WO2021037160A1 - Tumor enzyme-responsive recombinant pyroptosis protein delivery system and antitumor use thereof - Google Patents

Abstract

Provided is a fusion protein, a tumor enzyme-responsive recombinant pyroptosis protein delivery system, and antitumor use thereof. The fusion protein has the structure shown in formula I from the N-terminus to the C-terminus: Z0-Z1-Z2-Z3-Z4 (formula I), Z0 being an optional tag element; Z1 being the N-domain element of the pyrolysis protein; Z2 being a cell-penetrating peptide sequence element; Z3 being a peptide sequence element that can be specifically cleaved by a protease specifically expressed in a tumor microenvironment; Z4 being the C-domain element of the pyrolysis protein; "-" representing a peptide bond linking the elements; in the fusion protein, element Z4 inhibiting the activity of element Z1 by specifically binding to element Z1.

Description

Tumor enzyme-responsive recombinant pyrooptosis protein delivery system and its anti-tumor use Technical field

The present invention belongs to the field of biomedicine. More specifically, the present invention relates to a tumor enzyme-responsive recombinant pyroptosis protein delivery system and its anti-tumor use.

Background technique

Pyrodepressor protein is a family of proteins that can mediate pyrodesis of cells (gasdermin protein family), with 45% sequence homology among members, including GSDMA(1-3), GSDMB, GSDMC(1-4), GSDMD, DFNA5 (GSDME), DFNB59 (GSDMF).

Except for DFNB59, other pyro-apoptotic proteins have similar structures: two domains including N-domain and C-domain, and a linker that connects these two domains. Under normal circumstances, the two domains are tightly combined in a state of self-inhibition.

Once the linker is cleaved, the inhibitory effect of the C domain on the N domain is destroyed, and the active N domain is released. The active N domain binds to phosphatidylinositol (PI) or phosphatidylserine (PS) in the inner leaf of the cell membrane, or directly binds to the cardiolipin on the outer side of the bacterial plasma membrane, and accumulates in the cell membrane to form holes, resulting in cell swelling and cell membrane Rupture, release of contents, and cause an inflammatory response and ultimately lead to cell clearance. This process is called scorch of the cell.

Pyrolysis of cells is defined as a new, pro-inflammatory, programmed cell death method. Studies have found that pyrolysis and pyrolysis proteins are related to a variety of diseases. A large number of intracellular factors are released during the process of pyrolysis, including high mobility protein (HMGB1), lactate dehydrogenase (LDH), calreticulin (CRT), IL-1β, etc., so the process of pyrolysis can also be defined as secondary The process of sexual death.

While the pyrolysis of tumor cells causes inflammation, the immunogenic related molecules released can improve the recognition of dendritic cells (DC) to tumors and their ability to present antigens. DCs can activate cytotoxic T cells (cytotoxic T cells). T lymphocyte (CTL) specifically kills tumors and reduces the level of intracellular ATP.

However, there are still some insurmountable difficulties in the research of tumor cell pyrexia. The N-domain of pyrolysis protein must be in the cell to play a role, and it is difficult to ensure the efficiency of pyrolysis protein in the existing technology. In addition, how to target tumor cells with high specificity of the active pyroptosis protein, or how to enable the pyroptosis protein to be activated in the tumor microenvironment with high specificity, is also an urgent problem to be solved in this field.

Therefore, there is an urgent need in the art to develop a technical means that can enable pyroopterin to be activated in the tumor microenvironment with high specificity and enter tumor cells efficiently.

Summary of the invention

The purpose of the present invention is to provide a technical means capable of enabling pyrooptin to be activated in the tumor microenvironment with high specificity and to enter tumor cells efficiently.

In the first aspect of the present invention, a fusion protein is provided, and the fusion protein has the structure shown in formula I from the N-terminus to the C-terminus:

Z0-Z1-Z2-Z3-Z4 (Formula I)

Where

Z0 is an optional label element;

Z1 is the N domain element of pyrolysis protein;

Z2 is a penetrating peptide sequence element;

Z3 is a peptide sequence element that can be specifically cleaved by a protease specifically expressed in the tumor microenvironment;

Z4 is the C domain element of pyroopterin;

"-" indicates a peptide bond connecting the above-mentioned elements;

Wherein, in the fusion protein, the Z4 element inhibits the activity of the Z1 element by specifically binding to the Z1 element.

In another preferred embodiment, in the Z0 element, the tag is selected from the group consisting of His tag, GST tag, HA tag, c-Myc tag, Flag tag, or a combination thereof.

In another preferred embodiment, the pyroapone protein has the function of inducing the rupture of the cell membrane and releasing a large amount of content to cause the body's inflammatory response.

In another preferred embodiment, the pyroptosis protein is selected from the group consisting of human pyroptosis protein or murine pyroptosis protein.

In another preferred embodiment, the human pyroptosis protein is selected from the group consisting of GSDMA, GSDMB, GSDMC, GSDMD, DFNA5 (GSDME), DFNB59 (GSDMF), or a combination thereof.

In another preferred embodiment, the murine pyroptosis protein is selected from the following group: GSDMA1, GSDMA2, GSDMA3, GSDMC1, GSDMC2, GSDMC3, GSDMC4, or a combination thereof.

In another preferred embodiment, the pyroptin protein is GSDMA3.

In another preferred example, in the Z1, the N domain is an active protein domain that forms a hole in the cell membrane.

In another preferred example, in the Z1, the amino acid sequence of the N domain is selected from the following group:

(i) The sequence shown in SEQ ID NO:1;

(ii) On the basis of SEQ ID NO:1, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid residues are added to the N-terminus or C-terminus, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to obtain an amino acid sequence.

In another preferred example, in the Z2, the penetrating peptide has the function of carrying different components across the cell membrane.

In another preferred embodiment, in the Z2, the penetrating peptide is selected from the group consisting of cationic cell penetrating peptides (such as TAT), hydrophobic cell penetrating peptides, and amphiphilic cell penetrating peptides.

In another preferred embodiment, in the Z2, the penetrating peptide is TAT.

