EP-4741425-A1 - ANTI-TUMOR BACTERIA FOR REDUCING NEUTROPHIL LEVEL IN TUMOR

Abstract

Provided are a fusion protein, comprising an antibody that binds to a neutrophil or an antigen-binding fragment thereof, and a cytotoxin or an active fragment thereof. Also provided are modified bacteria having anti-tumor activity and expressing the fusion protein, the antibody or the antigen-binding fragment thereof, or the toxin or the active fragment thereof in a low-oxygen environment (such as a tumor). Also provided are a pharmaceutical composition comprising the modified bacteria and an anti-tumor use thereof.

Inventors

• LIU, Chenli
• MALEEHA, Saifi
• DONG, Yuxuan
• LI, YANG
• ZANG, Zhongsheng
• CHEN, Kening

Assignees

• Shenzhen Synthetica Pioneering Co., Ltd.

Dates

Publication Date

20260513

Application Date

20240708

Claims (20)

1. A fusion protein comprising an antibody that binds to a neutrophil or an antigen-binding fragment thereof and a cytotoxin or an active fragment thereof.
2. The fusion protein of claim 1, wherein the antibody is a nanobody.
3. The fusion protein of claim 2, wherein the antibody comprises the complementary determining regions (CDRs) selected from the group consisting of: CDR1 comprising residues 31-35 of SEQ ID NO: 19, CDR2 comprising residues 50-65 of SEQ ID NO: 19, and CDR3 comprising residues 98-104 of SEQ ID NO: 19; CDR1 comprising residues 31-35 of SEQ ID NO: 20, CDR2 comprising residues 50-67 of SEQ ID NO: 20, and CDR3 comprising residues 100-111 of SEQ ID NO: 20; and CDR1 comprising residues 31-35 of SEQ ID NO: 21, CDR2 comprising residues 50-64 of SEQ ID NO: 21, and CDR3 comprising residues 98-115 of SEQ ID NO: 21 o
4. The fusion protein of any one of claims 1-3, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20 and 21.
5. The fusion protein of any one of claims 1-4, wherein the cytotoxin is Pseudomonas exotoxin (PE).
6. The fusion protein of any one of claims 1-5, wherein the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25.
7. The fusion protein of any one of claims 1-6, wherein the fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27.
8. The fusion protein of any one of claims 1-7, further comprising a signal peptide.
9. The fusion protein of claim 8, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.
10. The fusion protein of any one of claims 1-9, further comprising a linker and/or a tag sequence.
11. An expression construct comprising a nucleotide sequence encoding an antibody that binds to neutrophils or antigen-binding fragment thereof operably linked to a strictly hypoxia inducible promoter.
12. The expression construct of claim 11, wherein the antibody is a nanobody.
13. The expression construct of claim 12, wherein the antibody comprises complementary determining regions (CDRs) selected from the group consisting of: CDR1 comprising residues 31-35 of SEQ ID NO: 19, CDR2 comprising residues 50-65 of SEQ ID NO: 19, and CDR3 comprising residues 98-104 of SEQ ID NO: 19; CDR1 comprising residues 31-35 of SEQ ID NO: 20, CDR2 comprising residues 50-67 of SEQ ID NO: 20, and CDR3 comprising residues 100-111 of SEQ ID NO: 20; and CDR1 comprising residues 31-35 of SEQ ID NO: 21, CDR2 comprising residues 50-64 of SEQ ID NO: 21, and CDR3 comprising residues 98-115 of SEQ ID NO: 21 o
14. The expression construct of any one of claims 11-13, wherein the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 20 and 21.
15. The expression construct of any one of claims 11-14, wherein the nucleotide sequence further encodes a signal peptide.
16. The expression construct of claim 15, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29.
17. The expression construct of any one of claims 11-16, wherein the nucleotide sequence further encodes a tag sequence.
18. An expression construct comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-10 operably linked to a strictly hypoxia inducible promoter.
19. The expression construct of any one of claims 11-18, wherein the strictly hypoxia inducible promoter is selected from the group consisting of pepTp, fnrSp, ysgAp, ssbp1, Hip1, BBa_I14018, BBa_R1074, Ptet-arcA and Ptet-Fnr promoters.
20. A modified bacterium, wherein the bacterium comprises the expression construct of any one of claims 11-19 as compared to an unmodified starting strain.

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. Further optimization for tumor therapy is imperative. Further, the inventors have found that neutrophils in the human body limit the application of bacterial therapy for tumor. Therefore, there is a need to optimize strains for bacterial therapy for tumor to reduce the effects of neutrophils. Summary of the Invention According to 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, and thus providing a more reliable anti-tumor efficacy and safety. In addition, exogenous proteins (such as antibodies against neutrophils or fusion proteins of the antibodies and cytotoxins) are expressed in the bacterium to inhibit the recruitment of neutrophils or kill neutrophils in tumors, thereby enhancing the anti-tumor efficacy of the bacterium. In a first aspect, the present invention provides a fusion protein comprising an antibody (e.g., a nanobody) that binds to a neutrophil or an antigen-binding fragment thereof and a cytotoxin or an active fragment thereof. In some embodiments, the antibody comprises complementary determining regions (CDRs) selected from the group consisting of CDR1 comprising residues 31-35 of SEQ ID NO: 19, CDR2 comprising residues 50-65 of SEQ ID NO: 19, and CDR3 comprising residues 98-104 of SEQ ID NO: 19; CDR1 comprising residues 31-35 of SEQ ID NO: 20, CDR2 comprising residues 50-67 of SEQ ID NO: 20, and CDR3 comprising residues 100-111 of SEQ ID NO: 20; and CDR1 comprising residues 31-35 of SEQ ID NO: 21, CDR2 comprising residues 50-64 of SEQ ID NO: 21, and CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20 and 21. In some embodiments, the cytotoxin is Pseudomonas exotoxin (PE). In some embodiments, the cytotoxin comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the fragment of the cytotoxin comprises the amino acid sequence of SEQ ID NO: 26 or 27. In some embodiments, the fusion protein further comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29. In some embodiments, the fusion protein further comprises a linker and/or a tag sequence. In a second aspect, the present invention provides an expression construct comprising a nucleotide sequence encoding an antibody (e.g., nanobody) that binds to neutrophils or antigen-binding fragment thereof operably linked to a strictly hypoxia inducible promoter. In some embodiments, the antibody comprises complementary determining regions (CDRs) selected from the group consisting of CDR1 comprising residues 31-35 of SEQ ID NO: 19, CDR2 comprising residues 50-65 of SEQ ID NO: 19, and CDR3 comprising residues 98-104 of SEQ ID NO: 19; CDR1 comprising residues 31-35 of SEQ ID NO: 20, CDR2 comprising residues 50-67 of SEQ ID NO: 20, and CDR3 comprising residues 100-111 of SEQ ID NO: 20; and CDR1 comprising residues 31-35 of SEQ ID NO: 21, CDR2 comprising residues 50-64 of SEQ ID NO: 21, and CDR3 comprising residues 98-115 of SEQ ID NO: 21. In some embodiments, the antibody comprises an amino acid sequence selected from SEQ ID NOs: 19, 20 and 21. In some embodiments, the nucleotide sequence further encodes a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 28 or 29. In some embodiments, the nucleotide sequence further encodes a tag sequence. In some embodiments, the nucleotide sequence encodes the fusion protein of the present invention. In a third aspect, the present invention provides a modified bacterium. In one embodiment, the bacterium compr