaccharide carba analog has at least 6 degrees of polymerization and preferably has a first analog monomer in which the C-1 of the first unit is linked to the C-6 of the second unit via a 1,6 linkage to the second analog monomer, where the 1,6 linkage includes a phosphonic acid moiety.

[0039] It should be noted that such derivatives are not only expected to mimic natural polysaccharides from MenA serogroups, but also to possess improved stability against natural CPS.

[0040] The term "oligosaccharide," in its sense as is commonly known in the art, includes polysaccharides having 3 to 10 monosaccharide units (see, for example, https: / / en.wikipedia.org / wiki / Oligosugar).

[0041] The term "oligomer" refers to carba-related polysaccharides that provide a cyclohexane skeleton by replacing the intraring oxygen with a methylene (-CH2-) group.

[0042] The degree of polymerization (DP) indicates the number of monomers linked together to provide the final oligomer. In this invention, unless otherwise specified, DP is represented by "n" in formulas (I) and (II).

[0043] The "average degree of polymerization" (avDP) indicates the average number of repeating units that make up an oligomer.

[0044] Unless otherwise specified, the term "conjugation" refers to the connection or linkage of the entities in question, in particular the oligomer of the present invention having n (i.e., DP) ≥ 6 and the selected protein.

[0045] As used herein, the term “alkyl” refers to a saturated, linear, or branched hydrocarbon moiety. The term “C1-C6-alkyl” refers to an alkyl moiety containing 1 to 6 carbon atoms.

[0046] As used herein, the term "haloalkyl" refers to a saturated, linear, or branched hydrocarbon moiety in which one or more hydrogen atoms are replaced by halogen atoms. In particular, when "haloalkyl" is said, it is a reference to "fluoroalkyl" where the halogen is fluoro. The term "C1-C6-haloalkyl" refers to an alkyl moiety containing 1 to 6 carbon atoms in which one or more hydrogen atoms are replaced by halogen atoms. Examples include -CF3, -CH2F, and -CH2CF3.

[0047] As used herein, phenyl may be substituted, in particular according to the definition of Z. The phenyl group may be substituted with one or more reactive functional groups to enable conjugation such as N3, NH2, SH, etc. Other suitable groups are well known to those skilled in the art.

[0048] As used herein, the term “protecting group” refers to any suitable protecting group for a given purpose. For the selection and use of such protecting groups, and for details of their use, refer to, for example, Greene, TW and Wuts, PGM, “Protective Groups in Organic Synthesis.” Suitable protecting groups are known to those skilled in the art.

[0049] As used herein, the term “pharmaceutically acceptable phosphate counterion” means any counterion suitable for a phosphate group, i.e., a metal cation that is within reasonable medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, or other problems or complications, and that has a reasonable benefit / risk ratio. A pharmaceutically acceptable phosphate counterion may be a Group 1 or Group 2 metal. A specific example of such a pharmaceutically acceptable phosphate counterion is sodium (Na). + ) and potassium (K + For example, when the oligomer or conjugate of the present invention is in a buffer solution, the counterion is preferably sodium.

[0050] In one embodiment, the present invention relates to an oligomeric conjugate comprising an oligomer of the following formula (Ia) or (Ib) conjugated serogroup A antigen. [ka] During the ceremony, In the oligomer, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and can be the same or different in each repeating unit; R yis H or -C(O)CH3, and can be the same or different in each repeating unit; R x or R y At least one of them is -C(O)CH3 in at least one repeating unit; Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 These are independently selected from the group consisting of H, linear or branched C1-C6 alkyl groups, and linear or branched C1-C6 haloalkyl groups; Z is (i) protecting group; (ii) Functional linkers for conjugation to proteins, (iii) A linear or branched C1-C6 alkyl, optionally substituted phenyl, -C(O)Y, or a linear or branched C1-C6-alkyl-X And, Y is H, a linear or branched C1-C6 alkyl group, or a protecting group. X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -SC≡N. In another embodiment, the conjugated serogroup A antigen is an oligomeric conjugate of the following formula (IIa) or (IIb). [ka] In the formula, in the oligomer, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and can be the same or different in each repeating unit; R y is H or -C(O)CH3, and can be the same or different in each repeating unit; Rx or R y At least one of them is -C(O)CH3 in at least one repeating unit; Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 These are independently selected from the group consisting of H, linear or branched C1-C6 alkyl groups, and linear or branched C1-C6 haloalkyl groups; Z is (i) a functional linker or bond; P stands for protein.

[0051] In a preferred embodiment, the oligomer is defined by formula (Ia).

[0052] As defined above, n is ≥ 6, preferably ≥ 8. In one embodiment, n is 8 to 30. In another embodiment, n is 8 to 20. In a particular embodiment, n is 8 to 15. In particular, n is 8 or 10. In one embodiment, n is 8. In one embodiment, n is 10.

[0053] In one embodiment, R is H or -P(O)(OR″)2, and at least one R″ is Na + In one embodiment, R is H.

[0054] In one embodiment, R is NHC(O)CH3.

[0055] In one embodiment, R′ is Na + Therefore, the oligomer of the present invention is defined according to formula (Ia') or (Ib'), preferably formula (Ia'). [ka]

[0056] Accordingly, in one embodiment, the oligomeric conjugate antigen of the present invention is defined according to formula (IIa') or formula (IIb'), preferably formula (IIa'). [ka]

[0057] As defined above, R x is H or -C(O)CH3, and can be the same or different in each repeating unit, R y is H or -C(O)CH3, and can be the same or different in each repeating unit, R x or R y At least one of them is -C(O)CH3 in at least one repeating unit. Therefore, it should be understood that the formulas defined in square brackets according to formulas (Ia), (IIa), (Ib), and (IIb) mean that each unit of the oligomer has this skeleton, but the monomer unit defined by the square brackets is R x and R y Given that different options for n and R may be selected for each repeating unit defined by square brackets, they do not necessarily have to be identical. Therefore, n and R do not necessarily have to be identical. x and R y It will be understood that different acetylation percentages can be achieved by the selection of H or -C(O)CH3 for . For example, each repeating unit of the oligomer defined by square brackets can be acetylated according to the level of acetylation, i.e., R x and R y Depending on the selection of H or -C(O)CH3 for each of these, they may be the same or different.

[0058] In one embodiment, the oligomer is R x is -C(O)CH3 in at least one repeating unit. In one embodiment, the oligomer has R in at least one identical repeating unit. x H is R y It is -C(O)CH3.

[0059] In one embodiment, in the oligomer, R in at least one identical repeating unit x is -C(O)CH3, and R y H is H.

[0060] In one embodiment, the oligomer is R x and R y In at least one identical repeating unit, both are -C(O)CH3.

[0061] In one embodiment, in the oligomer, R in at least one identical repeating unit x H is R y is -C(O)CH3, and in at least one other identical repeating unit, R x is -C(O)CH3, and R y H is H.

[0062] In one embodiment, the oligomer has at least four repeating units, and within the same repeating unit, R x H is R y is -C(O)CH3. In one embodiment, the oligomer has at least six repeating units, and within the same repeating unit, R x H is R y is -C(O)CH3. In one embodiment, the oligomer has at least eight repeating units, and within the same repeating unit, R x H is R y is -C(O)CH3. In one embodiment, the oligomer has at least 10 repeating units, and within the same repeating unit, R x H is R y It is -C(O)CH3.

[0063] In one embodiment, the oligomer has four repeating units, and within the same repeating unit, R x is -C(O)CH3, and R y H is H.

[0064] In one embodiment, the oligomer has four repeating units, and within the same repeating unit, R x H is R y is -C(O)CH3, and has four repeating units, within the same repeating unit, R x is -C(O)CH3, and R y H is H.

[0065] In one embodiment, the oligomer has R in all repeating units. x or R y The repeating unit may also be -C(O)CH3, in other words, the acetylation of repeating unit 3 or 4 on each repeating unit is a selectively acetylated unit.

[0066] In one embodiment, in the oligomer, R in at least one identical repeating unit x H is R y is -C(O)CH3, and in at least one identical repeating unit, R x is -C(O)CH3, and R y H is H, and in at least one identical repeating unit, R x and R y Both are -C(O)CH3.

[0067] As defined above, in total, R in the oligomer x and R y Approximately 50-90% of it is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is approximately 50-90%. In other words, in the oligomer of the present invention, R x At least one of and R y One of them is -C(O)CH3 in the same or different repeating units, and at position 3 (R y is -C(O)CH3) and 4th position (R x The total degree of acetylation at -C(O)CH3) is approximately 50-90%. To avoid misunderstanding, as stated above, R x and R y This can be the same or different in each repeating unit of the oligomer.

[0068] In another embodiment, taken together, R in the oligomer x and R y are such that about 60-80% is -C(O)CH3. In other words, the total amount of acetylation of the oligomer is about 60-80%. To avoid misunderstanding, as described above, R x and R y may be the same or different in each repeating unit of the oligomer.

[0069] In one embodiment, both R x and R y are -C(O)CH3 in at least one same repeating unit of the oligomer, preferably in about 40-50% of the repeating units of the oligomer; about 10-30% of the remaining repeating units may have -C(O)CH3 of either R x or R y , and the remaining repeating units in the oligomer have R x =R y =H.

[0070] As defined above, Az is an aza substituent selected from the group consisting of -NH(CO)R 1 , -N(R 1 )2 and -N3, and R 1 is independently selected from the group consisting of H, linear or branched C1-C6-alkyl and linear or branched C1-C6-haloalkyl. The nitrogen atom is directly connected to the carbacyclin repeating unit.

[0071] Examples of such Az substituents include -N3, -NH2, -NH-C1-C6 alkyl, -N-(C1-C6 alkyl)2 and -NH(CO)-C1-C6 alkyl. In one embodiment, -C1-C6 alkyl is -C1-C4 alkyl, especially -CH3. Thus, according to one embodiment, Az is -NH(CO)-C1-C6 alkyl, especially -NH(CO)-CH3, also denoted as -NHAc (Ac represents acetate, i.e., -C(O)CH3).

[0072] Z may have different meanings depending on whether or not the oligomer of the present invention is conjugated to a protein.

[0073] According to formula (Ia) or (Ib), the oligomer of the present invention is not conjugated to a protein. Therefore, as defined above, according to formula (Ia) or (Ib), Z is one of the following: (i) protecting group; (ii) linear or branched C1-C6 alkyl, optionally substituted aryl, -C(O)Y, or linear or branched C1-C6-alkyl-X, (iii) Functional linkers for conjugation to proteins.

[0074] Therefore, according to one embodiment, Z is a means for capping terminal sugar units so that they may be non-reactive or reactive, for example, for further chain elongation or subsequent modification.

[0075] If Z is intended to be a means for capping terminal carba-analog units, it may include a protecting group or a capping group, such as a linear or branched C1-C6 alkyl, an optionally substituted phenyl, C(O)-Y, or a linear or branched -C1-C6 alkyl-X, where X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -SC≡N, and Y is H, a linear or branched C1-C6 alkyl, or a protecting group.

[0076] As defined herein, Z may be a functional linker for conjugation to a protein. In this case, “functional linker” refers to any linker known in the art to be used to conjugate a sugar to a protein.

[0077] In one embodiment, X is -NH2.

[0078] In one embodiment, Z according to formula (Ia) or (Ib) is selected from -(CH2)6-NH2, -(CH2)4-NH2, -(CH2)3-NH2, and -(CH2)2-NH2, and the amino group may be protected by a suitable protecting group, such as -C(O)CH3 (for the selection and use of such protecting groups, and details of their use, refer to, for example, Greene, TW and Wuts, PGM, "protective groups in organic synthesis").

[0079] The oligomers of the present invention can be produced according to known synthetic methods in organic synthesis for the production of polysaccharide carba analogs. Generally, the production of the oligomers of the present invention can be achieved by linking at least six mannosamine carba analog constituent blocks in a desired manner by forming 1,6-alpha linkages between repeating units, thereby providing oligomers having at least 6 degrees of polymerization. As shown in formula (I), their monomers are linked via alpha-(1→6) phosphate linkages, and such linkages can be made using standard polymerization techniques, such as those described, among others, in Gao et al., Org. Biomol. Chem., 2012, 10, 6673.

[0080] The mannosamine carba analogue constituent block may have an acetate at position 3, or a protecting group that can be replaced with acetate at any step of the synthesis.

[0081] Alternatively, according to one embodiment, the present invention relates to a method for producing an oligomer of formula (I), comprising the following steps. a. Production of monomers having phosphodiester links; b. Extension reaction of the obtained monomer, for example, using phosphoramidite; c. O-acetylation of oligomers.

[0082] In one embodiment, R yIf the compound is C(O)CH3, steps (b) and (c) may be reversed so that O-acetylation occurs before the extension reaction.

[0083] More specifically, the process may include the steps shown in Scheme 1. [ka] Scheme 1: Method for producing oligosaccharides according to the present invention (a) TBAF, THF, 0℃ → room temperature, 92%. (b) MeONa, MeOH, room temperature, 85%. (c) DMTrCl, Et3N, DCM, room temperature, 91%. (d) 2-Cyanoethyl N,N-diisopropyl-chlorophosphoramidite, N,N-diisopropylethylamine, DCM, room temperature, 9 (94%). (e) I.11, DCI, MeCN, II.CSO, MeCN, III.TCA, DCM, H2O, 94%. (f) I.9, DCI, MeCN, II.CSO, MeCN, III.TCA, DCM, H2O, 16 (82%), 17 (95%), 18 (90%), 19 (92%), 20 (88%), 21 (86%), 22 (87%). (g) NH4OH, H2O, dioxane. (h)H2, Pd Black, H2O, AcOH, 1 (99%), 2 (76%), 3 (69%), 4 (39%), 5 (88%), 6 (83%), 7 (77%), 8 (44%), (i): (Boc)2O, NaHCO3, room temperature, 16 hours; (l): Ac2O / imidazole, 40℃, ~9 days; (NS): TFA, room temperature, 1 hour.

[0084] To avoid misunderstanding, Ac is intended to refer to the acetyl group, i.e., -C(O)CH3.

[0085] In particular, the use of phosphoramidite structural blocks is more effective in forming phosphodiester linkages. The inventors chose to use dimethoxytrityl (DMTr) ether to temporarily mask the function of the primary alcohol being extended. Each extension step is based on the repetition of a three-step procedure including coupling of the phosphoramidite with the growing chain alcohol, oxidation of the intermediate phosphite to the corresponding phosphodiester, and unmasking of the primary hydroxyl on the (n+1) oligomer. As shown in Scheme 1, the key structural block 9 is obtained from intermediate 10, and intermediate 10 is obtained in three steps from a known carba sugar 12 (see, e.g., Q. Gao et al., Org. Biomol. Chem., 2012, 10, 6673-6681). The latter carbamannose structural block can be prepared from commercially available 3,4,6-tri-O-acetyl-D-glucar according to prior art methodologies. Therefore, the primary silyl ether and acetyl ester were removed from compound 12 by the sequential action of tetrabutylammonium fluoride (TBAF) and NaOMe to obtain diol 14 in 85% yield. Next, the DMTr group was regioselectively introduced to obtain alcohol 10 in 91% yield. This compound was converted to the extended block phosphoramidite 9 by reaction with 2-cyanoethyl-N,N-diisopropyl-chlorophosphoramidite. The target oligomer was assembled using readily available constituent blocks. Its synthesis began with the attachment of an aminohexanol spacer to alcohol 10 using a known phosphoramidite 11. The constituent blocks were coupled in a two-step one-pot reaction using dicyanoimidazole (DCI) as an activator for the phosphoramidite activation. The phosphite formed in situ was oxidized using (1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). DCI (pKa 5.2) has low acidity and is suitable for use in combination with the acid-unstable DMTr group, so it is not used in the conventional tetrazole (pKa 5.2) a4.9) was preferred. CSO was used instead of iodine due to its high solubility in non-aqueous solvents such as acetonitrile. The crude phosphodiester product was treated with TCA to cleave the DMTr group. The product was purified by size exclusion chromatography (Sephadex LH-20) to obtain monomer 15 with spacers in 94% yield. All subsequent couplings were carried out according to the above procedure until a desired degree of polymerization of 8 or more was reached. For the elongation of longer oligomers, a larger amount of phosphoramidite 9 was used and the coupling reaction time was increased to ensure complete conversion of the alcohol. The yield for each elongation cycle was good to excellent, ranging from 82% to 95%. Starting from 10, octamer 22 was obtained in a total yield of 40%. Fragments 16-22 were deprotected using a two-step procedure. First, the cyanoethyl group (CE) was removed using aqueous ammonia (33%). Next, all residual protecting groups (benzyl ether and carboxybenzylcarbamate) on the phosphodiester thus formed were cleaved by hydrogenation with palladium black to obtain the target non-acetylated oligomers 1-8.

[0086] Non-acetylated oligomers 1-8 can be randomly O-acetylated at the 3rd and / or 4th positions, i.e., combined, the R in the oligomers x and R y Approximately 50-90% of these are -C(O)CH3. This can be achieved by (i) BOC protection of the free amine group; (ii) O-acetylation using, for example, Ac2O / imidazole; and (iii) deprotection to obtain acetylated oligomers 1c-8c or 1d-8d. Such acetylated oligomers are then activated with a linker group such as bis-succinimidyl adipate (also known as SIDEA) and CRM 197 It can be conjugated to proteins such as [list of proteins]. [ka] Scheme 2.3: Process leading to the production of the -O-acetylated monomer constituent block (a) K2CO3, MeOH; (b) PMBCH(OMe)2, PPTS; (c) BnBr, NaH; (d) DIBAL-H, DCM; (e) DMP, DCM; (f) PPh3CH3I, KHMDS, THF , -78℃; (g) m_dichlorobenzene, t, μwave; (h) NaBH4, EtOH / THF; (i) TDSCl, Im, DCM; (j) OsO4, TMANO, 3:1 acetone-H2O; (l) (MeO) 3Cme, PTSA, CAN, then 80% AcOH; (m)Tf2O, DCM / py, -20°C to room temperature; then NaN3, 19:1DMF-H2O; (n)PPh3, THF, 60°C, H2O; then Ac2O, MeOH; (o)NaOMe / MeOH; (p)TBSOTf, 2,6-lutidine, DCM; (q)DDQ, then Ac2O, py; (r)HF / pyridine, THF; (s)DMTrCl, pyridine, DCM.

[0087] Alternatively, the 3-O-acetylated monomer structural blocks and the 4-O-acetylated structural blocks can be produced by the process shown in Scheme 3 below. [ka] Scheme 3. Process leading to the production of 3-O-acetylated and 4-O-acetylated monomer constituent blocks (a') K2CO3, MeOH; (b') TDSCl, Imidazole, DMF, -30℃; (c') BnBr, NaH, DMF, 0℃; (d') TBAF, THF; (e') IBX, AcOEt; (f') PPh3CH3I, KHMDS, THF, -78℃ to room temperature; (g') 1,3-Dichlorobenzene, NaBH4, EtOH / THF, 230℃; (h (')TIPSCl, imidazole, DMF; (i')TiCl4, DCM / toluene 2:8, -70℃; (l')NapBr, NaH, DMF, 0℃; (m')Me3NO·2H2O, acetone / H2O3:1, OsO4; (n')(MeO)3CMe, PTSA, ACN; (o')Tf2O, DCM / Py, -20℃ to room temperature; then NaN3, 19:1 DMF-H2O;(p′)NaOMe, MeOH;(q′)TBSOTf, -10℃~70℃, Pyr, DMAP;(r′)Pd / C, H2, AcOH, then Ac2O, Pyr ;(s′)HFpyr, Pyr;(t′)DMTrCl, Pyr, 0℃;(r″)DDQ, DCM, H2O;(s″)PPh3, H2O, THF, then DMTrCl, Pyr.

[0088] Acetylated structural blocks 38, 55a, 55b and fully acetylated structural blocks (i.e., those having O-Ac groups at both the C3 and C4 positions of the same unit) can be converted to their oligomeric versions by conversion to phosphorymidates and subsequent coupling as described above in relation to compound 9.

[0089] A key prerequisite for the immunogenicity of the carba analogs of the present invention is their ability to mimic the corresponding MenA capsule sugar. To investigate this, competitive ELISA was performed using carba analogs with different degrees of polymerization.

[0090] The oligomers of the present invention can be introduced into a mammalian host, and preferably a human host, either alone, linked to a carrier protein, or as a homopolymer or heteropolymer of mannosecarba analog units. In certain embodiments, the oligomers of the present invention are used as protein conjugates. Accordingly, in further embodiments, the present invention includes conjugate derivatives comprising the oligomer of formula (I) of the present invention linked to a protein according to general formula (IIa) or (IIb). [ka] During the ceremony, n, Az, R, R′, R x , and R y This is as defined above; Z is a linker or bond; P stands for protein.

[0091] The oligomers of general formula (Ia) or (Ib) are particularly useful when conjugated to a protein via a Z moiety preferably connected to the C-1 carbon of the first repeating unit via a phosphate moiety. The oligomer-protein conjugate derivatives of formula (IIa) or (IIb) thus obtained may be useful in preparing compositions that can induce an immunogenic response in infants and possibly a cellular response that provides a memory effect to extend the efficacy of vaccination.

[0092] In one embodiment, the oligomeric conjugate is preferably defined by formula (IIa), i.e., the protein is conjugated at position 1 rather than position 6 of the carba analog.

[0093] A protein (or carrier protein) can affect the immunogenic response and even affect the exact nature of the antibodies resulting from treating a mammal with one or more compounds of the present invention when delivered as a conjugate. A suitable protein has a functional group that can react with the terminal portion of the Z moiety to form a conjugate derivative of the present invention. Preferably, the functional group is selected from -NH2 and -SH and can be connected to the Z moiety forming an amide bond or a thioether. More preferably, the protein has an -NH2 group suitable for forming an amide bond when reacting with Z.

[0094] Useful proteins are known in the art. However, in certain embodiments, P is an inactivated bacterial toxin selected from diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 , Escherichia coli (E. coli) ST and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is a hepatitis B virus core protein or SPR96 - 2021, or a meningococcus (N. meningitidis) serotype B antigen fHbp - 231 (i.e., a fusion protein of variants 2, 3 and variant I of factor H binding protein (fHbp) as defined in WO2015 / 128480, which is incorporated herein by reference).

[0095] In certain embodiments, P is TT, DT or CRM 197 is.

[0096] In certain embodiments, P is CRM 197 is.

[0097] As defined above, according to formula (IIa) or (IIb), Z is a linker or a bond. When Z is a linker, it can be derived from any suitable linker known in the art suitable for the conjugation of oligosaccharides to proteins.

[0098] In other words, Z in the unreacted form, i.e., when not linked to an oligomer and a protein, can be a functional linker (defined according to formula (Ia) and formula (Ib)) by having a functional group that enables it to act as a linker between the oligomer and the protein of the present invention. Preferably, Z is derived from a compound containing an amine, carboxylate, or hydroxyl group for coupling with a complementary group on a protein carrier, but other groups known in the art for providing a method of conjugating an oligosaccharide to a protein are also contemplated.

[0099] When the oligomer of the present invention is conjugated to a protein, the preferred Z moiety in formula (IIa) or (IIb) is derived from a linker that is an amine-substituted alkoxy group which may be in a protected form. In this form, the amine is acetylated or alkylated with a bifunctional reagent and the other end thereof is similarly connected to the protein.

[0100] In certain embodiments, according to formula (IIa) or (IIb), Z is derived from a linker that can be either a homobifunctional or heterobifunctional linker capable of linking the oligomer of the present invention to a protein. In this regard, bifunctional linkers suitable for use in the conjugates of the present invention include those known in the art such as dicarboxylic acids, preferably malonic acid, succinic acid, adipic acid, and suberic acid, or their activated forms. Alternatively, squarate esters can be used. These types of reagents are particularly convenient for linking a compound containing an amine in the spacer moiety to a protein. Preferably, the bifunctional linker is derived from adipic acid N-hydroxysuccinimide diester (SIDEA) and BS(PEG)5.

[0101] In some embodiments, Z is at least 2 or 3 atoms in length. Some non-limiting examples of linkers include -(CH2) m A, -Ph-A, -(CH2) a -Ph-(CH2) a-A and their substituted forms, etc., where each Ph represents a phenyl group that may be substituted, and a and m each independently represent an integer from 1 to 10. "A" represents a functional group or residue that can or can link proteins, such as -NH2, -OH or -SH, esters, amides or other carboxyl-containing groups, dienes or dienophiles, maleimides, alkynes, cycloalkynes, etc. Z can contain OR′, SR′ or N(R′)2, where each R′ is independently H or C1-C6-alkyl, acyl, aryl, arylalkyl, heteroacyl, heteroaryl, or heteroarylalkyl, and may further contain A.

[0102] In one embodiment, Z in formula (IIa) or (IIb) is a heterobifunctional linker having the following formula. [ka] During the ceremony, * This indicates a connection point. p is independently selected from 1 to 10; X is selected from -O-, -S-, and -NH-.

[0103] In one embodiment, Z is an equation * -(CH2)6NHCO(CH2)4CO * It holds.

[0104] In another embodiment, Z is a linker having the following formula: [ka] During the ceremony, * represents a connection point, and m is independently selected from 1 to 10.

[0105] In another embodiment, Z has the following formula. [ka]

[0106] The Z linker is usually introduced into the monomer that is linked to the protein before the extending monomer is connected, and optionally introduced in a protected form, so as not to affect or participate in the subsequent extension reaction.

[0107] Therefore, in certain embodiments, Z is a divalent linker having the following general formula. [Chemical formula] In the formula, r is an integer from 2 to 6, ( * ) represents the connection point to the oligomer, PG represents hydrogen or a protecting group, preferably selected from alkoxycarbonyl, methoxycarbonyl, t-butyloxycarbonyl or benzyloxycarbonyl. The protein is connected via an amine.

[0108] When present, PG can be preferably removed to enable the Z moiety to react with the protein to obtain its conjugate. Alternatively, PG can be removed, and the free amino group thus obtained can be further functionalized by introducing, for example, a further spacer moiety suitable for ligation to the protein.

[0109] In certain embodiments, an oligomer conjugate according to the following formula is provided. [Chemical formula] In the formula, n, Az, R, R′, R x , and R y are as defined above.

[0110] In certain embodiments of the present invention, an oligomer conjugate according to the following formula, i.e., where R′ is Na + is provided. [Chemical formula] In the formula, n, Az, R, R x , and R yThis is defined as above.

[0111] When this randomly acetylated oligomeric conjugate is incorporated into a vaccine composition, it exhibits higher acetylation percentage stability than the natural MenA conjugate, which is less than 5% of the acetylation that carba analogs may lose when formulated into a vaccine.

[0112] To avoid misunderstanding, it should be noted that the oligomers of the present invention can be conjugated to proteins by any suitable method known in the art, for example, according to the method reported in "The design of semi-synthetic and synthetic glycoconjugate vaccines", P. Constantino et al., Expert Opin. Drug. Discov.