In another preferred embodiment, in the Z2, the amino acid sequence of the penetrating peptide is selected from the following group:

(i) The sequence shown in SEQ ID NO: 3;

(ii) On the basis of SEQ ID NO: 3, one or more amino acid residues are replaced, deleted, changed or inserted to obtain an amino acid sequence.

In another preferred example, in the Z3, the protease specifically expressed in the tumor microenvironment is selected from the following group: asparagine endopeptidase (Legumain), matrix metalloprotease, or a combination thereof.

In another preferred embodiment, the matrix metalloproteinase is selected from the group consisting of MMP-2, MMP-7, MMP-9, MMP-12, or a combination thereof.

In another preferred example, in the Z3, the protease specifically expressed in the tumor microenvironment is asparagine endopeptidase.

In another preferred example, in the Z3, the peptide sequence is selected from: PTN, a substrate peptide sequence specifically recognized and cleaved by Legumain.

In another preferred example, in the Z3, the amino acid sequence of the peptide sequence is selected from the following group:

(i) Sequence PTN;

(ii) The amino acid sequence obtained by inserting one or more amino acid residues at the N-terminal or C-terminal on the basis of the sequence PTN.

In another preferred example, in the Z4, the amino acid sequence of the N domain is selected from the following group:

(i) The sequence shown in SEQ ID NO: 2;

(ii) On the basis of SEQ ID NO: 2, one or more amino acid residues are replaced, deleted, changed or inserted, or 1 to 30 amino acid residues are added to its N-terminal or C-terminal, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to obtain an amino acid sequence.

In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4.

In the second aspect of the present invention, an isolated polynucleotide is provided, which encodes the fusion protein according to the first aspect of the present invention.

In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO: 5.

In the third aspect of the present invention, a vector is provided, which contains the polynucleotide according to the second aspect of the present invention.

In another preferred embodiment, the vector is selected from the group consisting of pET vector, pMAL vector, and pGEX vector.

In another preferred embodiment, the vector is selected from the group consisting of pET28a, pMAL-2c, pGEX-4T-2, or a combination thereof.

In the fourth aspect of the present invention, a host cell is provided, the host cell contains the vector as described in the third aspect of the present invention, or the polynucleotide as described in the second aspect of the present invention is integrated into the genome.

In another preferred embodiment, the host cell is Escherichia coli.

In another preferred embodiment, the host cell is selected from the group consisting of BL21(DE3), Rosetta, Origami, or a combination thereof.

In the fifth aspect of the present invention, a method for producing the fusion protein according to the first aspect of the present invention is provided, which includes the steps:

Under conditions suitable for expression, the host cell according to claim 4 is cultured, thereby expressing the fusion protein according to the first aspect of the present invention.

In the sixth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) The fusion protein or its encoding gene according to the first aspect of the present invention;

(b) A pharmaceutically acceptable carrier.

In another preferred example, the content of the component (a) is 0.1-99.9% by weight, preferably 10-99.9% by weight, more preferably 70%-99.9% by weight.

In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral dosage form, an injection, or a topical pharmaceutical dosage form.

In another preferred embodiment, the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.

In another preferred embodiment, the pharmaceutical composition is a liquid composition.

In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the following group: infusion carrier and/or injection carrier, preferably, the carrier is one or more carriers selected from the following group : Normal saline, dextrose saline, or a combination thereof.

In another preferred embodiment, the pharmaceutical composition can be used alone or in combination with other anti-tumor drugs.

In the seventh aspect of the present invention, there is provided a fusion protein as described in the first aspect of the present invention, a polynucleotide as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, and a fusion protein as described in the present invention. The use of the host cell according to the fourth aspect of the invention is for preparing a preparation or pharmaceutical composition, and the preparation or pharmaceutical composition is used for one or more selected from the following group:

(a) Kill tumor cells in the tumor microenvironment;

(b) Increase the number of M1 macrophages in the tumor microenvironment, and reduce the number of M2 macrophages in the tumor microenvironment;

(c) Increase the level of anti-cancer factors, antigen presenting molecules, effector T cells, immunogenic cell death (ICD) related characteristic molecules (such as ATP, HMGB1, CRT), LDH and other pro-inflammatory factors in the tumor microenvironment ；

(d) Reduce the expression level of tumor proliferation and metastasis-related proteins and cancer cell factors.

In another preferred embodiment, the immunogenic cell death (ICD) related characteristic molecules are selected from the following group: ATP, HMGB1, CRT, or a combination thereof.

In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, colon cancer, prostate cancer, ovarian tumor, or a combination thereof.

In another preferred example, the tumor cells are 4T1 cells or CT26 colon cancer cells.

In another preferred embodiment, the anti-cancer factor is selected from the group consisting of TNF-α, IL-1β, IL-2, IFN-γ, or a combination thereof.

In another preferred embodiment, the antigen presenting molecule is MHC class I molecule or MHC class II molecule.

In another preferred embodiment, the effector T cells are selected from the group consisting of CD8 ⁺ T cells, CD4 ⁺ T cells, CD8 ⁺ &GranzymeB ⁺ T cells, CD8 ⁺ &IFN-γ ⁺ T cells, or a combination thereof.

In another preferred embodiment, the tumor proliferation and metastasis-related protein is MR or Legumain.

In another preferred embodiment, the cancer-promoting factor is TGF-β.

In the eighth aspect of the present invention, there is provided a method for treating tumors, comprising the steps of: administering the fusion protein according to the first aspect of the present invention and the polynucleoside according to the second aspect of the present invention to a subject in need Acid, the vector according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, or the pharmaceutical composition according to the sixth aspect of the present invention.

In another preferred embodiment, the subject includes humans or non-human mammals.

In another preferred embodiment, the non-human mammals include rodents (such as rats and mice) and primates (such as monkeys).

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them here.

Description of the drawings

Figure 1 shows the FPLC desalting column and molecular sieve purification diagram of recombinant pyopterin.

Among them, (A-C) are the FPLC chromatograms of GSDMA3, GSDMA3-PTN and GSDMA3-TAT-PTN after passing through the desalting column; (D-E) are the FPLC chromatograms of GSDMA3, GSDMA3-PTN and GSDMA3-TAT-PTN after molecular sieve purification.

