EP-4741507-A1 - MODIFIED BACTERIUM HAVING ANTI-TUMOR ACTIVITY AND BETTER SAFETY

Abstract

The present invention relates to a modified bacterium with anti-tumor activity, wherein the bacterium comprises a hypoxia regulated lipopolysaccharide (LPS) gene expression cassette comprising a gene involved in the biosynthesis or transport of LPS or a gene encoding murein-lipoprotein under the control of a first strictly hypoxia inducible promoter. The bacterium further comprises an essential gene expression cassette under the control of a strictly hypoxia inducible promoter, and the bacteria is deficient in at least one gene or functional expression product thereof that participates in or regulates an endogenous anti-oxidative stress response pathway. The present invention also relates to a pharmaceutical composition comprising the modified bacterium and anti-tumor use thereof.

Inventors

• LIU, Chenli
• WANG, Zuowei
• DONG, Yuxuan
• SHENG, Fangqian
• LU, WEIQI
• LI, YANG
• ZANG, Zhongsheng
• LI, WANLIN
• MALEEHA, Saifi

Assignees

• Shenzhen Synthetica Pioneering Co., Ltd.

Dates

Publication Date

20260513

Application Date

20240708

Claims (20)

1. A modified bacterium, wherein the bacterium comprises a hypoxia regulated lipopolysaccharide (LPS) gene expression cassette comprising a gene involved in the biosynthesis or transport of LPS or a gene encoding a murein-lipoprotein under the control of a first strictly hypoxia inducible promoter, as compared to an unmodified starting strain.
2. The modified bacterium of claim 1, wherein the gene involved in the biosynthesis of LPS is selected from the group consisting of a gene involved in the synthesis of O-antigen, a gene involved in the synthesis of core region, and a gene involved in the synthesis of lipid A.
3. The modified bacterium of claim 2, wherein the gene involved in the synthesis of O-antigen is selected from the group consisting of genes rfbN, rfbV, wzzB, gtrBb, gtrBa, and rfbP/wbaP.
4. The modified bacterium of claim 2, wherein the gene involved in the synthesis of core region is selected from the group consisting of genes rfaB, rfaC/waaC, rfaF, rfaG, yibD, rfaI, rfaJ, rfaK, rfaL, rfaP, rfaQ, rfaY, rfaZ, yijP, gmhA, yaeD, rfaE, and rfaD.
5. The modified bacterium of claim 2, wherein the gene involved in the synthesis of lipid A is selected from the group consisting of genes lpxA, lpxB, lpxC, lpxD, lpxH, lpxK, htrB, msbB/lpxM, lpxO, lpxP, yeU, kdtA, arnT, pagP, yjdB, yhjw, kdsA, kdsB, yrbI, and yrbH.
6. The modified bacterium of claim 1, wherein the gene involved in the transport of LPS is selected from the group consisting of genes msbA, Wzy, wzxE, lptA, lptB, lptC/yrbK, lptD, and lptG.
7. The modified bacterium of any one of claims 1-6, wherein the gene involved in the biosynthesis or transport of LPS or the gene encoding murein-lipoprotein is a naturally occurring gene in the chromosome of the bacterium, wherein the native promoter of the gene involved in the biosynthesis or transport of LPS or the gene encoding murein-lipoprotein is functionally replaced by the first strictly hypoxia inducible promoter, whereby the expression of the gene involved in the biosynthesis or transport of LPS or the gene encoding murein-lipoprotein in the bacterium is completely under the control of the first strictly hypoxia inducible promoter.
8. The modified bacterium of any one of claims 1-6, wherein the LPS gene expression cassette is exogenous, and the gene involved in the biosynthesis or transport of LPS or the gene encoding murein-lipoprotein naturally occurring in the chromosome of the bacterium is deleted or functionally inactivated, whereby the expression of the gene involved in the biosynthesis or transport of LPS or the gene encoding murein-lipoprotein in the bacterium is completely under the control of the first strictly hypoxia inducible promoter.
9. The modified bacterium of claim 8, wherein the exogenous LPS gene expression cassette is integrated into the bacterial chromosome.
10. The modified bacterium of claim 8, wherein the exogenous LPS gene expression cassette is outside of the bacterial chromosome.
11. The modified bacterium of claim 10, wherein the exogenous LPS gene expression cassette is present in the plasmid carried by the bacterium.
12. The modified bacterium of any one of claims 1-11, wherein the bacterium further comprises a hypoxia regulated essential gene expression cassette comprising an essential gene of the bacterium under the control of a second strictly hypoxia inducible promoter, and the bacterium is deficient in at least one gene involved in or regulating the endogenous anti-oxidative stress response pathway or a functional expression product thereof.
13. The modified bacterium of any one of claims 1-12, wherein the first and second strictly hypoxia inducible promoters are each independently selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA, and Ptet-Fnr.
14. The modified bacterium of any one of claims 1-12, wherein the first and second strictly hypoxia inducible promoters are each independently selected from the group consisting of fnrSp and ssbp1 promoters.
15. The modified bacterium of any one of claims 12-14, wherein the expression product of the essential gene is responsible for the synthesis of 2,6-diaminopimelic acid (DAP) within the bacterium, and when cultured under aerobic conditions, the bacterial growth is dependent on the additional DAP or an analog thereof added to the medium.
16. The modified bacterium of claim 15, wherein the essential gene is selected from dapA, dapB, dapD, dapE, argD, dapF, and any combination thereof.
17. The modified bacterium of claim 15, wherein the essential gene is selected from dapA and dapE.
18. The modified bacterium of any one of the claims 12-17, wherein the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family gene.
19. The modified bacterium of any one of claims 12-18, wherein the functional expression product of the gene involved in or regulating the endogenous anti-oxidative stress response pathway is an HtrA serine protease family related protein.
20. The modified bacterium of any one of claims 12-19, wherein the bacterium is deficient in the activity of HtrA serine protease.

