# US 2024/0200084 A1 · Autotransporter system

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2024/0200084 A1
- Document ID: US20240200084A1
- Status: Published application
- Kind: A1
- Application number: 18/010,792
- Filed: 2021-06-18
- Published: 2024-06-20
- Earliest claimed: 2020-06-19
- Assignees: Prokarium Limited
- Applicants: Prokarium Limited
- Inventors: Marc Biarnes Carrera; Annelise Soulier
- Web page: https://patentgrep.com/patent/US20240200084A1

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/74: Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- A61K 35/74: Bacteria (therapeutic use of a bacterial protein A61K38/00)
- A61K 38/20: Interleukins [IL]
- A61K 38/21: Interferons {[IFN]}
- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- C07K 14/21: from Pseudomonadaceae (F)
- C07K 14/245: Escherichia (G)
- C07K 14/255: Salmonella (G)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61K 39/0011: {Cancer antigens}
- A61K 2039/522: {avirulent or attenuated}
- A61K 2039/523: {expressing foreign proteins}
- A61P 35/00: Antineoplastic agents
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/02: containing a signal sequence
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/21: containing a His-tag
- C07K 2319/43: containing a FLAG-tag
- C07K 2319/50: containing protease site
- C12R 2001/42: Salmonella
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)
- 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)}
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention provides for a modified autotransporter and the use of genetically engineered microorganisms comprising said modified autotransporters in the treatment of infectious and neoplastic disease. The present invention therefore also relates to vaccine and immunotherapeutic compositions comprising said genetically engineered microorganism.

## Claims

### Claim 1

An autotransporter construct modified to permit insertion of a heterologous polynucleotide sequence that encodes a target polypeptide for translocation across the inner and outer membrane of a Gram-negative bacterium, the autotransporter comprising i) a polynucleotide sequence that encodes a N-terminal signal sequence; ii) a passenger region into which said heterologous polynucleotide sequence encoding the target polypeptide is to be inserted and iii) a polynucleotide sequence encoding a translocation domain; wherein the passenger region comprises a synthetic polynucleotide sequence flanked by restriction enzyme recognition sequences, wherein said synthetic polynucleotide sequence comprises a polynucleotide sequence that encodes a first polypeptide tag.

### Claim 2 (depends on 1)

The autotransporter construct of claim 1, wherein the passenger region further comprises a synthetic polynucleotide sequence that encodes a linker, preferably wherein the linker is a serine-glycine linker.

### Claim 3 (depends on 1)

The autotransporter construct of claim 1, wherein the passenger region further comprises a synthetic polynucleotide sequence that encodes a second polypeptide tag, preferably wherein the second polypeptide tag is a His tag.

### Claim 4 (depends on 1)

The autotransporter construct of claim 1, wherein the passenger region further comprises a synthetic polynucleotide sequence that encodes a cleavage site, preferably wherein the cleavage site is a caspase-3 cleavage site and/or an OmpT cleavage site.

### Claim 5 (depends on 1)

The autotransporter construct of claim 1, wherein the synthetic polynucleotide sequence that encodes the second polypeptide tag is upstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag.

### Claim 6 (depends on 1)

The autotransporter construct of claim 1, wherein the first polypeptide tag is any synthetic polynucleotide sequence that allows for the in-frame translation of the autotransporter, preferably the first polypeptide tag is a FLAG tag.

### Claim 7 (depends on 1)

The autotransporter construct of claim 1, wherein the synthetic polynucleotide sequence that encodes the second polypeptide tag is located upstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag, the synthetic polynucleotide sequence that encodes the linker is located downstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag and the synthetic polynucleotide sequence that encodes the cleavage site is located downstream of the synthetic polynucleotide sequence that encodes the linker.

### Claim 8 (depends on 1)

The autotransporter construct of claim 1, wherein the autotransporter to be modified is EstA, MisL, Hbp, AIDA-1, EstP or Pet.

### Claim 9 (depends on 1)

The autotransporter construct of claim 1, wherein the autotransporter construct further comprises a heterologous polynucleotide encoding a target peptide or protein.

### Claim 10 (depends on 1)

The autotransporter construct of claim 1, wherein the heterologous polynucleotide encodes for a therapeutic protein.

### Claim 11 (depends on 9)

The autotransporter construct of claim 9, wherein the heterologous polynucleotide encodes for an anti-cancer therapeutic or immunogenic molecule.

### Claim 12 (depends on 11)

The autotransporter construct of claim 11, wherein the anti-cancer therapeutic or immunogenic molecule is a cytokine, a chemokine, an antibody or a fragment thereof, a cytotoxic agent, a cancer antigen, or any combination thereof.

### Claim 13 (depends on 9)

The autotransporter construct of claim 9, wherein the heterologous polynucleotide encodes for IFNα, IFNβ, IL-18.

### Claim 14 (depends on 1)

A genetically engineered microorganism comprising the autotransporter construct of claim 1.

