CN-121991186-A - Cytokine-containing immunoregulatory bacterial protein and application thereof

Abstract

The invention provides an immunoregulation bacterial protein containing a cytokine module and application thereof, wherein the immunoregulation bacterial protein containing the cytokine module can specifically activate and promote immune cell proliferation, thereby enhancing host anti-tumor immune response and having good biological safety. The high-efficiency expression, purification and delivery of the protein can be realized through a genetic engineering strain system, the production process is stable, the large-scale implementation is convenient, the method is suitable for developing immunotherapeutic drugs and tumor auxiliary therapeutic preparations, and a new strategy is provided for clinical treatment.

Inventors

• WANG JUN
• WANG ZIYU

Assignees

• 中国科学院微生物研究所

Dates

Publication Date

20260508

Application Date

20260115

Claims (10)

1. 1. An immunomodulatory bacterial protein comprising a cytokine sequence, comprising any one of the amino acid sequences: (1) An amino acid sequence shown as SEQ ID NO. 1; (2) An amino acid sequence shown as SEQ ID NO. 2; (3) An amino acid sequence of the protein with the same function obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in the (1); (4) The amino acid sequence shown in (2) is obtained by replacing, inserting or deleting one or more amino acids to obtain the amino acid sequence of the protein with the same function.
2. 2. A nucleic acid molecule encoding an immunomodulatory bacterial protein comprising a cytokine module of claim 1.
3. 3. A fusion protein comprising the immunoregulatory bacterial protein comprising a cytokine sequence of claim 1 and a protein tag fused thereto.
4. 4. A composition comprising an immunomodulatory bacterial protein comprising a cytokine module according to claim 1, and preferably further comprising an additional anti-tumour agent.
5. 5. A biological material comprising the nucleic acid molecule of claim 2, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a vector, a microorganism, or a cell.
6. 6. Use of an immunomodulatory bacterial protein comprising a cytokine sequence according to claim 1, a nucleic acid molecule according to claim 2, a conjugate according to claim 3, a composition according to claim 4 or a biomaterial according to claim 5 for the preparation of a medicament for the treatment of a tumor.
7. 7. The use of claim 6, wherein the tumor comprises a intestinal tumor.
8. 8. Use of an immunomodulatory bacterial protein comprising a cytokine sequence according to claim 1, a nucleic acid molecule according to claim 2, a conjugate according to claim 3, a composition according to claim 4 or a biomaterial according to claim 5 for the preparation of a medicament, wherein the medicament is any one or more of the following (1) - (4): (1) A medicament for reducing the number of tumors; (2) A medicament for reducing tumor area; (3) A medicament for enhancing an immune response; (4) A medicament for alleviating weight loss in a patient suffering from a tumor.
9. 9. A biologic comprising the cytokine-containing immunomodulatory bacterial protein of claim 1, the nucleic acid molecule of claim 2, the conjugate of claim 3, the composition of claim 4, or the biologic of claim 5.
10. 10. A method for producing a cytokine-containing immunomodulatory bacterial protein of claim 1, wherein the microorganism or cell of claim 5 is cultured under conditions that allow expression of the cytokine-containing immunomodulatory bacterial protein of claim 1, and the cytokine-containing immunomodulatory bacterial protein is obtained from the resulting culture.

Description

Cytokine-containing immunoregulatory bacterial protein and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to an immunoregulatory bacterial protein containing a cytokine module and application thereof. Background Intestinal microorganisms produce various metabolites that regulate the host's immune and inflammatory response, maintaining intestinal homeostasis. Biological macromolecules such as DNA, RNA, proteins, polysaccharides and the like derived from intestinal flora have remarkable diversity and participate in the formation of a 'pathogen-associated molecular pattern' (Pathogen-associated molecular patterns, PAMPs), and also have profound effects on the host immune system. The presence of immunomodulatory proteins in bacteria similar to eukaryotes provides a new perspective for those skilled in the art to explore interactions between microbiomes and hosts. Human cytokines play a key role in regulating innate and acquired immunity, cell growth, differentiation, and death. However, many human cytokines face challenges such as low expression levels, low solubility, and lack of post-transcriptional modification when expressed in bacteria. The bacterially encoded cytokine-containing immunomodulatory bacterial proteins (immunomodulatory bacterial protein, CMCP) play an important role in host immunomodulation and disease progression. CMCP not only retains immunomodulatory activity, but is also compatible with bacterial expression systems, theoretically solving the problem of heterologous expression of traditional cytokines, and promoting the development of bacterial-based immunotherapy. By combining CMCP with different cytokines or immune checkpoint inhibitors, it is possible to achieve a wider application in various diseases such as autoimmune diseases, metabolic diseases and cancers. The identification and development of CMCP will provide important insight into the role of intestinal microbiome in immunomodulation, enriching the knowledge theory of microbial molecules. However, the research on CMCP by those skilled in the art is still in an initial exploration stage, and more efficient, controllable and safe CMCP needs to be developed for developing novel biomedical preparations. Disclosure of Invention In order to solve the problems in the prior art, the invention provides an immunoregulatory bacterial protein containing a cytokine module and application thereof. The invention utilizes the hidden Markov model to find 2 CMCPs related to diseases, namely bIL-1 and bIL-33, based on related metagenome data information of cancer patients and healthy people through abundance calculation and difference analysis. The invention realizes heterologous soluble expression of CMCP by using genetic engineering strain, determines the effect of CMCP on immune cells by ELISA, and can stimulate immune cells to generate immune response. The escherichia coli Nissle1917 (E. Coli Nissle1917, ecN) is further used as an engineering bacteria chassis to realize the induction expression and delivery of the CMCP, and the CMCP can directly reach the inside of intestinal tumor focus of animals, thereby achieving the effect of inhibiting the proliferation of intestinal tumor and enhancing the immune response. Specifically, the invention provides the following technical scheme: In a first aspect, the invention provides an immunomodulatory bacterial protein containing a cytokine sequence, which comprises any one of the following amino acid sequences (1) an amino acid sequence shown as SEQ ID NO. 1, (2) an amino acid sequence shown as SEQ ID NO. 2, (3) an amino acid sequence with the same functional protein, which is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as (1), and (4) an amino acid sequence with the same functional protein, which is obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown as (2). In a second aspect, the invention provides a nucleic acid molecule encoding said immunoregulatory bacterial protein comprising a cytokine sequence. The nucleic acid molecule provided by the invention preferably comprises any one of the following nucleotide sequences (1) a nucleotide sequence shown as SEQ ID NO. 5 or SEQ ID NO. 8, (2) a complete complementary sequence of the nucleotide sequence shown as (1), and (3) a nucleotide sequence which is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence of the nucleotide sequence shown as (1) and can code a protein with the same function. In a specific embodiment of the invention, when the nucleic acid molecule encodes the immunoregulatory bacterial protein containing a cytokine sequence as shown in SEQ ID NO. 1, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 5, the nucleotide sequence of the nucleic acid molecule is shown in the complete complement of the