Information rhzomorph array for resisting small cell lung cancer and application of information rhzomorph array

Abstract

The invention belongs to the field of biological medicine, and particularly relates to an information rhzomorph array for resisting small cell lung cancer and application of the information rhzomorph array. The invention provides polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30. The invention also provides a medicine for resisting small cell lung cancer. The medicine comprises fusion protein obtained by respectively connecting the channel structural domain of the coliform and the polypeptide with the amino acid sequence shown as SEQ ID NO: 1-30. Pharmacodynamic experiments prove that in each growth cycle, a mouse tumor-bearing model always cannot get rid of the recognition and killing of an information rhzomorph array composed of thirty fusion proteins on tumors. A guinea pig model experiment proves that no toxic or side effect is generated in an animal model when the information rhzomorph array is continuously used for 30 days. The information rhzomorph array can be used as an effective small cell lung cancer treatment medicine.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an anti-small cell lung cancer pheromone array and an application thereof. Background Art

[0002] There is still a lack of effective intervention methods to address the threat posed by the harmfulness and high incidence of small cell lung cancer, and new drugs are urgently needed.

[0003] Whether it is chemotherapy, radiotherapy, or biological therapy, many modern tumor treatment methods face two major defects that are difficult to overcome: (1) The course of tumors that have been alleviated after treatment is very likely to recur and metastasize, eventually leading to treatment failure; (2) Current anti-tumor treatment methods and drugs are highly toxic, and their toxic side effects often cause complications and even the death of patients. Even the currently highly promising tumor immunotherapy faces a similar dilemma: due to the inherent immunotoxicity of monoclonal antibodies, it is extremely difficult to use more than two monoclonal antibodies targeting different targets on the same patient at the same time. Even the latest antibody-drug conjugate (ADC) is difficult to use more than two ADCs on the same patient at the same time due to immune-related adverse events (irAE). Due to the defects of the above-mentioned drugs themselves, modern treatment methods are difficult to intervene simultaneously on 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 metabolism mechanism, or a certain signal transduction pathway. It is almost impossible to perform multi-target and multi-pathway intervention treatments on the same patient at the same time. This has caused limitations in treatment: once the tumor becomes resistant to or escapes from such single-pathway intervention, such single-pathway intervention treatments are blocked. Therefore, humans urgently need to develop a tumor intervention treatment method that can simultaneously perform multi-target and multi-pathway interventions to more effectively treat tumors.

[0005] Colicin is a classic example of bacteriocin. There are more than 20 types of colicins, which attack the genes, protein synthesis system, or cell membrane of other strains of E. coli. The channel-forming E1 family colicins that can form ion channels (forming ion channels on the cell membrane to kill E. coli) are composed of colicin E1, colicin Ia, colicin Ib, colicin A, colicin B, colicin N, etc.

[0006] Colicin E1, Ia, Ib, A, B, N, etc. are one of the regulatory forces that maintain the diversity and evolution of intestinal flora. Its bactericidal principle is that, taking colicin Ia as an example, it usually has three domains: a displacement domain, a receptor domain, and a channel-forming domain. The channel domain can form a voltage-activated ion channel on the bacterial cell membrane (lipid bilayer membrane). The channel domain of colicin Ia located at the carboxyl end is composed of 175 amino acids and 10 α-helices. Driven by hydrophilic and hydrophobic forces, it will be inserted into the inner membrane (cell membrane) of Escherichia coli without consuming energy to form an ion channel. The channel will open as soon as it senses a transmembrane potential of -50mv. Since the pore size of the channel is very large, about Almost all kinds of ions can leak out from this huge aqueous channel, causing the energy and ion reserves of bacteria to be exhausted, the cell membrane to rupture, the cell contents to leak, and the E. coli to die. This sterilization process is a physical process that can achieve the purpose of sterilization without changing or affecting the enzymes or metabolism required for bacterial growth, metabolism, and reproduction. Therefore, it has been effectively killing bacteria of the same species and strains for hundreds of millions of years until now.

