An information bacteriocin array against small cell lung cancer and application thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to an information bacteriocin array against small cell lung cancer and application thereof. Amino acid sequences such as SEQ ID NO: 1-30 are provided. A drug against small cell lung cancer is also provided, which comprises a fusion protein obtained by connecting a channel domain of colicin with polypeptides with amino acid sequences such as SEQ ID NO: 1-30. Pharmacodynamics experiments prove that in each growth cycle, a mouse tumor-bearing model cannot escape the recognition and killing of the information bacteriocin array composed of thirty kinds of fusion proteins. Guinea pig model experiments prove that the continuous use of the information bacteriocin array for 30 days does not produce any toxic side effects in the animal model. The information bacteriocin array can be used as an effective drug for treating small cell lung cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to an information bacteriocin array against small cell lung cancer and application thereof. BACKGROUND

[0002] 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.

[0003] No matter 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 is relieved after treatment is prone to recurrence and metastasis, ultimately leading to treatment failure; (2) the current anti-tumor treatment methods and drugs have great 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 against different targets in the same patient. Even the latest antibody drug conjugates (ADC) are difficult to use two or more ADCs in the same patient due to immune-related adverse events (irAE). Due to the defects of the above drugs, modern treatment methods are difficult to intervene in multiple tumor targets or along multiple tumor treatment pathways.

[0004] 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, and it is almost impossible to simultaneously intervene in multiple targets and multiple pathways in the same patient. This results in the limitation of treatment: once the tumor develops resistance or escapes from this type of single-pathway intervention, it blocks this type of single-pathway intervention treatment. Therefore, there is an urgent need to develop a tumor intervention treatment method that can simultaneously intervene in multiple targets and multiple pathways, so as to more effectively treat tumors.

[0005] Colicins are classic examples of bacteriocins. There are more than twenty kinds of colicins, which attack the gene, protein synthesis system, or destroy the cell membrane of other strains of Escherichia coli. Channel-forming E1 family colicins (which form ion channels on the cell membrane to kill Escherichia coli) are composed of colicin E1, colicin Ia, colicin Ib, colicin A, colicin B, and colicin N.

[0006] Colicin E1, Ia, Ib, A, B, N, etc. is one of the regulatory forces to maintain the diversity and evolution of intestinal flora. The bactericidal principle is that, taking colicin Ia as an example, it usually has three domains: translocation domain, receptor domain, and channel-forming domain. The channel-forming domain can form a voltage-activated ion channel on the cell membrane (lipid bilayer) of bacteria. The channel 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. Because the pore size of the channel is large, about Almost all kinds of ions can leak out of this huge aqueous pore, causing the energy and ion reserves of the bacteria to be depleted, the cell membrane to be broken, the cell contents to be leaked, and the E. coli to die. 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 allogeneic bacteria.

[0007] Colicin Ia is a model specimen of E1 group colicin, and its gene, protein structure and working mechanism are the most perfect and detailed among E1 group colicins. SUMMARY

[0008] The purpose of the present application is 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 cells and can efficiently kill lung cancer cells.

[0009] To achieve the above purpose, the antibody mimetic (Ab Mimetic) specific to lung cancer cells is designed innovatively. The antibody mimetic is selected from thirty 28-peptides, and the amino acid sequences are shown as SEQ ID NO: 1-30. The antibody mimetic is constructed based on the disclosed antibody sequence against lung cancer cells, which can recognize the corresponding cancer cell antigen. The channel domain of E1 group colicin which can form an ion channel is connected with the thirty antibody mimetics respectively, and the obtained fusion proteins are the active ingredients of the anti-small cell lung cancer drug. In this paper, the connection (fusion protein) of the antibody mimetic and the channel domain of colicin is also called "information colicin"; the combination of various information colicins is called information colicin array.

[0010] The present application provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30.

[0011] The present application also provides a use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 in the preparation of a medicament for treating small cell lung cancer.

