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WO2025078556A1 - Couplage de glucides amélioré - Google Patents

Couplage de glucides amélioré Download PDF

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Publication number
WO2025078556A1
WO2025078556A1 PCT/EP2024/078620 EP2024078620W WO2025078556A1 WO 2025078556 A1 WO2025078556 A1 WO 2025078556A1 EP 2024078620 W EP2024078620 W EP 2024078620W WO 2025078556 A1 WO2025078556 A1 WO 2025078556A1
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WO
WIPO (PCT)
Prior art keywords
gal
group
glcnac
fuc
compound
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Pending
Application number
PCT/EP2024/078620
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English (en)
Inventor
Peter Sondermann
Maria BRAEUTIGAM
Kathrin ROESCH
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Tacalyx GmbH
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Tacalyx GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Publication of WO2025078556A1 publication Critical patent/WO2025078556A1/fr
Pending legal-status Critical Current
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines

Definitions

  • Y is selected from the group consisting of an amino acid, peptide and a protein.
  • the chemistry used for generation of the covalent bond between sugar and carrier protein results in a non-native and due to the employed chemistry in general (often highly) immunogenic structure, e.g. represented by heterocyclic structures or aliphatic chains. Especially in the case of low immunogenic targets like carbohydrates this can lead to an immune response upon vaccination that might be dominated by the used linker while only a negligible or nonexistent immune response against the specific target is observed (for an overview see Adamo 2014).
  • the linker consists of the open chain form of the monosaccharide residue present on the reducing end of the oligosaccharide and exhibits a very low immunogenicity as this open-chain structure is tolerated by the immune system due to its continuous abundance in case it represents natural monosaccharide structures.
  • This reductive amination chemistry is also used in currently marketed pneumococcal conjugate vaccines like Prevnar® or Prevnar 13® (Turner 2017) indicating the safety and synthetic efficiency of this approach.
  • Prevnar® or Prevnar 13® Turner 2017
  • the coupling of the isolated pneumococcal carbohydrates to the carrier protein requires the oxidation of hydroxyl functions to carbonyl groups by periodate and that is accompanied by a ring opening that occurs in an undirected manner.
  • This oxidative destruction of at least one carbohydrate unit on the reducing end reduces the size of the structure against which relevant antibody might be raised and in addition can create irrelevant neo-epitopes (Poolman 2011).
  • G is a glycan comprising n monosaccharide units linked glycosidically; n is the number of monosaccharide units linked glycosidically, preferably n is 2 to 200, more preferably 2 to 100, most preferably 2 to 20 monosaccharide units; o is an integer, between 1 and 10, if Y is a Peptide or Protein an integer between 1 and 10 per 10 kDa of Peptide or Protein Y, preferably between 1 and 5; .
  • Y is selected from the group consisting of an amino acid, peptide and a protein.
  • Fig. 1 Immunization studies, Printing pattern for the glycan microarray.
  • Fig. 2 A) Binding of the sera of mice immunized with compound 5 or 6 to the target sTRA (13) or the truncated sTRA trisaccharide MLB-153 (17). B) Glycan array analysis of 5 mice immunized with glycoconjugate 5 (serum dilution 1 :100).
  • Fig. 3 Structurerelationship of sTRA, LSTa and TRA.
  • G is a glycan comprising n monosaccharide units which are linked glycosidically if n is larger than 1.
  • glycosidically linked means that, preferably a bond involving oxygen (ether bond), or other heteroatoms (for example such as N or S), or even carbon atoms is formed, involving a hemiketal or hemiacetal group of a saccharide.
  • ether bond oxygen
  • heteroatoms for example such as N or S
  • carbon atoms is formed, involving a hemiketal or hemiacetal group of a saccharide.
  • the glycosidic linkage if formed between a hemiketal or hemiacetal group of one saccharide and one other hydroxyl group of at least one other saccharide in form of an ether bond, to connect at least two saccharides, to form a glycan.
  • the monosaccharide units in the glycan may exhibit further functional groups attached to heteroatom containing groups, preferably hydroxyl groups, amino groups, or -SH groups, more preferably hydroxyl groups or amino groups, by substitution of one or more hydrogen atoms on these heteroatom containing groups not involved in the glycosidic bond.
  • heteroatom containing groups preferably hydroxyl groups, amino groups, or -SH groups, more preferably hydroxyl groups or amino groups, by substitution of one or more hydrogen atoms on these heteroatom containing groups not involved in the glycosidic bond.
  • protection groups for example such as -C(O)(Ci-Ce)alkyl, Benzyl (Bn), Benzoyl (Bz), Benzyloxycarbonyl- apparently (Cbz), Fluorenylmethoxycarbonyl (fmoc), preferably acetal (Ac).
  • a mammalian glycan is a glycan present in mammalian cells, preferably present on the surface of mammalian cells.
  • X is based on an aldose, preferably a D-aldose, linked glycosidically with G and wherein the aldehyde group of the aldose has been reacted with a primary amino group of group Y resulting in a product according to formula (I), wherein the aldehyde group is formally reduced to the corresponding secondary amino group in formula (I) preferably by reductive amination, resulting in a secondary amine group in formula (I).