[0007] Colicin Ia is the type specimen of the E1 colicins, and its gene, protein structure and working mechanism are the most complete and detailed among the E1 colicins. Summary of the invention

[0008] The purpose of the present invention is to provide a class of drugs against small cell lung cancer, which can specifically recognize typical surface antigens (proteins and / or hydrocarbons) of lung cancer cells and can effectively kill lung cancer cells.

[0009] To achieve the above-mentioned purpose, the present invention has innovatively designed an antibody mimetic (Ab Mimetic) specifically targeting lung cancer cells. The antibody mimetic is selected from thirty 28-peptides, and its amino acid sequence is shown in SEQ ID NO: 1-30. The antibody mimetic is constructed based on the publicly available anti-lung cancer cell antibody sequences and can recognize the corresponding cancer cell antigens. The channel domain of the E1 family colicin that can form an ion channel is connected to the thirty antibody mimics respectively, and the various fusion proteins obtained are the pharmacological ingredients of the anti-small cell lung cancer drug. In this article, the connector (fusion protein) of the antibody mimetic and the channel domain of colicin is also called "infocin"; the combination of various infocins is called infocin array.

[0010] The present invention provides polypeptides having amino acid sequences as shown in SEQ ID NOs: 1-30.

[0011] The present invention also provides use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 in preparing a drug against small cell lung cancer.

[0012] In the above use, the drug may be a preparation for treating small cell lung cancer.

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

[0014] In the above-mentioned drugs, the colicin includes colicin E1, Ia, Ib, A, B and N.

[0015] In the above-mentioned drug, the colicin is preferably colicin Ia, and the amino acid sequence of its channel domain (amino acids 346-626 of colicin Ia) is shown in SEQ ID NO:31.

[0016] In the above-mentioned drugs, the polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30 are respectively connected to the carboxyl terminus (C-terminus) and / or amino terminus (N-terminus) of the channel domain of colicin, preferably connected to the carboxyl terminus of the channel domain of colicin.

[0017] In the above-mentioned drug, the polypeptide is connected to the channel domain of colicin via a covalent bond.

[0018] In the above-mentioned drug, the arrangement of the polypeptide in the fusion protein can be N-terminus-SEQ ID NO:31-antibody mimetic-C-terminus, 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:10 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-mentioned drug, the amino acid sequence of the fusion protein may be the sequence shown in SEQ ID NO: 32-61.

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

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

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

[0023] In the above method, the polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30 are respectively connected to the carboxyl terminus (C-terminus) and / or amino terminus (N-terminus) of the channel domain of colicin, preferably to the carboxyl terminus of the channel domain of colicin.

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

[0025] The fusion protein of the present invention is an effective ingredient for small cell lung cancer and can be directly used as a drug for small cell lung cancer. The fusion protein can also be prepared into different dosage forms for small cell lung cancer by adding pharmaceutically acceptable excipients according to the needs of clinical use.

[0026] The pheromone of the present invention has a unique tumor-killing mechanism that destroys the integrity of the lipid bilayer membrane. H CDR1-V H FR2-V L The 28-peptide antibody mimetic structure constructed by the CDR3 primary structure sequence was based on the public antibody sequence against lung cancer cell antigens, and thirty antibody mimics that can recognize the corresponding antigens were constructed. The antibody mimics are selected from the Fab segments of antibodies that recognize lung cancer cell antigens. 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 is formed by linear connection of the primary structure of CDR3, and is composed of 28 amino acids. Antibody mimics were connected to the carboxyl or amino terminus of the channel domain of colicin Ia (amino acids 346-626 of colicin Ia), respectively, to construct a variety of pheromone, which are pheromone-1 (Pheromonicin-1, PMC-1) to pheromone-30 (Pheromonicin-30, PMC-30) connected by antibody mimics at the carboxyl terminus of the channel domain of colicin Ia, and antibody mimics connected to the amino terminus of PMC-1 to the amino terminus of PMC-30 at the amino terminus of the channel domain of colicin Ia.