[0012] In the use described above, the medicament can be a preparation for treating small cell lung cancer.

[0013] The present application also provides a medicament for treating small cell lung cancer, which comprises a fusion protein obtained by connecting a channel domain of colicin to a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30, respectively.

[0014] In the medicament described above, the colicin includes colicin E1, la, lb, A, B and N.

[0015] In the medicament described above, the colicin is preferably colicin la, and the amino acid sequence of the channel domain (amino acids 346-626 of colicin la) is as shown in SEQ ID NO: 31.

[0016] In the medicament described above, the polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 is connected to the carboxyl terminal (C terminal) and / or amino terminal (N terminal) of the channel domain of colicin, preferably to the carboxyl terminal of the channel domain of colicin.

[0017] In the medicament described above, the polypeptide and the channel domain of colicin are connected by a covalent bond.

[0018] In the above drug, the arrangement of the polypeptide 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.

[0019] In the above drug, the amino acid sequence of the fusion protein can be the sequence shown in SEQ ID NO: 32-61.

[0020] The present application also provides a method for preparing a drug for resisting small cell lung cancer, which comprises connecting polypeptides with amino acid sequences shown in SEQ ID NO: 1-30 to the channel domain of colicin, respectively, to obtain a fusion protein.

[0021] In the above method, the colicin includes colicin E1, la, lb, A, B, and N.

[0022] In the above method, the colicin is preferably colicin la, and the amino acid sequence of the channel domain (amino acids 346-626 of colicin la) is shown in SEQ ID NO: 31.

[0023] In the above method, the polypeptide having the amino acid sequence shown in SEQ ID NO: 1-30 is connected to the carboxy terminus (C-terminus) and / or the amino terminus (N-terminus) of the channel domain of the colicin, preferably to the carboxy terminus of the channel domain of the colicin.

[0024] In the above method, the amino acid sequence of the fusion protein can be the sequence shown in SEQ ID NO: 32-61.

[0025] The fusion protein in the present application is an effective component against small cell lung cancer and can be directly used as an anti-small cell lung cancer drug. Different dosage forms of the fusion protein against small cell lung cancer can also be prepared by adding pharmaceutically acceptable adjuvants according to the needs of clinical use.

[0026] The colicin of the present application has a unique tumor-killing mechanism of destroying the integrity of the lipid bilayer membrane. The present application applies the V H CDR1-V H FR2-V L The 28-peptide antibody mimetic structure constructed by the primary structure sequence of CDR3 can recognize the corresponding antigen. The antibody mimetic is selected from the Fab fragment of the antibody recognizing the lung cancer cell antigen, and the 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 according to V H CDR1-V H FR2-V LThe polypeptide linearly connected by the primary structure sequence of CDR3 is composed of 28 amino acids. The antibody mimics are connected to the carboxy terminal or amino terminal of the channel domain (amino acids 346-626 of colicin Ia) of colicin Ia, respectively, to construct a plurality of pheromonicins, which are pheromonicin-1 (PMC-1) to pheromonicin-30 (PMC-30) with the antibody mimics connected to the carboxy terminal of the channel domain of colicin Ia and PMC-1 amino terminal to PMC-30 amino terminal with the antibody mimics connected to the amino terminal of the channel domain of colicin Ia.

[0027] The beneficial effects and innovative points of the present application are as follows:

[0028] 1. Selecting the channel domain of colicin capable of forming an ion channel

[0029] The inventor has found through a large number of studies that colicin can form an ion channel on a variety of lipid bilayer membranes with different components and thicknesses. This suggests that if the inherent targeting of colicin can be changed (the inherent targeting can only recognize allogeneic E. coli), it is possible to recognize other bacteria, fungi, enveloped viruses, and even eukaryotic cells, so as to form an ion channel on the envelope or cell membrane (lipid bilayer membrane) of these living organisms to kill them.