[0113] The conjugation reaction can also be carried out using a conjugation method similar to that used for conjugating MenA sugars to carrier proteins, and for example, using the method described in WO2004 / 067030. In one embodiment, the oligomer of the present invention is conjugated using a conjugation procedure utilizing a di-N-hydroxysuccinimidyl adipate linker, for example, reported in Berti et al., ACSChem. Biol., 2012, 7, 1420-1428, using CRM 197 It can be coupled with [another substance]. After treatment with a selected linker in DMSO containing trimethylamine, the resulting activated oligomer can be purified by co-precipitation with acetone and used for conjugation. Therefore, CRM can be used in a 100:1 oligomer / protein molar ratio. 197By incubating overnight, the desired neoconjugate can be obtained. The conjugation can be conceived as activation of the oligomer of formula (Ia) / (Ib), followed by conjugation to a selected protein, or activation of the relevant protein functional group, and subsequently conjugation with the oligosaccharide of the present invention, typically via the Z moiety. Thus, according to one embodiment, the oligomer of the present invention is first activated with a suitable activator according to methods known in the art, and then coupled with the -NH2 residue of a selected protein.

[0114] In one embodiment, the Z group is activated by reaction with the first terminal portion of the linker, thereby allowing the other end of the linker to be linked to a selected protein. For example, and according to one embodiment, the process may include obtaining an activated ester of the starting oligomer by activating the oligomer of the present invention with SIDEA in the presence of triethylamine. Then, such an activated ester is subjected to CRM in the presence of a phosphonate buffer. 197 By reacting with this, the desired conjugate can be obtained.

[0115] After conjugation, the oligomer-protein conjugate can be purified by various techniques known in the art. One goal of the purification process is to remove unbound oligomers from the oligomer-protein conjugate. Typically, the conjugates of the present invention can be purified from unreacted proteins and oligomers by any number of standard techniques, such as size exclusion chromatography, density gradient centrifugation, hydrophobic interaction chromatography, or ammonium sulfate fractionation, as described, for example, Anderson, PW, et al. J. Immunol. (1986) 137:1181-1186 and Jennings, HJ et al., J. Immunol. (1981) 127:1011-1018.

[0116] In another embodiment, Z may be a monosaccharide, preferably a mannosamine as described below. Thus, in further embodiments, the present invention also relates to an oligomer having the following formula (III). [ka] In the formula, R, Az, and n are as defined above; Z is: [ka] and; P and the linker are defined above in relation to the definition of Z for equations (I) and (II).

[0117] For example, one example of a conjugate defined in this way is as follows: [ka]

[0118] According to this embodiment, the derivative of the present invention can be directly linked to a selected protein via the -O-linker Z moiety to produce a conjugate derivative having an -O-linker-P moiety directly attached to the carbon atom of the terminal monomer. As far as the linker is concerned, it can be any suitable divalent linker with linker Z as described above. Alternatively, Z can be an amine for conjugation to a protein derivatized with a linker having a keto or aldehyde group.

[0119] Conjugate serogroups C, W135, and Y antigenic components The immunogenic compositions of the present invention comprise capsular sugar antigens from meningococcal serogroups C, W135, and Y, respectively, wherein the antigens are conjugated to a carrier protein and / or are oligosaccharides. The capsular sugars can be used in the form of oligosaccharides. These are readily formed by fragmentation (e.g., hydrolysis) of purified capsular polysaccharides, which are then usually purified to the desired size.

[0120] To avoid confusion, the term "capsular polysaccharides / saccharides" (CPS) refers to sugars found on the outer surface of bacterial cells, specifically in a layer that is part of the outer layer of the bacterial cell itself. CPS are expressed on the outermost surface of a wide range of bacteria, and in some cases, in fungi as well.

[0121] The term "oligosaccharide," in its sense as is commonly known in the art, includes polysaccharides having 3 to 10 monosaccharide units (see, for example, https: / / en.wikipedia.org / wiki / Oligosugar).

[0122] Generally, conjugation enhances the immunogenicity of sugars by converting them from T-independent antigens to T-dependent antigens, thereby enabling priming for immunological memory. Conjugation is a well-known technique and is particularly useful in pediatric vaccines.

[0123] Techniques for producing capsular polysaccharides from Neisseria meningitidis have been known for many years (see, for example, WO2005 / 032583 and WO03 / 007985).

[0124] Typical carrier proteins include bacterial toxins such as diphtheria toxin and tetanus toxin, or toxoids or their mutants. 197A diphtheria toxin variant [Research Disclosure, 453077 (Jan 2002)] is useful and serves as a carrier in the Streptococcus pneumoniae vaccine sold under the trade name PREVNAR (trademark). Other suitable carrier proteins include the meningococcal (N. meningitidis) outer membrane protein complex [EP-A-0372501], synthetic peptides [EP-A-0378881, EP-A-0427347], heat shock proteins [WO93 / 17712, WO94 / 03208], pertussis proteins [WO98 / 58668, EP-A-0471177], cytokines [WO91 / 01146], lymphokines [WO91 / 01146], hormones [WO91 / 01146], growth factors [WO91 / 01146], artificial proteins containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens [Falugi et al. (2001) Eur J Immunol 31:3816-3824], e.g., N19 [Baraldo et al. (2004) Infect Immun 72(8):4884-7], protein D from Haemophilus influenzae [EP-A-0594610, Ruan et al. (1990) J Immunol 145:3379-3384], pneumolysin [Kuo et al. (1995) Infect Immun 63:2706-13] or its non-toxic derivative [Michon et al. (1998) Vaccine. 16:1732-41], pneumococcal surface protein PspA [WO02 / 091998], iron uptake protein [WO01 / 72337], toxin A or B from C. difficile [WO00 / 61761], recombinant Pseudomonas aeruginosa extraprotein A (rEPA) Examples include [WO00 / 33882].

[0125] Any suitable conjugation reaction can be used, and any suitable linker can be used as needed.

[0126] Glycans are typically activated or functionalized before conjugation. Activation may involve cyanylating reagents such as CDAP (e.g., 1-cyano-4-dimethylaminopyridinium tetrafluoroborate [Lee et al. (1996) Vaccine 14:190-198, WO95 / 08348, etc.]). Other preferred techniques include the use of carbodiimides, hydrazides, activated esters, norboranes, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU, etc.

[0127] Linking via linker groups can be prepared using any known procedure, e.g., the procedures described in US4,882,317 and US4,695,624. One type of linking involves the reductive amination of a polysaccharide, coupling the resulting amino group to one end of an adipic acid linker group, and then coupling the protein to the other end of the adipic acid linker group [Porro et al. (1985) Mol Immunol 22:907-919, EP0208375]. Other linkers include β-propionamide [WO00 / 10599], nitrophenyl-ethylamine [Gever et al. Med. Microbiol. Immunol, 165: 171-288 (1979)], haloacyl halide [US4,057,685], glycosidic bond [US4,673,574;US4,761,283;US4,808,700], 6-aminocaproic acid [US4,459,28], ADH [US4,965,338], and C4 to C 12 This includes parts such as [US4,663,160]. Instead of using a linker, direct linking can be used. Direct linking to a protein may involve oxidation of a polysaccharide followed by reduction and amination by the protein, as described, for example, in US4,761,283 and US4,356,170.

[0128] A preferred method involves introducing an amino group to the sugar (e.g., by replacing the terminal O group with -NH2), followed by derivatization with an adipic acid diester (e.g., adipic acid N-hydroxysuccinimide diester), and then reacting with a carrier protein. Another preferred reaction uses CDAP activation with a protein D carrier for MenC, for example.

[0129] Current serogroup C vaccines (Menjugate® [Costant et al. (1992) Vaccine 10:691-698, Jones (2001) Curr Opin Investig Drugs 2:47-49], Meningitec®, and NeisVac-C®) contain conjugated sugars. Menjugate® and Meningitec® are CRM 197 While other carriers have oligosaccharide antigens conjugated to them, NeisVac-C (trademark) uses a complete polysaccharide (de-O-acetylated) conjugated to a tetanus toxoid carrier.

[0130] The vaccine products marketed under the trade names MENVEO, MENACTRA, and NIMENRIX all contain conjugated capsular sugar antigens derived from serogroups Y, W135, C, and A.

[0131] In MENVEO (commonly known as the meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria CRM197 conjugate vaccine), each of the A, C, W135, and Y antigens is CRM 197 Conjugated by a career.

[0132] In preferred embodiments of the present invention, serogroup C, W135, and Y oligosaccharide antigen are each CRM 197 It is conjugated to. Preferably, each of the conjugated serogroups C, W135, and Y capsular glycoglycan antigens is the CRM of the approved MENVEO vaccine. 197 - Corresponds to the conjugate serogroups C, W135, and Y antigenic components.

[0133] In MENACTRA (commonly known as the meningococcal (groups A, C, Y, and W-135) polysaccharide diphtheria toxoid conjugate vaccine), each of the A, C, W135, and Y antigens is conjugated to a diphtheria toxoid carrier.

[0134] In preferred embodiments of the present invention, serogroup C, W135, and Y oligosaccharide antigens are each conjugated to a diphtheria toxoid carrier. Preferably, each of the conjugated serogroup C, W135, and Y oligosaccharide antigens corresponds to the diphtheria toxoid carrier-conjugated serogroup C, W135, and Y antigenic components of an approved MENACTRA vaccine.

[0135] In NIMENRIX (commonly known as a meningococcal polysaccharide group A, C, W-135 and Y conjugate vaccine), each of the A, C, W135, and Y antigens is conjugated to a tetanus toxoid carrier.

[0136] In a preferred embodiment of the present invention, serogroup C, W135, and Y oligosaccharide antigens are each conjugated to a tetanus toxoid carrier. Preferably, each of the conjugated serogroup C, W135, and Y oligosaccharide antigens corresponds to the tetanus toxoid carrier-conjugated serogroup C, W135, and Y antigenic components of an approved NIMENRIX vaccine.

[0137] Serogroup B antigenic components The BEXSERO vaccine product (also known as C4MenB) contains a preparation of OMV from the circulating strain of Neisseria meningitidis group B, NZ98 / 254, B:4:P1.7b,4. The same OMV is also contained in the MeNZB® vaccine, which is referred to herein as OMVnz. Furthermore, BEXSERO contains five meningococcal antigens: NHBA (287, subvariant 1.2), fHbp (741, subvariant 1.1), NadA (961, subvariant 3.1), GNA1030 (953), and GNA2091 (936). Four of these antigens exist as fusion proteins (NHBA-GNA1030 fusion protein (287-953) and GNA2091-fHbp (936-741) fusion protein). A 0.5 mL dose of BEXSERO® contains 50 μg each of NHBA, NadA, and fHbp adsorbed onto 1.5 mg of aluminum hydroxide adjuvant, along with 25 μg of OMV derived from Neisseria meningitidis NZ98 / 254 strain. BEXSERO is documented in the literature (see, for example, Bai et al. (2011) Expert Opin Biol Ther. 11:969-85 and Su & Snape (2011) Expert Rev Vaccines 10:575-88).

[0138] In a preferred embodiment, the serogroup B antigenic component of the immunogenic composition of the present invention comprises one or more protein antigenic components of BEXSERO.

[0139] In a preferred embodiment, the immunogenic composition of the present invention contains all of the above-mentioned BEXSERO meningococcal antigen components (protein antigen and OMV).

[0140] In a further preferred embodiment, the immunogenic composition of the present invention comprises a complete vaccine product marketed under the trade name BEXSERO.

[0141] In a further preferred embodiment, the immunogenic composition of the present invention comprises one or more fHbp antigens different from the fHbp v1.1 component of BEXSERO, preferably the additional fHbp antigen is in the form of an fHbp 231 fusion polypeptide. Preferably the additional fHbp antigen is an antigen disclosed in WO2020 / 030782. This fHbp antigenic component may be included in the immunogenic composition of the present invention as the sole MenB antigenic component of the composition, or more preferably, it may be included in addition to one or more BEXSERO antigens or complete BEXSERO vaccine products.

[0142] Lipoprotein factor H-binding protein (fHbp) is expressed on the surface of all MenB strains. fHbp binds to human complement regulatory protein factor H (hfH) to form a complex that protects bacteria from complement-mediated death, providing a survival mechanism for Neisseria meningitidis in the human bloodstream. Antibodies against fHbp have a dual role: they are bactericidal themselves, and by interfering with the binding of fHbp to hfH, they make the bacteria more susceptible to being killed. By reducing or eliminating the ability of fHbp to bind to hfH, the fHbp epitope is masked, and the formation of the protective complex between fHbp and hfH, which could block antibody binding, is prevented, thereby increasing the immunogenicity of the fHbp antigen.

[0143] fHbp exists in three different genes and immunogenic variants (v1, v2, and v3), and many subvariants exist. The majority of MenB strains not targeted by BEXSERO express fHbp in v2, v3, or v1 subvariants that are distantly related to variant 1.1 (variant 1.1 is the fHbp antigen included in BEXSERO).

[0144] WO2020 / 030782 discloses immunogenic mutant fHbp variant 1 (v1) polypeptides that, when combined with existing meningococcal vaccines, can provide improved coverage of N. meningitidis strains. In particular, these v1 polypeptides are sub-variants of fHbp variant 1 that are genetically diverse compared to the fHbp v1.1 antigen contained in BEXSERO.

[0145] Furthermore, the v1 polypeptide disclosed in WO2020 / 030782 is mutated to reduce its binding to hfH compared to the corresponding wild-type v1 polypeptide. In contrast, the fHbp v1.1 antigen in BEXSERO and the fHp v1.55 and v3.45 antigens in TRUMENBA bind to hfH.

[0146] The v1 polypeptide disclosed in WO2020 / 030782 can be provided alone or together with fHbp variants 2 and 3, which have been modified to improve stability and further reduce fHbp binding, as components of a fusion protein. By providing a single fusion protein containing these v2 and v3 antigens together with the v1 antigen of the present invention, the inventors improve strain coverage. For clarity, neither the v2 nor the v3 antigen is present in, for example, BEXSERO. The presence of the v2 and v3 antigens in the fusion protein of the present invention improves strain coverage compared to, for example, BEXSERO.

[0147] The v1 polypeptide and fusion protein, when used in combination with meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal outer membrane vesicles (for example, in combination with the BEXSERO composition), provide a combined immunogenic composition that exhibits increased immunogenicity (due to the addition / inclusion of unbound fHbp variants) and increased coverage of meningococcal (N. meningitidis) serotype B strains (due to the addition of new fHbp variants / subvariants) compared to BEXSERO alone.

[0148] Mutant v1.13 meningococcal fHbp polypeptide The inventors of WO2020 / 030782 identified residues within the fHbp v1.13 sequence that can be modified to reduce binding to hfH. Such variants are referred to herein as unbound (NB) variants. The inventors also identified combinations of mutations in the v1.13 sequence that are particularly useful for reducing binding to hfH. fHbp v1.13 is also known in the art as the fHbp variant B09.

[0149] The mature wild-type fHbp v1.13 lipoprotein derived from strain M982 (GenBank deposit number AAR84475.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined. TIFF0007877297000022.tif24162

[0150] The mature v1.13 lipoprotein differs from the full-length wild-type sequence in that it has an additional 19-residue N-terminal leader sequence from which the full-length polypeptide is cleaved. Therefore, the full-length wild-type fHbp v1.13 has the following amino acid sequence (the N-terminal leader sequence is shown in bold). TIFF0007877297000023.tif24162

[0151] The ΔG form of the mature v1.13 lipoprotein lacks the N-terminal polyglycine sequence of the mature polypeptide; that is, it lacks the first seven amino acids of SEQ ID NO: 1 and the first 26 amino acids of SEQ ID NO: 31. TIFF0007877297000024.tif13162TIFF0007877297000025.tif13162

[0152] Accordingly, in one embodiment, the serogroup B antigenic component of the immunogenic composition of the present invention includes a mutant v1.13 meningococcal fHbp polypeptide containing an amino acid sequence having at least k% sequence identity with SEQ ID NO: 2, wherein the amino acid sequence of the mutant v1.13 meningococcal fHbp polypeptide contains a substitution mutation in one or more residues E211, S216, or E232 of SEQ ID NO: 2.

[0153] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. It is preferably 80 (i.e., the mutant fHbp v1.13 amino acid sequence has at least 80% identity with SEQ ID NO: 2), more preferably 85, more preferably 90, and more preferably 95. Most preferably, the mutant fHbp v1.13 amino acid sequence has at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 2.

[0154] Preferably, the amino acid sequence differs from SEQ ID NO: 2 by at least one substitution of E211A, S216R, or E232A. More preferably, the amino acid sequence includes substitutions of (i) E211A and E232A, or (ii) multiple residues selected from E211A and S216R. More preferably, the amino acid sequence includes substitutions of residues E211A and S216R compared to SEQ ID NO: 2.

[0155] While we do not wish to be bound by theory, the substitution of glutamic acid (E) for alanine (A) at residue 211 of SEQ ID NO: 2 contributes to the discarding of fH binding by removing a negatively charged residue involved in hfH mobilization. The substitution of arginine (R) for serine (S) at residue 216 of SEQ ID NO: 2 replaces its wild-type amino acid with the corresponding residue from Neisseria gonorrhoeae that does not bind to hfH.

[0156] In a preferred embodiment, the mutant v1.13 polypeptide has the amino acid sequence of SEQ ID NO: 3 (v1.13 ΔG E211A / E232A) or SEQ ID NO: 4 (v1.13 ΔG E211A / S216R). More preferably, the mutant v1.13 polypeptide has the amino acid sequence of SEQ ID NO: 4.

[0157] The variant v1.13 polypeptide can induce antibodies capable of recognizing the wild-type meningococcal fHbp polypeptide of SEQ ID NO: 1 after administration to a host animal, preferably a mammal, more preferably a human. These antibodies are ideally bactericidal (see below).

[0158] Mutant v1.15 meningococcal fHbp polypeptide The inventors of WO2020 / 030782 also identified residues within the fHbp v1.15 sequence that can be modified to prevent binding to hfH. Such variants are referred to herein as non-binding (NB) variants. The inventors identified combinations of mutations in the v1.15 sequence that are particularly useful for preventing binding to hfH. fHbp v1.15 is also known in the art as the fHbp variant B44.

[0159] The mature wild-type fHbp v1.15 lipoprotein derived from strain NM452 (GenBank deposit number ABL14232.1) has the following amino acid sequence, with the N-terminal polyglycine signal sequence underlined. TIFF0007877297000026.tif23162

[0160] The mature v1.15 lipoprotein differs from the full-length wild-type sequence in that the full-length polypeptide has an additional 19 residues of N-terminal leader sequence that are cleaved from the mature polypeptide. Therefore, the full-length wild-type fHbp v1.15 has the following amino acid sequence (the N-terminal leader sequence is shown in bold). TIFF0007877297000027.tif23162

[0161] The ΔG form of mature v1.15 lipoprotein lacks the N-terminal polyglycine sequence; that is, it lacks the first 12 amino acids of SEQ ID NO: 5 and the first 31 amino acids of SEQ ID NO: 32. TIFF0007877297000028.tif23162

[0162] Therefore, in one embodiment, the serogroup B antigenic component of the immunogenic composition of the present invention includes an amino acid sequence having at least k% sequence identity with SEQ ID NO: 6, provided that the amino acid sequence of the mutant v1.15 meningococcal fHbp polypeptide includes a substitution mutation in one or more residues E214, S219, or E235 of SEQ ID NO: 6.

[0163] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. It is preferably 80 (i.e., the mutant fHbp v1.15 amino acid sequence has at least 80% identity to SEQ ID NO: 6), more preferably 85, more preferably 90, and more preferably 95. Most preferably, the mutant fHbp v1.15 amino acid sequence has at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6.

[0164] Preferably, the amino acid sequence differs from SEQ ID NO: 6 by at least one of the substitutions E214A, S219R, or E235A. More preferably, the amino acid sequence includes substitutions with residues selected from: (i) S219R, (ii) E214A and S219R, and (iii) E214A and E235A.

[0165] In a preferred embodiment, the mutant v1.15 polypeptide has the amino acid sequence of SEQ ID NO: 7 (v.1.15_S219R), SEQ ID NO: 8 (v1.15_E214A / S219R), or SEQ ID NO: 9 (v1.15_E214A / E235A).

[0166] The variant v1.15 polypeptide can induce antibodies capable of recognizing the wild-type meningococcal fHbp polypeptide of SEQ ID NO: 5 after administration to a host animal, preferably a mammal, more preferably a human. These antibodies are ideally bactericidal (see below).

[0167] Fusion polypeptide Furthermore, the disclosure in WO2020 / 030782 provides a fusion polypeptide containing all three v1, v2, and v3 meningococcal fHbp polypeptides, the variant fHbp sequence being in the order v2-v3-v1 from N to C-terminus. In a preferred embodiment, the serogroup B antigenic component of the immunogenic composition of the present invention comprises such an fHbp fusion polypeptide.

[0168] Preferably, the fHbp fusion polypeptide has the amino acid sequence of formula NH2-A-[-XL]3-B-COOH, where each X is a different variant fHbp sequence, L is an arbitrary linker amino acid sequence, A is an arbitrary N-terminal amino acid sequence, and B is an arbitrary C-terminal amino acid sequence.

[0169] The v1 fHbp polypeptide component of the fusion is either the mutant v1.13 fHbp polypeptide or the above-mentioned mutant v1.13 fHbp polypeptide.

[0170] The v2 and v3 fHbp polypeptide components of the fusion are preferably mutant v2 and v3 polypeptides having increased stability and hfH binding ability compared to wild-type v2 and v3 polypeptides. As described above, reducing the binding of fHbp to hfH is advantageous because it prevents the formation of a protective complex between fHbp and hfH that can mask the fHbp epitope, thereby increasing the immunogenicity of the polypeptide antigen.

[0171] Residues within the v2 and v3 sequences that can be modified to increase polypeptide stability and reduce binding to hfH have been identified and are described in detail in WO2015 / 128480.

[0172] The full-length wild-type fHbp v2 derived from strain 2996 has the following amino acid sequence (leader sequences are shown in bold, and polyglycine sequences are underlined). TIFF0007877297000029.tif24161

[0173] Mature lipoproteins lack the first 19 amino acids of sequence number 10. TIFF0007877297000030.tif24161

[0174] The ΔG form of sequence number 10 lacks the first 26 amino acids. TIFF0007877297000031.tif24161

[0175] In a preferred embodiment, the fusion polypeptide comprises a mutant v2 fHbp polypeptide having an amino acid sequence having at least k% sequence identity with SEQ ID NO: 12, wherein the v2 fHbp amino acid sequence includes substitution mutations at residues S32 and L123 of SEQ ID NO: 12. Preferably, the substitutions are S32V and L123R.

[0176] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. It is preferably 80 (i.e., the mutant fHbp v2 amino acid sequence has at least 80% identity with SEQ ID NO: 12), more preferably 85, more preferably 90, and more preferably 95.

[0177] In some embodiments, the fHbp v2 polypeptide contained in the fusion protein is cleaved at SEQ ID NO: 12. Compared to the wild-type mature sequence, SEQ ID NO: 12 is already cleaved up to the polyglycine sequence and at the N-terminus containing the polyglycine sequence (comparing SEQ ID NOs: 11 and 12), but SEQ ID NO: 12 may be cleaved at the C-terminus and / or further cleaved at the N-terminus.

[0178] In a preferred embodiment, the v2 fHbp polypeptide contained in the fusion protein contains or comprises the amino acid sequence of SEQ ID NO: 16.

[0179] The v2 fHbp polypeptide contained in this fusion protein exhibits higher stability than the same polypeptide under the same experimental conditions, but without sequence differences at residues S32 and L123, and is, for example, more stable than the wild-type meningococcal polypeptide consisting of SEQ ID NO: 10. The S32V mutation stabilizes the structure by introducing favorable hydrophobic interactions. The L123R mutation suppresses fH binding by introducing collisions with fH and unfavorable charges.

[0180] Stability enhancement can be evaluated using differential scanning calorimetry (DSC), as discussed, for example, in Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. Current Medicinal Chemistry 2005; 12:2011-20. DSC has previously been used to evaluate the stability of v2 fHbp (Johnson et al. PLoS Pathogen 2012;8:e1002981). Suitable conditions for DSC to evaluate stability include using 20 μM polypeptide in a buffer solution (e.g., 25 mM Tris) at pH 6-8 (e.g., 7-7.5) with 100-200 mM NaCl (e.g., 150 mM).

[0181] The increased stability is demonstrated by an increase of at least 5°C, e.g., at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or higher, in the thermal transition midpoint (Tm) of at least one peak compared to the wild type, as assessed by DSC. Wild-type fHbp exhibits two DSC peaks during unfolding (one in the N-terminal domain and one in the C-terminal domain), and the v2 polypeptide contained in the fusion protein of the present invention includes both such domains. “Increased stability” refers to an increase of at least 5°C in the Tm of the N-terminal domain. The Tm of the N-terminal domain can occur at 40°C or lower in the wild-type v2 sequence (Johnson et al., (2012) PLoS Pathogen 8: e1002981), while the C-terminal domain can have a Tm of 80°C or higher. Therefore, the mutant fHbp v2 amino acid sequence contained in the fusion protein of the present invention preferably has an N-terminal domain having a Tm of at least 45°C, for example, ≥50°C, ≥55°C, ≥60°C, ≥65°C, ≥70°C, ≥75°C, or ≥80°C.

[0182] The full-length wild-type fHbp v3 derived from strain M1239 has the following amino acid sequence (leader sequences shown in bold are indicated in bold, and polyglycine sequences are underlined). TIFF0007877297000032.tif24161

[0183] Mature lipoproteins lack the first 19 amino acids of sequence number 13. TIFF0007877297000033.tif24161

[0184] The ΔG form of sequence number 13 lacks the first 31 amino acids (i.e., it lacks the signal sequence and polyglycine sequence). TIFF0007877297000034.tif24161

[0185] In a preferred embodiment, the fusion polypeptide comprises a mutant v3 fHbp polypeptide having an amino acid sequence having at least k% sequence identity with SEQ ID NO: 15, wherein the v3 fHbp amino acid sequence includes substitution mutations at residues S32 and L126 of SEQ ID NO: 15. Preferably, the substitutions are S32V and L126R.

[0186] The value of k can be selected from 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. It is preferably 80 (i.e., the mutant fHbp v2 amino acid sequence has at least 80% identity with SEQ ID NO: 15), more preferably 85, more preferably 90, and more preferably 95.

[0187] In some embodiments, the fHbp v3 polypeptide contained in the fusion protein is cleaved at SEQ ID NO: 15. Compared to the wild-type mature sequence, SEQ ID NO: 15 is already cleaved up to the polyglycine sequence and at the N-terminus containing the polyglycine sequence (comparing SEQ ID NOs: 14 and 15), but SEQ ID NO: 15 may be cleaved at the C-terminus and / or further cleaved at the N-terminus.

[0188] In a preferred embodiment, the v3 fHbp polypeptide contained in the fusion protein contains or comprises the amino acid sequence of SEQ ID NO: 17.

[0189] The v3 fHbp polypeptide contained in this fusion protein exhibits higher stability than the same polypeptide under the same experimental conditions, but without sequence differences at residues S32 and L126, and is, for example, more stable than the wild-type meningococcal polypeptide consisting of SEQ ID NO: 13. The S32V mutation stabilizes the structure by introducing favorable hydrophobic interactions. The L126R mutation suppresses fH binding by introducing collisions with fH and unfavorable charges.