[0027] Beneficial effects and innovative features of the present invention:

[0028] 1. Select the channel domain of colicin that can form an ion channel

[0029] The inventors have found through extensive research that colicin can form ion channels on a variety of lipid bilayers of different compositions and thicknesses. This suggests that if the inherent targeting of colicin (which can only recognize E. coli of the same species or strain) can be changed, it is possible to recognize other bacteria, fungi, enveloped viruses, and even eukaryotic cells, thereby forming ion channels on the envelope or cell membrane (lipid bilayer) of these organisms to kill them.

[0030] In the information bacteria provided by the present invention, the antibody mimic is responsible for recognizing the target on the surface of the tumor cell membrane, while the channel domain of colicin Ia is responsible for destroying the integrity of the tumor cell membrane. It 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, almost all intracellular ions will leak out rapidly, and the transmembrane ion gradient will quickly become unbalanced, eventually leading to cell exhaustion and rapid death. Such a simple and direct physical killing method (passive leakage caused by ion concentration gradient difference) is currently unstoppable by the biochemical drug resistance mechanism (changing metabolic pathways, synthesizing new structural biological molecules, using energy-consuming pumps to pump drug molecules out of cells, etc.) widely used in tumor cells.

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

[0032] Tumor growth is a biological process coordinated and integrated among multiple genes, multiple proteins, and multiple signal transduction pathways. The rhythm of this process can be reflected by the four-dimensional spatial changes of various tumor cell surface antigens (the three-dimensional distribution of antigens on the cell surface that changes over time). In different growth cycles, various antigens appear alternately on the surface of tumor cells. Tumor growth is a highly ordered complex process. It is impossible to control it by intervening in a certain antigen, a certain signal transduction pathway, or a certain 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 at the same time to effectively intervene in the tumor growth process.

[0033] There are many kinds of antigens on the surface of tumor cells. In each process of tumor growth, these antigens appear alternately on the surface of tumor cells. That is to say, in a certain tumor growth process, antigens A and C may be dominant, but in the next growth process, antigens A and C may no longer be dominant, but antigens B and D may be dominant. In the next growth process, it may not be A and C, nor B and D, but other antigens. It is precisely because of the law that antigens on the surface of tumor cells change alternately with the growth cycle that the long-term use of single-target drugs to fight tumors will inevitably lead to drug resistance and escape, thus leading to treatment failure. With the change of the growth cycle, a single target may gradually weaken or even disappear, so the drug targeting this single target will become ineffective. In order to cope with this change, we need to design such a drug array, which consists of several drugs that can deal with multiple targets. When facing tumor cells with different growth cycles, there is always one or several drugs in the array that can recognize the antigens that are dominant in the corresponding growth cycle, so that the array can always attack the tumor efficiently and without off-target, and minimize the occurrence of drug resistance and escape, thereby effectively treating tumors.

[0034] In the Chinese patent with publication number CN1274829C, antibody mimics and wild-type colicin Ia (626 amino acid residues, 70 kDa) were used to recognize Epstein-Barr virus glycoprotein antigens and effectively kill malignant lymphosarcoma.

[0035] The efficacy of the information fungus array of the present invention was verified using two small cell lung cancer cell lines (NCI-H446 and DMS-153), wherein the information fungus array used was composed of thirty information fungi with amino acid sequences as shown in SEQ ID NO: 32-61. In vitro inhibition and killing experiments on small cell lung cancer cells showed that the information fungus array can effectively kill small cell lung cancer cells (Example 2). Pharmacodynamic experiments using small cell lung cancer models confirmed that in each growth cycle, the mouse tumor-bearing model could not get rid of the information fungus array's recognition and killing of the tumor, resulting in complete killing of cancer cells and the inability of the tumor model to grow. The information fungus array of the present invention showed extremely significant growth inhibition and killing effects on tumor models derived from the above two small cell lung cancer cell lines (Example 3).

