Pheromonicin array against small cell lung cancer and use thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a pheromonicin array against small cell lung cancer and the use thereof. Provided is a polypeptide having an amino acid sequence as shown in SEQ ID NOs: 1-30. Further provided is a drug against small cell lung cancer, which contains a fusion protein obtained by means of respectively connecting the channel domain of colicin to the polypeptide having the amino acid sequence as shown in SEQ ID NOs: 1-30. Pharmacodynamic experiments confirm that in each growth cycle, a tumor-bearing mouse model cannot escape the recognition and killing of tumors by the pheromonicin array consisting of 30 fusion proteins. Guinea pig model experiments confirm that the continuous use of the pheromonicin array for 30 days does not produce any toxic side effects in the animal model. The pheromonicin array can be used as an effective drug for treating small cell lung cancer.

Description

Information pheromone array against small cell lung cancer and application thereof

[0001] REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2024105757187, filed on May 10, 2024, entitled "Information pheromone array against small cell lung cancer and application thereof", and Chinese Patent Application No. 2025100221159, filed on January 07, 2025, entitled "Information pheromone array against small cell lung cancer and application thereof", the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine, and specifically relates to an information pheromone array against small cell lung cancer and application thereof. BACKGROUND

[0004] At present, there is still a lack of effective intervention means for the threat caused by the harmfulness and high incidence of small cell lung cancer, and new drugs are urgently needed.

[0005] No matter whether it is chemotherapy, radiotherapy, or biological therapy and many other modern tumor treatment methods, they all face two major defects that are difficult to overcome: (1) the tumor course that has been relieved after treatment is prone to recurrence and metastasis, ultimately leading to treatment failure; (2) the current anti-tumor treatment methods and drugs have relatively large toxicity, and their toxic side effects often cause complications, leading to the death of patients. Even the currently highly regarded tumor immunotherapy also faces similar difficulties: due to the inherent immunotoxicity of monoclonal antibodies, it is extremely difficult to use two or more monoclonal antibodies targeting different targets in the same patient. Even the latest antibody drug conjugates (ADC) also have difficulty in using two or more ADCs in the same patient due to immune-related adverse events (irAE). Due to the defects of the above-mentioned drugs, modern treatment methods are difficult to intervene in multiple tumor targets or multiple tumor treatment pathways at the same time.

[0006] Based on the above limitations, the current research and development of monoclonal antibodies and ADCs can only intervene in a certain target, a certain type of gene, a certain stage of tumor growth and metabolism mechanism, and a certain signal transduction pathway. It is almost impossible to simultaneously intervene in multiple targets and multiple pathways in the same patient. This results in limitations in treatment: once the tumor develops resistance or escapes from such single-pathway intervention, the single-pathway intervention treatment method is blocked. Therefore, there is an urgent need to develop a tumor intervention treatment method that can simultaneously intervene in multiple targets and multiple pathways, thereby more effectively treating tumors.

[0007] Colicins are classic examples of bacteriocins. There are more than twenty types of colicins, which attack the genetic, protein synthesis system, or destroy the cell membrane of other strains of E. coli. Channel-forming E1 family colicins, which can form ion channels on the cell membrane to kill E. coli, are composed of colicin E1, colicin Ia, colicin Ib, colicin A, colicin B, and colicin N.

[0008] Colicins E1, Ia, Ib, A, B, and N are one of the regulatory forces that maintain the diversity and evolution of intestinal flora. The bactericidal principle is that, for example, colicin Ia usually has three domains: a translocation domain, a receptor domain, and a channel-forming domain. The channel-forming domain can form a voltage-activated ion channel on the cell membrane (lipid bilayer membrane) of bacteria. The channel-forming domain at the carboxy terminus of colicin Ia is composed of 175 amino acids and 10 alpha helices. Driven by hydrophobic and hydrophilic forces, it can be inserted into the inner membrane (cell membrane) of E. coli without consuming energy to form an ion channel. The channel will open as soon as it senses a transmembrane potential of -50 mv. Since the pore size of the channel is very large, about Almost all ions can leak out of this huge aqueous pore, causing the depletion of the bacterium's energy and ion reserves, the rupture of the cell membrane, the leakage of cell contents, and the death of E. coli. Such a bactericidal process is a physical process that does not need to change or affect the enzymes or metabolism required for bacterial growth, metabolism, and reproduction to achieve the purpose of killing bacteria. Therefore, since hundreds of millions of years ago until now, it has been effectively killing allogenic bacteria.

[0009] Colicin Ia is a model example of E1 family colicins, and its gene, protein structure, and working mechanism are the most complete and detailed among E1 family colicins. SUMMARY

[0010] The present application aims to provide a kind of anti-small cell lung cancer drug, which can specifically recognize the typical surface antigen (protein and / or hydrocarbon) of lung cancer cell, and can efficiently kill lung cancer cell.