[0190] Stability enhancement can be evaluated using differential scanning calorimetry (DSC), as discussed, for example, in Johnson (2013) Arch Biochem Biophys 531:100-9 and Bruylants et al. (2005) Current Medicinal Chemistry 12:2011-20. DSC has previously been used to evaluate the stability of v3 fHbp (van der Veen et al. (2014) Infect Immun PMID 24379280). Suitable conditions for DSC to evaluate stability include using 20 μM polypeptide in a buffer solution (e.g., 25 mM Tris) at pH 6-8 (e.g., 7-7.5) with 100-200 mM NaCl (e.g., 150 mM).

[0191] The increased stability is demonstrated by an increase of at least 5°C, e.g., at least 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or higher, in the thermal transition midpoint (Tm) of at least one peak compared to the wild type, as assessed by DSC. Wild-type fHbp exhibits two DSC peaks during unfolding (one in the N-terminal domain and one in the C-terminal domain), and the v3 polypeptide contained in the fusion protein of the present invention includes both such domains. “Increased stability” refers to an increase of at least 5°C in the Tm of the N-terminal domain. The Tm of the N-terminal domain can occur at 60°C or lower in the wild-type v3 sequence (Johnson et al. (2012) PLoS Pathogen 8: e1002981), while the C-terminal domain can have a Tm of 80°C or higher. Therefore, the mutant fHbp v3 amino acid sequence of the present invention preferably has an N-terminal domain with a Tm of at least 65°C, e.g., ≥70°C, ≥75°C, or ≥80°C.

[0192] As described above, in a preferred embodiment, the fHbp fusion polypeptide has an amino acid sequence of the formula NH2-A-[-XL]3-B-COOH, where each X is a different variant fHbp sequence and L is an arbitrary linker amino acid sequence. In a preferred embodiment, the linker amino acid sequence "L" is a glycine polymer or a glycine-serine polymer linker.

[0193] Exemplary linkers include, but are not limited to, "GGSG," "GGSGG," "GSGSG," "GSGGG," "GGGSG," "GSSSG," and "GSGGGG." Other suitable glycine or glycine-serine polymer linkers will be apparent to those skilled in the art. In the preferred fusion polypeptide according to the present invention, the v2 and v3 sequences, and the v3 and v1 sequences, are linked by the glycine-serine polymer linker "GSGGGG."

[0194] In a preferred embodiment, the fusion polypeptide comprises or consists of one of the following amino acid sequences (glycine-serine linker sequences are underlined, and mutant residues are shown in bold).

[0195] fHbp23S_1.13_E211A / E232A (Sequence ID 18) TIFF0007877297000035.tif49165

[0196] fHbp23S_1.13_E211A / S216R(Sequence ID 19) TIFF0007877297000036.tif54165

[0197] fHbp_23S_1.15_S231R(Sequence ID 20) TIFF0007877297000037.tif40165TIFF0007877297000038.tif16166

[0198] fHbp_23S_1.15_E214A / S219R(Sequence ID 21) TIFF0007877297000039.tif55166

[0199] fHbp_23S_1.15_E214A / E235A(Sequence ID 22) TIFF0007877297000040.tif55166

[0200] In a preferred embodiment, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 19. In an alternative preferred embodiment, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 18.

[0201] The fusion polypeptide, after administration to a host animal, preferably a mammal, more preferably a human, can induce antibodies capable of recognizing wild-type Neisseria meningitidis fHbp polypeptides, particularly those of SEQ ID NOs: 31, 32, 10, and / or 13. These antibodies are ideally bactericidal (see below).

[0202] As described above, in a preferred embodiment, the fHbp fusion polypeptide has the amino acid sequence of formula NH2-A-[-XL]3-B-COOH, where each X is a different variant fHbp sequence and A is any N-terminal amino acid sequence. In a preferred embodiment, the fusion protein described herein further comprises the following N-terminal amino acid sequence, which is advantageous for enabling good expression of the fusion protein: TIFF0007877297000041.tif8153

[0203] Any of the fusion proteins disclosed herein (e.g., SEQ ID NOs: 18-22, 29, and 30) can be modified to include the amino acid sequence of SEQ ID NO: 34 at the N-terminus of the fusion polypeptide. That is, the amino acid sequence of SEQ ID NO: 34 is added to the N-terminus of the fHbp v2 component of the fusion polypeptide.

[0204] In a preferred embodiment, the serogroup B antigenic component of the immunogenic composition of the present invention comprises a complete BEXSERO vaccine product together with the fHbp fusion polypeptide as defined above. Most preferably, the fHbp fusion polypeptide is fHbp23S_1.13_E211A / S216R. Preferably, the serogroup B antigenic component is provided in a single complete liquid formulation.

[0205] Sterilization response The preferred v1.13, v1.15, and / or fusion polypeptides described above can induce a bactericidal antibody response against Neisseria meningitidis. The bactericidal antibody response is readily measurable in mice and is a standard indicator of vaccine efficacy (see, e.g., Pizza et al. (2000) Science 287:1816-1820, footnote 14; and further, WO2007 / 028408).

[0206] The above polypeptides can preferably induce a bactericidal antibody response against Neisseria meningitidis serogroup B strains expressing the v1.13fHbp sequence.

[0207] The preferred polypeptides described above can induce antibodies in mice that are bactericidal against Neisseria meningitidis strains expressing the v1.13fHbp sequence in serum bactericidal assays.

[0208] The above polypeptides can preferably induce a bactericidal antibody response against Neisseria meningitidis serogroup B strains expressing the v1.15fHbp sequence.

[0209] The preferred polypeptides described above can induce antibodies in mice that are bactericidal against Neisseria meningitidis strains expressing the v1.15fHbp sequence in serum bactericidal assays.

[0210] For example, immunogenic compositions containing these polypeptides can provide a serum bactericidal titer of ≥1:4 using the Goldschneider assay with human complement [Goldschneider et al. (1969) J. Exp. Med. 129:1307-26, Santos et al. (2001) Clinical and Diagnostic Laboratory Immunology 8:616-23, Frasch et al. (2009) Vaccine 27S:B112-6], and / or a serum bactericidal titer of ≥1:128 using juvenile rabbit complement.

[0211] polypeptide The above polypeptides can be prepared by various means, such as chemical synthesis (at least partially), digestion of longer polypeptides using proteases, translation from RNA, and purification from cell culture (e.g., recombinant expression or purification from Neisseria meningitidis culture). Heterologous expression in an Escherichia coli host is the preferred expression pathway.

[0212] The polypeptides of the present invention ideally consist of at least 100 amino acids, for example, 150aa, 175aa, 200aa, 225aa, or longer. They include mutant fHbp v1, v2, and / or v3 amino acid sequences, each of which is similarly at least 100 amino acids in length, for example, 150aa, 175aa, 200aa, 225aa, or longer.

[0213] fHbp is naturally a lipoprotein of Neisseria meningitidis. It has also been found to be lipid-modified when expressed in Escherichia coli with its native leader sequence or a heterologous leader sequence. The polypeptide of the present invention may have an N-terminal cysteine ​​residue, which may be lipid-modified, for example, containing a palmitoyl group, which may typically form tripalmitoyl-S-glyceryl-cysteine. In other embodiments, the polypeptide is not lipid-modified.

[0214] Polypeptides are preferably prepared in a substantially pure or substantially isolated form (i.e., substantially free from other Neisseria or host cell polypeptides). Generally, polypeptides are provided in an environment that does not exist in nature, e.g., an environment isolated from a naturally occurring environment. In some embodiments, polypeptides are present in a composition enriched with polypeptides compared to the starting material. Thus, purified polypeptides are provided, where purification means that the polypeptides are present in a composition substantially free from other expressed polypeptides, where substantially free means that more than 50% (e.g., ≥75%, ≥80%, ≥90%, ≥95%, or ≥99%) of the total polypeptides in the composition are the polypeptides of the present invention.

[0215] Polypeptides can take on various forms (e.g., natural, fused, glycosylated, non-glycosylated, lipid-based, disulfide-crosslinked, etc.).

[0216] When polypeptides are produced by translation within a biological host, a start codon is required, which in most hosts provides an N-terminal methionine. Therefore, the polypeptides of the present invention contain a methionine residue upstream of the sequence of the aforementioned sequence number, at least in the nascent stage.

[0217] The cleavage of the nascent sequence means that the mutant fHbp v1, v2, or v3 amino acid sequence itself provides the N-terminus of the polypeptide. However, in other embodiments, the polypeptide may include an upstream N-terminal sequence of the mutant fHbp v1, v2, or v3 amino acid sequence. In some embodiments, the polypeptide has a single methionine at the N-terminus immediately following the mutant fHbp v1, v2, or v3 amino acid sequence; in other embodiments, a longer upstream sequence may be used. Such upstream sequences may be short (for example, 40 or fewer amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include leader sequences that direct protein transport, or short peptide sequences that facilitate cloning or purification (e.g., histidine tags, i.e., His n Examples include (n=4, 5, 6, 7, 8, 9, 10 or more). Other suitable N-terminal amino acid sequences will be obvious to those skilled in the art.

[0218] Polypeptides may also contain amino acids downstream of the final amino acid of the mutant fHbp v1, v2, or v3 amino acid sequence. Such C-terminal elongations may be short (e.g., 40 or fewer amino acids, i.e., 39, 38, 37, 36, 35, 34, 33, 32, 31, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include sequences that direct protein transport, short peptide sequences that facilitate cloning or purification (e.g., histidine tags, i.e., His nExamples include sequences (n=4, 5, 6, 7, 8, 9, 10 or more) or sequences that enhance the stability of the polypeptide. Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art.

[0219] In some embodiments, the present invention excludes polypeptides containing histidine tags (see Johnson et al. (2012) PLoS Pathogen 8:e1002981 and Pajon et al. (2012) Infect Immun 80:2667-77), particularly C-terminal hexahistidine tags.

[0220] The term "polypeptide" refers to an amino acid polymer of any length. The polymer may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acids. The term also encompasses naturally occurring or interveningly modified amino acid polymers, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugation with labeling components. Furthermore, the definition includes polypeptides containing, for example, one or more analogues of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art. Polypeptides may exist as single-chain or associated chains.

[0221] Polypeptides can be bound to or immobilized on a solid support.

[0222] Polypeptides may contain detectable labels, such as radioactive labels, fluorescent labels, or biotin labels. This is particularly useful in immunoassay techniques.

[0223] Polypeptides typically consist of artificial amino acid sequences, i.e., sequences not found in any naturally occurring Neisseria meningitidis.

[0224] The affinity for factor H can be quantitatively evaluated by surface plasmon resonance using immobilized human fH (disclosed, for example, in Schneider et al. (2009) Nature 458:890-5). Affinity reduction (i.e., dissociation constant K) D Mutations that increase the (at least 10 times, ideally at least 100 times) are preferable (when measured under the same experimental conditions as the same polypeptide without mutation).

[0225] The immunogenic composition of the present invention The immunogenic composition of the present invention is a pentavalent composition containing antigenic components against five different meningococcal serotypes (A, B, C, W135, and Y). Each of these components is as defined above.

[0226] In a preferred embodiment, the pentavalent immunogenic composition of the present invention comprises the following: • Serum group A antigen, which is a synthetic analog of serogroup A capsule sugar conjugated to CRM 197 as defined above; • Serum group C antigen conjugated to CRM197 as defined above; • Serogroup W135 antigen conjugated to CRM197 as defined above; • Serum Y antigen conjugated to CRM197 as defined above; and • A combination of serogroup B antigens, including the antigen of the approved vaccine BEXSERO, along with the fHbp231 fusion protein defined above.

[0227] In a preferred embodiment, the pentavalent immunogenic composition of the present invention is provided as a completely liquid (aqueous) formulation. To avoid any doubt, this means that each component is in liquid form, and there are no components of the immunogenic composition in solid (lyophilized) form.

[0228] A further aspect of the present invention provides an immunogenic composition comprising the above-mentioned and at least one pharmaceutically acceptable excipient.

[0229] Generally, pharmaceutically acceptable excipients are substances that do not induce antibody production themselves, are not harmful to the patient receiving the composition, and can be administered without excessive toxicity. Pharmacologically acceptable carriers and excipients are those used in the art and may include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances such as wetting agents or emulsifiers and pH buffers may also be present in such vehicles according to the prior art.

[0230] The immunogenic composition may further contain an adjuvant. The adjuvant may be an aluminum-based adjuvant such as aluminum hydroxide or aluminum phosphate.

[0231] The immunogenic compositions of the present invention can be administered in combination with other pharmaceutically active substances or other vaccines. Compositions for administration may include other types of immunogenic compounds, such as complex carbohydrates that induce an immune response to provide protection against other Neisseria meningitidis pathogens.

[0232] A further aspect of the present invention provides a vaccine comprising the immunogenic composition described above.

[0233] The immunogenic composition described above is useful for immunizing mammals, preferably humans, against Neisseria meningitidis infection.

[0234] The immunogenic compositions of the present invention are used to immunize mammals against infections and / or diseases caused by Neisseria meningitidis serogroups A, B, C, W125 and / or Y, and recipients of such immunogenic compositions enhance the immune response that provides protection against infections and / or diseases caused by Neisseria meningitidis bacteria.

[0235] Therefore, the immunogenic composition according to the present invention is used in a preventive method to immunize subjects against infection and / or disease caused by Neisseria meningitidis. The immunogenic composition can also be used in therapeutic methods (i.e., for treating Neisseria meningitidis infection).

[0236] The present invention also provides a method for enhancing the immune response to Neisseria meningitidis infection in mammals in vivo, comprising administering the immunogenic composition of the present invention to the mammals. The present invention also provides polypeptides of the present invention for use in such a method.

[0237] The immune response is preferably defensive and preferably involves antibody and / or cell-mediated immunity. Preferably, the immune response is a bactericidal antibody response. This method can enhance the booster response. By enhancing the in vivo immune response, mammals can be protected from diseases caused by Neisseria (particularly meningococcal infection).

[0238] The present invention also provides a method for protecting mammals from Neisseria (e.g., meningococcal) infection, which includes administering the immunogenic composition of the present invention to mammals.

[0239] The immunological compositions of the present invention are preferably formulated as vaccine products suitable for therapeutic (i.e., to treat infection) or prophylactic (i.e., to prevent infection) use. Vaccines are typically preventive.

[0240] The mammal is preferably a human. The human may be an adult, a young adult, or a child (e.g., a toddler or infant). Vaccines intended for children may also be administered to adults to evaluate, for example, safety, dosage, immunogenicity, etc.

[0241] These uses and methods are, but are not limited, particularly useful for preventing / treating diseases including meningitis (especially bacterial, e.g., meningococcal meningitis) and bacteremia. For example, they are suitable for active immunization of individuals against invasive meningococcal disease caused by Neisseria meningitidis (specifically against serogroups A, B, C, W135, and Y).

[0242] While protection against Neisseria meningitidis can be measured epidemiologically, for example in clinical trials, it is convenient to use indirect means to confirm that an immunogenic composition induces a serum bactericidal antibody (SBA) response in the recipient. In an SBA assay, serum from the recipient of the composition is incubated with the target bacterium (Neisseria meningitidis in this invention) in the presence of complement (preferably human complement, but juvenile rabbit complement is often used instead), and SBA activity is determined by evaluating bacterial killing at various serum dilutions. The results observed in an SBA assay can be reinforced by performing a competitive SBA assay to provide further indirect evidence of the immunogenic activity of the target antigen. In a competitive SBA assay, serum from the recipient of an immunogenic composition containing the antigen is pre-incubated with the antigen and then incubated with the target bacterium in the presence of human complement. Next, bacterial toxicity is evaluated, and if the bactericidal antibodies in the recipient's serum did not bind to the target antigen during the pre-incubation phase and therefore could not bind to the surface antigen on the bacteria, they are reduced or eliminated.

[0243] It is not necessary for a composition to provide protection against each and all strains of Neisseria meningitidis, or for each and all recipients of a composition to be protected. Such universal protection is not the usual standard in this field. Rather, protection is typically selected, often by national criteria, evaluated against a panel of reference study strains that may change over time, and measured across the entire recipient population.

[0244] The preferred compositions of the present invention can confer antibody titers to patients that are superior to the criteria for seroprotection against each antigen component for an acceptable percentage of human subjects. Antigens with binding antibody titers that suggest the host is seroconverted to the antigen are well known, and such antibody titers are published by organizations, e.g., the WHO. Preferably, more than 80% of statistically significant samples of subjects are seroconverted, more preferably more than 90%, even more preferably more than 93%, and most preferably 96-100%.

[0245] The immunogenic composition may optionally contain an immunologically effective amount of immunogen, as well as any of the other specified components.

[0246] "Immunologically effective dose" means that the amount administered to an individual, either as a single dose or as part of a series, is effective for treatment or prevention.

[0247] The term "prevention" means that the progression of a disease is reduced and / or eliminated, or that the onset of the disease is eliminated. For example, the immune system of a target can be primed (e.g., by vaccination) to trigger an immune response that repels infection so that the onset of the disease is eliminated. Thus, the vaccinated target may still be susceptible to infection, but will be more repelled than the control target. This amount varies depending on the health and physical condition of the individual being treated, age, the taxonomic group of the individual being treated (e.g., non-human primates, primates, etc.), the antibody synthesis capacity of the individual's immune system, the desired degree of protection, the formulation of the vaccine, the medical condition assessment by the treating physician, and other relevant factors. A relatively wide range of reductions that can be determined by routine trials can be expected. The composition can be administered together with other immunomodulators.

[0248] Vaccine effectiveness The immunogenic composition for use in the present invention preferably has vaccine efficacy against at least 10%, for example, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, ≥85%, ≥90%, or more of at least one strain of Neisseria meningitidis.

[0249] The efficacy of the vaccine is determined by the reduction in the relative risk of meningococcal disease in subjects receiving the composition according to the present invention compared to subjects not receiving such a composition (e.g., unimmunized subjects, or subjects administered a placebo or negative control). Therefore, the incidence of meningococcal disease in a population immunized according to the present invention is compared to the incidence in a control population not immunized according to the present invention to provide a relative risk, and the efficacy of the vaccine is 100% minus this value.

[0250] Vaccine effectiveness is determined for populations, not individuals. Therefore, while it is a useful epidemiological tool, it does not predict individual protection. For example, individual subjects may be exposed to a very large amount of inoculation material containing infectious pathogens, or they may have other risk factors that make them more susceptible to infection, but this does not negate the validity or usefulness of the effectiveness measure. The size of the population immunized according to the present invention and for which vaccine effectiveness is measured is ideally at least 100 subjects, and possibly larger, e.g., at least 500 subjects. The size of the control group is also at least 100 subjects, e.g., at least 500 subjects.

[0251] Administration The compositions of the present invention are generally administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of tissue), or by rectal, oral, vaginal, topical, transdermal, nasal, ocular, ear, lung, or other mucosal administration. Administration by injection is preferred. Intramuscular administration into the thigh or upper arm is preferred. Injection may be performed via a needle (e.g., a subcutaneous injection needle), but needleless injections can be used instead.

[0252] Preferably, the composition of the present invention is contained in a single sealed container, preferably a vial or syringe.

[0253] Neisseria infection affects various parts of the body, and therefore the composition can be prepared in various forms. For example, the composition can be prepared as an injectable preparation, either as a liquid solution or a suspension. A solid form suitable for a solution or suspension in a liquid vehicle before injection can also be prepared. The composition can be prepared for topical administration, for example, as an ointment, cream, or powder. The composition can be prepared for oral administration, for example, as tablets or capsules, or as a syrup (sometimes flavored). The composition can be prepared using a fine powder or spray, for example, for pulmonary administration as an inhaler. The composition can be prepared as a suppository or pessary. The composition can be prepared for administration to the nose, ear, or eye, for example, as a droplet. Compositions suitable for parenteral injection are most preferred.

[0254] Most preferably, the immunogenic composition of the present invention is provided as a complete liquid formulation, that is, there are no antigenic components of the composition of the present invention in lyophilized form.

[0255] The present invention may be used to induce systemic immunity and / or mucosal immunity.

[0256] As used herein, “dose” of a composition refers to the volume of the composition suitable for administration to a subject as a single immunization. Human vaccines are typically administered in doses of about 0.5 ml, but divided doses may be administered (e.g., to children). The volume of the dose may further vary depending on the concentration of the antigen in the composition.

[0257] The composition may be provided as a "multi-dose" kit, i.e., a single container containing enough composition for multiple immunizations. The multi-dose kit may contain a preservative, or the multi-dose container may have a sterile adapter for dispensing individual doses of the composition.

[0258] Administration may involve a single-dose schedule, but is usually accompanied by a multi-dose schedule. Preferably, a schedule of at least three doses is given. The appropriate interval between priming doses can be routinely determined, for example, between 4 and 16 weeks, or between one or two months. For example, BEXSERO® may be administered after 2, 4, and 6 months, or after 2, 3, and 4 months, with any fourth dose administered after 12 months.

[0259] The subjects of immunization are people of any age, for example, 0-12 months, 1-5 years, 5-18 years, 18-55 years, or those who may be over 55 years old. Preferably, the subjects of immunization are adolescents (e.g., 12-18 years) or adults (18 years or older).

[0260] In some cases, the subjects may be adolescents or adults who were immunized against Neisseria meningitidis during childhood (e.g., before the age of 12) and who have received additional administration of the immunogenic composition according to the present invention.

[0261] Non-antigenic components The immunogenic compositions of the present invention generally contain a pharmaceutically acceptable carrier, which may be a substance that does not induce the production of antibodies harmful to the patient receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances such as wetting agents or emulsifiers and pH buffers may also be present in such vehicles. A thorough discussion of suitable carriers can be found in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472.

[0262] The composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered at pH 6 to pH 8, generally around pH 7. If the composition contains an aluminum hydroxide salt, it is preferable to use a histidine buffer (WO03 / 009869). The composition of the present invention may be isotonic with respect to humans.

[0263] Adjuvants that can be used in the composition of the present invention include, but are not limited to, insoluble metal salts, oil-in-water emulsions (e.g., MF59 or AS03, both containing squalene), saponins, non-toxic derivatives of LPS (e.g., monophosphoryl lipid A or 3-O-deacylated MPL), immunostimulant oligonucleotides, detoxifying bacterial ADP-ribosylated toxins, microparticles, liposomes, imidazoquinolones, or mixtures thereof. Other substances that act as immunostimulants are disclosed in Chapter 7 of Vaccine Design (1995) eds. Powell & Newman, ISBN:030644867X, Plenum.

[0264] The use of aluminum hydroxide and / or aluminum phosphate adjuvants is particularly preferred, and polypeptides are generally adsorbed to these salts. These salts include oxyhydroxides and hydroxyphosphates (see, for example, Chapters 8 and 9 of Vaccine Design (1995) eds. Powell & Newman, ISBN: 030644867X, Plenum). The salts can take any preferred form (e.g., gel, crystal, amorphous, etc.).

[0265] general In this disclosure and claims, the singular forms “a,” “an,” and “the” include the plural form unless the context clearly indicates otherwise. That is, unless otherwise specified, “a” means “one or more.”

[0266] When used in expressions such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," and "A" and "B." Similarly, when used in expressions such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0267] The term “comprising” encompasses both “including” and “consisting.” For example, a composition “comprising” X may consist solely of X or may include something additional, such as X + Y. References to “comprising” (or “comprises,” etc.) may be replaced with references to “consisting of” (or “consists of,” etc.). The term “consisting essentially of” limits the claims to the specified materials or steps and “not substantially affecting the basic and novel features” of the claimed invention.

[0268] Unless otherwise stated, the notations "A%-B%", "AB%", "A% to B%", "A to B%", "A%-B", and "A% to B" all have the usual and conventional meanings. In some embodiments, these notations are synonymous.

[0269] The terms “substantially” or “substantial” mean that the described or claimed condition functions as the described standard in all important aspects. Thus, “substantially not” means that a condition functions as a non-existence condition in all important aspects, even if the numerical value indicates the presence of some impurities or substances. “Substantially” generally means a value greater than 90%, preferably greater than 95%, and most preferably greater than 99%. Where a particular value is used in this specification and claims, unless otherwise stated, the term “substantially” means with an acceptable margin of error for that particular value.

[0270] The term "approximately" in relation to the number x is arbitrary and can mean, for example, x ± 10%.

[0271] Where this disclosure relates to an “epitope,” such epitope may be a B-cell epitope and / or a T-cell epitope, but is usually a B-cell epitope. Such epitopes can be identified empirically (e.g., using PEPSCAN (see, e.g., Geysen et al. (1984) PNAS USA 81:3998-4002 and Carter (1994) Methods Mol Biol 36:207-23) or similar methods), or they can be identified (e.g., Jameson-Wolf antigen index (Jameson, BA et al. 1988, CABIOS 4(1):181-186), matrix-based approaches (Raddrizzani & Hammer (2000) Brief Bioinform 1(2):179-89), MAPITOPE (Bublil et al. (2007) Proteins 68(1):294-304), TEPITOPE (De Lalla et al. (1999) J. Immunol. 163:1725-29 and Kwok et al. (2001)) Trends (Immunol 22:583-88), neural networks (Brusic et al. (1998) Bioinformatics 14(2):121-30), OptiMer & EpiMer (Meister et al. (1995) Vaccine 13(6):581-91 and Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610), ADEPT (Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7), Tsites (Feller & de la Cruz (1991) Nature 349(6311):720-1), hydrophilicity (Hopp (1993) Peptide Research 6:183-190), or antigen index (Welling et al. (1985) FEBS) It can be predicted using (Lett. 188:215-218).An epitope is a portion of an antigen that is recognized and binds to an antigen-binding site on an antibody or T cell receptor; these are also called "antigenic determinants."

[0272] As used herein, any reference to the “sequence identity percentage” between a query amino acid sequence and a target amino acid sequence is understood to refer to the value of identity calculated using a suitable algorithm or software program known in the art for performing pairwise sequence alignment.

[0273] A query amino acid sequence can be described by an amino acid sequence identified in one or more claims of this specification. The query sequence may be 100% identical to the target sequence, or may include amino acid changes (e.g., point mutations, substitutions, deletions, insertions, etc.) up to a certain integer compared to the target sequence, such that the identity percentage is less than 100%. For example, the query sequence may be at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the target sequence.

[0274] The preferred alignment tool used to perform alignment and calculate percentage (%) sequence identity is a local alignment tool such as the Basic Local Alignment Search Tool (BLAST) algorithm. Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov). Alignment can be determined by the Smith-Waterman homology search algorithm using affine gap search with gap-open penalty 12 and gap-expansion penalty 2, and a BLOSUM matrix 62. The Smith-Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2:482-489. Other preferred alignment tools are Water (EMBOSS) and Marcher (EMBOSS). Alternatively, a preferred alignment tool used to perform alignment and calculate percentage (%) sequence identity is a best-fit alignment tool, such as GENEPAST, also known as the KERR algorithm.