[0036] The above experiments confirmed that the pheromone array of the present invention showed an anti-tumor effect far superior to that of the single pheromone in the above patent (CN1274829C). In addition, the guinea pig model experiment confirmed that the continuous use of the pheromone array for 30 days did not produce any toxic side effects in the animal model (Example 4). Therefore, the pheromone array of the present invention can be used as an effective drug for the treatment of small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0038] Figure 2 This is a schematic diagram of the recombinant plasmid for preparing pheromone constructed in Example 1 of the present invention.

[0039] Figure 3 The SDS-PAGE electrophoresis results of three pheromones among the thirty pheromones prepared in Example 1 of the present invention. Lane 1: protein marker; Lanes 2-4: three pheromones in the pheromones array prepared in Example 1, all of which have a molecular weight of about 30 kDa; Lane 5: pheromones used as controls (containing full-length colicin Ia, with a molecular weight of 70 kDa); Lane 6: bovine serum albumin used as a standard control, with a molecular weight of 70 kDa.

[0040] Figure 4 The experimental results of Example 2 of the present invention using the pheromone array to attack small cell lung cancer cells (NCI-H446). Figure 4 a: Small cell lung cancer cells (NCI-H446) in the blank control group have a polygonal morphology. Figure 4b: After incubation with the infobacterium array (50 μg / ml) for 48-72 hours, the small cell lung cancer cells (NCI-H446) changed from the polygonal morphology of normal lung cancer cells to round, oval or long strips. Most of them could no longer adhere to the wall and their subcellular structures were completely changed. Propidium iodide staining (floating red spheres) indicated that the cell membrane of the dead cells was destroyed and propidium iodide entered the cells, staining them red. Under the continuous attack of the infobacterium array, the small cell lung cancer cells seen under the microscope had all lost their vitality.

[0041] Figure 5 The weight change of the tumor in Balb / C nude mice in Example 3 of the present invention was 3 days after inoculation of small cell lung cancer cells (NCI-H446) and then treated with pheromone array for 4 weeks. The blue triangle symbol represents the blank control group (CK); the purple 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 The weight change of the tumor in Balb / C nude mice in Example 3 of the present invention was 14 days after inoculation of small cell lung cancer cells (NCI-H446) and then treated with pheromone for 3 weeks. The blue triangle symbol represents the blank control group (CK), and the red square symbol represents the pheromone array treatment group (PMC-array).

[0043] Figure 7 The weight change of the tumor in the Balb / C nude mice in Example 3 of the present invention was 10 days after the inoculation of small cell lung cancer cells (DMS-153), and then treated with pheromone for 3 weeks and then stopped for 3 weeks. The blue triangle is the blank control group (CK), and the red square is the pheromone array treatment group (PMC-array). The red solid line indicates that the pheromone array was treated (administered) for 3 weeks, and the red blank line indicates that no treatment (drug withdrawal) was performed for 3 weeks.

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

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

[0046] Fig. 9 Pathological sections of the tumor anatomical specimens of the second experimental group in Example 3 of the present invention. Fig. 9 a: Pathological sections of tumors in the blank control group (small cell lung cancer mouse tumor model). Fig. 9 b: Pathological section of the tumor 6 days after treatment with the infobacterium array, showing coagulative necrosis of a large number of tumor cells (this is a typical case change of infobacterium killing tumor cells, Nat Biotech 2007). Fig. 9 c: Fig. 9 Partial enlargement of b. Fig. 9 d: After 3 weeks of treatment with pheromone array, the tumors in some mice gradually disappeared. The scale bar is 100 μm.

[0047] Fig.10 The results of the toxicity test of the pheromone array on normal guinea pigs in Example 4 of the present invention are shown in Table 1. There was no significant difference in the blood biochemical indexes between the blank control group (C, n=4) and the pheromone array treatment group (T, n=6). Fig.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 (serum total protein), ALB (albumin) and GLB (globulin) are g / dl; A / G represents the ratio of albumin to globulin; the concentration units of ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (alkaline phosphatase), LDH (lactate dehydrogenase), GGT (γ-glutamyl transpeptidase) and CK (creatine kinase) are Unit / L.