Figure 2 shows the characterization diagram of the recombinant protein.

Among them, lane M is Marker; lanes 1-3, lanes 4-6, and lanes 7-9 are the protein samples with three absorption peaks after GSDMA3-TAT-PTN, GSDMA3-PTN, and GSDMA3 are purified by Superdex 75.

Figure 3 shows the level of Legumain enzyme expression in cell lines cultured in vitro.

Figure 4 shows the in vitro Legumain cleavage experiment of the fusion pyrolysis protein.

Among them, lane M is Marker; lane 1 is GSDMA3-TAT-PTN after Legumain digestion; lanes 2 and 3 are GSDMA3-PTN after Legumain digestion; lane 4 is GSDMA3 after Legumain digestion.

Figure 5 shows the results of the cell uptake experiment.

Among them, (A) shows the results of the uptake of pyro-depth protein detected by flow cytometry after digestion; (B) shows the results of statistical analysis of the uptake results.

Figure 6 shows the results of the toxicity experiment of pyrooptin.

Among them, (A) shows the killing effect of pyrodepressin on 4T1 cells; (B) shows the killing effect of pyrodepressin on DC2.4 cells.

Figure 7 shows the changes in cell morphology after recombinant protein treatment.

Among them, (A-D) are the results of bright field photography of cells in the PBS, GSDMA3, GSDMA3-PTN, and GSDMA3-TAT-PTN administration treatment groups in order.

Figure 8 shows the experimental results of CRT eversion caused by the pyrooptin-mediated immunogenic death of tumor cells.

Among them, (A-D) are the results of PBS, GSDMA3, digested GSDMA3-PTN, and digested GSDMA3-TAT-PTN in order of treating tumor cells; (E) shows the statistical analysis of CRT.

Figure 9 (A) shows the released amount of HMGB1; (B) shows the amount of ATP released outside the cell.

Figure 10 shows the results of the in vitro sensitization experiment of DC.

Among them, (A) shows the ^{amount of CD80 +} DC cells after pyrolyst protein treatment; (B) shows the ^{amount of CD86 +} DC cells after pyrolyst protein treatment; (C) shows CD80 ⁺ /CD86 after pyrolyst protein treatment ^{+ The} amount of double positive DC cells.

Figure 11 shows the effect of pyroptin on antigen presentation.

Figure 12 shows the results of the pharmacodynamic experiment of recombinant pyrooptosis protein on murine 4T1 breast cancer in situ tumors.

Among them, (A) is the tumor volume change graph; (B) is the drug effect flow chart; (C) is the mouse body weight change curve; (D) shows the weight graph of the tumor at the experimental end point; (E) shows the experimental end point Tumor inhibition rate of each treatment group at time; (F) is the photo of the tumor at the end of the experiment (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001).

Figure 13 shows a schematic diagram of T cells and their granzymes in the spleen.

Among them, (AC) shows the spleen of the PBS group as an example, and the schematic diagram of the flow gate; (DG) shows the CD8 ⁺ T cells in the PBS group, GSDMA3 group, GSDMA3-PTN group and GSDMA3-TAT-PTN group in sequence. Changes in granzyme secretion.

Figure 14 shows the changes of T cells and their secreted factors in the spleen of each treatment group.

Among them, (A) shows the ^{change in the amount of CD4 +} T cells in the spleen; (B) shows the ^{change in the amount of CD8 +} T cells in the spleen; (C) shows the CD8 ⁺ &GranzymeB ⁺ T in the spleen of each group after treatment Cell changes; (D) shows the changes of CD8 ⁺ &IFN-γ ⁺ T cells in the spleen of each group after treatment.

Figure 15 shows the changes of T cells and their secreted factors in tumors after drug treatment.

Among them, (A) shows the ^{change in the amount of CD4 +} T cells in the tumor tissue; (B) shows the ^{change in the amount of CD8 +} T cells in the tumor tissue; (C) shows the CD8 ^{+ in each group of tumors after treatment} The changes of &Granzyme ⁺ T cells; (D) shows the changes of CD8 ⁺ &IFN-γ ⁺ T cells in the tumor tissues of each group after treatment.

Figure 16 shows the changes of T cells and their secreted factors in tumors after drug treatment.

Among them, (A) shows the ^{change in the amount of CD4 +} T cells in the lymph nodes; (B) shows the ^{change in the amount of CD8 +} T cells in the lymph nodes; (C) shows the CD8 ⁺ &Granzyme ⁺ T in each group of lymph nodes after treatment Cell changes; (D) shows the changes of CD8 ⁺ &IFN-γ ⁺ T cells in the lymph nodes of each group after treatment.

Figure 17 shows the changes of macrophages and NK cells in tumors after administration of treatment.

Among them, (A-B) shows the changes of M1 type macrophages; (C-D) shows the changes of M2 type macrophages; (E-F) shows the changes of NK cells. Among them (A, D, E) are the tumor cells in the GSDMA3-TAT-PTN treatment group as an example.

Figure 18 shows the changes in the expression of related proteins in tumor tissues in different treatment groups.

Figure 19 shows the changes of cytokines in tumor tissues.

Among them, A-B indicate the changes of IL-2 and TGF-β in the tumor tissue at the end of the experiment in turn.

Figure 20 shows the changes in the weight of the main organs.

Figure 21 shows a pathological section of the main organs.

Among them, the scale is 100μm.

Figure 22 shows the test results of liver function and kidney function of mice at the end of the experiment.

Among them, (A-C) respectively represent the changes of alanine aminotransferase, aspartate aminotransferase and total bilirubin content related to liver function; (D-F) represent the changes of serum urea, serum creatinine and serum uric acid content related to renal function, respectively.

detailed description

After extensive and in-depth research and extensive screening, the inventors developed a recombinant pyroptosis protein delivery system for the first time.