[0275] To calculate the identity percentage, the query and target sequences may be compared and aligned for the maximum correspondence over a specified region (which may be a region of at least approximately 40, 45, 50, 55, 60, 65 or more amino acids, and up to the full length of the target amino acid sequence). The specified region must include a region of the query sequence containing any specific point mutation in the amino acid sequence. Alternatively, the sequence identity percentage may be calculated over the "full length" of the target sequence. Any N-terminal or C-terminal amino acid stretches that may be present in the query sequence, such as a signal peptide or leader peptide, or in a C-terminal or N-terminal tag, should be excluded from the alignment.

[0276] With respect to polypeptide sequences, the term “fragment” means that the polypeptide is a fraction of a full-length protein. As used herein, a mutant polypeptide fragment also includes mutations. Fragments may have qualitative biological activity common to the full-length protein; for example, an “immunogenic fragment” contains or encodes one or more epitopes, such as an immunodominant epitope, which allows for the enhancement of the same or similar immune response to the fragment as to the full-length sequence. Polypeptide fragments generally have a deleted amino(N)-terminal and / or carboxy(C)-terminal portion compared to the native protein, but the remaining amino acid sequence of the fragment is identical to that of the native protein. A polypeptide fragment may contain, for example, approximately 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26, 28, 40, 45, 50, 55, 60, 70, 80, 90, 100, 150, 200, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, or 262 consecutive amino acids, for example, all integers between the reference polypeptide sequence, for example, 50-260, 50-255, 50-250, 50-200, or 50-150 consecutive amino acids in the reference polypeptide sequence. The term "fragment" explicitly excludes full-length fHbp polypeptides and their mature lipoproteins.

[0277] Following serogroups, meningococcal classification includes serotypes, serotypes, and then immunotypes. Standard nomenclature lists serogroups, serotypes, serotypes, and immunotypes, each separated by a colon, for example, B:4:P1.15:L3,7,9. Within serogroup B, some strains frequently cause disease (highly invasive), some cause more severe morphology than others (highly malignant), and others do not cause disease at all. Seven highly malignant strains are recognized: subgroups I, III, and IV-1, the ET-5 complex, the ET-37 complex, the A4 cluster, and strain 3. These are defined by polylocus enzyme electrophoresis (MLEE), although polylocus sequence typing (MLST) is also used to classify meningococci. The four main highly malignant clusters are the ST32, ST44, ST8, and ST11 complexes.

[0278] In this specification, the terms “increased stability,” “higher stability,” or “increased stability” mean that the mutant polypeptides disclosed herein have higher relative thermal stability (kcal / mol) compared to non-mutant (wild-type) polypeptides under the same experimental conditions. Increased stability can be evaluated using differential scanning calorimetry (DSC), as studied, for example, in Bruylants et al. (Differential Scanning Calorimetry in Life Sciences: Thermodynamics, Stability, Molecular Recognition and Application in Drug Design, 2005 Curr. Med. Chem. 12: 2011-2020) and Calorimetry Sciences' “Characterizing Protein stability by DSC” (Life Sciences Application Note, Doc. No. 20211021306 February 2006), or by differential scanning fluorescence (DSF). Increased stability can be characterized as an increase of at least about 5°C at the thermal transition midpoint (Tm), as evaluated by DSC or DSF. For example, see Thomas et al., Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen, 2013 Hum. Vaccin. Immunother. 9(4):744-752.

[0279] "Effective quantity" means a quantity sufficient to produce the referenced effect or outcome. "Effective quantity" can be determined empirically and conventionally using known techniques in relation to the stated purpose.

[0280] The “immunologically effective dose” or “therapeutic effective dose” means that the amount administered to an individual, either as a single dose or as part of a series of doses, is effective in treating or preventing disease. This dose may vary depending on the health and physical condition of the individual being treated, age, taxonomic group of the individual being treated (e.g., non-human primate, primate, etc.), the individual’s immune system’s ability to produce antibodies, the desired level of protection, the vaccine formulation, the medical condition assessment by the treating physician, and other relevant factors. The dose is expected to fall within a relatively broad range that can be determined by standard testing.

[0281] The term "treatment" means any one of the following: (i) prevention of infection or reinfection, as in the case of conventional vaccines; (ii) reduction of severity or elimination of symptoms; (iii) delay of symptom recurrence; and (iv) substantial or complete elimination of the pathogen or disorder in question in the subject. Therefore, treatment can be influenced prophylactically (before infection) or therapeutically (after infection).

[0282] The term "weight % (%w / w)" indicates, as shown, the weight percentage of a given compound relative to a different compound or to the total content of a composition.

[0283] Similarly, the term "volume % (%v / v)" indicates the volume percentage of a given compound relative to the total content of a different compound or composition, as shown.

[0284] All publications cited herein are incorporated herein in their entirety by reference.

[0285] array Sequence ID 1 [v1.13 mature polypeptide from strain M982] TIFF0007877297000042.tif22165

[0286] Sequence ID 2[v1.13ΔG] TIFF0007877297000043.tif22165

[0287] Sequence ID 3[v1.13ΔG(E211A / E232A)] TIFF0007877297000044.tif22165

[0288] Sequence ID 4[v1.13ΔG(E211A / S216R)] TIFF0007877297000045.tif12165TIFF0007877297000046.tif12165

[0289] Sequence ID 5 [v1.15 mature polypeptide from NM452 strain] TIFF0007877297000047.tif21165

[0290] Sequence ID 6[v1.15ΔG] TIFF0007877297000048.tif21165

[0291] Sequence ID 7[v1.15ΔG(S219R)] TIFF0007877297000049.tif21165

[0292] Sequence ID 8[v1.15ΔG(E214A / S219R)] TIFF0007877297000050.tif21165

[0293] Sequence ID 9[v1.15ΔG(E214A / E235A)] TIFF0007877297000051.tif21165

[0294] Sequence ID No. 10 [v2wt from 2996 strains] TIFF0007877297000052.tif21165

[0295] Sequence ID 11 [v2 mature polypeptide] TIFF0007877297000053.tif21165

[0296] Sequence ID 12[v2ΔG] TIFF0007877297000054.tif18165

[0297] Sequence ID 13 [v3wt from strain M1239] TIFF0007877297000055.tif20164

[0298] Sequence ID 14 [v3 maturation] TIFF0007877297000056.tif20164

[0299] Sequence ID 15[v3ΔG] TIFF0007877297000057.tif20164

[0300] Sequence ID 16[v2ΔGS32V / L123R] TIFF0007877297000058.tif17164

[0301] Sequence ID 17[v3ΔGS32V / L126R] TIFF0007877297000059.tif20164

[0302] Sequence ID 18[(23S_1.13_E211A / E232A)] TIFF0007877297000060.tif38165TIFF0007877297000061.tif11165

[0303] Sequence ID 19[23S_1.13_E211A / S216R] TIFF0007877297000062.tif53165

[0304] Sequence ID 20[23S_1.15_S219R] TIFF0007877297000063.tif53165

[0305] Sequence ID 21[23S_1.15_E214A / S219R] TIFF0007877297000064.tif53165

[0306] Sequence ID 22[23S_1.15_E214A / E235A] TIFF0007877297000065.tif22165TIFF0007877297000066.tif33165

[0307] Sequence ID 23 [v1.1ΔG+His tag] TIFF0007877297000067.tif22165

[0308] Sequence ID 24 [v1.13ΔG+His tag] TIFF0007877297000068.tif22165

[0309] Sequence ID 25[v1.13ΔG(E211A)] TIFF0007877297000069.tif22165

[0310] Sequence ID 26 [v1.13ΔG(S216R)] TIFF0007877297000070.tif22165

[0311] Sequence ID 27 [v1.15ΔG+His tag] TIFF0007877297000071.tif22165

[0312] Sequence ID 28 [v1.15ΔG(E214A)+His tag] TIFF0007877297000072.tif22165

[0313] Sequence ID 29 [fHbp231wt fusion polypeptide] TIFF0007877297000073.tif52165

[0314] Sequence ID 30 [fHbp231S fusion polypeptide] TIFF0007877297000074.tif52165

[0315] Sequence ID 31 [v1.13 full-length wt sequence] TIFF0007877297000075.tif23165

[0316] Sequence ID 32 [v1.15 full-length wt sequence] TIFF0007877297000076.tif23165

[0317] Sequence ID 33 [Mature fHbpv1.1] TIFF0007877297000077.tif23165

[0318] SEQ ID NO: 34 [Any N-terminal amino acid sequence] TIFF0007877297000078.tif6149

[0319] SEQ ID NO: 35 [SEQ ID NO: 34 + SEQ ID NO: 19; 23S_1.13_E211A / S216R with an additional N-terminal amino acid sequence] TIFF0007877297000079.tif53165

[0320] Modes for carrying out the invention The present invention can be further defined by reference to the following non-limiting embodiments. [Examples]

[0321] Synthesis and Characterization of Conjugate Serotype A Antigens General Procedure and Materials All chemicals (Acros, Biosolve, Sigma-Aldrich, and TCI) were used in their original condition upon arrival, and unless otherwise noted, all reactions were carried out under an argon atmosphere at ambient temperature (22°C). For TLC analysis, aluminum sheets (Merck, TLC Silica Gel 60 F254) were used, with the solution in H2SO4 / EtOH (20%) or (NH4)6Mo7O 24Solutions of 4H2O (25 g / L) and (NH4)4Ce(SO4)4·2H2O (10 g / L) in 10% H2SO4 aqueous solution, or a solution of KMnO4 (2%) and K2CO3 (1%) in H2O, were sprayed and heated at approximately 140°C. 40-63 μm 60 Å silica gel (SD Screening Devices) was used for column chromatography. NMR spectra (1H, 13 C and 31 P) was recorded using a Bruker AV-400liq, Bruker AV-500, or Bruker AV-600. High-resolution mass spectra were recorded by direct injection into a mass spectrometer with an electrospray ion source (Thermo Finnigan LTQ Orbitrap) in positive mode with a resolution of R=60000, using dioctyl phthalate as the rock mass (m / z=391.28428) at m / z 400 (mass range m / z=150–2000) and R=60000 (power supply voltage 3.5kV, sheath gas flow 10, capillary temperature 250°C).

[0322] abbreviation Acetic acid (ACOH) ACN = Acetonitrile DCM = Dichloromethane DMTrCl = 4,4′-dimethoxytrityl chloride alkyl = ethyl acetate THF = Tetrahydrofuran TBAF = Tetrabutylammonium fluoride

[0323] Example 1: Preparation of the oligomer of formula (Ia) of the present invention according to Scheme 1 Acetamide-3,4-di-O-benzyl-2-deoxy-6-O-texyldimethylsilyl-5a-carba-α-domannapyranose(13) Silyl ether 12 can be prepared according to the procedure described in Q. Gao et al. Org. Biomol. Chem., 2012, 10, 6673.

[0324] Silyl ether 12 (1.6 g, 2.7 mmol) was dissolved in dry THF (20 mL). The mixture was cooled to 0°C. 0.1 M TBAF / THF solution (4.1 mL, 4.1 mmol) was slowly added. The reaction mixture was warmed to room temperature and stirred for 3 hours. AcOH (0.31 mL) was added to the reaction mixture. The solution was extracted three times with DCM and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (siRNA / hexane) to obtain product 13 in 92% yield (1.1 g, 2.52 mmol). The spectral data were consistent with reported data.

[0325] 2-Acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranose(14) Alcohol 13 (1.12 g, 2.5 mmol) was dissolved in MeOH (32 mL). NaOMe (0.03 g, 0.5 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. Amberlite H+ resin was added until a neutral pH was reached. The suspension was filtered and concentrated under reduced pressure. 1 H NMR (400MHz, CDCl3)δ=1.70-1.85(m, 2H, H-5a), 1.90(s, 3H, AcNH), 2.19-2.23(m, 1H, H-5), 3.60-3.79(m, 3 H, H-6, H-1), 3.83-3.90(m, 1H, H-2), 3.91-3.99(m, 1H, H-4), 4.14-4.23(m, 1H, H-3), 4.33-4.41(m, 1H, CHH Bn), 4.54-4.72(m, 3H, CH2Bn, CHH Bn), 5.79(m, 1H, NHAc), 7.22-7.42(m, 10H, H arom ). 13 C NMR (100MHz, CDCl3)δ=23.5(CH3AcNH), 30.6(CH2C-5a), 39.5(CH C-5), 53.5(CH C-3), 64.1(CH2C-6), 67.9(CH C-4), 72.4(CH2Bn), 73.8(CH2Bn), 75.5(CH C-1), 79.0(CH C-4), 127.3-128.9(CH arom), 171.8 (C=OAcNH). HRMS:[C 23 H 29 NO5+H] + The required value was 400.21251, and the measured value was 400.21179.

[0326] 2-Acetamide-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5-carba-α-D-mannopyranose(10) Diol 14 (0.9 g, 2.25 mmol) was dissolved in dry DCM (30 mL). Et3N (1.9 mL, 13.5 mmol) was added to the mixture. DMTrCl (1.16 g, 3.38 mmol) was added. The reaction mixture was stirred for 2 hours. H2O was added to the reaction mixture and washed once with brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (siRNA / hexane) to obtain product 10 in 91% yield (1.6 g, 2.04 mmol). 1 H NMR (400MHz, CD3CN) δ=1.70-1.85(m, 1H, 5a′-H), 1.91(s, 3H, AcNH), 2.00-2.21(m, 2H, 5a-H, 5-H), 3.01-3.19(m, 1H, 6′-H ), 3.27-3.37(m, 1H, 6-H), 3.51-3.67(m, 1H, H-4), 3.73(s, 7H, H-3, 2×OMe), 4.06-4.20(m, 1H, H-1), 4.22-4.32(m, 1H, CHH Bn), 4.40-4.62(m, 3H, CH2Bn, H-2), 4.65-4.73(m, 1H, CHH Bn), 6.35-6.44(m, 1H, NHAc), 6.78-7.47(m, 23H, H arom ). 13 C NMR (100MHz, CD3CN)δ=23.2(CH3AcNH), 31.6(CH2C-5a), 38.6(CH C-5), 53.3(CH C-2), 55.8(2×CH3OMe), 64.6(CH2C-6), 67.6(CH C-1), 72.1(CH2Bn), 73.8(CH2Bn), 77.2(CH C-4), 79.8(CH C-3), 86.5(Cq DMTr), 113.9(CH arom), 127.3-130.7(CH arom ), 137.2-159.4(5×Cq DMTr), 171.1(C=OAcNH). HRMS:[C 44 H 47 [NO7+Na] + The required value was 724.32501, and the measured value was 724.32483.

[0327] 1-O-((N,N-diisopropylamino)-O-2-cyanoethyl-phosphorumidite))-2-acetamide-3,4-di-O-benzyl-2-deoxy-6-O-(bis(4-methoxyphenyl)(phenyl))-5a-carba-α-D-mannopyranose(9) Alcohol 10 (1.5 g, 2.14 mmol) was removed three times with ACN and dissolved in dry DCM (22 mL). Fresh activated MS3Å and DIPEA (0.6 mL, 3.2 mmol) were added to the mixture. 2-Cyanoethyl N,N-diisopropyl chlorophosphoramidite (0.6 mL, 2.6 mmol) was added to the mixture. The reaction mixture was stirred for 2 hours. H2O was added to this solution and washed once with a 1:1 brine / NaHCO3 solution. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone / Et3N) to obtain product 9 in 94% yield (1.81 g, 2.0 mmol) (mixture of diastereomers). 1¹H NMR (400MHz, CD3CN) δ = 1.04-1.24 (m, 12H, 4× isopropylamino), 1.70-1.85 (m, 1H, 5α′-H), 1.92 (s, 3H, AcNH), 2.00-2.21 (m, 2H, 5α-H, 5-H), 2.55-2.75 (m, 2H, CH2 cyanoethyl), 2.98-3 .10(m, 1H, 6′-H), 3.27-3.37(m, 1H, 6-H), 3.47-3.70(m, 3H, 2×CH isopropylamino, H-4), 3.70-3.88(m, 9H, H-3, CH2 cyanoethyl, 2×OMe), 4.06-4.20(m, 1H, H-1), 4.22-4.32(m, 1H, CHH Bn), 4.40-4.62(m, 3H, CH2 Bn, H-2), 4.65-4.73(m, 1H, CHH Bn), 6.35-6.44(m, 1H, NHAc), 6.78-7.47(m, 23H, H arom ). 13 ¹¹C NMR (100MHz, CD3CN) δ = 20.7 (CH2 cyanoethyl), 22.9 (CH3 AcNH), 24.5-24.7 (2×CH3 isopropylamino), 30.6 (CH2C-5a), 38.5 (CH3 C-5), 43.7 (2×CH isopropylamino), 51.7 (CH3 C-2), 55.5 (2×CH3 OMe), 59.1 (CH2 cyanoethyl), 64.2 (CH2C-6), 70.5 (CH3 C-1), 71.5 (CH2 Bn), 74.3 (CH2 Bn), 77.8 (CH3 C-4), 79.5 (CH3 C-3), 86.2 (Cq DMTr), 113.6 (CH3 arom ), 127.3-130.7(CH arom ), 136.8-159.2(5×Cq DMTr), 170(C=OAcNH). 31 P NMR (162MHz, CD3CN) δ=146.9, 147.26.

[0328] General procedure for phosphoramidite coupling, oxidation, and detritylation at typical scales (0.03-0.3 mmol) The starting alcohol was removed three times by co-distillation with ACN, and newly activated MS3Å and DCI (0.25 M / ACN solution, 1.5 equivalents) were added. The solution was stirred for 15 minutes. Phosphoramidite reagent (0.1-0.16 M / ACN solution, 1.3-3 equivalents) was added to the mixture and stirred until the starting materials were completely converted (approximately 2 hours). Subsequently, CSO (0.5 M / ACN solution, 2 equivalents) was added to the reaction mixture and stirred for 15 minutes. The mixture was diluted with HCl and washed with a 1:1 brine / NaHCO3 solution. The aqueous layer was extracted twice with HCl. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was removed three times by co-distillation with ACN and dissolved in DCM (5-10 mL). TCA (0.18 MDCM solution) was added to this solution and stirred for 1 hour. H2O was added to the reaction mixture and stirred for 15 minutes. The reaction mixture was washed with a 1:1 brine / NaHCO3 solution. The aqueous layer was extracted three times with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (DCM / acetone) or size exclusion chromatography (sephadex LH-20, MeOH / DCM 1:1).

[0329] 1-O-((2-acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzyl-carbamate(15) Using the general procedure described above, alcohol 10 (0.21 g, 0.3 mmol) was coupled with phosphoramidite 11 (2.5 mL 0.16 M / ACN solution, 0.45 mmol), followed by oxidation and detritylation. The crude product was purified by flash chromatography (DCM / acetone) to obtain product 15 in 94% yield (0.216 g, 0.282 mmol). 1¹H NMR (400MHz, CD3CN) δ = 1.24-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.70 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 4H, 5a′-H, AcNH), 1.96-2.02 (m, 2H, 5a-H, 5-H), 2.72-2.82 (m, 2H, CH2 shear) (Noethyl), 2.96 (bs, 1H, OH), 3.02-3.12 (m, 2H, CH2 hexyl spacer), 3.56-3.74 (m, 3H, H-6, H-4), 3.76-3.84 (m, 1H, H-3), 3.95-4.07 (m, 2H, CH2 hexyl spacer), 4.08-4.20 (m, 2H, CH2 cyanoethyl), 4.44-4.63 (m, 5H, H-1, H-2, CH2Bn, CHH Bn), 4.72-4.80 (m, 1H, CHH Bn), 5.03 (s, 2H, CH2Bn spacer), 5.70 (bs, 1H, NH), 6.49-6.60 (m, 1H, NHAc), 7.23-7.44 (m, 15H, H arom ). 13 ¹¹C NMR (100MHz, CD3CN) δ = 19.9 (CH2 cyanoethyl), 22.8 (CH3AcNH), 25.4 (CH2 hexyl spacer), 26.4 (CH2 hexyl spacer), 30.0 (CH2C-5a), 30.4 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 40.0 (CH2C-5), 41.0 (CH2 hexyl spacer), 51.1 (CH2C-2), 62.9 (CH2C-6), 63.0 (CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.8 (CH2 hexyl spacer), 72.2 (CH2Bn), 74.0 (CH2Bn), 75.1 (CH2C-1), 76.7 (CH2 C-4), 79.3(CHC-3), 128.1-129.1(CH arom ), 138.9-139.7(3×Cq Bn), 170.8(C=O AcNH). 31 P NMR (162MHz, CD3CN) δ=-2.40, -2.36. HRMS:[C 40 H 52 N3O 10 [P+H] +The required value was 766.34707, and the measured value was 766.34707.

[0330] 1-O-di-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(16) Using the general procedure described above, alcohol 15 (0.186 g, 0.24 mmol) was coupled with phosphoramidite 9 (2.3 mL 0.16 M / ACN solution, 0.37 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 16 in 82% yield (0.255 g, 0.199 mmol). 1 ¹H NMR (400MHz, CD3CN) δ = 1.25-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.71 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 8H, 2×5a′-H, 2×AcNH), 1.96-2.02 (m, 4H, 2×5a-H, 2×5-H), 2.70-2.81 (m, 4H, 2×CH2 cyanoethyl), 2.96 (bs, 1H, OH), 3.01-3.12 (m, 2 H, CH2 hexyl spacer), 3.56-3.87 (m, 6H, 2×H-6, 2×H-4), 3.94-4.28 (m, 8H, 2×H-3, CH2 hexyl spacer, 2×CH2 cyanoethyl), 4.29-4.85 (m, 12H, 2×H-1, 2×H-2, 4×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.75 (bs, 1H, NH), 6.52-6.62 (m, 1H, NHAc), 6.85-6.99 (m, 1H, NHAc), 7.21-7.41 (m, 25H, H arom ). 13¹³C NMR (100MHz, CD3CN) δ = 19.9-20.0 (2×CH2 cyanoethyl), 22.9-23.0 (2×CH3AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 29.1-29.2 (2×CH2C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.1-40.0 (2×CH C-5), 41.1 (CH2 hexyl spacer), 50.9-51.4 (2×CH C-2), 62.5-62.6 (2×CH2C-6), 63.0-63.2 (2×CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.9 (CH2 hexyl spacer), 72.1-72.3 (4×CH2Bn), 75.0-75.4 (2×CHC-1), 75.5-76.9 (2×CHC-4), 79.2-79.5 (2×CH C-3), 128.2-129.1 (CH arom ), 138.9-139.6(5×Cq Bn), 170.8(2×C=O AcNH). 31 P NMR (162MHz, CD3CN) δ=-2.60, -2.58, -2.34, -2.32, -2.22, -2.17. HRMS:[C 66 H 83 N5O 17 P2+H] + The required value was 1280.53320, and the measured value was 1280.53320.

[0331] 1-O-tri-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(17) Using the general procedure described above, alcohol 16 (0.215 g, 0.167 mmol) was coupled with phosphoramidite 9 (1.6 mL 0.16 M / ACN solution, 0.25 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 17 in 95% yield (0.285 g, 0.158 mmol). 1¹H NMR (400MHz, CD3CN) δ = 1.25-1.40 (m, 4H, 2×CH2 hexyl spacer), 1.40-1.51 (m, 2H, CH2 hexyl spacer), 1.58-1.71 (m, 2H, CH2 hexyl spacer), 1.80-1.92 (m, 12H, 3×5a′-H, 3×AcNH), 1.96-2.30 (m, 6H, 3×5a-H, 3×5-H), 2.68-2.83 (m, 6H, 3×CH2 cyanoethyl), 2.93 (bs, 1H, OH), 3.00-3.11 (m, 2 H, CH2 hexyl spacer), 3.59-3.89 (m, 9H, 3×H-6, 3×H-4), 3.96-4.22 (m, 11H, 3×H-3, CH2 hexyl spacer, 3×CH2 cyanoethyl), 4.31-4.86 (m, 18H, 3×H-1, 3×H-2, 6×CH2Bn), 5.03 (s, 2H, CH2Bn spacer), 5.78 (bs, 1H, NH), 6.55-6.65 (m, 1H, NHAc), 6.9-7.15 (m, 2H, 2×NHAc), 7.19-7.40 (m, 35H, H arom ). 13 ¹³C NMR (100MHz, CD3CN) δ = 20.0-20.1 (3×CH2 cyanoethyl), 22.9-23.0 (3×CH3AcNH), 25.5 (CH2 hexyl spacer), 26.5 (CH2 hexyl spacer), 28.9-29.2 (3×CH2C-5a), 30.1 (CH2 hexyl spacer), 30.5 (CH2 hexyl spacer), 38.0-40.0 (3×CH C-5), 41.1 (CH2 hexyl spacer), 50.8-51.4 (3×CH C-2), 62.5-63.0 (3×CH2C-6), 63.0-63.3 (3×CH2 cyanoethyl), 66.3 (CH2Bn spacer), 68.4 (CH2 hexyl spacer), 72.1-74.1 (6×CH2Bn), 75.2-75.5 (3×CH C-1), 75.5-76.1 (3×CH C-4), 79.3-79.5 (3×CH C-3), 128.2-129.1 (CH arom ), 138.9-139.7(7×Cq Bn), 170.9-171.2(3×C=O AcNH). 31P NMR (162MHz, CD3CN) δ=-2.82, -2.77, -2.62, -2.58, -2.36, -2.33, -2.24, -2.20, -2.16. HRMS:[C 92 H 11 4N7O 24 P3+H] + The required value was 1795.72333, and the measured value was 1795.22333.

[0332] 1-O-tetra-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(18) Using the general procedure described above, alcohol 17 (0.267 g, 0.148 mmol) was coupled with phosphoramidite 9 (1.4 mL 0.16 M / ACN solution, 0.22 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 18 in 87% yield (0.299 g, 0.129 mmol). 1 ¹H NMR (400MHz, (CD3)2CO) δ = 1.31-1.47 (m, 4H, 2×CH2 hexyl spacer), 1.47-1.57 (m, 2H, CH2 hexyl spacer), 1.62-1.75 (m, 2H, CH2 hexyl spacer), 1.85-2.02 (m, 16H, 4×5a′-H, 4×AcNH), 2.07-2.17 (m, 8H, 4×5a-H, 4×5-H), 2.82-3.00 (m, 8H, 4×CH2 cyanoethyl ), 3.08-3.18 (m, 2H, CH2 hexyl spacer), 3.66-4.01 (m, 12H, 4×H-6, 4×H-4), 4.04-4.36 (m, 14H, 4×H-3, CH2 hexyl spacer, 4×CH2 cyanoethyl), 4.40-4.94 (m, 24H, 4×H-1, 4×H-2, 8×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.39 (bs, 1H, NH), 7.17-7.42 (m, 45H, H arom ), 7.42-7.80(m, 4H, NHAc). 13¹³C NMR (100MHz, (CD3)2CO) δ = 20.0-20.1 (4×CH2 cyanoethyl), 23.1-23.2 (4×CH3AcNH), 25.8 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-29.8 (4×CH2C-5a), 30.8 (CH2 hexyl spacer), 30.8 (CH2 hexyl spacer), 38.3-40.3 (4×CH C-5), 41.4 (CH2 hexyl spacer), 51.2-51.5 (4×CH C-2), 62.6-63.4 (4×CH2C-6), 63.4-63.6 (4×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.8 (CH2 hexyl spacer), 72.0-75.0 (8×CH2Bn), 75.6-75.8 (4×CH C-1), 76.5-77.2 (4×CH C-4), 79.7-79.8 (4×CH C-3), 128.1-129.1 (CH arom ), 139.3-140.1(9xCq Bn), 170.7-171.2(4xC=O AcNH). 31 P NMR (162MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.91, -1.89. HRMS:[C 118 H 145 N9O 31 P4+2H] ++ The required value was 1155.45892, and the measured value was 1155.45892.