[0030] In the pheromonicin provided by the present application, the antibody mimic is responsible for recognizing the target on the surface of the tumor cell membrane, and the channel domain of colicin Ia is responsible for destroying the integrity of the tumor cell membrane, which forms a transmembrane ion channel on the tumor cell membrane. Since the diameter of the channel (lumen) is as large as 0.9-1.1 nanometers, once opened, it causes a sharp leakage of almost all intracellular ions to the outside of the cell, the transmembrane ion gradient difference is rapidly unbalanced, and finally leads to cell exhaustion and rapid death. Such a simple and direct physical killing method (passive leakage caused by ion concentration gradient difference) is invincible to the current biochemical drug resistance mechanism (changing metabolic pathways, synthesizing new structure biomolecules, using energy-consuming pumps to pump drug molecules out of cells, etc.) widely used by tumor cells.

[0031] 2. Selecting the corresponding tumor cell antigen as the target

[0032] ​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.

[0033] 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.

[0034] In Chinese Patent CN1274829C, the use of antibody mimics and wild-type colicin Ia (626 amino acid residues, 70 kDa) to recognize the information bacteriocin of EB virus glycoprotein antigen effectively killed malignant lymphosarcoma.

[0035] The information bacteriocin array of the present application was verified by using two small cell lung cancer cell lines (NCI-H446 and DMS-153). The information bacteriocin array used in the verification consisted 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 small cell lung cancer cells (Example 2). The pharmacodynamics experiment using a 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 above two small cell lung cancer cell lines (Example 3).

[0036] 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

[0037] Figure 1 Figure 1 is a schematic diagram of the structure of the information bacteriocin of the present application.

[0038] Figure 2 Figure 2 is a schematic diagram of the recombinant plasmid used to prepare the information bacteriocin constructed in Example 1 of the present application.

[0039] Figure 3 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.

[0040] Figure 4 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 4 a: 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) co-incubated with pheromone array (50 μg / ml) for 48-72 hours changed from the polygonal morphology of normal lung cancer cells to round, oval or elongated morphology. Most of them could no longer adhere to the wall, and the subcellular structure was completely changed. Propidium iodide staining (floating red spherical objects) indicated that the cell membrane of dead cells was destroyed, and propidium iodide entered the cell and stained it red. Under the continuous attack of pheromone array, all small cell lung cancer cells seen under the microscope had lost their vitality.

[0041] Figure 5 In Example 3 of this invention, Balb / C nude mice were inoculated with small cell lung cancer cells (NCI-H446) for 3 days, and then treated with a pheromone array for 4 weeks. The changes in tumor weight in the mice were recorded. The blue triangle symbol represents the blank control group (CK); the purple-red dot symbol represents the HB8627-pheromone treatment group (PMC-8627); and the red square symbol represents the pheromone array treatment group (PMC-array).

[0042] Figure 6 In Example 3 of this invention, Balb / C nude mice were inoculated with small cell lung cancer cells (NCI-H446) for 14 days, followed by treatment with pheromone for 3 weeks. The changes in tumor weight in the mice were then analyzed. The blue triangles represent the blank control group (CK), and the red squares represent the pheromone array treatment group (PMC-array).

[0043] Figure 7 In Example 3 of this invention, Balb / C nude mice were inoculated with small cell lung cancer cells (DMS-153) for 10 days, treated with pheromone for 3 weeks, and then the treatment was stopped for 3 weeks. The changes in tumor weight in the mice were recorded. The blue triangle represents the blank control group (CK), and the red square represents the pheromone array treatment group (PMC-array). The solid red line indicates treatment with the pheromone array (administration) for 3 weeks, and the blank red line indicates that the treatment was stopped for 3 weeks.

[0044] Figure 8 These are tumor anatomical specimens from the second experimental group in Example 3 of this invention. Top row: Tumors from three blank control groups.

[0045] (NCI-H446), with weights of 1683 mg, 301 mg, and 263 mg, respectively. Bottom row: Tumors (NCI-H446) from three cases in the pheromone array treatment group, with weights of 27 mg, 4 mg, and 4 mg, respectively.

