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

A pheromone array for small cell lung cancer and its application

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 2024105757187, filed on May 10, 2024, entitled "An Anti-Small Cell Lung Cancer Infectant Array and Its Application Thereof," and Chinese Patent Application No. 2025100221159, filed on January 7, 2025, entitled "An Anti-Small Cell Lung Cancer Infectant Array and Its Application Thereof," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine, specifically relating to an anti-small cell lung cancer pheromone array and its application. Background Technology

[0004] The threat posed by the harmfulness and high incidence of small cell lung cancer still lacks effective intervention methods, and new drugs are urgently needed.

[0005] Many modern cancer treatments, including chemotherapy, radiotherapy, and biotherapy, face two major drawbacks: (1) cancers that have improved after treatment are prone to relapse and metastasis, ultimately leading to treatment failure; (2) current anti-tumor treatments and drugs are highly toxic, and their side effects often cause complications leading to patient death. Even the currently promising cancer immunotherapy faces similar challenges: due to the inherent immunotoxicity of monoclonal antibodies, it is extremely difficult to use two or more monoclonal antibodies targeting different targets simultaneously in the same patient. Even the latest antibody-drug conjugates (ADCs) are difficult to use simultaneously in the same patient due to immune-related adverse events (irAEs). Due to the inherent defects of these drugs, modern treatments cannot simultaneously target multiple tumor targets or follow multiple cancer treatment pathways.

[0006] Due to the aforementioned limitations, current research and development of monoclonal antibodies and ADCs can only target a single point, a specific gene class, a specific stage of tumor growth and metabolism, or a single signal transduction pathway for intervention. It is virtually impossible to simultaneously perform multi-target, multi-pathway intervention in the same patient. This creates a limitation in treatment: once the tumor develops resistance to such single-pathway interventions or escapes, these single-pathway intervention methods become ineffective. Therefore, there is an urgent need to develop a tumor intervention therapy that can simultaneously target multiple points and utilize multiple pathways, thereby treating tumors more effectively.

[0007] Colicins are the classic examples of bacteriocins. There are more than twenty types of colicins, each attacking the genes and protein synthesis systems of other strains of E. coli, or disrupting the cell membrane of E. coli. The E1 family of channel-forming colicins, which can form ion channels (creating ion channels on the cell membrane, thereby killing E. coli), consists of colicin E1, colicin Ia, colicin Ib, colicin A, colicin B, and colicin N.

[0008] Escherichia coli E1, Ia, Ib, A, B, and N are among the regulatory forces that maintain the diversity and evolution of the gut microbiota. Their bactericidal mechanism lies in their structure. Taking coliform Ia as an example, it typically possesses three domains: a translocation domain, a receptor domain, and a channel-forming domain. The channel-forming domain can form a voltage-gated ion channel on the bacterial cell membrane (lipid bilayer). The channel domain at the carboxyl terminus of coliform Ia consists of 175 amino acids and 10 α-helices. Driven by hydrophilic-hydrophobic interactions, it inserts into the inner membrane (cell membrane) of *E. coli* without consuming energy to form an ion channel. This channel opens upon sensing a transmembrane potential of -50 mV. Due to the large pore size of this channel, it is approximately... Almost all types of ions can leak out from this huge aqueous channel, causing the bacteria to deplete their energy and ion reserves, rupture their cell membranes, and leak their cell contents, leading to the death of E. coli. This sterilization process is a physical process that can achieve sterilization without altering or affecting the enzymes or metabolism required for bacterial growth, metabolism, and reproduction. Therefore, it has been effective in killing bacteria of different species for hundreds of millions of years until now.

[0009] Coliformin Ia is the model organism of E1 coliforms, and its gene, protein structure, and working mechanism are the most well-understood and detailed among E1 coliforms. Summary of the Invention

[0010] The purpose of this invention is to provide a class of drugs for treating small cell lung cancer, which can specifically recognize typical surface antigens (proteins and / or hydrocarbons) of lung cancer cells and can efficiently kill lung cancer cells.

[0011] To achieve the above objectives, this invention innovatively designs antibody mimetic (Ab mimetic) specifically targeting lung cancer cells. The antibody mimetic is selected from thirty 28-peptides, whose amino acid sequences are shown in SEQ ID NO:1-30. The antibody mimetic is constructed based on publicly available anti-lung cancer cell antibody sequences and can recognize corresponding cancer cell antigens. The channel domains of E1 group coliforms, which can form ion channels, are linked to the thirty antibody mimetics respectively, resulting in various fusion proteins that are the active ingredients of anti-small cell lung cancer drugs. In this document, the linkers (fusion proteins) of the antibody mimetic and coliform channel domains are also referred to as "pheromone"; combinations of various pheromone are referred to as pheromone arrays.