[0048] Sequence Description

[0049] The amino acid sequences listed in the accompanying sequence listing are shown using the single-letter code for the amino acids, following the standard convention of starting with the amino terminus of the sequence and proceeding towards the carboxyl terminus.

[0050] SEQ ID NOs: 1-30 are amino acid sequences of thirty antibody mimetics 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 pheromones that are effective against small cell lung cancer. DETAILED DESCRIPTION

[0053] The following embodiments are provided:

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

[0055] 2. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 in the preparation of a drug against small cell lung cancer.

[0056] 3. The use described in Example 2, wherein the drug is a preparation for treating small cell lung cancer.

[0057] 4. A drug for treating small cell lung cancer, comprising a fusion protein obtained by connecting the channel domain of colicin and a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30.

[0058] 5. The drug of Example 4, wherein the colicin comprises colicin E1, Ia, Ib, A, B and N.

[0059] 6. The drug of Example 5, wherein the colicin is colicin Ia, and the amino acid sequence of its channel domain is shown in SEQ ID NO:31.

[0060] 7. The drug according to any one of Examples 4-6, wherein a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 is respectively connected to the carboxyl terminus and / or the amino terminus of the channel domain of colicin.

[0061] 8. The drug of Example 7, wherein the polypeptides having amino acid sequences as shown in SEQ ID NOs: 1-30 are respectively linked to the carboxyl termini of the channel domain of colicin.

[0062] 9. The drug of Example 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 drug against small cell lung cancer, comprising connecting polypeptides having amino acid sequences as shown in SEQ ID NOs: 1-30 to the channel domain of colicin to obtain a fusion protein.

[0064] 11. The method of Example 10, wherein the colicin comprises colicin E1, Ia, Ib, A, B and N.

[0065] 12. The method described in Example 11, wherein the colicin is colicin Ia, and the amino acid sequence of its channel domain is shown in SEQ ID NO:31.

[0066] 13. The method described in any one of Examples 10-12, wherein the polypeptides with amino acid sequences as shown in SEQ ID NOs: 1-30 are respectively linked to the carboxyl terminus and / or amino terminus of the channel domain of colicin.

[0067] 14. The method described in Example 13, wherein the polypeptides with amino acid sequences as shown in SEQ ID NO: 1-30 are respectively connected to the carboxyl terminus of the channel domain of colicin.

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

[0069] The present invention is further illustrated below in conjunction with examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0070] If not otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure. If not otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, and reference can be made to relevant experimental manuals, known documents or manufacturer specifications. Unless otherwise defined, the meanings of all technical and scientific terms used herein are the same as those generally understood by ordinary technicians in the field to which the present invention belongs.

[0071] Example 1

[0072] Preparation of pheromone array

[0073] Thirty pheromones with amino acid sequences as shown in SEQ ID NO: 32-61 were prepared. The pheromones are fusion proteins obtained by connecting an antibody mimetic to the carboxyl end of the channel domain of colicin Ia ( Figure 1 The amino acid sequence of the channel domain of colicin Ia is shown in SEQ ID NO:31. The amino acid sequences of thirty antibody mimetics that recognize relevant tumor antigens are shown in SEQ ID NO:1-30. Table 1 lists the primary structures of the thirty antibody mimetics (V H CDR1-V H FR2-V L CDR3), where the amino acids are represented by standard single letters and are arranged in order from the amino terminus to the carboxyl terminus.

[0074] Table 1: Thirty antibody mimetics that recognize relevant tumor antigens

[0075]

[0076]

[0077] The amino acid sequence of the channel domain of colicin Ia is as follows, wherein the amino acids are represented by standard single letters and are arranged in order from the amino terminus to the carboxyl 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.