[0046] Figure 9 The image shows a pathological section of a tumor anatomical specimen from the second experimental group in Example 3 of this invention. Figure 9 a: Pathological sections of tumors from the blank control group (a mouse tumor-bearing model of small cell lung cancer).Figure 9 b: Pathological section of tumor after 6 days treatment with information bacterioin array. A large number of tumor cells appeared coagulative necrosis (this is the typical case change of information bacterioin killing tumor cells, Nat Biotech 2007). Figure 9 c: Figure 9 b: Local magnification of b. Figure 9 d: After 3 weeks treatment with information bacterioin array, some tumor-bearing mice gradually disappeared. The scale length is 100 μm.

[0047] Figure 10 Toxicity test results of information bacterioin array on normal guinea pigs in Example 4 of the present application. There was no significant difference in blood biochemical indicators between the blank control group (C, n = 4) and the information bacterioin array treatment group (T, n = 6). 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 (alanine transaminase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), LDH (lactate dehydrogenase), GGT (gamma glutamyl transpeptidase) and CK (creatine kinase) are Unit / L.

[0048] Sequence Description

[0049] The amino acid sequences set forth 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 and proceeding toward the carboxy terminus of the sequence.

[0050] SEQ ID NO: 1-30 are the amino acid sequences of thirty antibody mimics that recognize relevant tumor antigens;

[0051] SEQ ID NO: 31 is the amino acid sequence of the channel domain of colicin Ia;

[0052] SEQ ID NO: 32-61 are the amino acid sequences of thirty information bacterioins against small cell lung cancer. DETAILED DESCRIPTION

[0053] The following examples are provided:

[0054] 1. A polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1-30.

[0055] 2. Use of a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1-30 in the preparation of a medicament for treating small cell lung cancer.

[0056] 3. The use according to embodiment 2, wherein the medicament is a preparation for treating small cell lung cancer.

[0057] 4. A medicament for resisting small cell lung cancer, 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.

[0058] 5. The medicament according to embodiment 4, wherein the colicin comprises colicin El, la, lb, A, B and N.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 10. A method for preparing a medicament for resisting small cell lung cancer, 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.

[0064] 11. The method according to embodiment 10, wherein the colicin comprises colicin El, la, lb, A, B and N.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 15. The method of embodiment 14, wherein the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 32-61.

[0069] 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.

[0070] Unless otherwise specified, the reagents used in the following examples are those conventionally used in the art, and are commercially available or prepared according to conventional methods in the art, at laboratory scale. Unless otherwise specified, the experimental methods and conditions used in the following examples are those conventionally used in the art, and can be found in relevant laboratory manuals, known literature 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.

[0071] Example 1

[0072] Preparation of bacteriocin array

[0073] Thirty bacteriocins with amino acid sequences as set forth in SEQ ID NO: 32-61 were prepared. The bacteriocins 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. Table 1 lists the primary structures of the thirty antibody mimics (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.

[0074] Table 1: Thirty antibody mimics recognizing relevant tumor antigens

[0075]

[0076]

[0077] 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. Table 1 lists the primary structures of the thirty antibody mimics (V

[0078] 346 nilnd rnpvvtedve

[0079] 361 gdkkiynae v aewdklrqrl ldarnkitsa esavnsarnn lsartneqkh andalnallk

[0080] 421 ekenirnqls ginqkiaeekr kqdelkatk dainfttefl ksvsekygak aeqlaremag

[0081] 481 qakgkkirnv eealktyeky radinkkinak draaiaaal esvklsdiss nlnrfsrglg

[0082] 541 yagkftslad witefgkavr tenwrplfvk tetiiagnaa talvalvfsi ltgsalgiig

[0083] 601 ygllmavtga lideslvekankfwgi (SEQ ID NO: 31)

[0084] Escherichia coli synthesizes two proteins, one molecule of colicin and one molecule of corresponding immune protein, when it synthesizes colicin in a physiological state. 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 Nuclease Gene 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. Figure 2 Thirty kinds of recombinant plasmids were obtained, and each of the recombinant plasmids was used to express one kind of colicin. DNA sequencing was performed on the thirty kinds of constructed recombinant plasmids.