[0333] 1-O-penta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(19) Using the general procedure described above, alcohol 18 (0.277 g, 0.120 mmol) was coupled with phosphoramidite 9 (1.1 mL 0.16 M / ACN solution, 0.18 mmol) for oxidation and detritylation. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 19 in 92% yield (0.31 g, 0.110 mmol).1 ¹H NMR (400MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.58 (m, 2H, CH2 hexyl spacer), 1.62-1.75 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 20H, 5×5a′-H, 5×AcNH), 2.07-2.19 (m, 10H, 5×5a-H, 5×5-H), 2.82-2.97 (m, 10H, 5×CH2 cyanoethyl) ), 3.08-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.02 (m, 15H, 5×H-6, 5×H-4), 4.04-4.36 (m, 17H, 5×H-3, CH2 hexyl spacer, 5×CH2 cyanoethyl), 4.38-4.95 (m, 30H, 5×H-1, 5×H-2, 10×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.43 (bs, 1H, NH), 7.16-7.41 (m, 55H, H arom ), 7.42-7.86(m, 5H, NHAc). 13 ¹³C NMR (100MHz, (CD3)2CO) δ = 19.8-20.0 (5×CH2 cyanoethyl), 23.0-23.1 (5×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.7 (CH2 hexyl spacer), 29.2-30.0 (5×CH2C-5a), 30.7 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (5×CH C-5), 41.2 (CH2 hexyl spacer), 51.0-51.4 (5×CH C-2), 62.5-63.2 (5×CH2C-6), 63.3-63.5 (5×CH2 cyanoethyl), 66.1 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.0-75.0 (10xCH2Bn), 75.6-75.8 (5×CH C-1), 76.5-77.2 (5×CH C-4), 79.7-79.8 (5×CH C-3), 128.0-129.0 (CH arom ), 139.2-140.0(11×Cq Bn), 170.7-171.2(5×C=OAcNH). 31P NMR (162MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.47, -2.42, -2.37, -2.30, -1.96, -1.88, -1.89, -1.86, -1.84, -1.79. HRMS:[C 144 H 176 N 11 O 38 P5+2H] ++ The required value was 1412.55219, and the measured value was 1412.55219.

[0334] 1-O-Hexa-((2-Acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(20) Using the general procedure described above, alcohol 19 (0.280 g, 0.099 mmol) was coupled with phosphoramidite 9 (1.24 mL 0.16 M / ACN solution, 0.20 mmol), followed by oxidation and detritylation. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 20 in 88% yield (0.29 g, 0.087 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 24H, 6×5a′-H, 6×AcNH), 2.07-2.30 (m, 12H, 6×5a-H, 6×5-H), 2.82-2.97 (m, 12H, 6×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 18H, 6×H-6, 6×H-4), 4.04-4.38 (m, 20H, 6×H-3, CH2 hexyl spacer, 6×CH2 cyanoethyl), 4.38-5.00 (m, 36H, 6×H-1, 6×H-2, 12×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 65H, H arom), 7.42-7.89(m, 6H, NHAc). 13 ¹³C NMR (100MHz, (CD3)2CO) δ = 19.9-20.0 (6×CH2 cyanoethyl), 23.0-23.1 (6×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (6×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (6×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (6×CH C-2), 62.5-63.4 (6×CH2C-6), 63.4-63.5 (6×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (12×CH2Bn), 75.6-75.8 (6×CH C-1), 76.5-77.2 (6×CH C-4), 79.7-79.8 (6×CHC-3), 128.1-129.1 (CH arom ), 139.2-140.0(13×Cq Bn), 170.7-171.2(6×C=O AcNH). 31 P NMR (162MHz, CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 170 H 207 N 13 O 45 P6+NH4] + 3356.312 was required, and the measured value was 3357.010.

[0335] To produce an oligomer with n=6, the general deprotection procedure described below can be performed after the above steps.

[0336] 1-O-epta-((2-acetamido-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(21) Using the general procedure described above, alcohol 20 (0.140 g, 0.042 mmol) was coupled with phosphoramidite 9 (0.8 mL 0.1 M / ACN solution, 0.84 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 21 in 86% yield (0.139 g, 0.036 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 28H, 7×5a′-H, 7×AcNH), 2.07-2.30 (m, 14H, 7×5a-H, 7×5-H), 2.82-2.97 (m, 14H, 7×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 21H, 7×H-6, 7×H-4), 4.04-4.38 (m, 23H, 7×H-3, CH2 hexyl spacer, 7×CH2 cyanoethyl), 4.38-5.00 (m, 42H, 7×H-1, 7×H-2, 14×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 75H, H arom ), 7.42-7.89(m, 7H, NHAc). 13¹³C NMR (125MHz, (CD3)2CO) δ = 19.9-20.0 (7×CH2 cyanoethyl), 23.0-23.1 (7×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (7×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (7×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (7×CH C-2), 62.5-63.4 (7×CH2C-6), 63.4-63.5 (7×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (14×CH2Bn), 75.6-75.8 (7×CH C-1), 76.5-77.2 (7×CH C-4), 79.7-79.8 (7×CH C-3), 128.1-129.1 (CH arom ), 139.2-140.0(15×Cq Bn), 170.7-171.2(7×C=O AcNH). 31 P NMR (202MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 196 H 238 N 15 O 52 P7+2H] ++ The required value was 1926,73908, and the measured value was 1926,73908.

[0337] 1-O-octa-((2-acetamide-3,4-di-O-benzyl-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)2-cyanoethyl)-6-hexyl-benzylcarbamate(22) n=8 Using the general procedure described above, alcohol 22 (0.105 g, 0.027 mmol) was coupled with phosphoramidite 9 (0.7 mL 0.1 M / ACN solution, 0.68 mmol), oxidized, and detritylated. The crude product was purified by size exclusion chromatography (sephadex LH-20, DCM / MeOH 1:1) to obtain product 22 in 87% yield (0.103 g, 0.023 mmol). 1 ¹H NMR (500MHz, (CD3)2CO) δ = 1.31-1.46 (m, 4H, 2×CH2 hexyl spacer), 1.46-1.57 (m, 2H, CH2 hexyl spacer), 1.63-1.74 (m, 2H, CH2 hexyl spacer), 1.84-2.02 (m, 32H, 8×5a′-H, 8×AcNH), 2.07-2.30 (m, 16H, 8×5a-H, 8×5-H), 2.82-2.97 (m, 16H, 8×CH2 cyanoethyl ), 3.09-3.18 (m, 2H, CH2 hexyl spacer), 3.67-4.04 (m, 24H, 8×H-6, 8×H-4), 4.04-4.38 (m, 26H, 8×H-3, CH2 hexyl spacer, 8×CH2 cyanoethyl), 4.38-5.00 (m, 48H, 8×H-1, 8×H-2, 16×CH2Bn), 5.05 (s, 2H, CH2Bn spacer), 6.42 (bs, 1H, NH), 7.16-7.41 (m, 85H, H arom ), 7.42-7.89(m, 8H, NHAc). 13¹³C NMR (125MHz, (CD3)2CO) δ = 19.9-20.0 (8×CH2 cyanoethyl), 23.0-23.1 (8×CH3AcNH), 25.7 (CH2 hexyl spacer), 26.8 (CH2 hexyl spacer), 29.2-30.2 (8×CH2C-5a), 30.4 (CH2 hexyl spacer), 30.7 (CH2 hexyl spacer), 38.2-40.2 (8×CH C-5), 41.3 (CH2 hexyl spacer), 51.0-51.4 (8×CH C-2), 62.5-63.4 (8×CH2C-6), 63.4-63.5 (8×CH2 cyanoethyl), 66.2 (CH2Bn spacer), 68.7 (CH2 hexyl spacer), 72.2-75.6 (16×CH2Bn), 75.6-75.8 (8×CH C-1), 76.5-77.2 (8×CH C-4), 79.7-79.8 (8×CH C-3), 128.1-129.1 (CH arom ), 139.2-140.0(17×Cq Bn), 170.7-171.2(8×C=O AcNH). 31 P NMR (202MHz, (CD3)2CO)δ=-2.84, -2.77, -2.68, -2.45, -2.42, -2.37, -2.31, -1.94, -1.81, -1.78. HRMS:[C 222 H 269 N 17 O 59 P8+2H] ++ The required value was 2184.33410, and the measured value was 2184.33410.

[0338] General procedure for deprotection at typical scales (5-40 μmol) The starting alcohol was dissolved in NH3 (30-33% aqueous solution, 1 mL per 10 μmol) and dioxane until completely dissolved. The reaction mixture was stirred for 2 hours. The mixture was concentrated under reduced pressure. 1 1H NMR and 311P NMR analysis showed complete conversion to a semi-protected intermediate. The crude product was dissolved in MilliQ H2O and eluted using a column containing Dowex Na+ cation exchange resin (type: 50WX4-200, stored in 0.5M NaOH / H2O solution, MilliQ H2O and MeOH flushed before use). The crude product was dissolved in MilliQ H2O (2 mL per 10 μmol). 4-5 drops of glacial acetic acid were added to the reaction mixture. The mixture was purged with Ar. 1 cup of Pd Black was added to the solution. The reaction mixture was purged with H2 for several seconds and stirred under an H2 atmosphere for 3 days. Celite was added to the mixture. The solution was filtered and concentrated under reduced pressure. The crude product was purified by size exclusion chromatography (Toyopearl HW-40). The pure compound was dissolved in MilliQ H2O, eluted using a column containing Dowex Na+ cation exchange resin (type: 50WX4-200, stored in 0.5M NaOH / H2O solution, MilliQ H2O and MeOH were flushed before use), and lyophilized.

[0339] 1-O-octa-(2-acetamide-2-deoxy-5a-carba-α-D-mannopyranosyl-1-O-phosphoryl)-6-hexylamine(8)n=8 Alcohol 22 (23.2 μmol) was deprotected using the general procedure described above. Pure oligomer 8 was obtained in 44% yield (25.9 mg, 10.2 μmol). 1 ¹H NMR (500MHz, D2O) δ = 1.33-1.43 (m, 4H, 2×CH2 hexyl spacer), 1.57-1.69 (m, 4H, 2×CH2 hexyl spacer), 1.73-2.08 (m, 48H, 8×5a′-H, 8×5a-H, 8×5-H, 8×AcNH), 2.92-3.00 (m, 2H (CH2 hexyl spacer), 3.48-3.68 (m, 8H, 8×H-4), 3.68-3.76 (m, 2H, CH2 hexyl spacer), 3.81-4.22 (m, 24H, 8×H-3, 8×H-6), 4.25-4.36 (m, 8H, 8×H-1), 4.37-4.53 (m, 8H, 8×H-2). 13¹³C NMR (126MHz, D2O) δ = 21.9 (8×CH3AcNH), 24.4 (CH2 hexyl spacer), 25.1 (CH2 hexyl spacer), 26.6 (CH2 hexyl spacer), 28.0 (8×CH2C-5a), 29.5 (CH2 hexyl spacer), 38.6 (8×CH₃C-5), 39.4 (CH₃Hexyl spacer), 53.5 (8×CH₃C-2), 61.9 (8×CH₃C-6), 66.2 (CH₃Hexyl spacer), 70.1 (8×CH₃C-1), 70.4 (8×CH₃C-4), 71.9 (8×CH₃C-3), 174.7 (8×C=O AcNH). 31 P NMR (202MHz, D2O) δ=0.25, 0.37, 0.41, 0.44, 0.48. HRMS:[C 78 H 145 N9O 57 P8+H] ++ The required value was 1183.83071, and the measured value was 1183.83071.

[0340] Production of randomly acetylated carbaoligomers according to the present invention 1. Amine protection as a Boc derivative Dried carba analogs DP6 (n=6), DP7 (n=7), and DP8 (n=8) were dissolved in H2O:dioxane in a 1:1 volume ratio. Then, NaHCO3 (2.95 equivalents) and (Boc)2O (1.13 equivalents) were added at 4°C. The reaction mixture was then maintained overnight at room temperature under magnetic stirring. The product was then purified using a Sephadex G10 column (eluent: H2O), and the fraction containing the compound was dried.

[0341] 2. Random O-acetylation The dried Boc-protected carba analog from step 1 was resuspended in acetonitrile, and acetic anhydride (3.6 equivalents per -OH group in the molecule) and imidazole (1.8 equivalents) were added. The reaction mixture was kept at 40°C, and the acetylation reaction time was extended until the target acetylation rate (%) (approximately 75%) was reached. 1 The results were monitored by 1H-NMR. Next, the crude acetylated compound was dried.

[0342] To avoid misunderstanding, "random O-acetylation" is R x and R y This means that the number of -C(O)CH3 groups is ultimately not controlled. However, using NMR techniques, the total O-acetylation rate (%) in the oligomer can be determined.

[0343] In this specification, oligomers are denoted as Ac-carbaMenA along with their corresponding degree of polymerization (DP), for example, as Ac-carbaMenA DP8.

[0344] 3.Boc deprotection The dried crude O-acetylated carba analog from step 2 was dissolved in CH2Cl2:TFA 4:1 (volume ratio), and the reaction mixture was maintained at room temperature under magnetic stirring for 1 hour. Next, the crude reaction mixture was dried, redissolved in H2O, and purified using a Sephadex G10 column (eluent: H2O).

[0345] NMR protocol for measuring acetylation rate (%) The sample was vacuum-dried, regenerated with 0.6 mL of D2O, and transferred to a 5 mm NMR tube. Proton NMR spectra were obtained using a standard one-dimensional pulse program at 400 MHz and 25 °C. Spectrum acquisition and processing were performed using TopSpin Bruker software.

[0346] The O-acetylation rate (%) in carba analogs was measured by integrating the H3+H4O-Ac (i.e., H of the acetate group) peak at 5–5.4 ppm and the triplet of CH2 adjacent to NH2 of the linker at approximately 3 ppm, where a value of 2 is given. As shown in Figure 1, assuming that the integral value of H3+H4O-Ac must be 12 in DP6 (14 in DP7 and 16 in DP8) when O-acetylation is 100%, the following proportions apply. 12:100 = 9.04:X (X = acetylation rate (%))

[0347] The final product 1The structures were characterized by 1H-NMR to confirm their identity, and the O-acetylation rate (%) of the synthesized sugars was determined (Figure 2 and Table 1).

[0348] Figure 2 shows the final randomly acetylated carba analog. 1 This shows the 1H NMR spectrum, and also the integral of the acetylation rate (%) measurement (n=8).

[0349] [Table 1]

[0350] For the same random acetylated carba analog of equation (Ia) with n=8, the distribution of acetyl groups between the 3rd and 4th positions is as follows: 31 The results were obtained by 1P NMR spectroscopy (101 MHz, D2O). The recorded spectra are shown in Figure 3. This indicates simultaneous acetylation occurring at the C3 and C4 positions in approximately 44% of cases (i.e., R in the same repeating unit of the oligomer). x and R y Both are -C(O)CH3. ) and acetylation at C3 or C4 up to about 28% (i.e., the same repeating unit, R x is -C(O)CH3, and R y H is, or R x H is R y This indicates that -C(O)CH3, and that 27% of the repeating units are not acetylated.

[0351] Example 2: Production of selectively acetylated carbamonomer constituent blocks by Scheme 2 (e.g., R x H is R y (is -C(O)CH3) D-glucar (23) [ka]

[0352] A mixture of 3,4,6-tri-O-acetyl-D-glucar (10.0 g, 36.7 mmol) was mixed with 150 mL of K2CO3 (508 mg, 3.67 mmol) / dried MeOH solution, and the mixture was stirred at room temperature under N2. After 1 hour, the reaction was completed, and the reaction was stopped with acetic acid to pH 7. The solvent was removed under reduced pressure, and the crude product of D-glucar, a clear oily substance, was used directly in the next step.

[0353] 4,6-O-(4-methoxybenzylidene)-D-glucar(24) [ka]

[0354] Crude compound 23 in dry DMF (100 mL) was mixed with anisaldehyde dimethyl acetal (9.40 mL, 55.1 mmol) under N2 conditions, followed by pyridine p-toluenesulfonate (922 mg, 3.67 mmol). The reaction was carried out on a rotary evaporator under reduced pressure (180 mbar) at 25-30°C for 2.5-3 hours. The DMF was then removed under reduced pressure, and the crude product was extracted with 100 mL of DCM. The organic layer was washed sequentially with 50 mL of NH4Cl, 50 mL of distilled water, and 50 mL of brine solution. Finally, the collected aqueous layer was extracted with 50 mL of DCM. The mixture was then dried over Na2SO4, and the solvent was removed under reduced pressure to obtain 4,6-O-(4-methoxybenzylidene)-D-glucar as a white powder in 45% yield.

[0355] δ 1 H(400MHz;CDCl3) 7.43(2H, td, J8.6, J4.7, 8-H), 6.90(2H, dt, J8.8, J4.9, 9-H), 6.33(1H, ddd, J6 .1, J1.6, J0.4, 1-H), 5.55 (1H, s, 7-H), 4.76 (1H, dd, J6.1, J2.0, 2-H), 4.49 (1H) , brd, J7.3, 3-H), 4.35(1H, dd, J10.3, J5.0, 5-H), 3.93-3.87(1H, m, 6-H), 3.83 -3.79(1H, m, 6-H), 3.80(3H, s, -OMe), 3.77-3.75(1H, m, 4-H), 2.47(1H, s, -OH).

[0356] δ 13 C(100MHz;CDCl3) 159.4(11-C), 143.3(1-C), 128.6(8-C), 126.7(9-C), 112.8(10-C), 102.7(2 -C), 100.9(7-C), 79.8(4-C), 68.9(5-C), 67.6(6-C), 65.7(3-C), 54.4(OMe).

[0357] 3-O-benzyloxy-4,6-O-(4-methoxybenzylidene)-D-glucar(25) [ka]

[0358] To a 350 mL solution of 24 (16.05 g, 60.7 mmol) DMF at 0°C, 7.29 g, 182 mmol of 60% sodium hydride / mineral oil was added in small increments (the mineral oil from the NaH could be washed off three times beforehand with dry n-hexane). After stirring at the same temperature for 30 minutes, the ice bath was removed. Benzyl bromide (14.4 mL, 121 mmol) was added, and the reaction mixture was stirred overnight, during which time it was heated to room temperature. Next, the reaction was stopped with methanol (20 mL), and the DMF was evaporated under reduced pressure. The organic phase was extracted with 100 mL of siRNA, and then the organic layer was washed with NH4Cl, NaHCO3, and brine (50 mL each). The organic layer was dried with Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by flash column chromatography (siRNA / hexane = 3:7) using silica gel to obtain 3-O-benzyloxy-4,6-O-(4-methoxybenzylidene)-D-glucar (18.43 g, 86%) as a white powder.

[0359] δ 1 H(400MHz;CDCl3) 7.42(2H, dt, J8.5, J4.6, 8-H), 7.37-7.23(7H, m, H arom ), 6.90(2H, dt, J8.9, J4.9, 9-H), 6.34(1H, dd, J6.2, J1.4, 1-H), 5.58(1H, s, 7-H), 4.81(1H, dd, J6.17, J2.06, 2-H), 4.79(1H, d, J12.110-H CH2Ph), 4.70(1H, d, J12.1, 10-H CH2Ph), 4.36-4.32(2H, m, 3-H, 6a-H), 4.00(1H, dd, J9.8, J7.4, 6b-H), 3. 88 (1H, td, J10.1, J4.7, 5-H), 3.81 (1H, t, J10.1, 4-H), 3.80 (3H, s, -OMe).

[0360] δ 13 C(100MHz;CDCl3) 160.2(11-C), 144.5(1-C), 138.6(13-C), 129.9(8-C), 129.9-127.2(C arom9, 14, 15, 16-C), 113.7(10-C), 102.4(2-C), 101.3(7-C), 80.1(5-C), 73.2(4-C), 72.1(6-C), 68.8(3-C), 68.4(12-C), 55.4(-OMe).

[0361] 3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-D-glucar(26) [ka]

[0362] Glucar 25 (780 mg, 2.20 mmol) was dissolved in DCM (20 mL), cooled to 0°C, and stirred at room temperature for 20 minutes. Next, 11.0 mL, 11.0 mmol, of 1 M DIBAL-H / hexane solution was added dropwise at 0°C. The mixture was stirred at 0°C for 2 hours. The reaction was stopped for 20 minutes with a distilled aqueous solution of potassium sodium tartrate tetrahydrate (1.5 g of tartrate in 7.5 mL of water), commonly known as Rochelle salt. Next, the mixture was extracted with DCM (30 mL), and the organic layer was washed twice with distilled water and brine (40 mL each). Finally, the aqueous layer was extracted with DCM (20 mL). The organic phase was collected and dried over Na2SO4. The solvent was removed under reduced pressure. The residue was purified by flash chromatography using silica gel (siRNA / hexane = 1:3) to obtain 3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-D-glucar as a white solid in 84% yield.

[0363] δ 1 H(400MHz;CDCl3) 7.34-7.20 (7H, m, H arom), 6.83(2H, dt, J8.7, J4.8, 9-H), 6.34(1H, dd, J6.1, J1.2, 1-H), 4.82(1H, dd, J6.1, J2.6, 2-H), 4.75(1H, d, J11.1, 10-H CH2Ph), 4.63(1H, d, J11.1, 10-H CH2Ph), 4.61(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.52(1H, d, J11.8, 7-H CH2Ph(4-OMe)), 4.19(1H, ddd, J6.3, J2.4, J2.3, 3-H), 3.87(1H, dt, J8.8, J4.2, 5-H), 3. 81-3.79(2H, m, 6-H), 3.77(1H, dd, J8.7, J6.3, 4-H), 3.71(3H, s, -OMe), 2.65(1H, s, -OH).

[0364] δ 13 C(100MHz;CDCl3) 159.2(11-C), 144.4(1-C), 138.1(13-C), 130.1(8-C), 129.7-127.6(C arom 9, 14, 15, 16-C), 113.7(10-C), 100.1(2-C), 77.5(5-C), 75.6(3-C), 74.1(4-C), 73.3(12-C), 70.4(7-C), 61.4(6-C), 55.1(-OMe).

[0365] 1,5-Anhydro-3-O-benzyloxy-4-O-(4-methoxybenzyloxy)-2,6,7-Trideoxy-D-arabinohepto-1,6-dienitol(28) [ka]

[0366] DMP (926 mg, 2.18 mmol) was added to a 6.1 mL dry DCM solution of the aforementioned alcohol 26 (650 mg, 1.82 mmol). The mixture was then stirred at room temperature (25°C) for 1 hour.

[0367] Meanwhile, an ylide was prepared with a dry THF solution (12.0 mL) of fresh PPh3CH3I (1.48 g, 3.65 mmol) at -78°C and stirred for 25 minutes. Next, KHMDS (7.3 mL, 3.65 mmol, 0.5 M / toluene solution) was added dropwise at -78°C. The mixture was stirred at -78°C for 20 minutes, at 0°C for 50 minutes, and finally at -78°C for 30 minutes to form the ylide.

[0368] Furthermore, the oxidation reaction was stopped for 10 minutes with a solution of Na2S2O3 (30 mL) and NaHCO3 (30 mL). Next, the aldehyde was work-treated with DCM (3 times with 40 mL), dried with Na2SO4, and the DCM was removed under reduced pressure.

[0369] Next, aldehyde (11.0 mL) in dry THF was added dropwise to the ylide at -78°C. The reaction mixture was stirred overnight. The mixture was treated with NH4Cl (20 mL) and DCM (50 mL). The organic layer was then extracted again with DCM (twice with 30 mL), washed with NaCl (80 mL), and dried over Na2SO4. The residue was purified by flash chromatography (nHexane / Ã=7:3) to obtain the alkene as a yellow oil in two steps with a yield of 83%.

[0370] δ 1 H(400MHz;CDCl3) 7.37-7.27 (4H, m, H arom), 7.24 (2H, dt, J8.6, J5.5, 9-H), 6.86 (2H, td, J8.7, J5.5, 10-H), 6.41 (1H, dd, J6. 1, J1.3, 1-H), 6.04(1H, ddd, J17.2, J10.6, J6.6, 6-H), 5.43(1H, dt, J2.9, J17.3, 7b -H), 5.31 (1H, dt, J2.6, J10.6, 7a-H), 4.88 (1H, dd, J6.2, J2.7, 2-H), 4.70 (1H, d, J1 0.9, 11-H, CH2Ph), 4.64 (1H, d, J11.7, 8-H, CH2Ph(4-OMe)), 4.62 (1H, d, J10.9, 11-H CH2Ph), 4.58(1H, d, J11.7, 8-H CH2Ph(4-OMe)), 4.31(1H, dd, J7.1, J8.0, 5-H), 4.19(1H, ddd, J6.2, J2.5, J1.5, 3-H), 3.79(3H, s, -OMe), 3.59(1H, dd, J8.6, J6.2, 4-H).

[0371] δ 13 C(100MHz;CDCl3) 159.4(12-C), 144.6(1-C), 138.5(14-C), 134.5(6-C), 130.3(9-C), 129.8-127.8(C arom 10, 15, 16, 17-C), 118.4(7-C), 113.9(11-C), 100.5(2-C), 78.2(5-C), 78.0(4-C), 75.5(3-C), 73.6(8-C), 70.8(13-C), 55.4(-OMe).

[0372] (3R,4R,5R)-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-5-(hydroxymethyl)cyclohexene(29) [ka]

[0373] Alkene 28 (200 mg, 0.57 mmol) was dissolved in m-DCB (1.43 mL, 0.4 M) at room temperature. Next, the Claisen rearrangement was performed under microwave conditions at 265°C for 10 minutes. Once the yellow solution of reactive aldehyde was consumed, it was immediately poured into a mixture of NaBH4 (86 mg, 2.27 mmol) in THF / EtOH (10 mL, 4:1) and stirred at room temperature for 1 hour (single spot on TLC, orange-red solution). The reaction was stopped with distilled water (10 mL). The aqueous phase was increased in volume with 10 mL of distilled water and extracted by DCM (three times with 20 mL). Finally, the organic layer was dried over Na2SO4. The residue was purified by flash chromatography (n-hexane / siRNA = 8:2) to obtain alcohol 7 as a colorless oil in two steps in yield.