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

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

[0014] In the above-mentioned uses, the drug may be a preparation for treating small cell lung cancer.

[0015] The present invention also provides a drug for treating small cell lung cancer, comprising a fusion protein obtained by linking a channel domain of coliform to a polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30.

[0016] In the aforementioned drugs, the coliforms include coliforms E1, Ia, Ib, A, B, and N.

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

[0018] In the above-mentioned drugs, the polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 are respectively linked to the carboxyl terminus (C-terminus) and / or amino terminus (N-terminus) of the channel domain of coliformin, preferably linked to the carboxyl terminus of the channel domain of coliformin.

[0019] In the aforementioned drug, the polypeptide is covalently linked to the channel domain of the coliformin.

[0020] In the aforementioned drug, the polypeptides in the fusion protein can be arranged in an N-terminal-SEQ ID NO:31-antibody mimic-C-terminal configuration, 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. 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 aforementioned drugs, the amino acid sequence of the fusion protein may be the sequence shown in SEQ ID NO:32-61.

[0022] The present invention also provides a method for preparing a drug for small cell lung cancer, which includes linking a polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30 to a channel domain of an escherichia coli to obtain a fusion protein.

[0023] In the above method, the coliforms include coliforms E1, Ia, Ib, A, B and N.

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

[0025] In the above method, the polypeptide with the amino acid sequence shown in SEQ ID NO:1-30 is respectively linked to the carboxyl terminus (C-terminus) and / or amino terminus (N-terminus) of the channel domain of coliformin, preferably linked to the carboxyl terminus of the channel domain of coliformin.

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

[0027] The fusion protein described in this invention is an effective component against small cell lung cancer and can be used directly as a drug against small cell lung cancer. Alternatively, depending on clinical needs, the fusion protein can be formulated into different dosage forms for treating small cell lung cancer by adding pharmaceutically acceptable excipients.

[0028] The pheromone of this invention possesses a unique tumor-killing mechanism that disrupts the integrity of the lipid bilayer. This invention utilizes the V-shaped pheromone designed by the inventors. H CDR1-V H FR2-V L The 28-peptide antibody mimicry structure constructed using the CDR3 primary structural sequence, based on publicly available antibody sequences against lung cancer cell antigens, resulted in the construction of thirty antibody mimicry capable of recognizing the corresponding antigens. These antibody mimicry are selected from the Fab segments of antibodies recognizing lung cancer cell antigens. H CDR1 (heavy chain antigen-binding region 1), V H FR2 (heavy chain skeleton region 2) and V L CDR3 (light chain antigen-binding region 3) and according to 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 involving the coordinated integration of multiple genes, proteins, and signaling 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). During different growth cycles, various antigens appear alternately on the tumor cell surface. Tumor growth is a highly ordered and complex process that cannot be controlled by intervening in a single antigen, signaling pathway, or gene-protein axis. Therefore, current therapies targeting single targets have struggled to control tumor growth. Simultaneous control of multiple factors is necessary to effectively intervene in the tumor growth process.

[0035] Tumor cells have many antigens on their surface. During various stages of tumor growth, these antigens appear alternately on the tumor cell surface. That is, in one stage of tumor growth, antigens A and C may be dominant, while in the next stage, antigens B and D may be dominant. In the next stage after that, neither A nor C, nor B and D, may be dominant, but other antigens may be dominant. It is precisely this alternating pattern of tumor cell surface antigens with the growth cycle that inevitably leads to drug resistance and evasion with long-term use of single-target drugs against tumors, resulting in treatment failure. With the change in the growth cycle, a single target may gradually weaken or even disappear, thus rendering drugs targeting that single target ineffective. To address this change, we need to design a drug array composed of several drugs that can target multiple targets. When facing tumor cells at different growth stages, this array always contains one or more drugs that can recognize the antigen that is dominant in the corresponding growth stage. This allows the array to consistently and efficiently attack the tumor without off-target effects, minimizing the occurrence of drug resistance and evasion, and thus effectively treating the tumor.

[0036] In Chinese patent CN1274829C, an informant that recognizes EB virus glycoprotein antigens using antibody mimicry and wild-type coliform Ia (626 amino acid residues, 70 kDa) effectively kills malignant lymphosarcoma.

[0037] The pharmacodynamics of the pheromone array of the present invention was validated using two small cell lung cancer cell lines (NCI-H446 and DMS-153). The pheromone array consisted of thirty pheromones 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 pheromone array could effectively kill small cell lung cancer cells (Example 2). Pharmacodynamic experiments using a small cell lung cancer model confirmed that, throughout each growth cycle, the mouse tumor-bearing model was unable to escape the recognition and killing of tumors by the pheromone array, resulting in complete killing of cancer cells and the inability of the tumor model to grow. The pheromone 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).