[0085] The thirty kinds of 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 the bacterial bodies were collected by centrifugation; the bacterial bodies were broken and resuspended in 50 mM boric acid buffer (pH 9), the supernatant was extracted by centrifugation, and streptomycin sulfate was added to the supernatant to precipitate the DNA; the supernatant was extracted by centrifugation again, the supernatant was 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 kinds of information bacteriocins, wherein the SDS-PAGE electrophoresis results of three kinds of information bacteriocins are shown in Figure 3 The yield of information bacteriocins can reach 5-12 mg / ml.

[0086] The thirty kinds of information bacteriocins 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 bacteriocin. The thirty kinds of information bacteriocins tested by LC-MS were mixed in equal amounts to obtain the information bacteriocin array of the present application.

[0087] Example 2

[0088] In vitro inhibition and killing experiment of information bacteriocin array on small cell lung cancer cells

[0089] 1. Purpose of the experiment

[0090] The in vitro inhibition and killing effect of the information bacteriocin array of the present application on small cell lung cancer cells was investigated.

[0091] 2. Experimental materials

[0092] Test drugs: (1) Information bacteriocin array of the present application prepared in Example 1, which is a mixture of thirty kinds of information bacteriocins with equal mass, and the amino acid sequences of which are shown in SEQ ID NO: 32-61, provided by Chengdu Finolink New Biotechnology Co., Ltd. (2) HB8627-information bacteriocin, provided by Chengdu Finolink New Biotechnology Co., Ltd. The HB8627-information bacteriocin 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.

[0093] The small cell lung cancer cell line NCI-H446, cell culture medium RMPI 1640, and microscope Leica DMi8 were provided by the Institute of Basic Medical Sciences, Peking Union Medical College.

[0094] 3. Experimental Location

[0095] Institute of Basic Medical Sciences, Peking Union Medical College.

[0096] 4. Experimental Methods

[0097] After incubating the pheromone array of the present invention with small cell lung cancer cells NCI-H446 for 48-72 hours, the survival status of the small cell lung cancer cells was detected, thereby verifying the inhibitory effect of the pheromone array of the present invention on small cell lung cancer cells.

[0098] The experiment included a blank control group, a positive control group, and a pheromone array treatment group, as detailed below:

[0099] (1) Blank control group: Normal cultured small cell lung cancer cells NCI-H446, without any treatment.

[0100] (2) Positive control group: HB8627-information (50 μg / mL culture medium) was incubated with small cell lung cancer cells NCI-H446 for 48-72 hours.

[0101] (3) Information pheromone array treatment group: The information pheromone array of the present invention (the total amount of thirty information pheromones is 50 micrograms / mL of culture medium, wherein the content of each information pheromone is the same, about 1.67 micrograms / mL of culture medium) is co-incubated with small cell lung cancer cells NCI-H446 for 48-72 hours.

[0102] The morphological changes of cells in each group were observed under a microscope, and the cell count was calculated. The efficiency of the pheromone array in inhibiting and killing small cell lung cancer cells in vitro was determined based on the cell survival and morphological differences between the blank control group and the pheromone array treatment group.

[0103] 5. Experimental Results

[0104] After small cell lung cancer cells were co-incubated with the pheromone array of this invention for 48-72 hours, significant changes in cell morphology were observed. Morphological and fluorescent staining results confirmed that all tumor cells had died. Figure 4 The small cell lung cancer cells in the blank control group had a polygonal morphology. Figure 4a) After co-incubation with the pheromone array for 48-72 hours, the small cell lung cancer cells have round, oval or long strip shape, most of which have lost the ability to adhere to the wall, and the subcellular structure has been completely changed; the results of propidium iodide staining suggest that the cell membrane of dead cells is destroyed, and propidium iodide enters the cell to dye it red; under the continuous attack of the pheromone array, the small cell lung cancer cells have completely lost their life activity under the microscope Figure 4 b).

