Granted Patent B2

US 12,357,661 · App. 17/037,455 · Granted Jul 15, 2025

Inventors: Christopher D. Thanos (Tiburon, CA); Laura Hix Glickman (Oakland, CA); Justin Skoble (Berkeley, CA); Alexandre Charles Michel Iannello (San Diego, CA)

Assignee: Actym Therapeutics, Inc.

A61K35/74A61K39/0275A61K40/11A61K40/17A61K40/24A61K40/42A61K40/4562A61P35/00C07K14/521C07K16/248C07K16/2818C07K16/2827C12N1/205C12N15/74A61K45/06A61K2239/50C12R2001/42

Abstract

Claims (51)

1. An immunostimulatory bacterium, comprising a plasmid encoding a therapeutic product under control of a eukaryotic promoter, wherein:

the therapeutic product is an anti-cancer product;

the bacterium comprises genome modifications whereby the bacterium is csgD − ;

the genome of the immunostimulatory bacterium is modified to eliminate flagella, whereby:

the ability of the immunostimulatory bacterium to infect epithelial cells is eliminated;

uptake by phagocytic cells is retained or increased; and

accumulation of the immunostimulatory bacterium in tumor-resident immune cells is increased compared to accumulation in tumor-resident immune cells in the absence of the genome modification(s); and

the wild-type bacterium comprises flagella.

2. The immunostimulatory bacterium of claim 1 that is a Gram-negative bacterium, wherein the genome of the immunostimulatory bacterium is modified, whereby lipopolysaccharide (LPS) of the bacterium is penta-acylated.

3. The immunostimulatory bacterium of claim 1 that is an adenosine auxotroph.

4. The immunostimulatory bacterium of claim 1 that comprises genome modifications, whereby the bacterium is pagP − .

5. The immunostimulatory bacterium of claim 1 that comprises genome modifications, whereby the immunostimulatory bacterium is pagP − and msbB − .

6. The immunostimulatory bacterium of claim 1 , wherein the anti-cancer therapeutic product is an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment.

7. The immunostimulatory bacterium of claim 6 , wherein the immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment is a cytokine.

8. The immunostimulatory bacterium of claim 6 , wherein the immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment is a chemokine.

9. The immunostimulatory bacterium of claim 6 , wherein the immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment is selected from among one or more of: IL-2, IL-7, IL-12p70 (IL-12p40+IL-12p35), IL-15, IL-36 gamma, IL-15/IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-2 that is modified so that it has attenuated binding or does not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate the recruitment or persistence of T cells, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

10. The immunostimulatory bacterium of claim 1 , wherein the therapeutic product is an antibody or antigen-binding fragment thereof.

11. The immunostimulatory bacterium of claim 10 , wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment that is selected from among a Fab, Fab′, F(ab′) 2 , single-chain Fv (scFv), Fv, dsFv, nanobody, diabody fragment, and a single-chain antibody.

12. The immunostimulatory bacterium of claim 10 , wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof or is a human antibody or antigen-binding fragment thereof.

13. The immunostimulatory bacterium of claim 1 , wherein nucleic acid encoding the therapeutic product is operatively linked for expression to a nucleic acid encoding a secretory signal, whereby, upon expression in a host, the therapeutic product is secreted.

14. The immunostimulatory bacterium of claim 1 , wherein the bacterium is auxotrophic for adenosine, or for adenosine and adenine.

15. The immunostimulatory bacterium of claim 1 that is aspartate-semialdehyde dehydrogenase” (asd − ), wherein the bacterium is asd − by virtue of disruption or deletion of all or a portion of the endogenous gene encoding aspartate-semialdehyde dehydrogenase (asd), whereby endogenous asd encoded in the bacterial genome is not expressed.

16. The immunostimulatory bacterium of claim 1 , wherein the plasmid is present in fewer than 150 copies.

17. The immunostimulatory bacterium of claim 1 , wherein the eukaryotic promoter is an RNA polymerase II promoter that is a viral promoter or a mammalian RNA polymerase II promoter.

18. The immunostimulatory bacterium of claim 17 , wherein the promoter is active in hypoxic conditions, or in conditions where the pH is less than 7.