[0038] The above experiments confirm that the pheromone array of the present invention exhibits a significantly superior antitumor effect compared to the single pheromone in the aforementioned patent (CN1274829C). Furthermore, guinea pig model experiments confirmed that 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 serve as an effective therapeutic agent for small cell lung cancer. Attached Figure Description

[0039] Figure 1 is a schematic diagram of the structure of the pheromone of the present invention.

[0040] Figure 2 is a schematic diagram of the recombinant plasmid for preparing pheromone constructed in Example 1 of the present invention.

[0041] Figure 3 shows the SDS-PAGE electrophoresis results of three of the thirty pheromones prepared in Example 1 of this invention. Lane 1: Protein Marker; Lanes 2-4: Three pheromones in the pheromone array prepared in Example 1, each with a molecular weight of approximately 30 kDa; Lane 5: The pheromone used as a control (containing whole coliform Ia, with a molecular weight of 70 kDa); Lane 6: Fetal bovine serum albumin used as a standard control, with a molecular weight of 70 kDa.

[0042] Figure 4 shows the experimental results of attacking small cell lung cancer cells (NCI-H446) with a pheromone array in Example 2 of this invention. Figure 4a: Small cell lung cancer cells (NCI-H446) in the blank control group, with a polygonal morphology. Figure 4b: Small cell lung cancer cells (NCI-H446) after being co-incubated with a pheromone array (50 μg / ml) for 48-72 hours changed from the polygonal morphology of normal lung cancer cells to a round, oval, or elongated morphology. Most of them could no longer adhere to the cell 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 the pheromone array, all small cell lung cancer cells observed under the microscope had lost their vitality.

[0043] Figure 5 shows the change in tumor weight in Balb / C nude mice after 3 days of inoculation with small cell lung cancer cells (NCI-H446) and 4 weeks of treatment with a pheromone array in Example 3 of this invention. Blue triangles represent the blank control group (CK); purple-red dots represent the HB8627-pheromone treatment group (PMC-8627); and red squares represent the pheromone array treatment group (PMC-array).

[0044] Figure 6 shows the change in tumor weight in Balb / C nude mice 14 days after inoculation with small cell lung cancer cells (NCI-H446) and 3 weeks after treatment with pheromone in Example 3 of the present invention. The blue triangle symbol represents the blank control group (CK), and the red square symbol represents the pheromone array treatment group (PMC-array).

[0045] Figure 7 shows the changes in tumor weight in Balb / C nude mice after 10 days of inoculation with small cell lung cancer cells (DMS-153) in Example 3 of this invention, followed by 3 weeks of treatment with pheromone and then a 3-week drug withdrawal period. 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 3 weeks of pheromone array treatment (drug administration), and the blank red line indicates no further treatment (drug withdrawal) for 3 weeks.

[0046] Figure 8 shows the tumor anatomical specimens of the second experimental group in Example 3 of the present invention. Top row: Tumors (NCI-H446) from three blank control groups, with weights of 1683 mg, 301 mg, and 263 mg, respectively. Bottom row: Tumors (NCI-H446) from three pheromone array treatment groups, with weights of 27 mg, 4 mg, and 4 mg, respectively.

[0047] Figure 9 shows pathological sections of tumor specimens from the second experimental group in Example 3 of this invention. Figure 9a: Pathological section of tumors from the blank control group (mouse model of small cell lung cancer). Figure 9b: Pathological section of tumors after 6 days of treatment with a pheromone array, showing coagulative necrosis of a large number of tumor cells (a typical case change of pheromone killing tumor cells, Nat Biotech 2007). Figure 9c: Enlarged view of a portion of Figure 9b. Figure 9d: After 3 weeks of treatment with a pheromone array, some tumors in mice gradually disappeared. Scale bar length is 100 μm.

[0048] Figure 10 shows the toxicity test results of the pheromone array on normal guinea pigs in Example 4 of the present invention. There were no significant differences in blood biochemical indicators between the blank control group (C, n=4) and the pheromone array treatment group (T, n=6). In Figure 10, the concentrations of UREA, CREA, UA, TBIL, and DBIL are in mg / dL; the concentrations of TP, ALB, and GLB are in g / dL; A / G represents the ratio of albumin to globulin; and the concentrations of ALT, AST, ALP, LDH, GGT, and CK are in Unit / L.

[0049] Sequence Description

[0050] The amino acid sequences listed in the accompanying sequence listing are displayed using single-letter codes for the amino acids, following the standard convention of starting from the amino terminus of the sequence and proceeding toward the carboxyl terminus.