[0374] δ 1 H(400MHz;CDCl3) 7.28-7.16 (7H, m, H arom ), 6.79(2H, brd, J8.3, 14-H), 5.67-5.64(1H, m, 1-H), 5.64-5.59(1H, m, 2-H), 4.88(1H, d, J11.3, 8-H CH2Ph), 4.64(1H, d, J11.3, 8-H CH2Ph), 4.56(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 4.48(1H, d, J11.7, 12-H CH2Ph(4-OMe)), 4.12(1H, brd, 4-H), 3.71(3H, s, -OMe), 3.57-3.47(3H, m, 3-H, 6-H), 2. 35(1H, s, -OH), 2.07-2.00(1H, m, 7-H), 1.97-1.88(1H, m, 5-H), 1.82-1.75(1H, m, 7-H)δ 13 C (100MHz; CDCl3).

[0375] δ 13 C(100MHz;CDCl3) 159.4(17-C), 138.5(9-C), 132.1(14-C), 130.5-128.0(C arom10, 11, 12, 15-C), 127.7(1-C), 126.1(2-C), 114.0(16-C), 82.3(3-C), 80.9(4) -C), 74.4(8-C), 71.1(13-C), 65.9(6-C), 55.4(-OMe), 40.7(5-C), 28.1(7-C).

[0376] 4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5-methylcyclohexene(30) [ka]

[0377] Alcohol 29 (715 mg, 2.02 mmol) was dissolved in dry THF (17 mL) at room temperature. Imidazole (125 mg, 1.83 mmol) was added, and the mixture was stirred at room temperature for 5 minutes, then at 0°C for 10 minutes. Next, texyldimethylsilyl chloride (1.19 mL, 6.05 mmol) was carefully added dropwise to form a white precipitate. The ice bath was removed at the time of the first precipitate, and the remaining TDSCl was slowly added to the mixture. The mixture was heated to room temperature and stirred overnight. The reaction was monitored by TLC (Pent / AcOEt3:1). The organic phase was extracted with ethyl acetate and then washed with distilled water (5 times). The residue was purified by flash chromatography (nHex / AcOEt95:5) to form compound 30 as a yellow oil in quantitative yield.

[0378] δ 1 H(400MHz;CDCl3) 7.37-7.16 (7H, m, H arom), 6.88-6.84(2H, m, 14-H), 5.75(1H, ddq, J9.0, J4.3, J2.4, 1-H), 5.64(1H, brd, 2-H), 4.91(1H, d, J11.0, 8-H CH2Ph), 4.68(1H, d, J11.0, 8-H CH2Ph), 4.64(1H, d, J11.3, 12-H CH2Ph(4-OMe)), 4.60(1H, d, J11.3, 12-H CH2Ph(4-OMe)), 4.16(1H, ddq, J7.1, J3.6, J1.8, 3-H), 3.86(1H, dd, J9.8, J4.8, 6-H), 3 .79(3H, s, -OMe), 3.64(1H, dd, J10.0, J6.6, 4-H), 3.63-3.58(1H, m, 6-H), 2.28-2.16(1 H, m, 7-H), 2.10 (1H, dt, J18.4, J5.3, 7-H), 1.91 (1H, ttd, J10.5, J5.1, J2.7, 5-H), 1.64 (1H, hept, J6.9, 17-H), 0.90 (6H, d, J6.9, 18-H), 0.87 (6H, s, 16-H), 0.13 (6H, s, 15-H).

[0379] δ 13 C(100MHz;CDCl3) 159.3(14-C), 139.3(9-C), 133.8(17-C), 131.0-128.0(C arom 10, 11, 12, 15-C), 127.6(1-C), 126.3(2-C), 113.9(16-C), 81.5(3-C), 79.7(4-C), 74.7(8-C), 71.5(13-C), 62.6(6-C), 5 5.4(-OMe), 41.4(5-C), 34.3(21-C), 28.7(7-C), 25.3(19-C), 20.5-20.3(20-C), 18.8-18.7(22-C), -3.27--3.46(18-C).

[0380] 4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-glucopyranose(31) [ka]

[0381] Compound 30 (230 mg, 0.46 mmol) was dissolved in a mixture of acetone (1.69 mL) and water (562 μL). A solution of OsO4 (4.5 mL of H2O and 537 μL of a solution of 250 mg of OsO4 dissolved in 18 mL of acetone) and TMANO (116 mg, 1.02 mmol) was added at room temperature. The reaction was carried out at 25 °C for 48 hours. A saturated aqueous solution of Na2S2O3 (2 mL) was added, and the mixture was stirred at room temperature to reduce OsO4. The organic phase was extracted with CHCl3 (15 mL), washed with brine (10 mL), and finally dried over Na2SO4. The crude product was purified by flash chromatography (nHex / AcOEt, 8.2) to form diol 31 as a colorless oil in 77% yield.

[0382] δ 1 H(400MHz;CDCl3) 7.37-7.15 (7H, m, H arom ), 6.87(2H, brd, J8.7, 14-H), 4.90(1H, d, J12, 8-H CH2Ph), 4.88(1H, d, J8, 12-H CH2Ph(4-OMe)), 4.69(1H, d, J10.9, 8-H CH2Ph), 4.61(1H, d, J11.1, 12-H CH2Ph(4-OMe)), 4.05(1H, brd, J2.7, 1-H), 3.96(1H, dd, J10.0, J3.3, 6-H), 3.78(3H, s, -OMe), 3.7 1(1H, t, J9.4, 3-H), 3.48(2H, t, J10.0, 6-H, 4-H), 3.43(1H, dd, J2.3, J9.4, 2-H), 2.64(1H, s, -OH) , 2.58 (1H, s, -OH), 2.09-2.03 (1H, m, 5-H), 1.77 (1H, dt, J14.5, J3.6, 7-H), 1.62 (1H, hept, J6.9, 1 7-H), 1.59-1.52(1H, m, 7-H), 0.88(6H, d, J6.9, 18-H), 0.85(6H, d, d1.2, 16-H), 0.07(6H, s, 15-H).

[0383] δ 13 C(100MHz;CDCl3) 159.5(14-C), 138.9(9-C), 130.9(17-C), 129.7-127.7(C arom 10, 11, 12, 15-C), 114.2(16-C), 83.4(3-C), 81.0(4-C), 75.1(13-C), 74.9(8-C), 74.6(2-C), 68.5(1-C), 62.1(6-C), 55 .3(-OMe), 38.9(5-C), 34.3(21-C), 30.4(7-C), 25.2(19-C), 20.5-20.4(20-C), 18.8-18.7(22-C), -3.35--3.56(18-C).

[0384] 1-O-acetyl-4-O-benzyl-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-glucopyranose(32) [ka]

[0385] Compound 31 (155 mg, 0.29 mmol) was dissolved in acetonitrile (2.9 mL) under nitrogen at room temperature. Trimethyl orthoacetate (115 μL, 0.88 mmol) and PTSA (5 mg, 0.03 mmol) were added sequentially to the mixture, and the mixture was stirred under nitrogen at room temperature for 60 minutes. After the reaction was complete, an 80% AcOH solution (AcOH 2.32 mL + H2O 0.58 mL) was added. The subsequent acetylation reaction was completely completed in 60 minutes. The organic phase was extracted with DCM (5 mL), washed with water (5 mL) and NaHCO3 (5 mL), and finally dried over Na2SO4. The residue was purified by flash chromatography (nHex / AcOEt) to obtain compound 32, selectively acetylated at the pseudoanomeric position, as a colorless oil in quantitative yield.

[0386] δ 1 H(400MHz;CDCl3) 7.39-7.13 (7H, m, H arom)、6.87(2H、dt、J8.7、J5.0、14-H)、5.26(1H、dd、J5.7、J3.0、1-H)、4.91(1H、d、J10.6、8-H CH2Ph)、4.90(1H、d、J10.9、12-H CH2Ph(4-OMe))、4.70(1H、d、J10.0、8-H CH2Ph)、4.68(1H、d、J10.5、12-H CH2Ph(4-OMe))、3.95(1H、dd、J10.0、J3.5、6-H)、3.80(3H、s、-OMe)、3.75(1H、t、J9.3、3-H)、3.58(1H、brd、J9.6、2-H)、3.53(1H、dd、J9.1、J10.1、4-H)、3.50(1H、dd、J9.8、J2.4、6-H)、2.28(1H、s、-OH)、2.08(3H、s、-OAc)、1.95-1.88(1H、m、5-H)、1.85(1H、dt、J14.8、J7.6、7-H)、1.61(1H、dt、J13.8、J6.9、7-H)、1.61(1H、hept、J6.9、17-H)、0.88(6H、d、J6.8、18-H)、0.84(6H、d、J1.7、16-H)、0.07(6H、d、J4.4、15-H)。

[0387] δ 13 C(100MHz;CDCl3) 170.9(C(O)、-OAc)、159.5(14-C)、138.7(9-C)、130.8(17-C)、129.8-127.9(C arom 10、11、12、15-C)、114.8(16-C)、84.0(3-C)、80.5(4-C)、75.4(13-C)、75.3(8-C)、73.4(2-C)、71.8(1-C)、61.8(6-C)、55.4(-OMe)、39.6(5-C)、34.3(21-C)、28.8(7-C)、25.3(19-C)、21.4(CH3、-OAc)、20.5-20.4(20-C)、18.8-18.7(22-C)、-3.28--3.53(18-C)。

[0388] 1-O-acetyl-2-azide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-mannopyranose [ka]

[0389] Compound 32 (220 mg, 0.38 mmol) was dissolved in a mixture of DCM / pyridine (5:1, 0.05 M) and stirred at 10°C for 10 minutes under nitrogen. Triflate anhydrous (355 μL, 2.11 mmol) was added dropwise at -10°C. The mixture was stirred sequentially for 30 minutes to slowly reach 0°C, and then stirred at 0°C for another 30 minutes. After the reaction was complete, the organic phase was washed with NaHCO3 and brine. The organic layer was dried over Na2SO4, and the resulting crude product was used directly in the next step after co-distillation with toluene (3 times). Next, the dried crude product was dissolved in DMF / H2O (19:1, 0.2 M) at 40°C. Sodium azide (125 mg, 1.92 mmol) and 15-crown-5 (15.2 μL, 0.08 mmol) were added at room temperature, and the reaction was carried out overnight at 40°C. After the triflate intermediate had completely disappeared, the solvent was removed by distillation, and the residue was finally purified by flash chromatography (nHex / siRNA) to form the title compound azide as a colorless oil in 82% yield.

[0390] δ 1 H(400MHz;CDCl3) 7.38-7.14 (7H, m, H arom), 6.86(2H, dt, J8.6, J4.9, 14-H), 4.98-4.94(1H, m, 1-H), 4.88(1H, d, J10.7, 8-H CH2Ph), 4.66(1H, d, J19.1, 12-H CH2Ph(4-OMe)), 4.63(1H, d, J19.5, 12-H CH2Ph(4-OMe)), 4.59(1H, d, J10.9, 8-H CH2Ph), 3.87-3.84(1H, m, 2-H), 3.84(1H, dd, J6.3, J2.7, 6-H), 3.80(3H, s, -OMe), 3.82-3.75(2H, m, 4-H, 3-H), 3.52(1H, dd, J9.9, J2.1, 6-H), 2.00 (3H, s, -OAc), 1.91-1.82(2H, m, 5-H, 7-H), 1.65-1.57(2H, m, 7-H, 17-H), 0 .89(6H, d, J6.9, 18-H), 0.85(6H, d, J1.2, 16-H), 0.07(6H, d, J4.1, 15-H).

[0391] δ 13 C(100MHz;CDCl3) 169.8(C(O), -OAc), 159.6(14-C), 138.9(9-C), 130.2(17-C), 129.8-127.8(C arom 10, 11, 12, 15-C), 114.0(16-C), 81.1(4-C), 77.0(3-C), 75.4(8-C), 72.9(13-C), 70.6(1-C), 62.2(6-C), 61.4(2-C), 55.4(-OMe) , 39.8(5-C), 34.4(21-C), 27.1(7-C), 25.3(19-C), 21.2(CH3, -OAc), 20.6-20.5(20-C), 18.8-18.7(22-C), -3.35--3.52(18-C).

[0392] 1-O-acetyl-2-acetamide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carba-α-D-mannopyranose(33) [ka]

[0393] To the mixture of azide (334 mg, 0.56 mmol) described above, PPh3 (366 mg, 1.40 mmol) and a catalytic amount of pyridine (13.6 μL, 0.17 mmol) were added, and the mixture was stirred at 60°C for 24 hours. After the starting materials had disappeared, the solvent was removed from the resulting amine by distillation, and then it was dissolved in pyridine (5.6 mL). Acetyl anhydride (1.06 mL, 11.2 mmol) was added, and the solution was stirred again for 24 hours. The crude product was purified by flash chromatography (nHex / AcOEt) to obtain acetamide 33 as a yellow oil in 75% yield.

[0394] δ 1 H(400MHz;CDCl3) 7.39-7.28 (5H, m, H arom ), 7.19(2H, dt, J9.4, J4.6, 13-H), 6.86(2H, dt, J9.4, J4.8, 14-H), 5.59( 1H, d, J8.1, NHAc), 5.12 (1H, td, J7.2, J3.9, 1-H), 4.71 (1H, d, J11.3, 8-H CH2Ph), 4.56(1H, d, J11.3, 8-H CH2Ph), 4.50(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 4.42(1H, td, J7.7, J4.1, 2-H), 4.36(1H, d, J11.2, 12-H CH2Ph(4-OMe)), 3.84(1H, dd, J2.4, J4.0, 3-H), 3.85-3.82(1H, m, 6-H), 3.80(3H, s, -OM e), 3.72(1H, t, J6.3, 4-H), 3.60(1H, dd, J9.9, J5.5, 6-H), 2.09-2.02(1H, m, 5-H), 2.01( 3H, s, -OAc), 1.90 (3H, s, -NHAc), 1.82 (2H, tdd, J14.2, J7.4, J4.6, 7-H), 1.66-1.57 (1H, hept, J6.9, 17-H), 0.89 (6H, d, J6.9, 18-H), 0.84 (6H, s, 16-H), 0.08 (6H, d, J6.2, 15-H).

[0395] δ 13 C(100MHz;CDCl3) 170.7(C(O), -NHAc), 170.1(C(O), -OAc), 159.6(14-C), 138.6(9-C), 130.0(15-C), 129.9(17-C), 128.6-127.8(C arom 10, 11, 12-C), 114.1(16-C), 78.7(3-C), 74.4(4-C), 73.6(8-C), 71.9(13-C), 69.6(1-C), 62.5(6-C), 55.4(-OMe), 50.6(2-C), 39. 9(5-C), 34.4(21-C), 27.1(7-C), 25.2(19-C), 23.5(CH3, -NHAc), 21.3(CH3, -OAc), 20.5(20-C), 18.8(22-C), -3.37--3.48(18-C).

[0396] 1-O-Terbutylsilyl-2-acetamide-4-O-benzyl-2-deoxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose(35) [ka]

[0397] Compound 33 (582 mg, 0.95 mmol) was dissolved in MeOH (9.5 mL). NaOMe (11 mg, 0.2 mmol) was added to the mixture. The reaction mixture was stirred at room temperature for 3 hours. Amberlite H was added until a neutral pH was reached. + An ion exchange resin was added. The suspension was filtered and concentrated under reduced pressure. The crude product was removed three times by co-distillation with toluene.

[0398] Under a flow of N2 gas, 4 mL of 34 (0.95 mmol) of DCM solution was placed in a flask. At 0°C, 2,6-lutidine (2.37 mmol), followed by TBSOTf (437 μL, 1.9 mmol) was added dropwise. The mixture was stirred and heated to room temperature. After completion, the reaction was cooled to room temperature, the reaction was stopped with MeOH, and the mixture was diluted with chloroform. The mixture was washed with 10% CuSO4 aqueous solution (twice), H2O, and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (nHex / Â) yielded the title compound 35 as an orange-red oil in 83% yield in two steps.

[0399] JDC Codee et al., J. Org. Chem, 2017, 82, 2, 848-868

[0400] δ 1 H(400MHz;CDCl3) 7.41-7.24 (5H, m, H arom)、7.19(2H、dt、J9.5、J4.6、13-H)、6.86(2H、dt、J9.4、J4.8、14-H)、5.57(1H、d、J5.7、NHAc)、4.93(1H、d、J10.6、8-H CH2Ph)、4.58(1H、d、J10.5、8-H CH2Ph)、4.56(1H、d、J11.1、12-H CH2Ph(4-OMe))、4.48(1H、d、J11.1、12-H CH2Ph(4-OMe))、4.27(1H、dd、J5.2、J2.3、2-H)、4.25-4.21(1H、m、1-H)、4.03(1H、dd、J9.6、J4.5、3-H)、3.97(1H、dd、J9.7、J3.6、6-H)、3.81(3H、s、-OMe)、3.54(1H、t、J9.9、4-H)、3.48(1H、dd、J9.7、J2.2、6-H)、2.09-2.02(1H、m、5-H)、2.01(3H、s、-NHAc)、1.78-1.69(1H、m、7-H)、1.69-1.59(1H、m、17-H)、1.52-1.45(1H、m、7-H)、0.93(6H、d、J6.9、18-H)、0.87(6H、s、16-H)、0.86(6H、s、20-H)、0.84(6H、s、16-H)、0.12(6H、d、J12.0、19-H)、0.09(6H、d、J9.4、15-H)。

[0401] δ 13 C(100MHz;CDCl3) 170.7(C(O)、-NHAc)、159.5(14-C)、139.1(9-C)、130.2(17-C)、130.0(15-C)、128.6-127.7(C arom 10、11、12-C)、114.0(16-C)、78.5(3-C)、77.6(4-C)、75.5(8-C)、71.4(13-C)、67.7(2-C)、62.6(6-C)、55.4(-OMe)、53.4(1-C)、38.6(5-C)、34.6(21-C)、30.4(7-C)、25.9(25-C)、25.2(19-C)、23.6(CH3-NHAc)、20.7-20.6(20-C)、18.9-18.8(22-C)、18.0(24-C)、-3.37--3.58(18-C)、-4.82--4.92(23-NS)。

[0402] 1-O-tert-butylsilyl-2-acetamide-4-O-benzyloxy-3-O-(4-methoxybenzyloxy)-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose [ka]

[0403] A 3.4 mL solution of 14 (71 mg, 0.10 mmol) in DCM was cooled to 0°C, and freshly prepared phosphate buffer (362 μL, pH 7.5, 10 mM) was added. Freshly prepared DDQ (50.0 mg, 0.22 mmol) was added in small amounts over 1 hour, and the mixture was then warmed to room temperature and stirred for 30 minutes. The mixture was diluted with NaHCO3, and the aqueous layer was extracted twice with DCM. The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. By purification by column chromatography (nHex / siRNA), compound 15 was obtained as an orange-red solid in 72% yield.

[0404] Dan Van Der Es, Thesis, 2016, Universiteit Leiden, pp160.

[0405] δ 1 H(400MHz;CDCl3) 7.41-7.27 (5H, m, H arom), 5.52 (1H, d, J5.4, NHAc), 4.73 (2H, s, 8-H CH2Ph), 4.26(1H, brd, J2.7, 1-H), 4.16(1H, dt, J9.0, J3.8, 3-H), 4.06(1H, dd, J9.0, J4.5, 2-H), 3.94(1H) , dd, J9.9, J3.7, 6-H), 3.53 (1H, dd, J10.0, J2.1, 6-H), 3.46 (1H, t, J9.5, 4-H), 2.73 (1H, s, -OH), 2.10-2.0 3(1H, m, 5-H), 2.00(3H, s, -NHAc), 1.81-1.69(1H, m, 7-H), 1.69-1.59(1H, m, 14-H), 1.51(1H, dt, J13.7, J 3.2, 7-H), 0.93 (6H, d, J6.9, 15-H), 0.88 (6H, s, 17-H), 0.87 (6H, s, 13-H), 0.14-0.04 (12H, m, 16-H, 12-H).

[0406] δ 13 C(100MHz;CDCl3) 170.1(C(O), -NHAc), 138.8(9-C), 128.7-127.7(C arom 10, 11, 12-C), 79.6(4-C), 74.8(8-C), 70.7(3-C), 67.6(1-C), 62.9(6-C), 56.5(2-C), 38.5(5-C), 34.6(16-C), 31.2(7-C), 25.9(20) -C), 25.3(14-C), 23.6(CH3, -NHAc), 20.7-20.6(15-C), 18.9-18.8(17-C), 18.0(19-C), -3.37--3.53(13-C), -4.80--4.90(18-C).

[0407] 1-O-tert-butylsilyl-2-acetamide-4-O-benzyloxy-6-O-texyldimethylsilyl-5a-carb-α-D-mannopyranose(36) [ka]

[0408] The alcohol (180 mg, 0.32 mmol) indicated above was dissolved in dry DCM (3.2 mL) at room temperature under nitrogen. Pyridine (257 μL, 3.18 mmol), acetic anhydride (601 μL, 6.36 mmol), and a catalytic amount of DMAP (7.8 mg, 0.06 mmol) were added in that order, and the mixture was stirred until the reaction was complete. The solution was stopped with MeOH and then concentrated under reduced pressure. Compound 36 was formed in quantitative yield as a yellow oil by flash chromatography (nHex / Â).

[0409] δ 1 H(400MHz;CDCl3) 7.37-7.13 (5H, m, H arom ), 5.44(1H, dd, J10.3, J4.5, 3-H), 5.27(1H, d, J7.4, NHAc), 4.70(2H, d, J10.9, 8-H CH2Ph), 4.61(1H, d, J10.9, 8-H CH2Ph), 4.31(1H, dt, J7.3, J3.8, 2-H), 4.10(1H, brd, J2.7, 1-H), 3.97(1H, dd, J9.8, J3.2, 6-H), 3.61( 1H, t, J10.3, 4-H), 3.46 (1H, dd, J9.8, J2.0, 6-H), 2.18-2.11 (1H, m, 5-H), 2.00 (3H, s, -NHAc), 1.98 (3H, s, -OAc), 1.79-1.70(1H, m, 7-H), 1.70-1.61(1H, m, 14-H), 1.52(1H, dt, J14.3, J2.8, 7-H), 0.95(6H, d, J6.9, 15-H), 0.90 (6H, s, 17-H), 0.88 (6H, s, 13-H), 0.13 (6H, d, J15.1, 16-H), 0.09 (6H, d, J14.8, 12-H).

[0410] δ 13 C(100MHz;CDCl3) 170.0(C(O), -NHAc), 169.8(C(O), -OAc), 138.7(9-C), 128.6-127.6(C arom10, 11, 12-C), 76.2(4-C), 75.1(8-C), 73.2(3-C), 68.1(1-C), 62.3(6 -C), 54.0(2-C), 38.7(5-C), 34.6(16-C), 30.6(7-C), 25.8(20-C), 25. 3(14-C), 23.6(CH3, -NHAc), 21.2(CH3, -OAc), 20.7-20.6(15-C), 19.0-18.9(17-C), 18.1(19-C), -3.41--3.62(13-C), -4.90--4.99(18-C).

[0411] 2-Acetamide-4-O-benzyloxy-5a-carba-α-D-mannopyranose(37) [ka]

[0412] Compound 36 (120 mg, 0.20 mmol) was dissolved in dry THF (2.0 mL) at 0°C. A 30% HF / Py solution (420 μL) was added dropwise, and the reaction mixture was slowly heated from 0°C to room temperature while stirring overnight. Next, the reaction was stopped with NaHCO3 (3 mL). The organic layer was extracted twice with siRNA, washed with brine, and dried over Na2SO4. The resulting crude compound 37 was filtered through silica to obtain a white solid in 60% yield.

[0413] δ 1 H (400MHz; CD3OD) 7.37-7.26 (5H, m, H arom), 5.33 (1H, dd, J8.4, J4.4, 3-H), 4.72 (2H, d, J11.4, 8-H CH2Ph), 4.66 (1H, d, J11.4, 8-H CH2Ph), 4.45 (1H, t, J4.8, 2-H), 4.10 (1H, brd, J2.7, 1-H), 3.87 (1H, q, J4.5, 1-H), 3.78-3.73 (2H, m, 4-H, 6-H), 3.68 (1H, dd, J10.6, J4.2 , 6-H), 2.17-2.09 (1H, m, 5-H), 2.04 (1H, s, -OH), 2.03 (1H, s-OH), 2.02 (3H, s, -NHAc), 1.98 (3H, s, -OAc), 1.83 (2H, dd, J7.8, J3.8, 7-H).

[0414] δ 13 C(100MHz; CDCl3) 173.6(C(O), -NHAc), 172.0(C(O), -OAc), 140.0(9-C), 129.3-128.6(C arom 10, 11, 12-C), 77.2(4-C), 74.9(8-C), 74.7(3-C), 68.2(1-C), 63.1(6-C) , 54.0(2-C), 40.7(5-C), 30.9(7-C), 22.5(CH3, -NHAc), 21.1(CH3, -OAc).

[0415] 2-アセトアミド-4-O-ベンジルオキシ-6-O-ジメトキシトリチル-5a-カルバ-α-D-マンノピラノース(38)

change

[0416] Compound 37 (15 mg, 42.7 μmol) was dissolved in dry DCM under nitrogen at room temperature. Dry pyridine (5.2 μL, 64.0 μmol) and DMTrCl (217 mg, 64.0 μmol) were added sequentially, and the mixture was stirred at room temperature for 3 hours. Next, H2O was added to the reaction product. The organic layer was washed once with brine, dried over Na2SO4, and concentrated under reduced pressure. By purification by flash chromatography (nHex / AcOEt, 0.1% TEA), compound 38 was obtained as a white solid in 74% yield.

[0417] δ 1 H (400MHz; CD3OD) 7.40-7.05 (14H, m, H) arom ), 6.79(4H, dd, J8.9, J1.7, 13-H), 5.24(1H, dd, J7.9, J4.3, 3-H), 4.53(1H, d, J11.3, 8-H CH2Ph), 4.38(1H, t, J4.8, 2-H), 4.31(1H, d, J11.3, 8-H CH2Ph), 3.79(1H, q, J5.2, 1-H), 3.72(3H, s, -OMe), 3.72(3H, s, -OMe), 3.61(1H, t, J8.1, 4-H), 3.34-3.26(1H, m, 6-H), 3.05(1H , t, J8.3, 6-H), 2.34-2.24(1H, m, 5-H), 2.08-1.98(1H, m, 7-H), 1.95(3H, s, -NHAc), 1.86(3H, s, -OAc), 1.85-1.79(1H, m, 7-H).

[0418] δ 13 C (100MHz; CD3OD) 173.6(C(O), -NHAc), 172.0(C(O), -OAc), 160.0(17-C), 146.7(9-C), 137.6(14-C), 137.5(14-C), 137.3(9-C), 131.4(18-C), 129.9-126.3(C arom10, 11, 12, 15, 19, 20, 21-C), 114.0(16-C), 87.2(13-C), 77.3(4-C), 74.5(3-C), 74.4(8-C), 68.0(1- C), 65.0(6-C), 55.7(-OMe), 54.1(2-C), 39.2(5-C), 31.9(7-C), 22.5(CH3, -NHAc), 21.1(CH3, -OAc).