[0011] To achieve the above-mentioned purpose, the present application innovatively designs antibody mimetics (Ab Mimetic) specific to lung cancer cells. The antibody mimetics are selected from thirty 28-peptides, and the amino acid sequences are shown in SEQ ID NO:1-30. The antibody mimetics are constructed based on the disclosed antibody sequences against lung cancer cells, and can recognize the corresponding cancer cell antigens. The channel domain of E1 group colicin, which can form an ion channel, is connected to the thirty antibody mimetics respectively, and the obtained fusion proteins are the active ingredients of the anti-small cell lung cancer drug. In this context, the connection (fusion protein) of the antibody mimetics and the channel domain of colicin is also called "information colicin", and the combination of various information colicins is called information colicin array.

[0012] The present application provides polypeptides with amino acid sequences shown in SEQ ID NO:1-30.

[0013] The present application also provides the use of polypeptides with amino acid sequences shown in SEQ ID NO:1-30 in the preparation of anti-small cell lung cancer drugs.

[0014] In the above use, the drug can be a preparation for treating small cell lung cancer.

[0015] The present application also provides an anti-small cell lung cancer drug, which comprises fusion proteins obtained by connecting the channel domain of colicin to polypeptides with amino acid sequences shown in SEQ ID NO:1-30 respectively.

[0016] In the above drug, the colicin includes colicin E1, Ia, Ib, A, B and N.

[0017] In the above drug, the colicin is preferably colicin Ia, and the amino acid sequence of the channel domain (amino acids 346-626 of colicin Ia) is shown in SEQ ID NO:31.

[0018] In the above drug, the polypeptides with amino acid sequences shown in SEQ ID NO:1-30 are connected to the carboxyl terminal (C terminal) and / or amino terminal (N terminal) of the channel domain of colicin respectively, preferably to the carboxyl terminal of the channel domain of colicin.

[0019] In the above drug, the polypeptides and the channel domain of colicin are connected by covalent bond.

[0020] In the above drug, the arrangement of the polypeptides in the fusion protein can be N-terminal-SEQ ID NO: 31-antibody mimetic-C-terminal, including: SEQ ID NO: 31-SEQ ID NO: 1, SEQ ID NO: 31-SEQ ID NO: 2, SEQ ID NO: 31-SEQ ID NO: 3, SEQ ID NO: 31-SEQ ID NO: 4, SEQ ID NO: 31-SEQ ID NO: 5, SEQ ID NO: 31-SEQ ID NO: 6, SEQ ID NO: 31-SEQ ID NO: 7, SEQ ID NO: 31-SEQ ID NO: 8, SEQ ID NO: 31-SEQ ID NO: 9, SEQ ID NO: 31-SEQ ID NO: 10, SEQ ID NO: 31-SEQ ID NO: 11, SEQ ID NO: 31-SEQ ID NO: 12, SEQ ID NO: 31-SEQ ID NO: 13, SEQ ID NO: 31-SEQ ID NO: 14, SEQ ID NO: 31-SEQ ID NO: 15, SEQ ID NO: 31-SEQ ID NO: 16, SEQ ID NO: 31-SEQ ID NO: 17, SEQ ID NO: 31-SEQ ID NO: 18, SEQ ID NO: 31-SEQ ID NO: 19, SEQ ID NO: 31-SEQ ID NO: 20, SEQ ID NO: 31-SEQ ID NO: 21, SEQ ID NO: 31-SEQ ID NO: 22, SEQ ID NO: 31-SEQ ID NO: 23, SEQ ID NO: 31-SEQ ID NO: 24, SEQ ID NO: 31-SEQ ID NO: 25, SEQ ID NO: 31-SEQ ID NO: 26, SEQ ID NO: 31-SEQ ID NO: 27, SEQ ID NO: 31-SEQ ID NO: 28, SEQ ID NO: 31-SEQ ID NO: 29, SEQ ID NO: 31-SEQ ID NO: 30.

[0021] In the above drug, the amino acid sequence of the fusion protein can be the sequence shown in SEQ ID NO: 32-61.

[0022] The application also provides a method for preparing an anti-small cell lung cancer drug, comprising connecting polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30 to the channel domain of colicin, respectively, to obtain a fusion protein.

[0023] In the method, the colicin includes colicin E1, Ia, Ib, A, B and N.

[0024] In the method, the colicin is preferably colicin Ia, and the amino acid sequence of the channel domain (amino acids 346-626 of colicin Ia) is as shown in SEQ ID NO: 31.

[0025] In the method, the polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30 are connected to the carboxy terminus (C-terminal) and / or amino terminus (N-terminal) of the channel domain of colicin, preferably to the carboxy terminus of the channel domain of colicin.

[0026] In the method, the amino acid sequence of the fusion protein can be the sequence as shown in SEQ ID NO: 32-61.

[0027] The fusion protein in the application is an effective component against small cell lung cancer and can be directly used as an anti-small cell lung cancer drug. According to the needs of clinical use, the fusion protein can be prepared into different dosage forms of anti-small cell lung cancer drugs by adding pharmaceutically acceptable excipients.