The present inventors took the cell pyrolysis and the pyrolysis protein Gasdermin A3 (GSDMA3) as the research object, and used genetic engineering technology to modify its structure, and introduced the Legumain substrate peptide sequence PTN between the two structural domains of the pyrolysis protein. In addition, the arginine-rich cationic penetrating peptide TAT (RKKRRQRRR) was selected and constructed in the adjacent position of the PTN sequence and close to the N-terminal side of the pyrolysis protein (N-GSDMA3-TAT-PTN-GSDMA3-C, or Marked as GSDMA3-TAT-PTN). The experimental results show that the GSDMA3-TAT-PTN protein of the present invention exhibits very good anti-tumor activity in in vivo and in vitro experiments.

The present invention has been completed on this basis.

Fusion protein of the present invention and its coding sequence

As used herein, the terms "fusion protein of the present invention", "recombinant pyroptosis protein" and "fusion pyroptosis protein" are used interchangeably and refer to the fusion protein according to the first aspect of the present invention, which has the ability to induce cell membrane rupture and release A large number of inclusions cause the function of the body's inflammatory response.

In the present invention, the provided fusion protein has the structure shown in formula I from N-terminus to C-terminus:

Z0-Z1-Z2-Z3-Z4 (Formula I)

Where

Z0 is an optional tag element; Z1 is the N-domain element of pyrooptin; Z2 is a penetrating peptide sequence element; Z3 is a peptide sequence element that can be specifically cleaved by a protease specifically expressed in the tumor microenvironment; Z4 is The C domain element of pyrolysis protein; "-" represents the peptide bond connecting the above elements;

Wherein, in the fusion protein, the Z4 element inhibits the activity of the Z1 element by specifically binding to the Z1 element.

In the present invention, the pyrolyst protein can be selected from human pyrolyt protein (such as GSDMA, GSDMB, GSDMC, GSDMD, DFNA5 (GSDME), DFNB59 (GSDMF), etc.) or murine pyrolyt protein (such as GSDMA1, GSDMA2, GSDMA3, GSDMC1, GSDMC2, GSDMC3, GSDMC4, etc.).

In a preferred embodiment, the pyroptin protein is GSDMA3.

In the fusion protein of the present invention, in the Z1, the N domain is an active protein domain that forms holes in the cell membrane; in the Z2, the penetrating peptide has the function of carrying different components through the cell membrane .

Preferably, in the Z2, the penetrating peptide is selected from the group consisting of cationic cell penetrating peptides (such as TAT), hydrophobic cell penetrating peptides, and amphiphilic cell penetrating peptides. In a preferred embodiment, the penetrating peptide is TAT.

In the fusion protein of the present invention, in the Z3, the protease specifically expressed in the tumor microenvironment is selected from the following group: asparagine endopeptidase (Legumain), matrix metalloproteinase (such as MMP-2, MMP- 7. MMP-9, MMP-12, etc.), or a combination thereof.

In a preferred embodiment, in the Z3, the protease specifically expressed in the tumor microenvironment is asparagine endopeptidase.

Preferably, in the Z3, the peptide sequence is selected from: a substrate peptide sequence PTN specifically recognized and cleaved by Legumain.

It should be understood that although the genes provided in the examples of the present invention are of murine origin, they are derived from other similar species (especially mammals) and are compatible with the sequence of the present invention (preferably, the sequence is shown in SEQ ID NO: 5). ) The gene sequence of recombinant pyroopterin protein with certain homology (conservation) is also included in the scope of the present invention, as long as those skilled in the art can easily obtain information from other sources after reading this application according to the information provided in this application. The sequence is isolated from species (especially mammals).

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include: DNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the fusion protein may be the same as the coding region sequence shown in SEQ ID NO: 5 or a degenerate variant.

The polynucleotide encoding the fusion protein includes: only the coding sequence of the fusion protein; the coding sequence of the fusion protein and various additional coding sequences; the coding sequence (and optional additional coding sequence) of the fusion protein and non-coding sequences.

The term "polynucleotide encoding a fusion protein" may include a polynucleotide encoding the fusion protein, or a polynucleotide that also includes additional coding and/or non-coding sequences. The present invention also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs and derivatives of polyglycosides or polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the fusion protein encoded by it. Features.

The present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) methylphthalamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, More preferably, the hybridization occurs when 95% or more occurs.

The full-length nucleotide sequence or fragments thereof encoding the fusion protein of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and a commercially available DNA library or a cDNA prepared by a conventional method known to those skilled in the art can be used. The library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order. Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. It is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. At present, the DNA sequence encoding the fusion protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the fusion protein sequence of the present invention through chemical synthesis.

The present invention relates to a recombinant pyroptin fusion protein used in an anti-tumor drug delivery system. In a preferred embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4. The polypeptide of the present invention can effectively induce the rupture of the cell membrane and release a large amount of contents to cause inflammation in the body.

The present invention also includes 50% or more of the sequence shown in SEQ ID NO: 4 of the present invention (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98 % Or more, such as 99%) homologous polypeptides or proteins with the same or similar functions.

The "same or similar function" mainly refers to: "effectively induce the rupture of the cell membrane and release a large amount of content to cause the body's inflammatory response".

The fusion protein of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The fusion protein of the present invention can be a natural purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, plant, insect, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the fusion protein of the present invention may be glycosylated or non-glycosylated. The fusion protein of the present invention may also include or not include the initial methionine residue.

The present invention also includes other polypeptide fragments and analogs having the activity of the fusion protein of the present invention. As used herein, the terms "fragment" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the fusion protein of the present invention.

The polypeptide fragments, derivatives or analogues of the present invention may be: (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues The base may or may not be encoded by the genetic code; or (ii) a polypeptide with a substitution group in one or more amino acid residues; or (iii) the mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, For example, polyethylene glycol) fused to form a polypeptide; or (iv) additional amino acid sequence is fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or Fusion protein). According to the definition herein, these fragments, derivatives and analogs belong to the scope well known to those skilled in the art.