[0105] 6、Experimental conclusion

[0106] The pheromone array of the present application has a strong effect of inhibiting and killing small cell lung cancer cells.

[0107] Example 3

[0108] Pharmacodynamic experiment of the pheromone array on a mouse tumor-bearing model

[0109] 1、Experimental purpose

[0110] To verify the killing effect of the pheromone array of the present application on a mouse tumor-bearing model.

[0111] 2、Experimental materials

[0112] 60 Balb / C immunodeficient nude mice (half male and half female) were obtained from Beijing Vantoll Life Experimental Animal Technology Co., Ltd.

[0113] 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.

[0114] Test drugs: (1) The pheromone array of the present application prepared in Example 1, which is a mixture of equal amounts of thirty kinds of pheromones with amino acid sequences as shown in SEQ ID NO: 32-61, was provided by Chengdu Finolink New Biotechnology Co., Ltd. (2) HB8627-pheomone, provided by Chengdu Finolink New Biotechnology Co., Ltd. The HB8627-pheomone 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.

[0115] 3、Experimental method

[0116] (1) Establishment of a mouse tumor-bearing model

[0117] The Balb / C nude mice were injected with the cultured small cell lung cancer cells in the armpit, with a dose of 0.1 ml 5×10 7 After 3-14 days of inoculation, the tumor-bearing model animals were randomly grouped, and the drug treatment was started.

[0118] (2) Drug administration method and dose

[0119] The Balb / C nude mice in the first experimental group were inoculated with the small cell lung cancer cell strain NCI-H446, and the intraperitoneal administration was started after 3 days of inoculation. The blank control group (n=6), the positive control group (n=4), and the information bacterin array treatment group (n=6) were set, wherein 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 bacterin, 1 mg per day, twice a day, 0.5 mg / time. The information bacterin array treatment group: intraperitoneal injection of the information bacterin array of the application, 1 mg per day, twice a day, 0.5 mg / time. After 28 days of continuous administration, dissection was performed.

[0120] The Balb / C nude mice in the second experimental group were inoculated with the small cell lung cancer cell strain NCI-H446, and the intraperitoneal administration was started after 14 days of inoculation. The blank control group (n=10) and the information bacterin array treatment group (n=10) were set, 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 bacterin array treatment group: intraperitoneal injection of the information bacterin array of the application, 2 mg per day, three times a day, about 0.7 mg / time. After 21 days of continuous administration, dissection was performed.

[0121] The Balb / C nude mice in the third experimental group were inoculated with the small cell lung cancer cell strain DMS-153, and the intraperitoneal administration was started after 10 days of inoculation. The blank control group (n=10) and the information bacterin array treatment group (n=10) were set, 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 bacterin array treatment group: intraperitoneal injection of the information bacterin array of the application, 2 mg per day, three times a day, about 0.7 mg / 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.

[0122] (3) Body weight detection

[0123] The electronic digital display scale was used for body weight detection 3 days before inoculation of the animals and every day after administration.

[0124] (4) Observation of tumor-bearing lesion

[0125] After dissection, the tumor-bearing pathological conditions of the mice were observed. The tumor-bearing, weighing, tumor-fixed pathological sections were observed. After observing the pathological sections, the pathological changes of each group of tumor-bearing were scored.

[0126] (5) Result statistical method

[0127] The paired t test statistical analysis method was used to evaluate the changes of the tumor size and weight of each group.

[0128] 4、Experimental results

[0129] The experimental results of the three experimental groups are shown in Table 2.