[0028] The colicin of the application has a unique tumor-killing mechanism of destroying the integrity of the lipid bilayer membrane. The application uses the V H CDR1-V H FR2-V L The 28-peptide antibody mimetic structure constructed by the primary structure sequence of CDR3 is based on the disclosed antibody sequence of lung cancer cell antigen, and thirty antibody mimetics that can recognize the corresponding antigen are constructed. The antibody mimetics are selected from the Fab fragments of antibodies that recognize lung cancer cell antigens, V H CDR1 (heavy chain antigen binding region 1), V H FR2 (heavy chain framework region 2), and V L CDR3 (light chain antigen binding region 3) and V H CDR1-V H FR2-V LThe polypeptide, composed of 28 amino acids, is a linearly linked primary structure of CDR3. Various pheromone molecules were constructed by attaching antibody mimics to the carboxyl or amino terminus of the channel domain of colicin Ia (amino acids 346-626 of colicin Ia). These are pheromone-1 (PMC-1) to pheromone-30 (PMC-30) attached to the carboxyl terminus of the channel domain of colicin Ia, and pheromone-1 to PMC-30 attached to the amino terminus of the channel domain of colicin Ia.

[0029] The beneficial effects and innovative points of this invention are as follows:

[0030] 1. Select the channel domain of coliforms, which can form ion channels.

[0031] Through extensive research, the inventors discovered that coliformin can form ion channels on various lipid bilayers of different compositions and thicknesses. This suggests that if the inherent targeting of coliformin (which can only recognize different strains of E. coli) can be altered, it may be possible to recognize other bacteria, fungi, enveloped viruses, and even eukaryotic cells, thereby forming ion channels on the envelopes or cell membranes (lipid bilayers) of these organisms to kill them.

[0032] In the informational serotonin provided by this invention, the antibody mimic is responsible for recognizing target sites on the surface of tumor cell membranes, while the channel domain of coliformin Ia is responsible for disrupting the integrity of the tumor cell membrane, forming a transmembrane ion channel on the tumor cell membrane. Because the diameter of this channel (lumen) is large enough... (0.9-1.1 nanometers) Once activated, it causes a rapid leakage of almost all intracellular ions to the extracellular space, quickly disrupting the transmembrane ion gradient and ultimately leading to cell exhaustion and rapid death. This simple and direct physical killing method (passive leakage caused by ion concentration gradient) is something that current biochemical drug resistance mechanisms widely used in tumor cells (altering metabolic pathways, synthesizing new biomolecules, and using energy-consuming pumps to pump drug molecules out of the cell, etc.) cannot resist.

[0033] 2. Select corresponding tumor cell antigens as targets.

[0034] Tumor growth is a biological process that is coordinated by multiple genes, multiple proteins, and multiple signaling pathways. The rhythm of this process can be reflected by the four-dimensional spatial changes of various tumor cell surface antigens (three-dimensional spatial distribution of antigens on the cell surface changes over time). During different growth cycles, various antigens appear on the tumor cell surface in turn. Tumor growth is a highly ordered complex process that cannot be controlled by intervention in a single antigen, a single signaling pathway, or a single gene-protein axis. Therefore, current treatments targeting a single target have been difficult to control the tumor growth process. It is necessary to control multiple factors simultaneously to effectively intervene in the tumor growth process.

[0035] There are many antigens on the surface of tumor cells. During various processes of tumor growth, these antigens appear on the tumor cell surface in turn, that is, in a certain tumor growth process, antigens A and C may be dominant, but in the next growth process, antigens A and C may not be dominant, and antigens B and D may be dominant, and in the next growth process, neither A and C nor B and D may be dominant, and other antigens may be dominant. Due to this law of alternation of tumor cell surface antigens with growth cycle, the use of single-target drugs to fight tumors for a long time will inevitably lead to drug resistance and escape, resulting in treatment failure. With the change of growth cycle, a single target may gradually weaken or even disappear, so the drug targeting this single target will also be ineffective. In order to cope with this change, we need to design an array of drugs that can deal with multiple targets, and when facing tumor cells of different growth cycles, there is always one or more drugs in the array that can recognize the dominant antigens in the corresponding growth cycle, so that the array can always efficiently and on-target attack tumors, maximize the avoidance of drug resistance and escape, and effectively treat tumors.

[0036] In Chinese Patent CN1274829C, phorin effectively killed malignant lymphosarcoma by using antibody mimics and wild-type colicin Ia (626 amino acid residues, 70 kDa) to recognize EB virus glycoprotein antigens.

[0037] The information bacteriocin array of the present application was verified for its efficacy using two small cell lung cancer cell lines (NCI-H446 and DMS-153), wherein the information bacteriocin array used was composed of thirty information bacteriocins with amino acid sequences as shown in SEQ ID NO: 32-61. The in vitro killing experiment of small cell lung cancer cells showed that the information bacteriocin array could effectively kill the small cell lung cancer cells (Example 2). The pharmacodynamic experiment using the small cell lung cancer model confirmed that the tumor-bearing mouse model could not escape the recognition and killing of the tumor by the information bacteriocin array in each growth cycle, resulting in complete killing of the cancer cells and failure of the tumor model to grow. The information bacteriocin array of the present application showed a highly significant growth inhibition and killing effect on the tumor models derived from the two small cell lung cancer cell lines (Example 3).