In the present invention, the fusion protein variant is the amino acid sequence shown in SEQ ID NO: 4, after several (usually 1-10, preferably 1-8, more preferably 1-4 , Preferably 1-2) the derived sequence obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually within 10, preferably within 5) at the C-terminus and/or N-terminus , More preferably within 3) amino acids. For example, when the protein is substituted with amino acids with similar or similar properties, it usually does not change the function of the protein. Adding one or several (such as 1-3) amino acids at the C-terminus and/or N-terminus is usually also Does not change the function of the protein. These conservative variants are best produced by substitutions according to Table 1.

Table 1

Initial residue	Representative substitution	Preferred substitution
Ala(A)	Val; Leu; Ile	Val
Arg(R)	Lys; Gln; Asn	Lys
Asn(N)	Gln; His; Lys; Arg	Gln
Asp(D)	Glu	Glu

Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
Glu(E)	Asp	Asp
Gly(G)	Pro; Ala	Ala
His(H)	Asn; Gln; Lys; Arg	Arg
Ile(I)	Leu; Val; Met; Ala; Phe	Leu
Leu(L)	Ile; Val; Met; Ala; Phe	Ile
Lys(K)	Arg; Gln; Asn	Arg
Met(M)	Leu; Phe; Ile	Leu
Phe(F)	Leu; Val; Ile; Ala; Tyr	Leu
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr; Phe	Tyr
Tyr(Y)	Trp; Phe; Thr; Ser	Phe
Val(V)	Ile; Leu; Met; Phe; Ala	Leu

The present invention also includes analogs of the claimed protein. The difference between these analogs and the natural SEQ ID NO: 4 may be the difference in the amino acid sequence, the difference in the modified form that does not affect the sequence, or both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs having residues different from natural L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the protein of the present invention is not limited to the representative proteins listed above.

Modified (usually not changing the primary structure) forms include: chemically derived forms of proteins in vivo or in vitro, such as acetate or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation modifications during protein synthesis and processing. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine).

Pharmaceutical composition and its application method

The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the following active ingredients: the fusion protein or its coding gene as described in the first aspect of the present invention.

As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce function or activity on humans and/or animals and can be accepted by humans and/or animals.

As used herein, "pharmaceutically acceptable" ingredients are substances that are suitable for humans and/or mammals without excessive side effects (such as toxicity, irritation, and allergic reactions), that is, substances that have a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, and includes various excipients and diluents.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually the pharmaceutical preparation should match the mode of administration. For example, it can be prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition should be manufactured under aseptic conditions.

The effective amount of the active ingredient of the present invention can vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, through clinical trials). The factors include, but are not limited to: the pharmacokinetic parameters of the active ingredients such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, and administration The way and so on. For example, due to the urgent requirement of treating the condition, several divided doses can be given every day, or the dose can be reduced proportionally.

The pharmaceutically acceptable carriers of the present invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or Its combination. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

The main advantages of the present invention include:

1) The present invention uses genetic engineering technology to transform the pyrooptosis protein, which can realize the multifunctional delivery of the pyroptosis protein. The penetrating peptide sequence is introduced into the pyroptosis protein, which gives the pyroptosis protein the ability to enter the cell, thereby exerting the pyroptosis effect.

2) The present invention applies the pyroopterin to induce immunogenic cell death (ICD) and tumor immunotherapy.

3) The present invention uses tumor-related enzymes such as Legumain-PTN as a responsive design for activating pyroapone proteins, which has a wide range of applications, improves the selectivity of tumor killing effects, and reduces its toxic side effects.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are percentages by weight and parts by weight.

method

Using genetic engineering technology, the prokaryotic expression plasmid pET28a-GSDMA3 was used as a template for subcloning and construction. Part of the amino acid sequence was replaced with Legumain-specific digested substrate peptide sequence PTN, and incorporated into the cell-penetrating peptide sequence TAT. Constructed into pET28a-PTN-GSDMA3, pET28a-PTN-TAT-GSDMA3 plasmids. The recombinant plasmid is transformed in vitro and expressed in Escherichia coli with high efficiency. The protein’s His-tag is used for preliminary purification, and then further purified by molecular sieve, and finally the recombinant protein is obtained.

Construction of recombinant pyroptosis protein pellet

Using pET28a-GSDMA3 as a template for subcloning construction, construct pET28a-PTN-GSDMA3, pET28a-PTN-TAT-GSDMA3 plasmids.

Expression and purification of recombinant fusion protein

The plasmid is transferred to competent cells for culture, and then the culture is expanded and the protein expression is induced, and the obtained protein is purified. First, use the His-tag that comes with the protein to purify the Ni binding column, and use

Purifier 10 (FPLC) purifies the preliminarily purified protein by molecular sieve (Superdex 75), and uses SDS-PAGE electrophoresis to detect the purity of the protein.

Cell level experiment

Use FITC dye to label the protein, and then use Legumain digestion medium prepared in vitro to digest the recombinant protein, and then perform the cell uptake experiment on the recombinant protein after digestion. Detect the killing ability of the recombinant protein after digestion on tumor cells and observe it with a microscope. Detection of changes in the amount of ICD-related molecules (including CRT and HMGB1, etc.) released during the process of cell pyrolysis caused by the recombinant protein. Observe the effect of recombinant protein on the sensitization and presentation of DC.

Animal level experiment

Construct 4T1 breast cancer subcutaneous tumor in situ model, when the tumor volume reaches about 40mm ³ , use recombinant pyrooptosis protein for treatment, conduct pharmacodynamic research, and perform eyeball blood collection at the end of the experiment (for liver function and Renal function test), the mice were then euthanized, and the tumors and organs (heart, liver, spleen, lung, kidney) of the mice were dissected. They were weighed and photographed. A part of the tumor was immersed in 4% paraformaldehyde and fixed for subsequent slice experiments and biosafety (pathological slices, etc.) testing experiments; the other part was used to study the spleen, lymph nodes, and tumor tissues in vivo The changes of T cells and secreted factors, and the changes of tumor tissue macrophages and NK cells. WB was used to detect the changes of legumain, MR, TGF-β, and TNF-α in tumor tissues after treatment, and ELISA kits were used to detect cytokines (TGF-β, TNF-α) in tumor tissues of each group after treatment. The change situation.