[0078] 346nilnd rnpvvtedve

[0079] 361gdkkiynaev aewdklrqrl ldarnkitsa esavnsarnn lsartneqkh andalnallk

[0080] 421ekenirnqls ginqkiaeekrkqdelkatk dainfttefl ksvsekygak aeqlaremag

[0081] 481qakgkkirnv eealktyeky radinkkinakdraaiaaal esvklsdiss nlnrfsrglg

[0082] 541yagkftslad witefgkavr tenwrplfvk tetiiagnaa talvalvfsi ltgsalgiig

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

[0084] When Escherichia coli synthesizes colicin under physiological conditions, it synthesizes two proteins, one molecule of colicin and one molecule of corresponding immune protein. The function of the immune protein is to prevent the synthesized colicin from forming ion channels on the cell membrane of Escherichia coli itself. After the synthesized colicin leaves the Escherichia coli (secreted outside the cell), the colicin and the immune protein will separate. Therefore, when using engineered bacteria to synthesize information bacteria, it is necessary to synthesize the corresponding immune protein at the same time. The coding genes of the structural protein and immune protein of colicin Ia are registered in the GenBank accession number of the National Center for Biotechnology Information (NCBI) of the United States as M13819. Thirty information bacteria with amino acid sequences shown in SEQ ID NO:32-61 were prepared using pET11a plasmid and Escherichia coli B834 (DE3). Nosai Gene Company was commissioned to synthesize the pET11a recombinant plasmid for expressing information bacteria, which contains genes encoding the channel domain of colicin Ia, genes encoding antibody mimics, and genes encoding immune proteins ( Figure 2 ). A total of 30 recombinant plasmids were obtained, each of which was used to express a pheromone. DNA sequencing was performed on the 30 recombinant plasmids constructed.

[0085] Thirty recombinant plasmids identified by sequencing were transfected into competent Escherichia coli B834 (DE3) cells respectively; the B834 engineered bacteria containing the recombinant plasmids were placed in LB liquid medium (containing 100 μg / ml ampicillin) for proliferation, and then the bacteria were collected by centrifugation; the bacteria 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 DNA; the supernatant was extracted by centrifugation again, and the supernatant was dialyzed in 50 mM boric acid buffer (pH 9), then passed through an agarose (CM-Sepharose) gel column, and eluted with a 0.3 M NaCl solution to obtain thirty pheromones, of which the SDS-PAGE electrophoresis results of three pheromones are shown as follows: Figure 3 As shown in the figure, the yield of pheromone can reach 5-12 mg / ml.

[0086] The prepared thirty pheromones were tested using liquid chromatography-mass spectrometry (LC-MS), confirming that the amino acid residue of the antibody mimetic was located at the carboxyl end of the pheromones. The thirty pheromones tested by LC-MS were mixed in equal mass to obtain the pheromones array of the present invention.

[0087] Example 2

[0088] In vitro inhibition and killing experiment of small cell lung cancer cells by pheromone array

[0089] 1. Experimental purpose

[0090] The in vitro inhibitory and killing effect of the pheromone array of the present invention on small cell lung cancer cells was investigated.

[0091] 2. Experimental Materials

[0092] Test drugs: (1) The pheromone array of the present invention prepared in Example 1, which is composed of an equal mass mixture of thirty pheromone species with amino acid sequences as shown in SEQ ID NO: 32-61, provided by Chengdu Fino Lixin Biotechnology Co., Ltd. (2) HB8627-pheromone, provided by Chengdu Fino Lixin Biotechnology Co., Ltd. The HB8627-pheromone 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] 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 the pheromone array of the present invention was co-incubated 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 and killing effect of the pheromone array of the present invention on small cell lung cancer cells.

[0098] The blank control group, positive control group, and pheromone array treatment group were set up in the experiment, as follows:

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

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

[0101] (3) Pheromone array treatment group: The pheromone array of the present invention (the total amount of thirty pheromone species is 50 μg / ml of culture medium, wherein the content of each pheromone is the same, about 1.67 μg / ml of culture medium) was 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 number of cells was calculated. The efficiency of the information bacteria array of the present invention 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 information bacteria array treatment group.