[0038] The above experiments confirmed that the information bacteriocin array of the present application showed a much better anti-tumor effect than the single information bacteriocin in the above patent (CN1274829C). In addition, the guinea pig model experiment confirmed that the continuous use of the information bacteriocin array for 30 days did not produce any toxic side effects in the animal model (Example 4). Therefore, the information bacteriocin array of the present application can be used as an effective treatment for small cell lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a schematic diagram of the structure of the information bacteriocin of the present application.

[0040] Figure 2 is a schematic diagram of the recombinant plasmid used to prepare the information bacteriocin constructed in Example 1 of the present application.

[0041] Figure 3 is the SDS-PAGE electrophoresis result of three information bacteriocins among the thirty information bacteriocins prepared in Example 1 of the present application. Lane 1: protein Marker; Lanes 2-4: three information bacteriocins in the information bacteriocin array prepared in Example 1, each with a molecular weight of about 30 kDa; Lane 5: information bacteriocin as a control (containing full-length colicin Ia with a molecular weight of 70 kDa); Lane 6: bovine serum albumin as a standard control, with a molecular weight of 70 kDa.

[0042] Figure 4 is the experimental result of attacking small cell lung cancer cells (NCI-H446) with the information bacteriocin array in Example 2 of the present application. Figure 4a: small cell lung cancer cells (NCI-H446) in the blank control group, with a polygonal shape. Figure 4b: small cell lung cancer cells (NCI-H446) after being incubated with the information bacteriocin array (50 μg / ml) for 48-72 hours, changed from the polygonal shape of normal lung cancer cells to round, oval or long strip shape, most of which have lost adhesion, and the subcellular structure has been completely changed; iodinated propylamine staining (red floating spherical objects) indicates that the cell membrane of dead cells is destroyed, and iodinated propylamine enters the cell to stain it red; under the continuous attack of the information bacteriocin array, the small cell lung cancer cells observed under the microscope have completely lost life activity.

[0043] Figure 5 is the weight change of the tumor carried by the mouse after 3 days of inoculating small cell lung cancer cells (NCI-H446) in Balb / C nude mice in Example 3 of the present application, and treating for 4 weeks with the information bacteriocin array. The blue triangular symbol represents the blank control group (CK); the purple red dot symbol represents the HB8627-information bacteriocin treatment group (PMC-8627); and the red square symbol represents the information bacteriocin array treatment group (PMC-array).

[0044] Figure 6 is the weight change of the tumor carried by the mouse after 14 days of inoculating small cell lung cancer cells (NCI-H446) in Balb / C nude mice in Example 3 of the present application, and treating for 3 weeks with the information bacteriocin. The blue triangular symbol represents the blank control group (CK); and the red square symbol represents the information bacteriocin array treatment group (PMC-array).

[0045] Figure 7 is the weight change of the tumor carried by the mouse after 10 days of inoculating small cell lung cancer cells (DMS-153) in Balb / C nude mice in Example 3 of the present application, and treating for 3 weeks with the information bacteriocin, and then stopping the treatment for 3 weeks. The blue triangle represents the blank control group (CK); and the red square represents the information bacteriocin array treatment group (PMC-array). The red solid line represents treating (administering) with the information bacteriocin array for 3 weeks; and the red blank line represents stopping the treatment for 3 weeks.

[0046] Figure 8 is the tumor dissection specimen of the second experimental group in Example 3 of the present application. The upper row: three tumors (NCI-H446) of the blank control group, with weights of 1683 mg, 301 mg and 263 mg, respectively. The lower row: three tumors (NCI-H446) of the information bacteriocin array treatment group, with weights of 27 mg, 4 mg and 4 mg, respectively.

[0047] Figure 9 is the pathological section of tumor dissection specimen of the second experimental group in Example 3 of the present application. Figure 9a: the pathological section of tumor of the blank control group (mouse tumor model of small cell lung cancer). Figure 9b: the pathological section of tumor after 6 days of treatment with the information bacteriocin array, a large number of tumor cells appear coagulative necrosis (this is the typical case change of information bacteriocin killing tumor cells, Nat Biotech 2007). Figure 9c: the local enlarged view of Figure 9b. Figure 9d: after 3 weeks of treatment with the information bacteriocin array, some of the tumor-bearing mice gradually disappeared. The scale length is 100 μm.

[0048] Figure 10 is the experimental result of the toxicity of the information bacteriocin array on normal guinea pigs in Example 4 of the present application. There is no significant difference in blood biochemical indicators between the blank control group (C, n = 4) and the information bacteriocin array treatment group (T, n = 6). In Figure 10, the concentration units of UREA (urea), CREA (creatinine), UA (uric acid), TBIL (total bilirubin) and DBIL (direct bilirubin) are mg / dl; the concentration units of TP (total serum protein), ALB (albumin) and GLB (globulin) are g / dl; A / G represents the ratio of albumin and globulin; the concentration units of ALT (glutamic-pyruvic transaminase), AST (glutamic-oxaloacetic transaminase), ALP (alkaline phosphatase), LDH (lactate dehydrogenase), GGT (γ-glutamyl transpeptidase) and CK (creatine kinase) are Unit / L.

