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CN-122003250-A - Improved carbohydrate coupling

CN122003250ACN 122003250 ACN122003250 ACN 122003250ACN-122003250-A

Abstract

The invention relates to a compound (G-X-NH) o -Y of formula (I), wherein G is a glycan comprising n monosaccharide units connected by glycosidic bonds, n is an integer of monosaccharide units connected by glycosidic bonds, preferably n is an integer of 2 to 200, more preferably 2 to 100, most preferably 2 to 20 monosaccharide units, o is an integer of 0 to 10, if Y is a peptide or protein, is an integer of 1 to 10, preferably an integer of 1 to 5 per 10kDa peptide or protein Y, X is based on an aldose, is connected by glycosidic bonds to G and wherein the aldehyde group of the aldose has reacted with a primary amino group of the group Y to give a product of formula (I), wherein the aldehyde group of the aldose has been formally reduced to the corresponding secondary amino group in formula (I), preferably by reductive amination, to give a secondary amino group in formula (I), and Y is selected from amino acids, peptides and proteins.

Inventors

  • P. Sandman
  • M. Brodigam
  • K. Lech

Assignees

  • 塔卡莱克斯公司

Dates

Publication Date
20260508
Application Date
20241011
Priority Date
20231012

Claims (12)

  1. 1. A compound of formula (I) (G-X-NH) o -Y (I) Wherein, the G is a glycan comprising n monosaccharide units connected by glycosidic linkages; n is an integer of monosaccharide units connected by glycosidic linkages, preferably n is 1 to 200, more preferably 2 to 100, most preferably 2 to 20 monosaccharide units; o is an integer from 1 to 10, and if Y is a peptide or protein, it is an integer from 1 to 10, preferably an integer from 1 to 5, per 10kDa of peptide or protein Y; X is linked to G by a glycosidic bond based on an aldose, and wherein the aldehyde group of the aldose has reacted with a primary amino group of the group Y to give a product of formula (I), wherein the aldehyde group of the aldose has been formally reduced, preferably by reductive amination, to the corresponding secondary amino group in formula (I) to give a secondary amino group in formula (I); Y is selected from amino acids, peptides and proteins.
  2. 2. The compound according to claim 1, wherein G is a mammalian glycan, preferably a glycan consisting of N monosaccharide units independently selected from the group consisting of arabinose, fructose, fucose, galactose, galactosamine, N-acetylgalactosamine, glucose, glucosamine, N-acetylglucosamine, glucuronic acid, mannose, muramic acid, neuraminic acid, sialic acid, rhamnose, ribose and xylose.
  3. 3. The compound of claim 1 or 2, wherein the glycan is present on the surface of a human cancer cell.
  4. 4. A compound according to any one of claims 1 to 3, wherein G is selected from: * Popular names have not been defined.
  5. 5. A compound according to any one of claims 1 to 4, wherein the aldose is preferably selected from hexose, pentose, tetrose or triose, more preferably, I) The hexose is selected from glucose, galactose, mannose, allose, altrose, idose, talose, or Ii) the pentose is selected from ribose, arabinose, xylose and lyxose, or Iii) The tetrose is selected from erythrose and threose; iv) the triose is glyceraldehyde.
  6. 6. The compound of any one of claims 1 to 5, wherein X is based on D-glucose.
  7. 7. The compound according to any one of claims 1 to 6, wherein Y comprises a lysine side chain within the peptide or protein, which provides a primary amino group for the-NH-group in formula (I).
  8. 8. The compound according to any one of claims 1 to 6, wherein Y is a protein selected from CRM 197 , keyhole Limpet Hemocyanin (KLH), diphtheria Toxoid (DT), tetanus Toxoid (TT), haemophilus influenzae (H. influenza) protein D (HiD), meningococcal Outer Membrane Protein Complex (OMPC), Q.beta.protein, bovine Serum Albumin (BSA), immunoglobulin G (IgG).
  9. 9. A pharmaceutical composition comprising a compound of any one of claims 1 to 8 and at least one pharmaceutically acceptable carrier.
  10. 10. A compound according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for use as a medicament.
  11. 11. Use of a compound according to any one of claims 1 to 8 in a study.
  12. 12. Use of a compound according to any one of claims 1 to 11 for the production of antibodies specific for the compound.