[0103] 5. Experimental results

[0104] After incubating small cell lung cancer cells with the information bacteria array of the present invention for 48-72 hours, the cell morphology showed significant changes, and the morphological and fluorescent staining results confirmed that all tumor cells were dead ( Figure 4 The small cell lung cancer cells in the blank control group had a polygonal morphology ( Figure 4a). After incubation with the infobacterium array for 48-72 hours, the small cell lung cancer cells have round, oval or long strip shapes, most of them can no longer adhere to the wall, and the subcellular structure has completely changed; the propidium iodide staining results indicate that the cell membrane of the dead cells is destroyed, and the propidium iodide enters the cells and dyes them red; under the continuous attack of the infobacterium array, the small cell lung cancer cells seen under the microscope have all lost their vitality ( Figure 4 b).

[0105] 6. Experimental conclusion

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

[0107] Example 3

[0108] Pharmacodynamics experiment of pheromone array on mouse tumor-bearing model

[0109] 1. Experimental purpose

[0110] Verify the killing effect of the pheromone array of the present invention on the mouse tumor-bearing model.

[0111] 2. Experimental Materials

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

[0113] 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 invention prepared in Example 1, which is composed of an equal mass mixture of thirty pheromone species with amino acid sequences as shown in SEQ ID NO: 32-61, provided by Chengdu Fino Lixin Biotechnology Co., Ltd. (2) HB8627-pheromone, provided by Chengdu Fino Lixin Biotechnology Co., Ltd. The HB8627-pheromone 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 methods

[0116] (1) Establishment of mouse tumor model

[0117] Balb / C nude mice were inoculated with cultured small cell lung cancer cells by axillary injection at a dose of 0.1 ml and 5×10 7 Cells / mouse. 3-14 days after inoculation, the tumor-bearing mice were randomly divided into groups and drug treatment was started.

[0118] (2) Administration and dosage

[0119] The Balb / C nude mice in the first experimental group were inoculated with the small cell lung cancer cell line NCI-H446, and intraperitoneal administration began 3 days after inoculation. A blank control group (n=6), a positive control group (n=4) and a pheromone array treatment group (n=6) were set up, where n represents the number of nude mice. Blank control group: intraperitoneal injection of normal saline, 1 ml / each mouse / day. Positive control group: intraperitoneal injection of HB8627-pheromone, 1 mg per day, twice a day, 0.5 mg / time. Infobacterium array treatment group: intraperitoneal injection of the pheromone array of the present invention, 1 mg per day, twice a day, 0.5 mg / time. Dissection was performed after 28 consecutive days of administration.

[0120] The Balb / C nude mice in the second experimental group were inoculated with the small cell lung cancer cell line NCI-H446, and intraperitoneal administration began 14 days after inoculation. A blank control group (n=10) and a pheromone array treatment group (n=10) were set up, where n represents the number of nude mice. Blank control group: intraperitoneal injection of normal saline, 1 ml / each mouse / day. Infobacterium array treatment group: intraperitoneal injection of the pheromone array of the present invention, 2 mg per day, three times a day, about 0.7 mg / time. Dissection was performed after 21 days of continuous administration.

[0121] The Balb / C nude mice in the third experimental group were inoculated with the small cell lung cancer cell line DMS-153, and intraperitoneal administration began 10 days after inoculation. A blank control group (n=10) and an infographic array treatment group (n=10) were set up, where n represents the number of nude mice. Blank control group: intraperitoneal injection of normal saline, 1 ml / each mouse / day. Infobacterium array treatment group: intraperitoneal injection of the infographic array of the present invention, 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 21 days after drug withdrawal.

[0122] (3) Weight measurement

[0123] The body weight of the animals was measured using an electronic digital scale 3 days before inoculation and every day after administration.

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

[0125] After dissection, the tumor-bearing lesions of the mice were observed. The tumors were separated, weighed, and fixed for pathological section observation. After pathological observation and reading of the slides, scores were given according to the pathological changes of the tumors in each group.

[0126] (5) Statistical method of results

[0127] Paired t test statistical analysis method was used to evaluate the changes in tumor size and weight in 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 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 pheromone array treatment group (PMC-array) was 3.33 mg ( Figure 5 ).

