Carrier-protein polysaccharide conjugation methods
Inventors
FAIRMAN, Jeffery C. • Kapoor, Neeraj • PILL, Lucy Estella • Snyder, Scott
Assignees
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Publication Number
US-12252454-B2
Publication Date
2025-03-18
Expiration Date
Abstract
The present disclosure provides methods of preparing heteroaryl-containing compounds, wherein an azide-alkyne cycloaddition is accelerated in the presence of lauryldimethylamine oxide (LDAO). The present disclosure further provides conjugates of polypeptides and antigens prepared using such methods.
Core Innovation
The disclosure describes the preparation of a heteroaryl-containing compound by contacting an azide compound with an alkyne compound in the presence of lauryldimethylamine oxide (LDAO), water, and DMSO. The LDAO is used at a concentration of up to 2% v/v, and the azide compound is defined as a compound of formula (I) while the alkyne compound is defined as a compound of formula (II) or (III).
The reactant structures are defined through extensive variable substitution definitions, including R1, R2, and R3 classes for the alkyne compound, with substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, and specified (CH2)a-carbonyl-containing substituent classes. In a biomolecular embodiment, the alkyne compound can include a first polypeptide comprising at least one non-natural amino acid residue (nnAAr) of formula (IV), where Z is absent or a 5-membered or 6-membered aryl or heteroaryl ring and W1 is selected from C1-C10 alkylene, —NH—, —O—, and —S—.
The polypeptide includes nnAAr residues connected through parameters that link to X1, with X1 being a polysaccharide or a second polypeptide, and an optional linker L1 connecting the polypeptide and X1 through selected linker chemistries. The document further specifies antigen-containing embodiments in which X1 is a polysaccharide, including Streptococcus pneumoniae capsular polysaccharide serotypes, and polypeptide embodiments include sequences such as SEQ ID NO:1 and SEQ ID NO:2.
Claims Coverage
The provided material identifies one independent claim describing a method of preparing a heteroaryl-containing compound. The claim includes four inventive features: LDAO-mediated azide-alkyne coupling under specified medium conditions; defined formula-based azide and alkyne reactants; polypeptides bearing nnAAr residues of formula (IV); and linkage to polysaccharides or a second polypeptide through X1 and an optional linker L1.
LDAO-mediated azide-alkyne coupling in water and DMSO
Contacting an azide compound with an alkyne compound in the presence of lauryldimethylamine oxide (LDAO), water, and DMSO to form the heteroaryl-containing compound, wherein the LDAO is at a concentration of up to 2% v/v.
Azide compound of formula (I) and alkyne compound of formula (II) or (III)
Employing the azide compound as a compound of formula (I) and the alkyne compound as a compound of formula (II) or (III), with R1, R2, and R3 for the alkyne independently selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl, and specified (CH2)a-carbonyl-containing substituent classes.
Polypeptide-bearing nnAAr alkyne embodiment with defined nnAAr formula (IV)
Defining an embodiment where R1 is a first polypeptide comprising at least one non-natural amino acid residue (nnAAr) of formula (IV), with Z absent or a 5-membered or 6-membered aryl or heteroaryl ring and W1 selected from C1-C10 alkylene, —NH—, —O—, and —S—, and with R8 H or an amino acid residue of the first polypeptide and R9 OH or an amino acid residue of the first polypeptide.
Polysaccharide or second polypeptide linkage via X1 and optional linker L1
Linking the polypeptide embodiment to a moiety defined by X1, where X1 is a polysaccharide or a second polypeptide, and optionally including linker L1 selected from substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, and multiple specified bond types and carbonyl-containing linker structures.
Across the identified independent claim, the core inventive coverage centers on forming a heteroaryl-containing compound by LDAO-mediated azide-alkyne coupling in water and DMSO, using formula (I) azide and formula (II)/(III) alkyne reactants, and optionally incorporating an alkyne embodiment that contains a first polypeptide with nnAAr of formula (IV) linked to a polysaccharide or a second polypeptide through X1 and an optional linker L1.
Stated Advantages
Faster metal-free click for bioconjugation.
Avoids copper toxicity associated with copper-catalyzed click.
Antibody titer improvement asserted for LDAO-inclusive conjugation versus absence of LDAO.
Higher molecular weight conjugates are obtained when conjugation uses LDAO, compared to without LDAO.
Antibody titer upon challenge is higher with LDAO than for the same conjugate prepared without LDAO.
Conjugate molecular weight increases when LDAO is used, including at least 1.3× and up to 2.5× compared with the same conjugate prepared without LDAO.
Documented Applications
Preparing heteroaryl-containing polypeptide/antigen conjugates by conjugating nnAAr azides (within a polypeptide) to antigens or polysaccharides using an alkyne component that participates in the triazole-forming chemistry.
Constructing bioconjugates involving carrier proteins and antigens, polysaccharides, or glycans.
Antigen-containing embodiments where X1 is a polysaccharide, including Streptococcus pneumoniae capsular polysaccharide serotype sets.
Conjugates that involve an antigen-containing polysaccharide and/or a second polypeptide conjugated through nnAAr-containing first polypeptides.
Immunization-related use where antibody titer upon challenge is measured for conjugates generated using LDAO-enabled azide-alkyne chemistry.
Conjugate embodiments that include an antibody-titer improvement upon challenge.
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