[0419] Example 3: Preparation of acetylated oligomeric conjugates - CRM-MenA DP6 (without OAC) and CRM-MenA DP8 (without OAc) The starting oligomers (DP6 and DP8) were vacuum-dried and dissolved in a 1:9 H2O:DMSO solution to a final amino group concentration of 40 mmol / mL. These were then reacted with a 12-fold molar excess of di-N-hydroxysuccinimidyl adipic acid linker (SIDEA) in the presence of triethylamine in a 5-fold molar excess compared to the amino groups. The reaction was maintained at room temperature for 3 hours with gentle stirring. The activated oligosaccharides were precipitated with 4 times the volume of ethyl acetate, and then purified by washing the pellet 10 times with 1 mL of the same solvent. Finally, the pellet was vacuum-dried, and the content of the introduced N-hydroxysuccinimidyl ester groups was determined.

[0420] The conjugates were prepared in 50 mM NaH2PO4 pH 7 using an active ester (AE) to protein molar ratio of 40:1, and allowed to stand overnight at room temperature with gentle stirring. The conjugates were purified by tangential flow filtration (Vivaspin) using a 30 kDa cutoff and PBS pH 7.2 as the buffer. The conjugates were characterized for total protein content by SDS-PAGE and micro-BCA (Smith, PK, et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85), and for total sugar content by MALDI analysis. As shown in Table 2 below, sugar / protein molar ratios of 16.9 and 10.4 were determined by MALDI TOF MS for two conjugates from carba DP6 and DP8, respectively.

[0421] [Table 2]

[0422] Sodium dodecyl sulfate-polyacrylamid gel electrophoresis (SDS-PAGE). SDS-PAGE was performed on precast 3-8% polyacrylamide gels (NuPAGE® Invitrogen). For electrophoresis, 5 μg of protein was loaded for each sample and the electrophoresis was performed for approximately 40 minutes in Tris-acetate SDS running buffer (NuPAGE® Invitrogen) using an electrophoresis chamber at 150 V. Samples were prepared by adding 3 μL of NuPAGE® LDS sample buffer. After electrophoresis, the gels were washed three times with H2O and stained with Coomassie.

[0423] Example 4: Preparation of the oligomer conjugate of the present invention according to formula (IIa) The randomly produced O-acetylated carba analogs were activated with di-N-hydroxysuccinimidyl adipate relinker (SIDEA), and the activation rates (%) obtained for the oligosaccharides were estimated to be 56% for DP6OAc, 79% for DP7OAc, and 84% for DP8OAc.

[0424] Activated oligosaccharides (i.e., activated O-acetylated carba analogs) were freeze-dried and prepared for the conjugation process. Conjugates were obtained by applying the chemistry reported in Figure 4 and the same figure showing SDS-PAGE characterization, and the smear of the conjugates could be observed.

[0425] As shown in Table 3, purified complex carbohydrates (i.e., those containing random O-acetylated carbaenids) were characterized in terms of protein content using MicroBCA and sugar content using HPAEC-PAD.

[0426] [Table 3]

[0427] Example 5: In vitro selection of oligomer length A key prerequisite for the immunogenicity of carba analogs is their ability to mimic natural MenA CPS. To investigate this property in vitro, the binding of oligomers of different lengths to the bactericidal anti-MenA mAb (JW-A1) was tested in comparison with moderate-length oligomers containing CPS and avDP~15, according to the methods described in Giuntini, S. et al, PLoS One, 2012, e34272; Tsang, RS et al., Clin. Diagn. Lab. Immunol. 2005, 12, 152-156; and Reyes, F. et al., Biologicals, 2013, 41, 275-278.

[0428] Surprisingly, as shown in Figure 5A, DP4-7 did not recognize mAb, but coupling was confirmed in DP8. However, the IC was four orders of magnitude higher compared to natural CPS. 50 The results showed that de-oacetylation of MenA CPS reduced its recognition, demonstrating the specificity of the mAb to the acetylated epitope.

[0429] If carbaDP8 acts as an inhibitor equivalent to OAc CPS, MenA-CRM 197 Similar behavior was observed when inhibitor assays were performed using anti-MenA polyclonal serum produced by immunization of mice with conjugates (Figure 5B).

[0430] This supports the idea that carba DP8 readily binds to anti-MenA antibodies. Next, different fragments were tested against anti-OAc MenA serum. In this case, de-OAc CPS(IC) 50 = 1.86 × 10 -6 ) inhibits similarly to the Ac equivalent, indicating serum specificity to the CPS skeleton independently of the acetylation pattern. Most importantly, recognition was observed for all carba DP4-8 analogs, indicating that they are all similar to the deacetylated MenA CPS moiety.

[0431] In particular, the coupling affinity (IC) of the carba DP8 50 = 1.59 × 10 -2 mM) is DP7 (IC 50 = 1.84 × 10 -1 The result was an order of magnitude higher than mM. Based on this result, DP8 and DP6 were selected as conjugations to carrier proteins, and their ability to induce antibodies in mice was compared. The former clearly bound to the anti-MenA antibody, while the latter showed no recognition at all.

[0432] Example 6: CRM of Carba MenA DP6 and DP8 197 Conjugation to Using a di-N-hydroxysuccinimidyl adipic acid linker, the carbaMenA DP6 and DP8 compounds were converted to CRM using the conjugation procedure previously reported by Adamo et al. (ACS Chem. Biol., 2012, 7, 1420-1428) and Adamo et al. (J. Carbohydr. Chem., 2011, 30, 249-280). 197 The compounds were coupled. Conjugates produced by this method are known not to induce anti-linker antibodies (Adamo et al., Chem. Sci., 2014, 5, 4302-4311). After treating the amines of DP6 and DP8 compounds with a spacer in triethylamine-containing DMSO, the resulting activated oligomers were purified by co-precipitation with acetone and used for conjugation. CRM was performed in a buffered pH 7.2 solution at a 100:1 glycan / protein molar ratio (measured by NHS quantification, corresponding to approximately 40-50 active ester:protein). 197 By incubating them overnight, the desired neo-complex carbohydrates were obtained, evaluated by SDS-PAGE gel electrophoresis, and purified by dialysis against a 30 kDa MW cutoff membrane. The sugar / protein molar ratios of 16.9 and 10.4 were determined for the two conjugates from carba DP6 and DP8, respectively, by MALDI TOF MS.

[0433] Calva MenA CRM 197 Immunogenicity of conjugates To investigate the immunogenicity of the conjugate carba MenA DP6 and DP8, eight BALB / c female mice were immunized with neocomplex carbohydrates according to the method described in detail below. Conjugates prepared from sized MenA polysaccharides avDP8.5 and ~15 according to previously reported methods were used as controls.

[0434] Mice were administered three subcutaneous (sc) doses (2 μg based on glucose) at two-week intervals. The neo-complex carbohydrate induced an immune response in week 3, as observed by assaying serum conjugate-induced samples against the same product coated on ELISA plates. In the serum dilutions tested, anti-CRM 197 No antibodies were detected at all. Each conjugate yielded an antibody that recognized the conjugate antigen, and the specificity of this recognition was confirmed by competitive ELISA. Binding of anti-carbaMenA DP8 serum was significantly inhibited by the non-conjugated octamer compared to its conjugated form due to multivalent exposure to the antigen. Furthermore, this binding was inhibited by de-OAc CPS by approximately 25%, and nearly equally by spontaneously acetylated forms, suggesting the possibility that the resulting antibodies recognize the capsule structure.

[0435] To measure the ability of induced antibodies to cross-react with acetyl-desorbed CPS structures, serum assays were performed against non-acetylated CPS conjugates in human serum albumin (HSA). The carbaMenA conjugate clearly showed an anti-desorbed OAc CPS immune response, but the IgG titer was lower than that induced by the control conjugates avDP8.5 and ~15 (p ≤ 0.003, Figure 6B). There was no statistically significant difference in the responses of the two carba analog conjugates. However, the number of responder mice to the DP8 conjugate was greater than that to the DP6 conjugate.

[0436] When ELISA was performed using acetylated CPS as a coating reagent, the differences between carbaMenA DP6 and DP8 neocomplex carbohydrates and the control-induced anti-natural MenA IgG titers were even more pronounced (p<0.0001, Figure 6A).

[0437] Next, the functionality of induced antibodies in pooled serum was evaluated by measuring complement-mediated lysis of acetylated CPS-expressing bacteria, following the methods reported in Gao, Q et al, CS Chem. Biol, 2013, 8, 2561-2567; and Adamo, R. J, Glycoconj. 2014, 31, 637-647. This assay is considered a predictor of human protection for meningococcal vaccines.

[0438] This revealed that pooled serum from responder mice produced using carbaMenA DP6 and DP8 conjugates showed low bactericidal activity (1024 vs. 512, respectively). When serum bactericidal activity (SBA) was measured using human complement, carbaDP8-CRM 197 The titer was 128, which was significantly lower than that of serum SBA produced by the benchmark vaccine at the natural MenA CPSavDP ~15 standard. These results (shown in Figure 6A) are consistent with observations reported in the literature that antibodies induced by the de-OAc MenA CPS conjugate have little functionality (Berry, D. S et al., Infect, Immun, 2002, 70, 3707-3713).

[0439] In summary, this data indicates that carbaMenA DP8 is an effective and stable mimetic of MenA CPS capable of binding anti-MenA CPS antibodies. The carbaMenA DP8 conjugate induced antibodies capable of activating human complement deposition, while carbaMenADP6 did not. This highlights the DP8 molecule as a lead antigen. However, the carbaMenA DP8 neocomplex carbohydrate vaccine induced only very low levels of bactericidal anti-MenA antibodies. Therefore, considering the inconsistency of O-acetylation at positions 3 and 4 of MenA CPS, the inventors attempted to further enhance similarity to natural polysaccharides and increase the production of protective antibodies by randomly O-acetylating the carbaMenA DP8 lead molecule.

[0440] Example 7: Optimization of sugar-mimicking vaccine candidate To introduce the acetyl ester into carbaMenA DP8, a Boc protecting group was first temporarily introduced to the amine group of the linker. Next, the resulting compound was carefully acetylated by Ac2O / imidazole treatment to reach a 75% acetylation level, similar to that of natural CPS. Then, Boc deprotection was performed to obtain Ac-carbaMenA DP8 ready for conjugation. NMR analysis revealed that acetyl was present at either the C-3 or C-4 position, and that co-acetylation at C-3 / 4 occurred at up to approximately 44%. To investigate this compound as an antigen, the inventors first evaluated its binding to anti-MenA CPS mAb using competitive surface plasmon resonance (SPR) experiments. This SPR was optimized to achieve higher sensitivity compared to conventional standard ELISA.

[0441] Ac-CarbaMenA DP8 reacts with di-N-hydroxysuccinimidyl adipate and spacer, and CRM 197 A two-step procedure used for non-acetylated oligomers, involving incubation, is used in CRM 197 It was conjugated. The purified neo-complex carbohydrate was used as a comparison with avDP~15CRM. 197 It was used in an immunization study with 10 BALB / c female mice using a conjugate. After three subcutaneous injections (2 μg based on glucose), serum was collected and analyzed for bactericidal IgG content. As shown in Figure 6C, Ac-carbaDP8-CRM 197 This is the non-acetylated form, carbaDP8-CRM 197 Compared to (shown in Figure 6A), its inhibitory ability was more than four orders of magnitude higher, and its binding to mAbs was almost equivalent to that of the natural avDP8 and avDP~15 oligomers.

[0442] Surprisingly, Ac-Calva MenA DP8-CRM 197The conjugate induced higher levels of anti-MenA CPS antibodies compared to the control. SBA titers analyzed in individual mice also showed that the synthetic antigen could induce rabbit complement-mediated bactericidal killing of MenA bacteria in a statistically non-inferior manner compared to the vaccine benchmark (Figure 6D). Analysis of pooled serum confirmed that the human complement-mediated bactericidal activity was comparable between Ac-carbaMenA DP8 and native avDP~15, thus demonstrating that Ac-carbaMenA DP8 is a truly potent mimetic of MenA CPS and can be used to produce a stabilized neo-complex carbohydrate vaccine.

[0443] Example 8: Immunological evaluation of randomly O-acetylated carbaMenA DP6 and DP8 analogues To investigate the immunogenicity of conjugated carba DP6 and DP8 analogs with and without random acetylation, a group of eight BALB / c female mice were immunized with neocomplex carbohydrates. MenA polysaccharides of conjugate size were used as a control. Mice were immunized with three subcutaneous (sc) doses (2 μg based on glucose) at two-week intervals. When the anti-MenA CPS response was evaluated, the data showed no response for conjugates obtained with carba MenA glycoantigens without O-acetylation, for both glycan lengths 6 (n=6) and 8 (n=8). Conversely, carba MenA conjugates obtained after random O-acetylation of oligomers induced a significantly higher response to natural MenA CPS compared to the non-acetylated vaccine (Table 4). For comparison, the response induced by the O-acetylated vaccine was lower than that of the benchmark MenA-CRM197 conjugate, but only twice as low as DP8, which gave a better response among those tested.

[0444] The vaccine formulations used in the CarbaMenA conjugate were as follows: 324.96 μL of AlPO4 (4.43 mg / mL, containing 2 mg / mL of NaCl) was added to the target conjugate. The volume was adjusted to 1.2 mL with pH 7.2 PBS buffer to achieve an AlPO4 concentration of 1.2 mg / mL. Finally, the solution was diluted 1:1 by volume with PBS to a volume of 2.4 mL with a final AlPO4 concentration of 0.6 mg / mL. 200 μL of the formulation was injected per mouse. This procedure was used for MenA-CRM from the stock solution. 197 It was also used in the formulation of the drug.

[0445] Table 4 shows the ELISA responses after 2 doses and 3 doses. As can be seen from the table, groups 2 and 3 are according to the present invention. For group 2, an oligomeric conjugate with n=6 and random acetylation as described above was used. For group 3, an oligomeric conjugate with n=8 and random acetylation as described above was used. The acetylation level of the conjugates in groups 2 and 3 was approximately 75%.

[0446] [Table 4]

[0447] Figures 8A and 8B show the ELISA titers after two and three doses. The p-value indicates a comparison between the benchmark natural MenA-CRM197 and the other groups.

[0448] The above random O-acetylated carbaMenADP8 analogs of the present invention are all CRM 197 A second immunological trial was conducted by comparing carbaMenADP8, which is conjugated to and selectively O-acetylated only at position 3 with approximately 70% O-acetylation, with the MenA vaccine as a positive control.

[0449] Three groups of 10 Balb / C mice were immunized with the above conjugates. Mice were immunized by subcutaneous (sc) administration (2 μg based on glucose; 200 μL / mouse) three times, with a 2-week interval between doses. The vaccine formulation used for the carba MenA conjugate was the same as that reported above for the first immunological study. When the anti-MenA CPS response was evaluated, the data (summarized in Table 5) showed that after the third immunization, the total IgG response for 3O-acetylated carba MenA DP8 was approximately 10 times lower compared to the MenA vaccine benchmark. Conversely, the random O-acetylated carba MenA DP8 conjugate of the present invention induced a significantly higher response to natural MenA CPS compared to the 3O-acetylated conjugate and was substantially equivalent to that of the MenA vaccine benchmark (see Figure 7).

[0450] [Table 5]

[0451] Figure 9 shows the CRM of selectively 3-O-acetylated carbaMenA DP8 and randomly acetylated carbaMenA DP8 of the present invention after three administrations. 197 - Indicates the human complement-mediated serum bactericidal titer induced by the conjugate. MenA-CRM 197 The vaccine was used as a positive control.

[0452] O-acetylated carba MenA-CRM 197 SBA titers induced by the conjugate were statistically equivalent to those of the MenA vaccine after three doses, but 3O-acetylated carbaMenA-CRM 197 When measured in both rabbit infant complement and human complement, the conjugate induced significantly lower serum SBA titers compared to the vaccine benchmark.

[0453] The results reported in Figure 10 and Table 6 indicate that anti-MenA antibodies may be bactericidal against MenA strains, particularly natural MenA-CRM. 197The vaccine and the vaccine obtained using two O-acetylated synthetic carbaenid analogs (Group 2 (DP6) and Group 3 (DP8)) maintained significant bactericidal activity even when tested with human complement. The DP8 O-acetylated synthetic carbaenid analog (Group 3) according to the present invention shows better bactericidal activity than the DP6 O-acetylated synthetic carbaenid analog (Group 2). Figure 10 shows the SBA titers obtained in rabbit (rSBA) and human (hSBA) complement after two and three doses.

[0454] [Table 6]

[0455] conclusion Based on the data obtained, it can be concluded that the carbaMenA oligomer of the present invention can be used in the development of a more stable version of the MenA vaccine, and that the OAc portion, combined with the oligomer length, is key to inducing a functional immune response against the MenA strain.

[0456] method Preparation of neo-complex carbohydrates. Amino-oligosaccharides are vacuum-dried and dissolved in a 1:9H2O:DMSO solution to obtain a final amino group concentration of 40 mmol / mL. -1 The mixture was then reacted with a 12-fold molar excess of di-N-hydroxysuccinimidyl adipate linker (SIDEA) in the presence of triethylamine in a 5-fold molar excess compared to the amino groups. The reaction mixture was maintained at room temperature for 3 hours with gentle stirring. The resulting activated oligosaccharide was precipitated with 4 volumes of ethyl acetate, and then purified by washing the pellet 10 times with 1 mL of the same solvent. Finally, the pellet was vacuum-dried, and the content of the introduced N-hydroxysuccinimidyl ester groups was measured.

[0457] The conjugate was prepared by gently stirring overnight at room temperature in 50 mM NaH2PO4 pH7 with an active ester (AE) to protein molar ratio of 40:1. The conjugate was purified by tangential flow filtration (Vivaspin) using PBS pH7.2 as the buffer, with a 30 kDa cutoff. The total protein content of the conjugate was characterized by micro-BCA (Smith, PK, et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85), and the total sugar content was characterized by MALDI analysis.

[0458] Mouse immunization and ELISA analysis: All mice were housed under conditions free from specific pathogens. Antigen preparations were prepared under sterile conditions. Groups of 10 BALB / c mice were immunized on days 1, 14, and 28, and blood samples were collected on day 0 (pre-immunization), day 27 (after second dose), and day 42 (after third dose). The vaccine was administered as a glucose dose, with a glucose dose of 2 μg / mouse / dose. The adjuvant AlPO4 was added in 0.12 mg of Al 3+ The drug was used at the specified dose. Antibody responses induced by the complex carbohydrate were measured by ELISA. In this analysis, preimmune serum was used as a negative control. 5 μg / mL in 100 μL / well of PBS buffer at pH 8.2. -1 The plates were coated with HSA-de-OAc or MenA CPS by adding a polysaccharide solution and then incubating overnight at 4°C

[46] . HSA-de-OAc MenA CPS, CRM 197 Conjugate and CRM 197 This is 2 μg / mL in pH 7.2 PBS buffer. -1The plates were coated with the specified protein concentration. The coating solution was removed from the plates by washing three times with PBS buffer (TPBS) containing 0.05% Tween20 (Sigma). Next, a blocking step was performed by adding 100 μL / well of 3% BSA solution in TPBS and incubating the plates at 37°C for 1 hour. The blocking solution was removed from the plates by washing three times with TPBS. 200 μL / well of undiluted serum (1:25 for pre-immuno-negative control, 1:200 / 1:500 for reference serum and 1:25 / 1:200 for test serum) was added to the first well of each row of the plate, and 100 μL of TPBS was dispensed into the other wells. Next, eight 2x dilution series were prepared along each row by moving 100 μL of serum solution from well to well. After the initial antibody dilution, the plates were incubated at 37°C for 2 hours. The plates were washed three times with TPBS, and 100 μL / well of TPBS solution of secondary antibody alkaline phosphate conjugate (anti-mouse IgG 1:10000, Sigma-Aldrich) was added. The plates were incubated at 37°C for 1 hour. After three further washes with TPBS, 100 μL / well of p-NPP(Sigma) in 0.5 M di-ethanolamine buffer pH 9.6 was added. Finally, the plates were incubated at room temperature for 30 minutes and read at 405 nm using a Spectramax 190 plate reader. Serum titer was expressed as the reciprocal of the serum dilution corresponding to a cutoff OD=1.

[0459] Each immunization group was expressed as the geometric mean (GMT) and the 95% confidence interval (CI) of the single mouse titer. Statistical and graphical analyses were performed using GraphPad Prism software.

[0460] In vitro bactericidal assay: Functional antibodies induced by vaccine immunization were analyzed by measuring complement-mediated lysis of Neisseria meningitidis using an in vitro sterilization assay (Jackson, L. A et al., Clin. Infect. Dis., 2009, 49, e1-10). Commercially available lot of infant rabbit complement (Peel Freeze Biological, code 31061) was used as the source of active complement for rSBA, and plasma was used as the source of complement for hSBA.

[0461] Meningococcal (N. meningitidis) strains were grown overnight on chocolate agar plates at 37°C in 5% CO2. Colonies were inoculated into Mueller-Hinton broth containing 0.25% glucose and incubated at 37°C with shaking until the OD600 reached 0.05–0.08. When the OD600 of the bacterial suspension reached 0.25–0.27, the bacteria were diluted with assay buffer (DPBS containing 1% BSA and 0.1% glucose) to a working dilution ratio (approximately 10%). 4 CFU·mL -1The test serum was diluted using the following method: The total volume in each well was 0 μL, followed by 25 μL of a serial 2-fold dilution of the test serum, 12.5 μL of bacteria at the working dilution, and 12.5 μL of complement source. The test serum was pooled and heat-inactivated at 56°C for 30 minutes. Negative controls included complement serum without the test serum, complement serum containing the test serum, heat-inactivated complement, and bacteria incubated separately. Immediately after adding the baby rabbit complement, the negative controls were seeded onto Mueller-Hinton agar plates using the tilt method (time 0). Microtiter plates were incubated at 37°C for 1 hour, and then each sample was spot-added in double rows onto Mueller-Hinton agar plates, with the controls seeded using the tilt method (time 1). The agar plates were incubated overnight at 37°C, and colonies (surviving bacteria) corresponding to time 0 and time 1 were counted. Serum bactericidal titer was defined as the serum dilution in which, after incubation of bacteria in the reaction mixture for 60 minutes, the colony-forming units (CFU) / mL decreased by 50% compared to the control CFU / mL at time 0. Typically, bacteria incubated in the presence of complement in the test pool or without individual mouse serum (negative control) decreased during the 60-minute incubation period. -1 It showed an increase of 150-200%. The reference strain for meningococcal serotype A was F8238 (Mak, PA, Santos, GF, Masterman, KA, Janes, J., Wacknov, B., Vienken, K., Giuliani, M., Herman, AE, Cooke, M., Mbow, ML, Donnelly, J., Clin. Vacc. Immunol., 2011, 18, 1252-1260.).

[0462] statistical method For data obtained from ELISA, a nonparametric t-test was performed, and the Mann-Whitney hypothesis was used to determine the two control groups (CRM). 197 -MenA avDP15 and CRM 197- The ELISA was performed using GraphPad software to compare ranks between MenA DP6OAc or DP8OAc. ELISA data were reported as geometric mean with a CI of 95%. Additionally, fixed effects such as group (all except 4 and 5), time, and group / time interactions were analyzed. 10 An analysis of variance (ANOVA) model was fitted to the antibody titers. Since identical variances were not assumed between groups, a heterogeneous variance model was used. For each endpoint, this model was used to estimate the geometric mean and its 95% confidence interval, as well as the geometric mean ratio (O-acetylated formulation vs. benchmark) and its 95% confidence interval. In contrast, for SBA data, since there was only a single observation for each group at each time point (serum pool), only graph analysis was performed.

[0463] Protocol for the quantification of hydrolyzed MenA and carbaMenA oligomers in the final conjugate. The amount of monomers released over time from the MenA and carbaMenA conjugates of the present invention was quantified using HPAEC-PAD. The reported titers were obtained by hydrolyzing the sample in a dry heat oven at 110°C for 2 hours with HCl at a final concentration of 6 M. After incubation, the sample was dried in a Speedvac system, redissolved in water, and filtered through 0.45 μm. Quantification was performed using a standard curve prepared for the range of 0.5–5.0 μg / mL for carbaMenA DP7 treated as the sample, quantified by NMR. Analysis was performed on an ICS5000 system (Dionex-Themo Fisher) equipped with a guarded CarboPac PA1 column. Elution was performed using a sodium acetate gradient in the presence of 100 mM sodium hydroxide at 1.0 mL / min, and peaks were detected by pulsed integrated current measurement using a fourth-harmonic waveform for carbohydrates. Results were prepared using Chromeleon® 7.2 Chromatography Data System (CDS) software.

[0464] Example 9: Comparison of ABCWY combination vaccine and CalvaMenA-BCWY combination vaccine To investigate the MenA carba (random OAc) response in combination with the MenBCWY antigen, four groups of Balb / C mice were immunized with the vaccine formulations shown in Table 7 below. The mice were immunized three times subcutaneously (sc) at two-week intervals (days 1, 14, and 28) with 2 μg of the formulation (200 μL / mouse), and blood samples were collected on day 0, day 27, and day 42.

[0465] The vaccine formulation used in the carbaMenA conjugate was the same as that reported above for the initial immunological trial.

[0466] * B NG This indicates that the MenB antigen component of the composition consists of the BEXSERO vaccine antigen, along with an additional fHbp fusion protein corresponding to the 231.13 fusion protein identified above as SEQ ID NO: 35.

[0467] [Table 7]

[0468] Groups 1 and 2 were administered a vaccine formulation containing solid (lyophilized) MenA component. Groups 3 and 4 were administered a complete liquid formulation. For clarity, MenA carba corresponds to carba MenA.

[0469] Total IgG was measured by HT-ELISA in single and pooled serum after three doses, and in pooled serum after two doses. As shown in Figure 11, the IgG titer induced by carbaMenA is equivalent to that of MenA combined with BCWY antigen.

[0470] An important point is that the first column in Figure 11 (MenAB) NG The data shown in CWY) pertains to the freeze-dried MenA component mixed with the liquid BCWY component, and is MenA carba(random OAc) + MenB NGThe data regarding CWY pertains to the complete liquid formulation (without lyophilized MenA component).

[0471] When the functional antibody response was measured using both rSBA and hSBA, as shown in Figure 12, B NG Antibody functionality induced by carbaMenA in combination with CWY is benchmarked against combination AB. NG It is equivalent to CWY.

[0472] Therefore, the immunogenic composition according to the present invention has the advantage of being fully effective in liquid formulation without compromising the immunoefficacy of a benchmark pentavalent composition incorporating a lyophilized MenA component that requires regeneration with a BCWY component before administration.

[0473] Example 10: Comparison of ABCWY combination vaccine and CalvaMenA-BCWY combination vaccine in rats method Manufacturing of Neo-Complex Carbohydrates To introduce acetyl esters into carbaMenA DP8 and DP10, the inventors first temporarily attached a Boc protecting group to the amine group of the linker. Next, the resulting compounds were carefully acetylated by Ac2O / imidazole treatment to reach a 75% acetylation level, similar to that of natural CPS. Subsequently, Boc deprotection was performed to obtain Ac-carbaMenA DP10 and Ac-carbaMenA DP10, which were ready for conjugation.