[0049] SEQUENCE DESCRIPTION

[0050] The amino acid sequences set out in the accompanying sequence listing are shown using the one-letter code for amino acids, following the standard convention of starting at the amino terminus of the sequence and proceeding toward the carboxy terminus.

[0051] SEQ ID NOs: 1-30 are the amino acid sequences of thirty antibody mimics that recognize relevant tumor antigens;

[0052] SEQ ID NO: 31 is the amino acid sequence of the channel domain of colicin Ia;

[0053] SEQ ID NOs: 32-61 are the amino acid sequences of thirty information bacteriocins against small cell lung cancer. DETAILED DESCRIPTION

[0054] The following examples are provided:

[0055] 1. A polypeptide having an amino acid sequence as set forth in SEQ ID NOs: 1-30.

[0056] 2. Use of a polypeptide having an amino acid sequence as set forth in SEQ ID NOs: 1-30 in the preparation of a medicament for the treatment of small cell lung cancer.

[0057] 3. The use according to embodiment 2, wherein the medicament is a preparation for treating small cell lung cancer.

[0058] 4. An anti-small cell lung cancer medicament comprising a fusion protein obtained by connecting a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 to a channel domain of colicin, respectively.

[0059] 5. The medicament according to embodiment 4, wherein the colicin comprises colicin El, la, lb, A, B and N.

[0060] 6. The medicament according to embodiment 5, wherein the colicin is colicin la, and the amino acid sequence of the channel domain of the colicin la is as shown in SEQ ID NO: 31.

[0061] 7. The medicament according to any one of embodiments 4-6, wherein the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 is connected to the carboxyl terminal and / or amino terminal of the channel domain of the colicin, respectively.

[0062] 8. The medicament according to embodiment 7, wherein the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 is connected to the carboxyl terminal of the channel domain of the colicin.

[0063] 9. The medicament according to embodiment 8, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 32-61.

[0064] 10. A method for preparing an anti-small cell lung cancer medicament, comprising connecting a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 to a channel domain of colicin, respectively, to obtain a fusion protein.

[0065] 11. The method according to embodiment 10, wherein the colicin comprises colicin El, la, lb, A, B and N.

[0066] 12. The method according to embodiment 11, wherein the colicin is colicin la, and the amino acid sequence of the channel domain of the colicin la is as shown in SEQ ID NO: 31.

[0067] 13. The method according to any one of embodiments 10-12, wherein the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 is connected to the carboxyl terminal and / or amino terminal of the channel domain of the colicin, respectively.

[0068] 14. The method according to embodiment 13, wherein the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1-30 is connected to the carboxyl terminal of the channel domain of the colicin.

[0069] 15. The method of embodiment 14, wherein the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 32-61.

[0070] The present application is further illustrated by the following examples. It is to be understood that the following examples are merely illustrative of the present application and do not limit the scope of the application.

[0071] Unless otherwise indicated, the reagents used in the following examples are of standard quality and are commercially available or are prepared by conventional procedures known in the art, and are used at the laboratory scale. Unless otherwise indicated, the experimental methods and conditions used in the following examples are conventional in the art and are well known to those skilled in the art, and are described in relevant laboratory manuals, known references or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0072] Example 1

[0073] Preparation of information phage array

[0074] Thirty information phages with amino acid sequences set forth in SEQ ID NO: 32-61 were prepared. The information phages are fusion proteins obtained by linking antibody mimics to the carboxy terminus of the channel domain of colicin Ia (Figure 1). The amino acid sequence of the channel domain of colicin Ia is set forth in SEQ ID NO: 31. The amino acid sequences of the thirty antibody mimics recognizing relevant tumor antigens are set forth in SEQ ID NO: 1-30. The primary structures of the thirty antibody mimics are listed in Table 1 (V H CDR1-V H FR2-V L CDR3), wherein the amino acids are in standard single letter designation and are arranged in order from the amino terminus to the carboxy terminus.

[0075] Table 1: Thirty antibody mimics recognizing relevant tumor antigens

[0076] The amino acid sequence of the channel domain of colicin Ia is as follows, wherein the amino acids are in standard single letter designation and are arranged in order from the amino terminus to the carboxy terminus, wherein the number at the beginning of each line indicates the position of the first amino acid in the amino acid sequence of colicin Ia.