### Claim 15 (depends on 14)

The genetically engineered microorganism of claim 14, wherein the genetically engineered microorganism is an attenuated Gram-negative bacterium.

### Claim 16 (depends on 14)

The genetically engineered microorganism of claim 14, wherein the genetically engineered microorganism is a Salmonella spp, preferably Salmonella enterica, even more preferably Salmonella enterica serovar Typhi or Salmonella enterica serovar Typhimurium.

### Claim 17 (depends on 14)

The genetically engineered microorganism of claim 14, wherein the genetically engineered microorganism is derived from Salmonella spp. and comprises an attenuating mutation in a Salmonella Pathogenicity Island 2 (SPI-2) gene and an attenuating mutation in a second gene, preferably wherein the second gene is an aro gene.

### Claim 18 (depends on 14)

The genetically engineered microorganism of claim 14, wherein the genetically engineered microorganism is Salmonella enterica serovar Typhi ZH9.

### Claim 19 (depends on 14)

The genetically engineered microorganism of claim 14, wherein the genetically engineered microorganism is derived from Salmonella enterica serovar Typhi, wherein said strain comprises a modification in which the lipopolysaccharide 02 O-antigens and/or the flagella proteins of Salmonella enterica serovar Paratyphi A are expressed.

### Claim 20 (depends on 1)

A vaccine composition comprising the autotransporter construct of claim 1.

### Claim 21 (depends on 20)

The vaccine composition of claim 20, wherein the vaccine composition further comprises an adjuvant, pharmaceutically acceptable carrier or excipient.

### Claim 22 (depends on 20)

A method of prophylactic or therapeutic treatment of an infectious disease or a neoplastic disease in a subject, the method comprising administering to the subject the vaccine composition of claim 20.

### Claim 23 (depends on 22)

The method of claim 22, wherein the infectious disease is a viral infection, a bacterial infection, a fungal infection and/or a parasitic infection.

### Claim 24 (depends on 22)

The method of claim 22, wherein the neoplastic disease is associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

### Claim 25 (depends on 1)

An immunotherapeutic composition comprising the modified autotransporter of claim 1.

### Claim 26 (depends on 25)

The immunotherapeutic composition of claim 25, wherein the immunotherapeutic composition is given in combination with a checkpoint inhibitor, an antigen-specific T cell, a therapeutic antibody, a cancer vaccine or other cellular component of the immune system.

### Claim 27 (depends on 25)

A method of prophylactic or therapeutic treatment of an infectious disease in a subject, the method comprising administering to the subject the immunotherapeutic composition of claim 25.

### Claim 28 (depends on 27)

The method of claim 27, wherein the infectious disease is a viral infection, a bacterial infection, a fungal infection and/or a parasitic infection.

### Claim 29 (depends on 25)

A method of prophylactic or therapeutic treatment of a neoplastic disease in a subject, the method comprising administering to the subject the immunotherapeutic composition of claim 25.

### Claim 30 (depends on 29)

The method of claim 29, wherein the neoplastic disease is associated with a cancer selected from prostate cancer, liver cancer, renal cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, brain cancer, hepatocellular cancer, lymphoma, leukaemia, gastric cancer, cervical cancer, ovarian cancer, thyroid cancer, melanoma, carcinoma, head and neck cancer, skin cancer or sarcoma.

### Claim 31

A method for modifying a Gram-negative bacterial autotransporter, comprising: i) removing the passenger domain from the passenger region, ii) introducing a synthetic polynucleotide sequence that encodes a first polypeptide tag in the passenger region flanked by restriction enzyme recognition sequences, iii) introducing a synthetic polynucleotide sequence that encodes a second polypeptide tag within the passenger region upstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag and is positioned outside of the restriction enzyme recognition sequence boundaries, iv) introducing a synthetic polynucleotide sequence that encodes a linker, said sequence being downstream of the synthetic polynucleotide sequence that encodes the first polypeptide tag and is positioned outside of the restriction enzyme recognition sequence boundaries, and v) introducing a synthetic polynucleotide sequence that encodes for a cleavage site within the passenger region downstream of the synthetic polynucleotide sequence that encodes for a linker.

### Claim 32 (depends on 31)

The method of modifying a Gram-negative bacterial autotransporter of claim 31, wherein the first polypeptide tag is a FLAG tag or any other tag that allows in-frame translation of the autotransporter.

### Claim 33 (depends on 31)

The method of modifying a Gram-negative bacterial autotransporter of claim 31, wherein the second polypeptide tag is a His tag.

### Claim 34 (depends on 31)

The method of modifying a Gram-negative bacterial autotransporter of claim 31, wherein the linker is a serine-glycine linker.

### Claim 35 (depends on 31)

The method of modifying a Gram-negative bacterial autotransporter of claim 31, wherein the cleavage site is a caspase-3 cleavage site and/or an OmpT cleavage site.