Example 1: Purification results of FPLC

The proteins (GSDMA3, GSDMA3-PTN and GSDMA3-TAT-PTN) that have been preliminarily purified by a nickel column are passed through a desalting column (HiTrap Desalting) (Figure 1A-C) and a gel exclusion chromatography column (Superdex 75) (Figure 1D-F) Purification. The peak shapes and peak positions of the three proteins are basically the same, which preliminarily shows that the protein modified by genetic engineering in this experiment did not affect its structure and function.

Example 2: Purification and identification of fusion protein

The protein samples of the first three absorption peaks of Superdex 75 were prepared and verified by SDS-PAGE electrophoresis. The results are shown in Figure 2. Lanes 1-9 are the proteins of the three absorption peaks of GSDMA3-TAT-PTN, GSDMA3-PTN and GSDMA3, respectively. The band of GSDMA3 conforms to the theoretical molecular weight of about 55kDa (lane 7); the band of GSDMA3-PTN (lane 4) basically maintains the same level as the band of GSDMA3, which shows that the molecular weight of the two is close to and theoretically consistent; the recombinant protein GSDMA3 -The TAT-PTN band (lane 1) has a significant upward shift; while the other lanes have mixed bands, which indicates that the protein purity of the first absorption peak is better, while the other two absorption peaks have a large amount of mixed protein , So all subsequent experiments use the protein with the first absorption peak.

Example 3: WB detects the expression level of Legumain in cells

Western Blot was used to detect the expression level of Legumain in RAW 264.7 and 4T1 cells induced into M2 macrophages. As shown in Figure 3, Legumain is highly expressed in tumor-associated macrophages. In this study, 4T1 cells were cultured in vitro and it was found that Legumain was indeed low in expression.

Example 4: In vitro Legumain cleavage experiment of the fusion pyrolysis protein

Figure 4 shows the results of digestion of GSDMA3-TAT-PTN, GSDMA3-PTN and GSDMA3 in turn. After the substrate peptide PTN is cleaved by Legumain, the N-domain and C-domain of the recombinant pyrolysis protein are released. The fragments produced by GSDMA3-PTN and GSDMA3-TAT-PTN proteins are similar in size, so the band positions are close, and the results show that the recombination The protein can be cleaved by Legumain enzyme.

Example 5: Cellular uptake

The FITC-labeled recombinant protein was cleaved and activated by Legumain enzyme in advance, and then the uptake experiment of 4T1 cells was performed. The experimental results are shown in Figure 5, the digested GSDMA3-TAT-PTN has the best ingestion effect. The average fluorescence intensity values of GSDMA3, digested GSDMA3-TAT-PTN and GSDMA3-PTN are about 51.8, 109 and 67.4 respectively. The uptake efficiency of GSDMA3-TAT-PTN is 1.6 times that of GSDMA3-PTN and 2.1 that of GSDMA3. Times. This indicates that the active fragment produced by GSDMA3-TAT-PTN after being activated by Legumain enzyme cleavage has a higher cell entry efficiency under the action of the penetrating peptide TAT.

Example 6: Cytotoxicity test

4T1 and DC2.4 cells were used to detect the toxicity of pyroptin. For 4T1 cells: with the increase of protein concentration in the experimental concentration range, the survival rate of the cells decreased. Among them, the recombinant protein GSDMA3-TAT-PTN after restriction digestion was significantly better than the other two groups in killing 4T1 tumor cells (Figure 6A) . All of the pyrolyzed proteins had no killing effect on DC2.4 cells within the tested concentration range (Figure 6B), and had good biocompatibility, so the pyrolyzed proteins would not affect the function of antigen presenting cells.

Example 7: Bright field imaging

4T1 cells were treated with three groups of pyroptin proteins for 48h, as shown in Figure 7: normal 4T1 cells would grow into a network in sheets (Figure 7A); after treatment with GSDMA3 and digested GSDMA3-PTN, the morphology of the cells changed. There are also a large number of cells that are absorbing water and swelling (Figures 7B and 7C), which is consistent with their cell killing effect and the results of the uptake experiment. After the digested GSDMA3-TAT-PTN treats the cells, a large number of cells have died, and have been washed away during the washing process. In the remaining fixed cells, a large number of swollen cells can also be seen, and there are basically no complete cells. Exist (Figure 7D).

Example 8: Calreticulin (CRT) measurement experiment

Figure 8 shows the change in the amount of extracellular CRT. The tumor cells in the control group will have a negative peak on the left and a positive peak on the right (Figure 8A). After 4T1 cells are treated with pyroptin, the positive peaks of all groups are significantly increased. After digestion, the negative peak of the cells treated with GSDMA3-TAT-PTN was significantly decreased, and the positive peak was significantly increased (Figures 8B, 8C and 8D). The average fluorescence intensities of GSDMA3-TAT-PTN, GSDMA3-PTN and GSDMA3 after digestion were 132, 87 and 80.7, respectively. The intensities of GSDMA3-TAT-PTN were 1.5 times and 1.6 times that of the latter two, respectively.

Example 9: HMGB1 migration and extracellular ATP release experiment

As shown in Figure 9, the GSDMA3 and GSDMA3-PTN administration groups can increase the release of HMGB1, but there is no significant difference from the PBS group. The GSDMA3-TAT-PTN administration group can greatly increase the release of HMGB1, and the release concentration of HMGB1 is 1.9 times and 2.4 times that of the GSDMA3 group and the GSDMA3-PTN group, respectively, with significant differences. The extracellular ATP content of all experimental groups increased significantly. Among them, the extracellular ATP content of the GSDMA3-TAT-PTN treatment group was significantly different from the other treatment groups, indicating that ATP was released from the inside of the cell to the outside of the cell, which is considered to be The hole formed by the pyrolysis protein is released to the outside of the cell or the membrane channel activated by the pyrolysis is released to the outside of the cell.