[0077] 346 nilnd rnpvvtedve

[0078] 361 gdkkiynaev aewdklrqrl ldarnkitsa esavnsarnn lsartneqkh andalnallk

[0079] 421 ekenirnqls ginqkiaeekrkqdelkatk dainfttefl ksvsekygak aeqlaremag

[0080] 481 qakgkkirnv eealktyeky radinkkina kdraaiaaal esvklsdiss nlnrfsrglg

[0081] 541 yagkftsladwitefgkavr tenwrplfvktetiiagnaa talvalvfsi ltgsalgiig

[0082] 601 ygllmavtga lideslveka nkfwgi(SEQ ID NO:31)

[0083] When Escherichia coli synthesizes colicin in a physiological state, it synthesizes two proteins, one molecule of colicin and one molecule of a corresponding immune protein. The immune protein functions to prevent the synthesized colicin from forming an ion channel on the cell membrane of the Escherichia coli itself. When the synthesized colicin exits the Escherichia coli (is secreted outside the cell), the colicin and the immune protein are separated. Therefore, when colicin is synthesized using an engineered bacterium, the corresponding immune protein needs to be synthesized at the same time. The genes encoding the structural protein of colicin la and the immune protein have the GenBank accession number M13819 at the National Center for Biotechnology Information (NCBI). Thirty kinds of colicin were prepared using the amino acid sequences shown in SEQ ID NOs: 32-61 using the pET11a plasmid and Escherichia coli B834(DE3). The pET11a recombinant plasmid for expressing the colicin was synthesized by commissioning the Nuseigen company, and it contains a gene encoding the channel domain of colicin la, a gene encoding an antibody mimic, and a gene encoding an immune protein (FIG. 2). Thirty kinds of recombinant plasmids were obtained, each of which was used to express one kind of colicin. DNA sequencing was performed on the thirty kinds of constructed recombinant plasmids.

[0084] The thirty recombinant plasmids identified by sequencing were respectively transfected into E. coli B834 (DE3) competent cells; the B834 engineering bacteria containing the recombinant plasmids were placed in LB liquid medium (containing 100 μg / ml ampicillin) for proliferation, and then centrifuged to collect the bacterial bodies; the bacterial bodies were broken and resuspended in 50 mM boric acid buffer (pH 9), the supernatant was extracted by centrifugation, and DNA was precipitated by adding streptomycin sulfate to the supernatant; the supernatant was extracted by centrifugation again, and then dialyzed in 50 mM boric acid buffer (pH 9), and then passed through a Sepharose (CM-Sepharose) gel column and eluted with 0.3 M NaCl solution to obtain thirty information bacterins, and the SDS-PAGE electrophoresis results of three information bacterins are shown in FIG. 3. The yield of information bacterins can reach 5-12 mg / ml.

[0085] The thirty information bacterins prepared were tested by liquid chromatography-mass spectrometry (LC-MS), which confirmed that the amino acid residues of the antibody mimetic were located at the carboxyl end of the information bacterin. The thirty information bacterins tested by LC-MS were mixed in equal amounts to obtain the information bacterin array of the present application.

[0086] Example 2

[0087] In vitro killing experiment of small cell lung cancer cells by information bacterin array

[0088] 1. Purpose of the experiment

[0089] The in vitro killing effect of the information bacterin array of the present application on small cell lung cancer cells was investigated.

[0090] 2. Experimental materials

[0091] Test drugs: (1) Information bacterin array of the present application prepared in Example 1, which is a mixture of thirty information bacterins with amino acid sequences as shown in SEQ ID NO: 32-61 in equal amounts, provided by Chengdu Finolink New Biotechnology Co., Ltd. (2) HB8627-information bacterin, provided by Chengdu Finolink New Biotechnology Co., Ltd. The HB8627-information bacterin is described in the 2007 Nature Biotechnology paper "Qiu, XQ, et al. Small antibody mimetic comprising two complementarity-determining regions and a framework region for tumor targeting. Nature Biotechnology, 2007, 25(8): 921-929", the entire contents of which are incorporated herein by reference.

[0092] The small cell lung cancer cell line NCI-H446, the cell culture solution RMPI 1640 and the microscope Leica DMi8 are provided by the Institute of Basic Medical Sciences, Peking Union Medical College.

[0093] 3. Experimental site

[0094] Institute of Basic Medical Sciences, Peking Union Medical College.

[0095] 4. Experimental method

[0096] After the information bacteriocin array of the application is incubated with the small cell lung cancer cell NCI-H446 for 48-72 hours, the survival state of the small cell lung cancer cell is detected, so as to verify the inhibition and killing effect of the information bacteriocin array of the application on the small cell lung cancer cell.

[0097] In the experiment, a blank control group, a positive control group and an information bacteriocin array treatment group are set, and the details are as follows:

[0098] (1) Blank control group: the small cell lung cancer cell NCI-H446 is normally cultured without any treatment.

[0099] (2) Positive control group: the HB8627-information bacteriocin (50 micrograms per milliliter of culture solution) is incubated with the small cell lung cancer cell NCI-H446 for 48-72 hours.

[0100] (3) Information bacteriocin array treatment group: the information bacteriocin array of the application (the total amount of thirty information bacteriocins is 50 micrograms per milliliter of culture solution, and the content of each information bacteriocin is the same, about 1.67 micrograms per milliliter of culture solution) is incubated with the small cell lung cancer cell NCI-H446 for 48-72 hours.