[0474] The amino-oligosaccharide was vacuum-dried and dissolved in a 1:9H2O:DMSO solution to obtain a final amino group concentration of 40 mmol / mL. -1The mixture was then reacted with a 12-fold molar excess of di-N-hydroxysuccinimidyl adipate linker (SIDEA) in the presence of triethylamine in a 5-fold molar excess compared to the amino groups. The reaction mixture was maintained at room temperature for 3 hours with gentle stirring. The resulting activated oligosaccharide was precipitated with 4 times the volume of ethyl acetate, and then purified by washing the pellet 10 times with 1 mL of the same solvent. Finally, the pellet was vacuum-dried, and the content of the introduced N-hydroxysuccinimidyl ester groups was measured.

[0475] The conjugate was prepared in 100 mM NaH2PO4 pH7 using the active ester (AE):protein molar ratio reported in the table below (Table 8).

[0476] [Table 8]

[0477] The reaction was carried out overnight at room temperature with gentle stirring. The conjugate was purified by tangential flow filtration (Vivaspin) using a 30 kDa cutoff and 10 mM NaH2PO4 pH 7.2 buffer. The total protein content of the conjugate was determined by micro-BCA (Smith, PK, et al. (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85), and the characteristics were determined by SDS-PAGE and Western blotting (Figure 13).

[0478] [Table 9]

[0479] Rat immunization All rats were housed under conditions free from specific pathogens. Antigen preparations were prepared under sterile conditions. Ten CD(SD) sprague-dolly rats were immunized on days 1, 22, and 36, and blood samples were taken on day 0 (pre-immunization) and day 49 (post-third dose). The vaccine was administered intramuscularly (IM) at a dose of 1 / 5 of the human dose of ACWY-7B, i.e., a 1:5 dilution (1:5dil) (1 / 5HD). The adjuvant AlOH was used at a dose of 3 mg / mL.

[0480] [Table 10]

[0481] MenACWY RAT ELISA (conventional HT-ELISA): Plates were coated with 5 μg / mL solutions of each polysaccharide (A, C, W135, Y) in PBS 1 × pH 8.2 and incubated at +2 to 8°C. After washing (PBS 1 × Tween 20), the plates were blocked by adding 200 μL of Smartblock (Candor Bioscience) and incubated at room temperature for 2 hours. After washing, the plates were sealed with a Liquid Plate Sealer (Candor Bioscience) and incubated at room temperature for 2 hours. Finally, the plates were aspirated and stored in a refrigerator at 2 to 8°C.

[0482] The samples were diluted in a PBS 1×BSA 1% pH 7.4 solution at initial dilutions of 1:100 (MenA and MenY), 1:500 (MenC), and 1:200 (MenW135), and then in a further 5-fold 2-fold dilution series.

[0483] Next, the plate was incubated at 30°C for 90 minutes, washed according to the method described above, and 100 μL of secondary antibody (anti-RAT total IgG alkaline phosphatase conjugate) solution was added. Then, the plate was incubated at 30°C for 60 minutes.

[0484] After washing, 100 μL of substrate (para-nitrophenyl phosphate) was added to the plate, incubated at 30°C for 30 minutes, and then read at 405 nm.

[0485] Bexsero+NG RAT ELISA (conventional HT-ELISA): Plates were coated with 0.15 μM solutions of recombinant proteins (287-953, 936-741, 961c, 741-231.16) in 1× PBS and 2 μg / mL of Tris 100 mM pH 9.0 for OMV-NZ, and incubated overnight at +2 to 8°C. After washing (PBS 1× Tween 20), the plates were blocked by adding 200 μL of Smartblock (Candor Bioscience) and incubated at room temperature for 2 hours. After washing, the plates were sealed with a Liquid Plate Sealer (Candor Bioscience) and incubated at room temperature for 2 hours. Finally, the plates were aspirated and stored in a refrigerator at 2 to 8°C.

[0486] Samples were diluted in a PBS 1×BSA 1% pH 7.4 solution at initial dilutions of 1:1000 (936-741), 1:500 (287-953, 961c and OMV-NZ), and 1:1000 (741-231.13), followed by five further 2-fold dilutions.

[0487] Next, the plate was incubated at 37°C for 90 minutes, washed according to the method described above, and 100 μL of a solution of the secondary antibody (anti-RAT total IgG alkaline phosphatase conjugate) was added. Then, the plate was incubated at 37°C for 60 minutes.

[0488] After washing, 100 μL of substrate (para-nitrophenyl phosphate) was added to the plate, incubated at 37°C for 25-30 minutes, and then read at 405 nm.

[0489] Total IgG was measured by HT-ELISA on single serum samples after the third administration. As shown in Figure 14A, the IgG titer against MenA PS was similar in rat serum immunized with ABNGCWY combined with BNGCWY or with calvaMenA, and was higher in rats immunized with calvaMenA than in rats immunized with MenA-CRM.

[0490] Figure 14B shows that equivalent IgG titers were obtained compared to Men CWY PS measured in rat serum immunized with ABNGCWY or carbaMenA in combination with BNGCWY.

[0491] Figure 14C shows that comparable IgG titers were obtained for Bexsero antigen and 231.13_NB fusion protein when measured in rat serum immunized with ABNGCWY or carbaMenA in combination with BNGCWY.

[0492] CarbaMenA formulations induced higher anti-PS MenA IgG titers than the MenA-CRM group. No significant results were observed when comparing MenCWY_7B-carbaMenA with a standard pentavalent formulation.

[0493] In vitro bactericidal assay: On day 1, Neisseria meningitidis was streaked onto chocolate agar polyvitex plates (BIOMERIEUX 43101) to isolate from the parent culture medium, and incubated at 37°C in 5% CO2 for 16 (±2) hours. On day 2, the bacteria were harvested from the agar plates and resuspended in Mueller-Hinton medium until the optical density (OD600) reached 0.05, and incubated at 37°C in 5% CO2 until the OD reached 0.25 (10 9 The cells were grown by shaking at 135 rpm until the concentration reached a level equivalent to CFU / mL, and then used in the assay.

[0494] Next, the bacteria were placed in a reaction buffer containing 5 U / mL heparin, 10 mM MgCl2, and 1.5 mM CaCl2 (Dulbecc's phosphate-buffered saline, 0.1% glucose, and 1% bovine serum albumin) for 10 minutes. 5 Diluted to CFU / mL.

[0495] Diluted serum series (2-fold) was mixed with 20 μL of working buffer and bacteria (3 / 5 × 10⁻¹⁶). 4 SBA was prepared in a 96-well microplate with a final volume of 40 μL / well by mixing 10 μL of CFU / mL with 10 μL of activated plasma replacement solution (plasma stored at -80°C and thawed immediately before use). Human plasma obtained from volunteer donors with informed consent was selected to be used as a complement source containing specific meningococcal strains only if it did not significantly reduce the number of colony-forming units of the strain when added to the assay solution at a concentration of 50%.

[0496] The sterilization assay includes the following two internal controls: 1) Complement-dependent controls to evaluate bacterial killing by complement alone in the absence of antibodies; these reactions include only bacteria and active complement. 2) Complement-independent controls to evaluate sterilization by serum alone in the presence of thermoactivated complement; these reactions include bacteria, serum samples, and thermoactivated complement.

[0497] The reaction mixture was incubated in 5% CO2 at 37°C for 60 minutes (T60).

[0498] On a T60 thermometer, 100 μL of thawed TSB / 0.7% agar was added to each well and allowed to solidify for 10 minutes. A second layer of 50 μL of thawed agar was added to each well and allowed to solidify for another 10 minutes. Next, the plate was covered and incubated overnight at 37°C. After incubation overnight at 37°C in 5% CO2, the microplate was placed on an AxioLab system (MicroTechniX BVBA), and images of each well were obtained and automatically saved to a file sharing system for both raw and analyzed images. Image analysis was performed using AvioVision Rel. 4.84.8.2, and the colony count was automatically obtained. Bactericidal titer (hSBA titer) was determined as the reciprocal of the serum dilution rate that resulted in at least a 50% decrease in colony-forming units (CFUs) compared to the number of CFUs present in a control reaction without serum. Interpolated SBA titers were used for statistical analysis.

[0499] Functional antibody responses were measured by hSBA, and as shown in Figures 15A, 15B, and 15C, the antibody functionality induced by carbaMenA in combination with BNGCWY was equivalent to that of the benchmark combination ABNGCWY. Furthermore, equivalent hSBA titers were also induced by other CWY antigens and proteins using Bexsero (Figures 15B and 15C).

[0500] The carba MenA formulation showed a lower hSBA titer for MenA 3125 strain compared to MenA-CRM. No difference was detected between the MenACWY_7B fHbp 1X formulation and the MenCWY_7B-carba MenA formulation.

[0501] The MenACWY-7B fHbp 1X formulation showed superior hSBA titer against MenA F8238 strain compared to the MenCWY_7B-carbaMenA formulation. No difference was observed between the MenA-CRM formulation and the carbaMenA formulation.

[0502] Embodiments of the present invention are described in the following numbered paragraphs. 1. An aqueous immunogenic composition that, after administration to a subject, can induce a bactericidal immune response against serogroups A, B, C, W135, and Y of Neisseria meningitidis, wherein the composition i. Conjugate serogroup A antigen, ii. Conjugate serogroup C antigen, iii. Conjugate serogroup W135 antigen, iv. Conjugate serogroup Y antigen; and v. One or more polypeptide antigens from serogroup B Includes, (ii), (iii), and (iv) are capsular sugar antigens, and (i) is a synthetic analog of serogroup A capsular sugar. 2. The composition according to paragraph 1, wherein the conjugate serogroup A antigen is an oligomeric conjugate and comprises an oligomer of the following formula (Ia) or (Ib). [ka] [In the formula, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and can be the same or different in each repeating unit; R y is H or -C(O)CH3, and can be the same or different in each repeating unit; R x or R y At least one of them is -C(O)CH3 in at least one repeating unit; Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 These are independently selected from the group consisting of H, linear or branched C1-C6 alkyl groups, and linear or branched C1-C6 haloalkyl groups; Z is (i) protecting group; (ii) Functional linkers for conjugation to proteins, (iii) A linear or branched C1-C6 alkyl, optionally substituted phenyl, -C(O)Y, or a linear or branched C1-C6-alkyl-X And, Y is H, a linear or branched C1-C6 alkyl group, or a protecting group. X is -NH2, -N3, -C≡CH, -CH=CH2, -SH, or -SC≡N. 3. The composition according to paragraph 1 or 2, wherein the conjugate serogroup A antigen is a conjugate of the following formula (IIa) or (IIb), preferably the following formula (IIa). [ka] [In the formula, in the oligomer, n is ≥ 6; R is H or -P(O)(OR″)2, and R″ is H or a pharmaceutically acceptable phosphate counterion; R′ is H or a pharmaceutically acceptable phosphate counterion; R x is H or -C(O)CH3, and can be the same or different in each repeating unit; R y is H or -C(O)CH3, and can be the same or different in each repeating unit; R x or R y At least one of them is -C(O)CH3 in at least one repeating unit; Az is -NH(CO)R 1 , -N(R 1 ) an aza substituent selected from the group consisting of 2 and -N3, R 1 These are independently selected from the group consisting of H, linear or branched C1-C6 alkyl groups, and linear or branched C1-C6 haloalkyl groups; Z is (i) a functional linker or bond; P is a protein. 4. In the oligomer, R x The composition of paragraph 2 or 3, wherein at least one repeating unit is -C(O)CH3. 5. One of the compositions from paragraphs 2 to 4, wherein in the oligomer, n is 6 to 30. 6. One of the compositions from paragraphs 2 to 4, wherein in the oligomer, n is 8 to 20. 7. One of the compositions from paragraphs 2 to 4, wherein in the oligomer, n is 8 to 15. 8. A composition from any one of paragraphs 2 to 4, wherein in the oligomer, n is 8 or 10. 9. A composition according to any one of paragraphs 2 to 8, wherein Az in the oligomer is -NHC(O)CH3. 10. In the oligomer, at least one identical repeating unit R x and R y A composition according to any one of paragraphs 2 to 9, wherein both are -C(O)CH3. 11. In the oligomer, at least one identical repeating unit R x H is R y A composition according to any one of paragraphs 2 to 10, wherein is -C(O)CH3. 12. In the oligomer, at least one identical repeating unit R x is -C(O)CH3 and R y A composition according to any one of paragraphs 2 to 11, wherein H is present. 13. In the oligomer, R x and R y A composition according to any one of paragraphs 2 to 12, wherein both are -C(O)CH3 in at least one identical repeating unit. 14. In the oligomer, at least one identical repeating unit R x H is R y is -C(O)CH3, and R is in at least one identical repeating unit. x is -C(O)CH3 and R y A composition according to any one of paragraphs 2 to 13, wherein H is present. 15. In the oligomer, at least one identical repeating unit R x H is R y is -C(O)CH3, and R is at least one identical repeating unit. x is -C(O)CH3 and R y H is and R is in at least one identical repeating unit. x and R y A composition according to any one of paragraphs 2 to 14, wherein both are -C(O)CH3. 16. R is present in 40-50% of the repeating units of the oligomer. x and R y A composition according to any one of paragraphs 2 to 15, wherein both are -C(O)CH3. 17. R x or R y One of them is -C(O)CH3, and the remaining repeating unit in the oligomer is R x =R y A composition according to paragraph 16 having =H. 18. R in the oligomer x and R y A composition according to any one of paragraphs 2 to 17, wherein approximately 50-90% of the composition is -C(O)CH3. 19. R in each repeating unit x H is and R in the oligomer y A composition according to any one of paragraphs 2 to 18, wherein at least 80% of it is -C(O)CH3. 20. P is diphtheria toxoid (DT), tetanus toxoid (TT), CRM 197 A composition according to any one of paragraphs 2 to 19, wherein P is an inactivated bacterial toxin selected from Escherichia coli (E. coli) ST and Pseudomonas aeruginosa exotoxin (rEPA), or P is a polyamino acid such as poly(lysine:glutamic acid), or P is hepatitis B virus nucleoprotein or SPR96-2021. 21. P is CRM 197 The composition is one of the compositions described in paragraphs 2 to 20. 22. A composition from any one of paragraphs 2 to 21, wherein Z is a linker having the following formula. [ka] [In the formula, * The symbol indicates a connection point. p is independently selected from 1 to 10; X is selected from -O-, -S-, and -NH-. 23. A composition from any one of paragraphs 2 to 21, wherein Z is a linker having the following formula. [ka] [In the formula, m is independently selected from 1 to 10] 24. A composition according to any one of paragraphs 2 to 23, wherein the oligomer conjugate has the following structure. [ka] [In the formula, n, Az, R, R x and R y This is defined in any one of paragraphs 2-19. 25. The conjugate serogroups C, W135, and Y antigens are diphtheria toxoid, tetanus toxoid, Haemophilus influenzae protein D, and CRM 197 A composition according to any one of the paragraphs above, conjugated to a carrier protein selected from the above. 26. The serogroups C, W135, and Y antigens are CRM 197 A composition according to paragraph 25, which is conjugated to the above. 27. A composition according to any one of the preceding paragraphs, wherein the one or more polypeptide antigens from serogroup B include one or more meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal outer membrane vesicles (OMVs). 28. A composition according to paragraph 27 comprising v1.13 meningococcal fHbp polypeptide in at least one identical repeating unit having at least 80% sequence identity with SEQ ID NO: 2, wherein the amino acid sequence includes one or more substitutional mutations in residues S216, E211, or E232 of SEQ ID NO: 2. 29. The composition according to paragraph 28, wherein the amino acid sequence differs from that of Sequence ID No. 2 by at least one substitution S216R, E211A, and E232A. 30. The amino acid sequence is as follows: (i) E211A and S216R, (ii) E211A and E232A A composition according to paragraph 29, comprising substitution with multiple residues selected from. 31. A composition according to any one of paragraphs 28 to 30, wherein the v1.13 meningococcal fHbp polypeptide has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. 32. A composition according to paragraph 27, comprising a fusion polypeptide containing v1, v2, and v3 meningococcal fHbp polypeptides in the order v2-v3-v1 from the N-terminus to the C-terminus, wherein the v1 fHbp polypeptide is a mutant v1.13 fHbp polypeptide as defined in any of paragraphs 28 to 31. 33. (a) The v2 fHbp polypeptide is a mutant v2 fHbp polypeptide having an amino acid sequence that is at least 80% sequence-identical to SEQ ID NO: 12, wherein the v2 fHbp amino acid sequence includes substitution mutations at residues S32 and L123 of SEQ ID NO: 12, and the substitutions are S32V and L123R; (b) The v3 fHbp polypeptide is a mutant v3 fHbp polypeptide having an amino acid sequence that is at least 80% sequence-identical to SEQ ID NO: 15, wherein the v3 fHbp amino acid sequence includes substitution mutations at residues S32 and L126 of SEQ ID NO: 15, and the substitutions are S32V and L126R. Composition according to paragraph 32. 34. (a) The v2 fHbp polypeptide contains or consists of the amino acid sequence of SEQ ID NO: 16; and / or (b) The v3 fHbp polypeptide contains or consists of the amino acid sequence of SEQ ID NO. 17, Composition according to paragraph 33. 35. A composition according to any one of paragraphs 32 to 34, wherein the v2 and v3 fHbp amino acid sequences and the v3 and v1 fHbp amino acid sequences are linked by a glycine-serine linker, and preferably the v2 sequence has an N-terminal leader sequence corresponding to SEQ ID NO: 18. 36. A composition according to any one of paragraphs 32 to 35, wherein the fHbp fusion polypeptide comprises the amino acid sequence of any of SEQ ID NOs. 19 to 23. 37. The composition according to paragraph 36, wherein the fHbp fusion polypeptide further comprises any N-terminal amino acid sequence of SEQ ID NO: 34. 38. The composition according to paragraph 36, wherein the fHbp fusion polypeptide has the sequence of SEQ ID NO: 35. 39. A composition according to any one of paragraphs 28 to 36, wherein the composition further comprises meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal outer membrane vesicles (OMV). 40. A composition according to any one of the preceding paragraphs, further comprising an adjuvant. 41. A composition according to paragraph 40, wherein the adjuvant is aluminum hydroxide. 42. A composition according to any one of the paragraphs, wherein the composition comprises BEXSERO. 43. A composition according to any one of the paragraphs, contained in a single sealed container, preferably a vial or syringe. 44. A method for inducing an immune response in a mammal, comprising administering an immunogenic composition according to any of paragraphs 1 to 43, wherein the mammal is optionally a human. 45. A method for treating or preventing an infection and / or disease caused by Neisseria meningitidis in a mammal requiring treatment, comprising administering to the mammal an immunologically effective amount of a composition according to any of paragraphs 1 to 43, wherein the mammal is optionally a human. 46. ​​Immunogenic compositions for use in medical applications, according to any of paragraphs 1 to 43. 47. An immunogenic composition according to any of paragraphs 1 to 43, for use as a vaccine. 48. An immunogenic composition according to any one of paragraphs 1 to 43 for use in a method for eliciting an immune response in a mammal, wherein the mammal is a human. 49. A composition according to any one of paragraphs 1 to 43 for use in the immunization of a mammal against meningococcal (N. meningitidis) infection, wherein the mammal is a human. 50. Use of compositions defined in any of paragraphs 1 to 43 in the manufacture of a pharmaceutical product for use in the treatment or prevention of infection and / or disease caused by Neisseria meningitidis.

Claims

1. An aqueous immunogenic composition that, after administration to a subject, can induce a bactericidal immune response against serogroups A, B, C, W135, and Y of Neisseria meningitidis, wherein the composition i. Conjugate serogroup A antigen, ii. Conjugate serogroup C antigen, iii. Conjugate serogroup W135 antigen, iv. Conjugate serogroup Y antigen; and v. One or more polypeptide antigens from serogroup B Includes, (ii), (iii), and (iv) are capsular sugar antigens, and (i) is a synthetic analog of serogroup A capsular sugar. An aqueous immunogenic composition wherein the conjugate serogroup A antigen is an oligomeric conjugate and comprises an oligomer of the following formula (Ia) or (Ib). 【Chemistry 1】 [In the formula, n is ≥ 6; R is H or -P(O)(OR'') ２ And R″ is H or a pharmaceutically acceptable phosphate counterion; R' is H or a pharmaceutically acceptable phosphate counterion; R ｘ is H or -C(O)CH ３ And each repeating unit can be the same or different; R ｙ is H or -C(O)CH ３ And each repeating unit can be the same or different; R ｘ or R ｙ at least one of which is -C(O)CH ３ in at least one repeating unit; R ｘ is -C(O)CH ３ in at least one repeating unit; in at least one same repeating unit, both R x and R y are -C(O)CH 3; Az is -NH(CO)R １ , -N(R １ ) ２ and -N ３ an aza substituent selected from the group consisting of R １ H is independent, and C is linear or branched. １ -C ６ - Alkyl and linear or branched C １ -C ６ - Selected from the group consisting of haloalkyl groups; Z is (i) a protecting group; (ii) Functional linkers for conjugation to proteins, (iii) Linear or branched C １ -C ６ Alkyl, -C(O)Y, or linear or branched C １ -C ６ -Alkyl-X And, Y is H, a linear or branched C １ -C ６ - Alkyl or protecting group, X is -NH ２ , -N ３ , -C≡CH, -CH=CH ２ [, -SH or -S-C≡N]

2. The composition according to claim 1, wherein in the oligomer of formula (Ia) or (Ib), Az is -NH(CO)CH3.

3. The composition according to claim 1 or 2, wherein the conjugate serogroup A antigen is a conjugate of the following formula (IIa) or (IIb). 【Chemistry 2】 [In the formula, n, R, R', R ｘ , R ｙ Az is as defined in claim 1 or 2, Z is a functional linker or bond.

4. The composition according to any one of claims 1 to 3, wherein in the oligomer, n is 6 to 30, 8 to 20, 8 to 15, 8, or 10.

5. In the aforementioned oligomer, (i) R ｘ H is R ｙ ga-C(O)CH ３ That is, (ii) R ｘ ga-C(O)CH ３ And R ｙ H is (iii) R ｘ H is R ｙ ga-C(O)CH ３ And R in at least one identical repeating unit. ｘ ga-C(O)CH ３ And R ｙ H is (iv) R ｘ H is R ｙ ga-C(O)CH ３ Therefore, R is the unit of at least one identical repeating unit. ｘ ga-C(O)CH ３ And R ｙ H is and R is in at least one identical repeating unit. ｘ and R ｙ Both are -C(O)CH ３ That is, (v) 40-50% of the repeating units of the oligomer, R ｘ and R ｙ Both are -C(O)CH ３ That is, (vi) R in the oligomer ｘ and R ｙ Approximately 50-90% of it is -C(O)CH ３ is, and / or, (vii) R in each repeating unit ｘ H is and R in the oligomer ｙ At least 80% of it is -C(O)CH ３ That is, The composition according to any one of claims 1 to 4.

6. (v) The composition according to claim 5, wherein in 40 to 50% of the repeating units of the oligomer, both R x and R y are -C(O)CH3, and in 10 to 20% of the remaining repeating units of the oligomer, either R x or R y is -C(O)CH3, and the remaining repeating units in the oligomer have R x = R y = H.

7. The protein P of the conjugates of formulas (IIa) and (IIb) is diphtheria toxoid (DT), tetanus toxoid (TT), CRM １９７ The composition according to any one of claims 2 to 6, wherein the protein P is an inactivated bacterial toxin selected from Escherichia coli (E. coli) ST and Pseudomonas aeruginosa exotoxin (rEPA), or the protein P is a polyamino acid such as poly(lysine:glutamic acid), or the protein P is hepatitis B virus nucleoprotein or SPR96-2021.

8. Z is a linker having the following equation: 【Transformation 3】 [In the formula, ＊ The symbol indicates a connection point. p is independently selected from 1 to 10; X is selected from -O-, -S-, and -NH-, or The composition according to any one of claims 1 to 7, wherein Z is a linker having the following formula. 【Chemistry 4】 [In the formula, m is independently selected from 1 to 10]

9. The composition according to any one of claims 1 to 8, wherein the oligomer conjugate has the following structure. 【Transformation 5】 [In the formula, n, Az, R, R ｘ and R ｙ This is defined as in any one of claims 1 to 6.

10. The aforementioned conjugate serogroups C, W135, and Y antigens are diphtheria toxoid, tetanus toxoid, Haemophilus influenzae (H. influenzae) protein D, and CRM １９７ The composition according to any one of claims 1 to 9, which is conjugated to a carrier protein selected from.

11. The composition according to any one of claims 1 to 10, wherein the one or more polypeptide antigens from serogroup B include one or more meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal outer membrane vesicles (OMVs).

12. The composition according to claim 11, comprising v1.13 meningococcal fHbp polypeptide in at least one identical repeating unit having at least 90% sequence identity with SEQ ID NO: 2, wherein the amino acid sequence includes substitutions at the residues E211A and S216R.

13. The composition according to claim 12, wherein the v1.13 meningococcal fHbp polypeptide has the amino acid sequence of SEQ ID NO:

4.

14. The composition according to claim 11, comprising a fusion polypeptide containing v1, v2, and v3 meningococcal fHbp polypeptides in the order v2-v3-v1 from the N-terminus to the C-terminus, wherein the v1 fHbp polypeptide is a mutant v1.13 fHbp polypeptide as defined in claim 12 or 13.

15. A fusion polypeptide comprising v1, v2 and v3 meningococcal fHbp polypeptides, (a) The v2 fHbp polypeptide is a mutant v2 fHbp polypeptide having an amino acid sequence that is at least 90% sequence-identical to SEQ ID NO: 12, wherein the v2 fHbp amino acid sequence includes substitution mutations at residues S32 and L123 of SEQ ID NO: 12, and the substitutions are S32V and L123R; (b) The v3 fHbp polypeptide is a mutant v3 fHbp polypeptide having an amino acid sequence that is at least 90% sequence-identical to SEQ ID NO: 15, wherein the v3 fHbp amino acid sequence includes substitution mutations at residues S32 and L126 of SEQ ID NO: 15, and the substitutions are S32V and L126R. The composition according to claim 14.

16. The composition according to claim 14 or 15, wherein the fHbp fusion polypeptide comprises the amino acid sequence of SEQ ID NO:

19.

17. The composition according to any one of claims 12 to 16, wherein the composition further comprises meningococcal NHBA antigen, meningococcal NadA antigen, meningococcal fHbp antigen, and meningococcal outer membrane vesicles (OMV).

18. The composition according to any one of claims 1 to 17, further comprising an adjuvant.

19. The composition according to claim 18, wherein the adjuvant is aluminum hydroxide.

20. The composition according to any one of claims 1 to 19, contained in a single sealed container.

21. A composition according to any one of claims 1 to 20, which is an immunogenic composition for use in medical applications.

22. A composition according to any one of claims 1 to 20, which is an immunogenic composition for use as a vaccine.

23. A composition according to any one of claims 1 to 20 for use in the immunization of mammals against meningococcal (N. meningitidis) infection.

Images