Description

Technical Field The present invention relates to a modified bacterium with anti-tumor activity, a pharmaceutical composition comprising the modified bacterium and the anti-tumor use thereof. Background Cancer has become one of the most important diseases threatening human life and health, but existing treatments (radiotherapy, chemotherapy, surgery and targeted drugs) all have shortcomings and there is an urgent need to develop new treatment approaches. Traditional bacterial tumor therapies, represented by Coley's toxin, have a history of 150 years, but are known for their unstable efficacy and safety concerns. The development of genetic engineering technology has made great progress in reducing the toxicity of bacterial strains, and a series of clinical trials have demonstrated that the attenuated engineered bacteria for human tumor therapy are safe but with limited efficacy, i.e., are unable to combine safety and therapeutic efficacy, thus failing to satisfy the needs of clinical tumor therapy. In addition, bacteria carrying intact lipopolysaccharide (LPS) can activate the immune response in normal tissues and organs after the administration to a patient. On the one hand, such an immune response will lead to the loss of anti-tumor bacteria during the delivery, and on the other hand, it will cause side effects to the body, for example, resulting in the loss of body weight in patients. In 2016, researchers obtained attenuated Salmonella strains by the knockout of the msbB gene in the Salmonella LPS pathway and the murein-lipoprotein synthesis gene lppAB, or the knockout of both the two genes. However, previous studies showed that the therapeutic effect of attenuated Salmonella strain VNP20009 (msbB-, purI-) with msbB knockout on tumor is not significant. Therefore, the further optimization for tumor therapy with bacteria is imperative. Summary of the Invention In the present invention, a strictly anaerobic bacterium is constructed by means of synthetic biological genetic circuits, which shows therapeutic efficacy against tumors after being administered to an animal body or a human body, and can be cleared by normal tissues and organs in a short period of time, thereby reducing the toxic and side effects on the animal body or the human body due to the long-term retention of the bacterium in vivo. The bacterium of the present invention is deficient in the wild-type genes related to the biosynthesis and transport of LPS, and the deficient genes are conditionally expressed in the bacterium, thereby achieving site-specific production of complete LPS in tumors, enabling the induction of an immune response in tumors, meanwhile, avoiding the immune response of normal tissues against LPS, and thus, the bacterium has more reliable anti-tumor efficacy and safety. The present invention provides a modified bacterium, wherein the bacterium comprises a hypoxia regulated lipopolysaccharide (LPS) gene expression cassette, and the LPS gene expression cassette comprises a gene involved in the biosynthesis or transport of LPS or a gene encoding murein-lipoprotein under the control of a first strictly hypoxia inducible promoter, as compared with an unmodified starting strain. In one embodiment, the bacterium comprise a hypoxia regulated essential gene expression cassette comprising an essential gene of the bacterium under the control of a second strictly hypoxia inducible promoter, and the bacterium is deficient in at least one gene required for survival in macrophages or a functional expression product thereof, as compared to an unmodified starting strain. In one embodiment, the bacterium comprise a hypoxia regulated essential gene expression cassette comprising an essential gene of the bacterium under the control of a second strictly hypoxia inducible promoter, and the bacterium is deficient in at least one gene involved in or regulating the endogenous anti-oxidative stress response pathway or a functional expression product thereof, as compared to an unmodified starting strain. In one embodiment, the bacterium further comprise a hypoxia regulated essential gene expression cassette comprising an essential gene of the bacterium under the control of a second strictly hypoxia inducible promoter and a pH regulated expression cassette comprising a gene encoding a bacteria-derived hemolysin protein under the control of a promoter that is active under acid pH condition, and the bacterium is deficient in at least one gene involved in or regulating the endogenous anti-oxidative stress response pathway or a functional expression product thereof, as compared to an unmodified starting strain. In one embodiment, the bacterium comprise a hypoxia regulated essential gene expression cassette comprising an essential gene of the bacterium under the control of a second strictly hypoxia inducible promoter and a pH regulated expression cassette comprising a gene encoding a bacteria-derived hemolysin protein under the control of a promoter that is