Example 10: Dendritic cell sensitization experiment in vitro

As shown in Figure 10, after treating BMDC with pyrolysis protein, the amount of CD80 ⁺ and CD86 ⁺ DC cells basically maintained an upward trend, but ^{there was no significant difference in the amount of CD80 +} DC cells between the groups (Figure 10A). The number of CD86 ⁺ DC cells can reach the level of the positive control group (Figure 10B), and the number of CD80 ⁺ /CD86 ⁺ double-positive DCs in the protein treatment group can also reach the level of the positive control group. Among them, the DCs treated with GSDMA3-TAT-PTN The double positive amount of cells was the highest (Figure 10C).

Example 11: The effect of recombinant pyroopterin on antigen presentation

As shown in Figure 11, compared with the PBS group, all protein treatment groups can increase the expression intensity of MHC-I molecules (Figure 11A). Among them, the amount of GSDMA3 and GSDMA3-TAT-PTN treated DC is about the same as that of the PBS group. Twice, forming a significant difference. The amount of MHC-II molecules in the GSDMA3 treatment group was slightly lower than that in the PBS group, but there was no significant difference (Figure 11B). The GSDMA3-PTN and GSDMA3-TAT-PTN treatment groups can significantly increase the amount of MHC-II, and the GSDMA3-TAT-PTN treatment group has the best enhancement effect.

Example 12: Pharmacodynamic Study of Recombinant Pyroopterin on Subcutaneous Xenograft Tumor Model

The pharmacodynamic results are shown in Figure 12: Figure 12B is a flowchart. The tumor volume of each treatment group increased, but the pyrooptin treatment group could slow down the growth trend (Figure 12A), and the tumor inhibition rates of the three groups at the experimental end point could reach 21%, 34% and 62% respectively (Figure 12E). Among them, there is a significant difference between the GSDMA3-TAT-PTN treatment group and the control group. The body weight of the mice did not change much during the protein treatment (Figure 12C), indicating that there were no obvious side effects. Figures 12D and 12F are after the tumor was dissected at the end of the experiment (day 31) and weighed and photographed. It was found that the tumors in the pyroopterin treatment group were reduced, and the tumors in the GSDMA3-TAT-PTN group were the smallest, which was similar to other groups. There is a significant difference than that.

Example 13: Changes of T cells and cytokines secreted by them in the body after treatment

13.1 Spleen

Figure 13 (A-D) takes the mouse spleen cells in the PBS treatment group as an example, showing a schematic diagram of the entire flow cytometry gate. Figures (13E-F) show the changes in the amount of granzyme secreted in the spleen of mice after GSDMA3, GSDMA3-PTN, and GSDMA3-TAT-PTN treatment. The results of the secretion of granzyme and its interferon in tumors and lymph nodes are also analyzed with reference to this section.

Figure 14 shows the statistical results of T cells, granzyme and interferon in the spleen. After treatment, the ^{amount of CD4 +} T cells in each group did not change significantly (Figure 14A and 14B). The GSDMA3-TAT-PTN treatment group had the ^{largest number of CD8 +} T cells and secreted the most granzyme and interferon (Figure 14B). , 14C and 14D), which indicates that the treatment of tumor-bearing mice with GSDMA3-TAT-PTN in this experiment can effectively activate T cells in the spleen and release a large amount of cytokines to kill the tumor.

13.2 Tumor

The changes of T cells and their secreted factors in the tumor tissue at the end of the experiment are shown in Figure 15. After protein drug treatment, ^{there were no significant changes in CD4 +} T cells and CD8 ⁺ T cells in each group (Figures 15A and 15B). ^{However, the expression of granzyme B in mouse tumor CD8 +} T cells in the GSDMA3-TAT-PTN treatment group increased significantly, while there was no significant change in the other treatment groups (Figure 15C), which indicates that the GSDMA3-constructed in this study TAT-PTN has a very good tumor killing effect. ^{The interferon secreted by CD8 +} T cells of all tumors treated with pyroostatin was significantly increased, which formed a significant contrast with the PBS group, in which GSDMA3-TAT-PTN secreted the most amount (Figure 15D).

13.3 Lymph nodes

Figure 16 shows the statistical results of T cells, granzyme and interferon in the axillary lymph nodes around the tumor. GSDMA3-TAT-PTN treatment group and the other groups as compared to CD4 ⁺ T no significant change, but CD4 ⁺ T cells and the PBS group as compared to significantly reduce GSDMA3 and GSDMA3-PTN treatment groups (FIG. 16A); and all Compared with the PBS group, CD8 ⁺ T cells in the protein treatment group increased significantly (Figure 16B). The expression levels of granzyme B and interferon in CD8 ⁺ T cells of all pyrostatin treatment groups increased, and the expression levels of the GSDMA3-TAT-PTN treatment group increased significantly (Figure 16C and 16D).

Example 14: Changes of macrophages and NK cells in tumors

14.1 Macrophages

As shown in Figures 17A and 17B: M1 type macrophages of all protein treatment groups increased, and M1 type macrophages of tumors in the GSDMA3-TAT-PTN group increased significantly. Compared with the PBS group, all protein treatment groups down-regulated M2 type macrophages (Figure 17C and 17D), and the M2 type macrophages of the GSDMA3-PTN and GSDMA3-TAT-PTN treatment groups decreased significantly.

14.2 NK cells

Figure 17 (E-F) shows that NK cells in the tumor tissue of the GSDMA3-TAT-PTN treatment group increased significantly, while there was no significant change in the other groups.

Example 15: Changes of legumain, MR, TGF-β, and TNF-α in tumor tissues of each group

As shown in Figure 18, using the WB results to analyze the expression of related proteins in tumor tissues at the end of the experiment, the intratumoral pro-inflammatory factor (TNF-α) increased significantly after GSDMA3-TAT-PTN treatment, and at the same time, related proteins that are conducive to tumor proliferation and metastasis The expression levels of (MR, Legumain) and factor (TGF-β) decreased significantly. This indicates that the recombinant protein constructed in this study has a very good anti-tumor effect.

Example 16: Changes in cytokines in tumor tissues

The ELISA kit was used to check the secretion of cytokines in the tumor tissues of each group at the end of the experiment, as shown in Figure 19. It was found that the secretion of cancer cell cytokines (TGF-β) decreased in each treatment group, combined with the result analysis in Figure 18: GSDMA3-TAT-PTN treatment group can enhance the body’s immunity and cell killing and reduce tumor metastasis, and has a good anti-tumor effect. effect.