Description

Improved carbohydrate coupling Technical Field The present invention relates to a compound of formula (I) (G-X-NH)o-Y (I) Wherein G is a glycan comprising 2 to 200, more preferably 2 to 100, most preferably 2 to 20 monosaccharide units connected by glycosidic linkages; o is an integer from 1 to 10, and if Y is a peptide or protein, it is an integer from 1 to 10, preferably from 1 to 5, per 10kDa of peptide or protein Y; x is based on an aldose, linked to G by a glycosidic bond and wherein the aldehyde group of the aldose has reacted with a primary amino group of the group Y to give a product of formula (I), wherein the aldehyde group of the aldose has been formally reduced, preferably by reductive amination, to the corresponding secondary amino group in formula (I) to give a secondary amino group in formula (I), and Y is selected from amino acids, peptides and proteins. Background Immunization with non-protein targets requires their covalent binding to the respective carrier proteins, providing enhanced half-life of the target molecule and immunostimulation of the receiving organism. Coupling of hapten-like structures (e.g., synthetic carbohydrates) to proteins is typically performed by random coupling to specific groups (e.g., free SH groups), N-glycosylation sites, or primary amino groups including lysine residue side chains. Coupling with primary amines generally requires a functional group on the carbohydrate that is capable of effecting covalent coupling with an amino group. Examples are linkers with primary amine or thiol functionality. The functional group of such carbohydrates is typically introduced at the reducing end of the carbohydrate at the beginning of the synthesis. Chemical reactions used to create covalent bonds between sugars and carrier proteins can create unnatural and, due to the chemical reactions used, generally (often highly) immunogenic structures, such as represented by heterocyclic structures or fatty chains. Particularly in the case of low immunogenic targets (such as carbohydrates), this may lead to an immune response after vaccination that may be dominated by the linker used, whereas an immune response against a specific target is very little or absent (see Adamo 2014 for an overview). Frequently used and highly immunogenic linker molecules are shown in the following figures, which are the coupling of oligosaccharides to carrier proteins: linker "A" is a triazole group Linker "B" aliphatic group (Wu 2004) Linker "C" maleimide group (Buskas 2004) Currently, various methods for chemically linking non-protein targets to protein carriers are described in the literature (Berti 2018). However, glycan-protein conjugates for immunization are most often obtained by applying "click" chemistry, using maleimide linkers or other organic moieties, which lead to the presence of, for example, triazole groups (linker "a"), alkyl chains (linker "B"), or other cyclic moieties (linker "C"), etc., which are strong immunogens due to their hydrophobic and/or rigid structures themselves. Other methods of coupling carbohydrates are described in a recent overview (Mettu 2020). Among them, reductive amination reactions that couple carbohydrates to carrier proteins have been used for decades (e.g., as described in Anderson 1985), and remain in optimization in terms of reaction conditions and yields (e.g., see GILDERSLEEVE 2008). The basis of this reaction is the balance between the cyclic hemiacetal form (endohemiacetal) and the open-chain aldehyde form of the carbohydrate molecule at its reducing end. The latter forms imine ions (schiff bases) with the terminal amino groups on the protein, followed by irreversible reduction to secondary amines, as shown in the following figure: Thus, the linker consists of an open-chain form of monosaccharide residues present at the reducing end of the oligosaccharide and exhibits very low immunogenicity, since such open-chain structures, in the case of representing natural monosaccharide structures, are tolerated by the immune system due to their sustained high presence. This reductive amination chemistry is also used for pneumococcal conjugate vaccines currently on the market, such as Prevnar ® or Prevnar 13 ® (Turner 2017), indicating the safety and synthetic efficiency of the method. However, since the carbohydrate is isolated from the pathogen, it must be functionalized. Thus, coupling of an isolated pneumococcal carbohydrate to a carrier protein requires oxidation of the hydroxyl functionality to carbonyl by periodate, and this is accompanied by ring opening occurring in a non-directional manner. This oxidative destruction of at least one carbohydrate unit at the reducing end reduces the size of the structure that may produce the relevant antibody, and in addition, creates an unrelated neoepitope (Poolman 2011). In contrast to vaccines based on capsular polysaccharides (e.g. Prevnar ®, which consist of a large number of repeat units), small carbohydrates with very