19. An immunostimulatory bacterium, comprising a plasmid encoding an anti-cancer therapeutic product, wherein:

the immunostimulatory bacterium is a Salmonella species;

the immunostimulatory bacterium is an adenosine auxotroph;

the immunostimulatory bacterium comprises genome modifications, whereby the bacterium is fliC − /fljB − and thereby lacks flagellin and flagella; and

the anti-cancer therapeutic product encoded on the plasmid is expressed under the control of a eukaryotic promoter or eukaryotic regulatory sequences.

20. The immunostimulatory bacterium of claim 1 , wherein the bacterium prior to the genome modifications is an attenuated bacterium.

21. The immunostimulatory bacterium claim 1 , wherein the bacterium prior to the genome modifications is a wild-type strain.

22. The immunostimulatory bacterium of claim 1 , wherein the bacterium prior to the genome modifications is a strain of Salmonella .

23. The immunostimulatory bacterium of claim 22 , wherein the bacterium prior to the genome modifications is a Salmonella typhimurium strain.

24. The immunostimulatory bacterium of claim 23 , wherein the bacterium prior to the genome modifications is a Salmonella typhimurium strain selected from among strains designated as YS1646 (ATCC #202165), RE88, SL7207, χ 8429 , χ 8431, χ 8468, and wild-type strain ATCC 14028.

25. A pharmaceutical composition, comprising the immunostimulatory bacterium of claim 1 in a pharmaceutically acceptable vehicle.

26. An isolated eukaryotic cell, comprising the immunostimulatory bacterium of claim 1 .

27. The cell of claim 26 that is an immune cell, a stem cell, a tumor cell, or a primary cell line.

28. The cell of claim 26 that is a hematopoietic cell.

29. The cell of claim 26 that is a T-cell.

30. An immunostimulatory bacterium, comprising genome modifications whereby the bacterium lacks flagella and is pagP − /msbB − , wherein the wild-type bacterium comprises flagella.

31. The immunostimulatory bacterium of claim 30 , comprising a plasmid encoding a therapeutic product under control of a eukaryotic promoter.

32. The immunostimulatory bacterium of claim 30 , comprising a plasmid encoding a cytokine.

33. The immunostimulatory bacterium of claim 32 , wherein the cytokine is IL-15 or IL-15/IL-15R alpha chain complex.

34. An immunostimulatory bacterium, comprising a plasmid encoding a therapeutic product under control of a eukaryotic promoter, wherein:

the therapeutic product is an anti-cancer product that is an immunostimulatory protein that confers or contributes to an anti-tumor immune response in the tumor microenvironment;

the genome of the immunostimulatory bacterium is modified, whereby the bacterium is pagP − /ImsbB − , and lacks flagella; and

the wild-type bacterium has flagella.

35. The immunostimulatory bacterium of claim 34 that is a Salmonella strain that comprises genome modifications whereby the bacterium is purI − , asd − , and csgD − .

36. The immunostimulatory bacterium of claim 33 , wherein the therapeutic product is selected from among one or more of: IL-2, IL-7, IL-12p70, IL-15, IL-36 gamma, IL-15/IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-2 that is modified so that it has attenuated binding or does not bind to IL-2Ra, CXCL9, CXCL10, CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate the recruitment or persistence of T cells, CD40, CD40 ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

37. The immunostimulatory bacterium of claim 35 , wherein the therapeutic product is selected from among one or more of: IL-2, IL-7, IL-12p70,IL-15, IL-36 gamma, IL-15/IL-15R alpha chain complex, IL-18, IL-21, IL-23, IL-2 that is modified so that it has attenuated binding or does not bind to IL-2Ra, CXCL9, CXCL10,CXCL11, interferon-α, interferon-β, interferon-γ, CCL3, CCL4, CCL5, proteins that are involved in or that effect or potentiate the recruitment or persistence of T cells, CD40, CD40ligand (CD40L), CD28, OX40, OX40 ligand (OX40L), 4-1BB, 4-1BB ligand (4-1BBL), members of the B7-CD28 family, CD47 antagonists, TGF-beta polypeptide antagonists, and members of the tumor necrosis factor receptor (TNFR) superfamily.

Assignments (1)

Continuity (8)

References Cited (400)