Example 17: Biosafety Evaluation

17.1 Organ coefficient

The main organs (heart, liver, lung, kidney, spleen) of the mice at the end of the experiment were taken out and weighed. As shown in Figure 20, mice bearing 4T1 breast cancer had obvious splenomegaly, while GSDMA3- The spleen of the mice in the TAT-PTN treatment group was smaller than that of the PBS group at the end of treatment, and there was no significant difference in other organs in each group.

17.2 Pathological Sections

The main organs (heart, liver, spleen, lung, and kidney) of the mice at the end of the experiment were subjected to pathological analysis, as shown in Figure 21. It was found that the alveoli in the GSDMA3 treatment group were deformed, and the alveoli in the other groups were normal in shape without obvious cell shedding; while other organs in each group had no obvious lesions, indicating that the recombinant protein has good biological safety and no obvious damage to the organs.

17.3 Biochemical indicators

Blood was taken at the end of the experiment to detect liver and kidney functions. The results are shown in Figure 22: AST in the GSDMA3-TAT-PTN treatment group was significantly lower than the other two groups, but there was no significant difference compared with the PBS group. Based on the analysis of the increased expression of AST in acute, chronic hepatitis and toxic hepatitis, GSDMA3-TAT-PTN treatment does not cause liver damage. There were no significant changes in other indicators in each group, which indicated that the use of pyroapone protein treatment did not cause liver and kidney damage and inflammation.

discuss:

The pyrolysis protein does not have a toxic effect when it is not activated by cutting. Once it is activated by cutting, it will release the toxic end, leading to perforation of the cell membrane, release of the contents, and inflammatory reaction, thereby mediating cell death, that is, pyrolysis. It is a class of promising biological macromolecular prodrugs, but it is an intracellular protein that must overcome the cell membrane barrier to enter the cytoplasm to play a role. In addition, the role of pyrolysis protein lacks cell specificity and is likely to cause toxic side effects.

When designing a recombinant pyroptosis protein delivery system, the inventors took into account the high expression of Legumain in the tumor microenvironment (TME), and chose a drug delivery strategy targeting TME activation. Through genetic engineering recombination technology, a TME-responsive drug delivery strategy was prepared. Activate functional pyro-apoptosis protein to enhance the effect of tumor site protein and reduce the toxic side effects of normal tissues.

The invention utilizes the specific and highly expressed protease Legumain enzyme in TME, and realizes the anti-tumor effect of the pyrostat protein mediated by tumor enzyme activation by introducing the Legumain substrate peptide sequence PTN between the two structural domains of the pyrostat protein.

In the present invention, GSDMA3 of the pyrodepressin family is selected for research, and its N-domain is the active domain, which can cause cell pyrolysis. In this study, genetic engineering technology was used to insert PTN into the linker sequence between the C-domain and N-domain of GSDMA3, so that it can be cleaved in TME to release the active N-domain. However, the efficiency of protein itself is not high, and the N-domain needs to enter the cell to play a role. Therefore, this study introduced a penetrating peptide sequence between the N-domain and PTN to promote the penetration of the cell through the penetrating peptide. After entering the cell, the N-domain forms a hole in the cell membrane, leading to cell death. At the same time, a large amount of intracellular ATP, HMGB1, LDH, IL-1β, calreticulin, etc. are released outside the cell to activate immune cells and inhibit tumor proliferation.

The GSDMA3-TAT-PTN protein provided by the present invention exhibits very good anti-tumor activity in in vivo and in vitro experiments, which shows the effectiveness of the design of the pyrostat protein delivery strategy using tumor enzyme activation and fusion of penetrating peptides.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. A fusion protein, characterized in that the fusion protein has a structure shown in formula I from N-terminus to C-terminus:

   Z0-Z1-Z2-Z3-Z4 (Formula I)

   Where

   Z0 is an optional label element;

   Z1 is the N domain element of pyrolysis protein;

   Z2 is a penetrating peptide sequence element;

   Z3 is a peptide sequence element that can be specifically cleaved by a protease specifically expressed in the tumor microenvironment;

   Z4 is the C domain element of pyroopterin;

   "-" indicates a peptide bond connecting the above-mentioned elements;

   Wherein, in the fusion protein, the Z4 element inhibits the activity of the Z1 element by specifically binding to the Z1 element.
2. The fusion protein according to claim 1, wherein the pyroopterin protein has the function of inducing the rupture of the cell membrane and releasing a large amount of content to cause the body's inflammatory response.
3. The fusion protein according to claim 1, wherein the pyrolysis protein is GSDMA3.
4. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO: 4.
5. An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1.
6. A vector, characterized in that it contains the polynucleotide according to claim 5.
7. A host cell, characterized in that the host cell contains the vector according to claim 6, or the polynucleotide according to claim 5 is integrated into the genome.
8. A method for producing the fusion protein according to claim 1, characterized in that it comprises the steps of:

   Under conditions suitable for expression, the host cell according to claim 7 is cultured to express the fusion protein according to claim 1.
9. A pharmaceutical composition, characterized in that it comprises:

   (a) The fusion protein of claim 1 or its encoding gene;

   (b) A pharmaceutically acceptable carrier.
10. A use of the fusion protein according to claim 1, the polynucleotide according to claim 5, the vector according to claim 6 and the host cell according to claim 7, characterized in that it is used for A preparation or pharmaceutical composition is prepared, and the preparation or pharmaceutical composition is used for one or more selected from the following group:

    (a) Kill tumor cells in the tumor microenvironment;

    (b) Increase the number of M1 macrophages in the tumor microenvironment, and reduce the number of M2 macrophages in the tumor microenvironment;

    (c) Increase the level of anti-cancer factors, antigen presenting molecules, effector T cells, immunogenic cell death (ICD) related characteristic molecules (such as ATP, HMGB1, CRT), LDH and other pro-inflammatory factors in the tumor microenvironment ；

    (d) Reduce the expression level of tumor proliferation and metastasis-related proteins and cancer cell factors.

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