  • Y comprises a lysine side chain within a peptide or a protein which provides the primary amino group for the group -NH- in formula (I).
  • the invention is further directed to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of claims as described above and at least one pharmaceutically acceptable carrier.
  • the invention is directed to the use of the compound as described above, in medicine.
  • the invention is directed to the use of the compounds as described above in research. [0047] Further, the invention is directed to the use of the compounds to generate antibodies directed against said compounds.
  • the antibodies may be generated by methods as known to the person skilled in the art (Dubel & Reichert 2014).
  • inventive compounds may be prepared using procedures for carbohydrate, peptide and protein synthesis commonly known to the person skilled in the art, optionally including solid phase synthesis and/or protecting group chemistry. Different synthetic approaches may be chosen resulting in the inventive compounds. However, preferably all approaches may comprise a reductive amination step, which preferably comprises the intermediate formation of an imine, to connect the group Y with the linker X, comprising a reduction agent which reduces an imine to the corresponding amine but not an aldehyde to the corresponding alcohol. Reduction agents which exhibt this selectivity for imine reduction are for example NaCNBHs, sodium triacetoxyborohydride, certain BHs-amine complexes, hydrogenium in combination with suitable metal catalysts (Tripathi 2008).
  • the temperature of the reaction vessel was adjusted to -20 °C. During that time the resin was swollen in 2 ml DCM. When the reaction vessel temperature reached -20 °C, TMSOTf solution (1mL) was added dropwise to the reaction vessel. The reaction was bubbled with Argon for 3 min before the solution was drained and the resin was washed with 2 mL DCM for 25 s.
  • Modul C2 was used for BB2 and BB4.
  • the building block solution (0.096 mmol of BB in 1 ml DCM per glycosylation) was delivered into the reaction vessel. After the set temperature is reached, the activator solution was added dropwise to the reaction vessel (1.0 mL, 0.15 mmol). The reaction was bubbled for 20 min, before the solution was drained and the resin was washed with DCM, DCM/Dioxane and again DCM (twice, each with 2 mL for 25 s). Then, the cycle of glycosylation was repeated. The reaction vessel temperature was set to 25 °C for the next reaction step after the completion of the second glycosylation.
  • the resin was washed three times with DMF (2 mL, 25 s) while the reaction vessel was adjusted to 25 °C.
  • the Fmoc deprotection solution (2 mL) was delivered into reaction vessel. After 5 min of Argon bubbling, the solution was drained from the reaction vessel.
  • the resin was washed with DMF (3x, 2 mL, 25 s) and DCM (5x, 2 mL, 25 s). The reaction was set to -20 °C if another glycosylation followed.
  • Modul F Methanolysis on resin [0060] The resin was suspended in anhydrous tetrayhdrofuran (THF, 4 mL). Sodium metoxide (0.5 M in methanol, 0.4 mL) was added. The suspension was shaken at room temperature for 24 h (in a 5 mL plastic syringe). After the reaction the resin was washed three times with methanol (4 mL) and DCM (4 mL).
  • the linkers used in the glycan array to immobilize the glycans are the same as the linker used for the glycoconjugate 5.
  • a FACS analysis confirmed that none of the mice showed a sTRA specific immune response. Cell binding was not observed (data not shown).
  • Sera samples were spun down and diluted 1 :100 in phosphate-buffered saline (PBS) containing 2% FBS and incubated with 1x10 A 5 MCF7 WT cells for 30 min on ice. The cells were then spun down, washed with PBS-FBS and stained with donkey anti-mouse IgG (H+L) CF488A (Biotium) 1 :1000 in PBS/FBS for 30 min on ice. Afterwards cells were stained with live/dead stain eFlour780 1 :1000 (Invitrogen) in PBS for 10 min on ice. Then washed with PBS/FBS and acquired on an Attune (Invitrogen) flow cytometer.
  • PBS phosphate-buffered saline

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Saccharide Compounds (AREA)

Abstract

L'invention concerne un composé de formule (I) (G-X-NH)o-Y dans laquelle G est un glycane comprenant n motifs monosaccharidiques liés par voie glycosidique ; n est le nombre entier de motifs monosaccharidiques liés par voie glycosidique, de préférence n vaut de 2 à 200, de manière mieux préférée de 2 à 100, de manière la mieux préférée de 2 à 20 motifs monosaccharidiques ; o est un nombre entier compris entre 1 et 10, si Y est un peptide ou une protéine, un nombre entier compris entre 1 et 10 pour 10 kDa de peptide ou de protéine Y, de préférence entre 1 et 5 ; X étant basé sur une aldose, liée par voie glycosidique à G et le groupe aldéhyde de l'aldose ayant été mis à réagir avec un groupe amino primaire du groupe Y conduisant à un produit selon la formule (I), le groupe aldéhyde de l'aldose ayant été formellement réduit au groupe amino secondaire correspondant dans la formule (I) de préférence par amination réductrice, conduisant à un groupe amine secondaire dans la formule (I) ; et Y étant choisi dans le groupe constitué par un acide aminé, un peptide et une protéine.
PCT/EP2024/078620 2023-10-12 2024-10-11 Couplage de glucides amélioré Pending WO2025078556A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
LU505267 2023-10-12
LULU505267 2023-10-12