[0101] The morphological changes of the cells in each group are observed under a microscope, and the number of cells is calculated. According to the survival state and morphological differences of the cells in the blank control group and the information bacteriocin array treatment group, the efficiency of the information bacteriocin array of the application in inhibiting and killing the small cell lung cancer cell in vitro is determined.

[0102] 5. Experimental results

[0103] After the small cell lung cancer cells were incubated with the information pheromone array of the present application for 48-72 hours, the cell morphology changed significantly, and the morphological and fluorescent staining results confirmed that all the tumor cells had died (Figure 4). The small cell lung cancer cells in the blank control group had a polygonal morphology (Figure 4a). After the small cell lung cancer cells were incubated with the information pheromone array for 48-72 hours, the cells had a round, oval or long strip morphology, most of which could not adhere to the wall, and the subcellular structure was completely changed; the results of propidium iodide staining suggested that the cell membrane of the dead cells was destroyed, and propidium iodide entered the cells and dyed them red; under the continuous attack of the information pheromone array, the small cell lung cancer cells observed under the microscope had completely lost their life activity (Figure 4b).

[0104] 6. Experimental conclusion

[0105] The information pheromone array of the present application has a strong effect of inhibiting and killing small cell lung cancer cells.

[0106] Example 3

[0107] Pharmacodynamic experiment of the information pheromone array on a mouse tumor-bearing model

[0108] 1. Experimental purpose

[0109] To verify the killing effect of the information pheromone array of the present application on a mouse tumor-bearing model.

[0110] 2. Experimental materials

[0111] 60 Balb / C immunodeficient nude mice (half male and half female) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0112] The small cell lung cancer cell lines NCI-H446 and DMS-153 were obtained from the Institute of Basic Medical Sciences, Peking Union Medical College.

[0113] Test drugs: (1) The information bacterioxin array of the present application prepared in Example 1, which is a mixture of equal amounts of thirty kinds of information bacterioxins with amino acid sequences as shown in SEQ ID NOs: 32-61, was provided by Chengdu Finolink New Biotechnology Co., Ltd. (2) HB8627-information bacterioxin, which was provided by Chengdu Finolink New Biotechnology Co., Ltd. The HB8627-information bacterioxin is described in the paper "Qiu, XQ, et al. Small antibody mimetic comprising two complementarity-determining regions and a framework region for tumor targeting. Nature Biotechnology, 2007, 25(8): 921-929" published in 2007, the entire contents of which are incorporated herein by reference.

[0114] 3. Experimental methods

[0115] (1) Establishment of tumor-bearing mouse model

[0116] Balb / C nude mice were injected with cultured small cell lung cancer cells in the armpit, with a dose of 0.1 ml 5x10 7 After 3-14 days of inoculation, the tumor-bearing mouse model animals were randomly divided into groups, and the drug treatment was started.

[0117] (2) Drug administration method and dose

[0118] Balb / C nude mice in the first experimental group were inoculated with small cell lung cancer cell line NCI-H446, and intraperitoneal administration was started 3 days after inoculation. A blank control group (n=6), a positive control group (n=4), and an information bacterioxin array treatment group (n=6) were set up, where n represents the number of nude mice. The blank control group: intraperitoneal injection of normal saline, 1 ml per mouse per day. The positive control group: intraperitoneal injection of HB8627-information bacterioxin, 1 mg per day, twice a day, 0.5 mg each time. The information bacterioxin array treatment group: intraperitoneal injection of the information bacterioxin array of the present application, 1 mg per day, twice a day, 0.5 mg each time. Dissection was performed after 28 days of continuous administration.

[0119] The second experimental group of Balb / C nude mice were inoculated with small cell lung cancer cell line NCI-H446, and intraperitoneal administration was started 14 days after inoculation. A blank control group (n=10) and an information bacterioin array treatment group (n=10) were set up, wherein n represents the number of nude mice. The blank control group: intraperitoneal injection of normal saline, 1 ml per mouse per day. The information bacterioin array treatment group: intraperitoneal injection of the information bacterioin array of the application, 2 mg per day, three times a day, about 0.7 mg each time. After 21 days of continuous administration, dissection was performed.

[0120] The third experimental group of Balb / C nude mice were inoculated with small cell lung cancer cell line DMS-153, and intraperitoneal administration was started 10 days after inoculation. A blank control group (n=10) and an information bacterioin array treatment group (n=10) were set up, wherein n represents the number of nude mice. The blank control group: intraperitoneal injection of normal saline, 1 ml per mouse per day. The information bacterioin array treatment group: intraperitoneal injection of the information bacterioin array of the application, 2 mg per day, three times a day, about 0.7 mg each time. After 21 days of continuous administration, 6 mice were randomly selected from each group for dissection, and the remaining 4 mice in each group were dissected after 21 days of drug withdrawal.

[0121] (3) Body weight detection

[0122] The body weight of the animals was detected by electronic digital display before inoculation for 3 days and every day after administration.

[0123] (4) Observation of tumor-bearing pathological changes

[0124] After dissection, the tumor-bearing pathological changes of the mice were observed. The tumor-bearing, weighing, tumor fixation and pathological section observation were performed. After reading the pathological observation film, the pathological changes of each group of tumor-bearing were scored.

