# US 10,864,266 B2 · Modified lipopolysaccharide glycoform and method of use

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- Number: US 10,864,266 B2
- Document ID: US10864266B2
- Status: Granted
- Kind: B2
- Application number: 16/690,739
- Filed: 2019-11-21
- Published: 2020-04-23
- Granted: 2020-12-15
- Earliest claimed: 2014-05-09
- Assignees: The Trustees of Princeton University
- Applicants: The Trustees of Princeton University; President and Fellows of Harvard College
- Inventors: Thomas J. Silhavy; Marcin Grabowicz; Daniel Kahne; Matthew Lebar; Dorothee Andres
- Web page: https://patentgrep.com/patent/US10864266B2

## CPC

- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- C07H 13/06: Fatty acids
- C07H 15/04: attached to an oxygen atom of the saccharide radical
- C07K 4/00: Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
- C12N 9/00: Enzymes; Proenzymes; Compositions thereof (preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; {enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113}); Processes for preparing, activating, inhibiting, separating or purifying enzymes (preparation of malt C12C1/00)
- C12N 9/93: {Ligases (6)}
- C12N 15/04: Fungi
- C12P 21/005: {Glycopeptides, glycoproteins}
- A61K 2039/55516: {Proteins; Peptides}
- A61K 2039/55572: {Lipopolysaccharides; Lipid A; Monophosphoryl lipid A}
- A61K 2039/55583: {Polysaccharides}
- A61K 2039/55594: {from bacteria}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present disclosure generally relates to genetic engineering of bacteria. More particularly, the present disclosure describes genetic engineering of E. coli to create mutant O-antigen ligase, as well as novel lipopolysaccharide molecules resulting from that genetic engineering. Methods for using those novel molecules are also described.

## Claims

### Claim 1

A vaccine adjuvant comprising at least one of a lipopolysaccharide isolated from a mutant O-antigen ligase or lipopolysaccharide derivative isolated from the mutant O-antigen ligase, the mutant O-antigen ligase comprising either an isolated protein that has the amino acid sequence SEQ ID NO: 1, or
an isolated protein having at least 90% sequence identity to SEQ ID NO: 2 and having an amino acid substitution of phenylalanine to serine at the phenylalanine homologous to position 332 of SEQ ID NO: 2,
wherein the isolated protein has at least one of 0-antigen ligase activity or the activity to ligate lipid A to disaccharide pentapeptide (DPP).

### Claim 2

A vaccine adjuvant comprising a lipopolysaccharide derivative molecule prepared by the process comprising the steps of:
providing an E. coli strain adapted to express an LPS derivative molecule;
modifying the E. coli strain by creating a mutant 0-antigen ligase selected from the group consisting of:
a mutant O-antigen ligase comprising an isolated protein that has the amino acid sequence SEQ ID NO: 1, and
a mutant O-antigen ligase comprising an isolated protein having at least 90% sequence identity to SEQ ID NO: 2 and having an amino acid substitution of phenylalanine to serine at the phenylalanine homologous to position 332 of SEQ ID NO: 2, wherein the protein has at least one of 0-antigen ligase activity or the activity to ligate lipid A to disaccharide pentapeptide (DPP);
providing a growth media for the modified E. coli strain; and
allowing the modified E. coli strain to produce an LPS* derivative molecule, wherein the LPS* derivative molecule includes at least one non-native sugar not present in an LPS molecule it derives from.