# US 12,005,109 B2 · Multi-functionalized nOMV conjugates

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- Number: US 12,005,109 B2
- Document ID: US12005109B2
- Status: Granted
- Kind: B2
- Application number: 16/636,560
- Filed: 2018-08-08
- Published: 2022-01-13
- Granted: 2024-06-11
- Earliest claimed: 2017-08-10
- Assignees: Glaxosmithkline Biologicals SA
- Applicants: Glaxosmithkline Biologicals SA
- Inventors: Renzo Alfini; Roberta Di Benedetto; Francesca Micoli; Allan James Saul
- Web page: https://patentgrep.com/patent/US12005109B2

## CPC

- A61K 39/0275: {Salmonella}
- A61K 39/095: Neisseria
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- A61K 39/0258: {Escherichia}
- A61K 39/116: Polyvalent bacterial antigens
- A61K 39/385: Haptens or antigens, bound to carriers
- A61K 47/6911: {the form being a liposome}
- A61P 31/04: Antibacterial agents
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- A61K 2039/6018: {Lipids, e.g. in lipopeptides}
- A61K 2039/6068: {Other bacterial proteins, e.g. OMP}
- A61K 2039/6087: {Polysaccharides; Lipopolysaccharides [LPS]}
- A61K 2039/627: {characterised by the linker}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention is in the field of conjugating native, non-detergent extracted, outer membrane vesicles (nOMV) to multiple antigens to form multi functionalized nOMV-antigen conjugated derivatives, which are particularly useful for immunogenic compositions and immunisation; processes for the preparation and use of such conjugates are also provided.

## Claims

### Claim 1

An immunogenic conjugate comprising an isolated, intact native outer membrane vesicle (nOMV), having at least a surface saccharide moiety connected to an antigen, and having at least a surface protein residue connected to a different antigen through a bivalent linker;
wherein said bivalent linker has the general formula (I):
X-L-X′ (I)
wherein:
X and X′ are different to each other or the same, and are a functional group able to selectively react with the nOMV protein residue on one hand and with the antigen on the other hand;
-L- is a bivalent linear or branched C1-C15 alkyl or alkenyl group optionally substituted, and optionally interrupted by one or more heteroatom selected from:
oxygen (—O—), sulfur (—S—), and nitrogen (NH— or optionally substituted —N— group);
wherein X and X′ are N-hydroxysuccinimide ester derivatives selected from at least one of:
wherein the * represents the point of contact with the -L- group in formula (I);
wherein said nOMV is prepared from S. sonnei, S. flexneri, meningococcus, or Salmonella bacteria;
wherein said antigen is selected from a group consisting of: Neisseria meningitidis fHbp, SsIE, and FdeC;
wherein SsIE comprise an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 15;
wherein FdeC comprise an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity to SEQ ID NO: 16; and
wherein Neisseria meningitidis fHbp comprise an amino acid sequence comprising SEQ ID NO: 8, SEQ ID NO: 1, or of SEQ ID NO: 9.

### Claim 2 (depends on 1)

The conjugate according to claim 1, wherein said bivalent linker is a homobifunctional linker having the formula X═X′.

### Claim 3 (depends on 1)

The conjugate according to claim 1, wherein said bivalent linker is BS3 or SIDEA.

### Claim 4 (depends on 1)

The conjugate according to claim 1, wherein said nOMV is obtained by a detergent free process, being released into the fermentation broth and purified using a centrifugation and subsequent filtration; or being released into the fermentation broth and purified using two consecutive Tangential Flow Filtration (TFF) steps.

### Claim 5 (depends on 1)

The conjugate according to claim 1, wherein the antigens are selected from an immunogenic polypeptide or a capsular polysaccharide.

### Claim 6 (depends on 1)

The conjugate according to claim 1, wherein said nOMV and the antigens connected through a surface saccharide moiety and through a bivalent linker respectively are derived from different bacterial strain.

### Claim 7 (depends on 1)

The conjugate according to claim 1,
wherein said antigen is selected from a group consisting of: Neisseria meningitidis fHbp, Plasmodium falciparum CSP, Plasmodium falciparum Pfs25, Plasmodium falciparum RO6C, Plasmodium falciparum R010C, Escherichia coli CTF1232, S. typhi Vi saccharide, Neisseria meningitidis NHBA, Poly-rhamnose oligosaccharide, Escherichia coli FdeC, Escherichia coli SslE, Synthetic or native GAS PS, Synthetic or native GBS PS, Neisseria meningitidis ser A saccharide, Neisseria meningitidis ser C saccharide, Haemophilus influenzae type b, Haemophilus influenzae type a, and Streptococcus pneumoniae saccharide.

### Claim 8 (depends on 1)

The conjugate according to claim 1, wherein said bivalent linker is selected from a group consisting of: BS3, BS(PEG)5, and SIDEA.

### Claim 9 (depends on 1)

The conjugate according to claim 1,
wherein said antigen/linker is selected from a group consisting of: (NANP)3, MenC/SIDEA or BS3, MenA/SIDEA or BS3, Neisseria meningitidis fHbp/BS3, Native or synthetic GAS PS/BS3, Hib PS/SIDEA or BS3, Hia PS/SIDEA or BS3, FdeC/BS3, and SsIE/BS3.

### Claim 10 (depends on 1)

The conjugate according to claim 1, wherein said nOMV is produced from wild type bacteria or from genetically-modified bacterial strains that are mutated to enhance vesicle production.

### Claim 11 (depends on 1)

A process for preparing the conjugate according to claim 1, comprising the steps of:
i) activating at least a nOMV saccharide moiety, generally bond to the nOMV surface, and
ii) connecting the thus obtained activated saccharide to at least one antigen to obtain an antigen-nOMV intermediate;
iii) reacting at least a nOMV surface protein residue of said antigen-nOMV intermediate with the first terminal portion of a bivalent linker, to obtain an antigen-nOMV-linker intermediate, and
iv) connecting the thus obtained antigen-nOMV-linker intermediate to at least a different antigen via the second terminal portion of the bivalent linker, thus obtaining the conjugate;
or
i) reacting at least a nOMV surface protein residue with the first terminal portion of a bivalent linker to obtain a nOMV-linker intermediate, and
ii) connecting said nOMV-linker intermediate to at least one antigen via the second terminal portion of the bivalent linker, thus obtaining a nOMV-linker-antigen intermediate,
iii) activating at least a nOMV saccharide moiety of the thus obtained nOMV-linker-antigen intermediate, and
iv) connecting the thus obtained activated saccharide to at least a different antigen, to obtain the conjugate.

### Claim 12 (depends on 1)

An immunogenic composition comprising a conjugate according to claim 1 and at least one pharmaceutically acceptable carrier or excipient.

### Claim 13 (depends on 1)

A medicament comprising the immunogenic conjugate according to claim 1, and at least one pharmaceutically acceptable carrier or excipient.

### Claim 14 (depends on 13)

A method for inducing an immune response in a vertebrate in need thereof, wherein the method comprises administering the medicament according to claim 13 to the vertebrate.