[0131] The experimental results of the second experimental group showed that the average tumor weight of the blank control group (CK) was 409 mg, and the average tumor weight of the pheromone array treatment group (PMC-array) was 16 mg ( Figure 6 ).

[0132] The experimental results of the third experimental group showed that after 21 days of continuous administration, the average tumor weight of the blank control group (CK) was 123.83 mg (n=6), and the average tumor weight of the information bacteria array treatment group (PMC-array) was 11 mg (n=6). After 21 days of continuous administration and then 21 days of drug withdrawal, the average tumor weight of the blank control group (CK) was 202.75 mg (n=4), and the average tumor weight of the information bacteria array treatment group (PMC-array) was 7.25 mg (n=4) ( Figure 7 ).

[0133] Table 2

[0134]

[0135]

[0136] *Tumor weight is 0, indicating that no tumor was found after the mouse was dissected.

[0137] 5. Experimental conclusion

[0138] The pheromone array of the present invention exhibited a 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 tumor in the blank control group continued to grow during the same period) ( Figure 7). Tumor autopsy results ( Figure 8 ) and pathological section observation results ( Fig. 9 ) also confirmed that the information bacteria array effectively eliminated the small cell lung cancer tumors produced by vaccination. The weight of the experimental mice increased, confirming that the information bacteria array did not cause toxic damage to the tested animals.

[0139] Example 4

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

[0141] 1. Experimental Materials

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

[0143] Test drug: The pheromone array of the present invention prepared in Example 1 is composed of an equal mass mixture of thirty pheromone species with amino acid sequences as shown in SEQ ID NO: 32-61, and is provided by Chengdu Finolyxin Biotechnology Co., Ltd.

[0144] 2. Experimental Grouping

[0145] Guinea pigs were divided into two groups: blank control group (n=4) and pheromone array treatment group (n=6), with a total of 10 guinea pigs.

[0146] 3. Experimental methods

[0147] (1) Administration and dosage

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

[0149] Pheromycin array treatment group: intraperitoneal injection of the pheromone array of the present invention, 3 mg / mouse / day.

[0150] All the above groups were given the drug once a day for 30 consecutive days, and then the animals were killed after blood was collected for examination.

[0151] (2) Weight measurement

[0152] The body weight of the animals was measured using an electronic digital scale 3 days before inoculation and every 7 days after administration.

[0153] (3) Blood biochemical test

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

[0155] 4. Experimental results

[0156] Compared with the control group, the animals injected with pheromone array showed no changes in appetite and behavior, and the results of blood biochemical indexes ( Fig.10) had no difference (no damage to liver, kidney or immune function was observed).

[0157] 5. Experimental conclusion

[0158] 30-day treatment with the pheromone array (intraperitoneal injection) did not produce any toxic side effects in the experimental animals.

Claims

1. A polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30.

2. Use of a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30 in the preparation of a drug against small cell lung cancer.

3. The use according to claim 2, wherein the drug is a preparation for treating small cell lung cancer.

4. A drug for treating small cell lung cancer, comprising a fusion protein obtained by connecting the channel domain of colicin and a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-30.

5. The medicament of claim 4, wherein the colicin comprises colicin E1, Ia, Ib, A, B and N.

6. The drug of claim 5, wherein the colicin is colicin Ia, and the amino acid sequence of its channel domain is shown in SEQ ID NO:

31.

7. The drug according to any one of claims 4 to 6, wherein the polypeptides having amino acid sequences as shown in SEQ ID NOs: 1 to 30 are respectively linked to the carboxyl terminus and / or the amino terminus of the channel domain of colicin.

8. The drug of claim 7, wherein the polypeptides having amino acid sequences as shown in SEQ ID NOs: 1-30 are respectively linked to the carboxyl termini of the channel domain of colicin.

9. The drug of claim 8, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 32-61.

10. A method for preparing the drug against small cell lung cancer according to any one of claims 4 to 9, comprising connecting the polypeptides with amino acid sequences as shown in SEQ ID NOs: 1 to 30 to the channel domain of colicin to obtain fusion proteins.

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