Publications (1)

Publication Number Publication Date
WO2025078556A1 true WO2025078556A1 (fr) 2025-04-17

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Country Status (1)

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WO (1) WO2025078556A1 (fr)

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Handbook of therapeutic antibodies", 2014, JOHN WILEY & SONS
ADAMO R.HU Q.-YTOROSANTUCCI A.CROTTI S.BROGIONI G.ALLAN M.CHIANI P.BROMURO C.QUINN D.TONTINIA M.: "Deciphering the structure-immunogenicity relationship of anti-Candida glycoconjugate vaccines", CHEM. SCI., vol. 5, 2014, pages 4302, XP055371764, DOI: 10.1039/C4SC01361A
ANDERSON P.PICHICHERO M. E.INSEL R. A.: "Immunogens Consisting of Oligosaccharides from the Capsuleof Haemophilus influenzae Type b Coupled to Diphtheria Toxoidor the Toxin Protein CRM197", J. CLIN. INVEST., vol. 76, 1985, pages 52
BERTI F.ADAMO R.: "Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification", CHEM. SOC. REV., vol. 47, 2018, pages 9015, XP055702534, DOI: 10.1039/C8CS00495A
BUSKAS T.LI YBOONS G.-J.: "The Immunogenicity of the Tumor-Associated Antigen Lewisy May Be Suppressed by a Bifunctional Cross-Linker Required for Coupling to a Carrier Protein", CHEM. EUR. J., vol. 10, 2004, pages 3517, XP055085926, DOI: 10.1002/chem.200400074
GILDERSLEEVE J. C.OYELARAN O.SIMPSON J. T.ALLRED B.: "Improved Procedure for Direct Coupling of Carbohydrates to Proteins via Reductive Amination", BIOCONJUG CHEM, vol. 19, 2008, pages 1485, XP055980988, DOI: 10.1021/bc800153t
GILDERSLEEVE JEFFREY C. ET AL: "Improved Procedure for Direct Coupling of Carbohydrates to Proteins via Reductive Amination", BIOCONJUGATE CHEMISTRY, vol. 19, no. 7, 1 July 2008 (2008-07-01), US, pages 1485 - 1490, XP055980988, ISSN: 1043-1802, DOI: 10.1021/bc800153t *
KOSHIDA SHUHEI ET AL: "Synthesis of oligomeric assemblies of a platelet-binding key disaccharide in heparin and their biological activities", TETRAHEDRON LETTERS, vol. 42, no. 7, 28 February 2001 (2001-02-28), pages 1289 - 1292, XP085047408, ISSN: 0040-4039, DOI: 10.1016/S0040-4039(00)01826-8 *
METTU R.CHEN C.-Y.WU1 C.-Y.: "Synthetic carbohydrate-based vaccines: challenges and opportunities", J. BIOMED. SCI.,, vol. 27, 2020, pages 9
POOLMAN J.FRASCH C.NURKKA A.KAYHTY H.BIEMANS R.SCHUERMAN L.: "Impact of the conjugation method on the immunogenicity of Streptococcus pneumoniae serotype 19F polysaccharide in conjugate vaccines", CLIN. VACCINE IMMUNOL., vol. 18, 2011, pages 327, XP055035142, DOI: 10.1128/CVI.00402-10
TRIPATHI R. P.VERMA S. S.PANDEY J.TIWARI V. K.: "Recent Development on Catalytic Reductive Amination and Applications", CURR. ORG. CHEM., 2008, pages 1093, XP055116094, DOI: 10.2174/138527208785740283
TURNER A. E.B.GERSON J. E.SO H. Y.KRASZNAI D. J.HILAIRE A. J. ST.GERSON D. F.: "Novel polysaccharide-protein conjugates provide an immunogenic 13-valent pneumococcal conjugate vaccine for S. pneumoniae", SYNTHETIC AND SYSTEMS BIOTECHNOLOGY, vol. 2, 2017, pages 49, XP055668061, DOI: 10.1016/j.synbio.2016.12.002
WU X.LING C.-C.BUNDLE D. R. A: "New Homobifunctional p-Nitro Phenyl Ester Coupling Reagent for the Preparation of Neoglycoproteins", ORGANIC LETTERS, vol. 6, pages 4407, XP008121791, DOI: 10.1021/OL048614M

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