[0125] (5) Statistical method of results

[0126] The paired t test statistical analysis method was used to evaluate the changes of tumor size and weight in each group.

[0127] 4. Experimental results

[0128] The experimental results of the three experimental groups are shown in Table 2.

[0129] The experimental results of the first experimental group showed that after 28 days of continuous administration, the average tumor weight of the blank control group (CK) was 129.33 mg, the average tumor weight of the positive control group (PMC-8627) was 89.25 mg, and the average tumor weight of the information bacterioin array treatment group (PMC-array) was 3.33 mg (Figure 5).

[0130] The experimental results of the second experimental group show that the average tumor weight of the blank control group (CK) is 409 mg, and the average tumor weight of the phagemid array treatment group (PMC-array) is 16 mg (Figure 6).

[0131] The experimental results of the third experimental group show that after 21 days of continuous administration, the average tumor weight of the blank control group (CK) is 123.83 mg (n = 6), and the average tumor weight of the phagemid array treatment group (PMC-array) is 11 mg (n = 6). After 21 days of continuous administration and 21 days of drug withdrawal, the average tumor weight of the blank control group (CK) is 202.75 mg (n = 4), and the average tumor weight of the phagemid array treatment group (PMC-array) is 7.25 mg (n = 4) (Figure 7).

[0132] Table 2

[0133] * The tumor weight is 0, indicating that no tumor was found after dissection of the mouse.

[0134] 5、Experimental Conclusion

[0135] The phagemid array of the present application shows strong killing effect on the nude mouse tumor model (human small cell lung cancer). After three weeks of drug withdrawal, no tumor growth was observed (the tumors of the blank control group in the same period continued to grow) (Figure 7). The tumor dissection results (Figure 8) and pathological section observation results (Figure 9) also confirm that the phagemid array effectively eliminates the small cell lung cancer tumor inoculated. The experimental mice gained weight, confirming that the phagemid array does not cause toxic damage to the test animals.

[0136] Example 4

[0137] Toxicity experiment of phagemid array on normal guinea pigs

[0138] 1、Experimental materials

[0139] Experimental animals: 10 guinea pigs (half male and half female) from Beijing Vivotec Laboratories Animal Technology Co., Ltd.

[0140] Test drug: the phagemid array of the present application prepared in Example 1, which is a mass mixture of thirty kinds of phagemids with amino acid sequences as shown in SEQ ID NO: 32-61, provided by Chengdu Finolife New Biotechnology Co., Ltd.

[0141] 2、Experimental grouping

[0142] Guinea pig grouping: blank control group (n = 4), phagemid array treatment group (n = 6), a total of 10.

[0143] 3、Experimental method

[0144] (1) Administration method and dosage

[0145] Control group: intraperitoneal injection of normal saline, 2 ml per mouse per day.

[0146] Information pheromone array treatment group: intraperitoneal injection of the information pheromone array of the present application, 3 mg per mouse per day.

[0147] Each group was administered once a day, and after 30 days of continuous administration, blood samples were taken for examination and the animals were sacrificed.

[0148] (2) Body weight detection

[0149] The body weight of the animals was detected using an electronic digital display scale 3 days before inoculation and every 7 days after administration.

[0150] (3) Blood biochemical detection

[0151] The blood samples were sent to the Animal Hospital of China Agricultural University for detection, and the changes in blood biochemical indicators were compared.

[0152] 4. Experimental results

[0153] Compared with the control group, the animals injected with the information pheromone array showed no changes in appetite and behavior, and the detection results of blood biochemical indicators (Figure 10) showed no differences (no damage to liver, kidney, and immune function).

[0154] 5. Experimental conclusion

[0155] The 30-day information pheromone array treatment (intraperitoneal injection) did not produce toxic side effects in experimental animals.

Claims

1. A polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30.

2. Use of a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30 in the preparation of a medicament for treating small cell lung cancer.

3. The use of claim 2, wherein the medicament is a preparation for treating small cell lung cancer.

4. A medicament for treating small cell lung cancer, comprising a fusion protein obtained by linking a channel domain of colicin to a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30, respectively.

5. The medicament of claim 4, wherein the colicin comprises colicin El, la, lb, A, B and N.

6. The medicament of claim 5, wherein the colicin is colicin la, and the amino acid sequence of the channel domain of the colicin la is as set forth in SEQ ID NO:

31.

7. The medicament of any one of claims 4-6, wherein the polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30 is linked to the carboxy terminus and / or the amino terminus of the channel domain of the colicin, respectively.

8. The medicament of claim 7, wherein the polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30 is linked to the carboxy terminus of the channel domain of the colicin.

9. The medicament of claim 8, wherein the amino acid sequence of the fusion protein is as set forth in any one of SEQ ID NOs: 32-61.

10. A method for preparing the medicament for treating small cell lung cancer of any one of claims 4-9, comprising linking a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-30 to a channel domain of colicin, respectively, to obtain a fusion protein.

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