[0130] The experimental results of the first experimental group show that after 28 days of continuous administration, the average tumor weight of the blank control group (CK) is 129.33 mg, the average tumor weight of the positive control group (PMC-8627) is 89.25 mg, and the average tumor weight of the information bacterioin array treatment group (PMC-array) is 3.33 mg( Figure 5 )。

[0131] 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 information bacterioin array treatment group (PMC-array) is 16 mg( Figure 6 )。

[0132] 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 information bacterioin 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 information bacterioin array treatment group (PMC-array) is 7.25 mg (n=4)( Figure 7 )。

[0133] Table 2

[0134]

[0135]

[0136] The tumor weight is 0, indicating that no tumor was found after dissection of the mouse.

[0137] 5、Experimental conclusion

[0138] The information bacterioin array of the present application shows a strong killing effect on the nude mouse tumor-bearing model (human small cell lung cancer). After three weeks of drug withdrawal, no tumor growth was observed (the tumor of the blank control group in the same period was continuously growing) Figure 7). Tumor anatomy results Figure 8 ) and pathological section observation results Figure 9 ) also confirmed that the pheromone array effectively eliminated the inoculated small cell lung cancer. The experimental mice gained weight, confirming that the pheromone array did not cause toxic damage to the test animals.

[0139] Example 4

[0140] Toxicity experiment of pheromone array on normal guinea pigs

[0141] 1. Experimental materials

[0142] Experimental animals: 10 guinea pigs (half male and half female) were from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.

[0143] Test drug: the pheromone array of the application prepared in Example 1, which was a mass mixture of thirty kinds of pheromones with amino acid sequences as shown in SEQ ID NO: 32-61, was provided by Chengdu Finolife New Biotechnology Co., Ltd.

[0144] 2. Experimental grouping

[0145] Guinea pig grouping: blank control group (n=4), pheromone array treatment group (n=6), a total of 10.

[0146] 3. Experimental method

[0147] (1) Dosing method and dose

[0148] Blank control group: intraperitoneal injection of normal saline, 2 ml per mouse per day.

[0149] Pheromone array treatment group: intraperitoneal injection of the pheromone array of the application, 3 mg per mouse per day.

[0150] Each group was dosed once a day, and after 30 days of continuous dosing, blood was collected for testing and the animals were sacrificed.

[0151] (2) Body weight detection

[0152] The body weight of the animals was detected by electronic digital display weighing before inoculation 3 days and every 7 days after dosing.

[0153] (3) Blood biochemical detection

[0154] The blood samples were sent to the Animal Hospital of China Agricultural University for testing, and the changes in blood biochemical indicators were compared.

[0155] 4. Experimental results

[0156] Compared with the control group, the animals injected with the pheromone array had no changes in appetite and behavior, and the detection results of blood biochemical indicators Figure 10) no difference (no damage to liver, kidney, immune function, etc.).

[0157] 5. Experimental Conclusion

[0158] 30-day information phage array treatment (intraperitoneal injection) did not produce toxic side effects in experimental animals.

Claims

1. A polypeptide composition consisting of thirty polypeptides, the amino acid sequences of which are shown in SEQ ID NO: 1-30.

2. Use of the polypeptide composition of claim 1 in the preparation of a medicament for combating 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 combating small cell lung cancer, comprising thirty fusion proteins obtained by linking the channel domain of colicin Ia with thirty polypeptides having amino acid sequences shown in SEQ ID NO: 1-30, respectively, wherein the thirty polypeptides are linked to the carboxyl terminus of the channel domain of colicin Ia, respectively.

5. The medicament of claim 4, wherein the amino acid sequence of the channel domain of colicin Ia is shown in SEQ ID NO:

31.

6. The medicament of claim 5, wherein the amino acid sequences of the thirty fusion proteins are shown in SEQ ID NO: 32-61.

7. A method for preparing the medicament for combating small cell lung cancer of any one of claims 4-6, comprising linking the carboxyl terminus of the channel domain of colicin Ia with thirty polypeptides having amino acid sequences shown in SEQ ID NO: 1-30, respectively, to obtain thirty fusion proteins.

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