[0051] SEQ ID NO: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 NO:32-61 is the amino acid sequence of thirty pheromones that are effective against small cell lung cancer. Detailed Implementation

[0054] The following examples are provided:

[0055] 1. A polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30.

[0056] 2. Use of polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 in the preparation of drugs for small cell lung cancer.

[0057] 3. The use described in Example 2, wherein the drug is a preparation for treating small cell lung cancer.

[0058] 4. A drug for treating small cell lung cancer, comprising a fusion protein obtained by linking a channel domain of coliform to a polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30.

[0059] 5. The drug described in Example 4, wherein the coliform includes coliform E1, Ia, Ib, A, B and N.

[0060] 6. The drug described in Example 5, wherein the coliformin is coliformin Ia, and the amino acid sequence of its channel domain is shown in SEQ ID NO:31.

[0061] 7. The drug described in any of Examples 4-6, wherein the polypeptide with the amino acid sequence shown in SEQ ID NO:1-30 is respectively linked to the carboxyl terminus and / or amino terminus of the channel domain of the coliform.

[0062] 8. The drug described in Example 7, wherein the polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 are respectively linked to the carboxyl terminus of the channel domain of the coliform.

[0063] 9. The drug described in Example 8, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO:32-61.

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

[0065] 11. The method described in Example 10, wherein the coliform includes coliform E1, Ia, Ib, A, B and N.

[0066] 12. The method described in Example 11, wherein the coliform is coliform Ia, and the amino acid sequence of its channel domain is shown in SEQ ID NO:31.

[0067] 13. The method described in any of Examples 10-12, wherein the polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 are respectively linked to the carboxyl terminus and / or amino terminus of the channel domain of the coliform.

[0068] 14. The method described in Example 13, wherein the polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 are respectively linked to the carboxyl terminus of the channel domain of coliform.

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

[0070] The invention is further illustrated below with examples. It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0071] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, commercially available or prepared according to conventional methods in the art, and should be of laboratory purity. Unless otherwise specified, the experimental methods and conditions used in the following examples are conventional experimental methods and conditions in the art, and can be found in relevant experimental manuals, public 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 invention pertains.

[0072] Example 1

[0073] Preparation of pheromone arrays

[0074] Thirty pheromone proteins with amino acid sequences as shown in SEQ ID NO:32-61 were prepared. These pheromone proteins were fusion proteins obtained by linking antibody mimics to the carboxyl terminus 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 mimics recognizing relevant tumor antigens are shown in SEQ ID NO:1-30. Table 1 lists the primary structures (V...) of the thirty antibody mimics. H CDR1-V H FR2-V L CDR3), in which amino acids are represented by standard single letters and arranged in order from the amino terminus to the carboxyl terminus.

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

[0076] The amino acid sequence of the channel domain of colicin Ia is as follows, where the amino acids are represented by standard single letters and arranged in order from the amino terminus to the carboxyl terminus. The first number 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 under physiological conditions, it synthesizes two proteins: one molecule of colicin and one molecule of the corresponding immunoprotein. The immunoprotein's role is to prevent the synthesized colicin from forming ion channels on the *E. coli* cell membrane. Once the synthesized colicin leaves the *E. coli* (is secreted extracellularly), the colicin and immunoprotein separate. Therefore, when synthesizing pheromones using engineered bacteria, the corresponding immunoprotein must be synthesized simultaneously. The genes encoding the structural protein and immunoprotein of colicin Ia are accessed in GenBank by the National Center for Biotechnology Information (NCBI) under the number M13819. Thirty pheromones with amino acid sequences as shown in SEQ ID NO:32-61 were prepared using the pET11a plasmid and *E. coli* B834(DE3). The pET11a recombinant plasmid for expressing the pheromones was synthesized by Norsei Genetics, containing genes encoding the channel domain of colicin Ia, genes encoding antibody mimics, and genes encoding the immunoprotein (Figure 2). Thirty recombinant plasmids were obtained, each used to express a specific pheromone. The DNA of these thirty recombinant plasmids was sequenced.

[0084] Thirty recombinant plasmids, identified through sequencing, were transfected into *E. coli* B834(DE3) competent cells. The B834 engineered bacteria containing the recombinant plasmids were multiplied in LB broth (containing 100 μg / ml ampicillin), and the cells were collected by centrifugation. The cells were lysed and resuspended in 50 mM borate buffer (pH 9), and the supernatant was extracted by centrifugation. Streptomycin sulfate was added to the supernatant to precipitate DNA. The supernatant was extracted again by centrifugation, dialyzed against 50 mM borate buffer (pH 9), and then passed through an agarose gel column and eluted with 0.3 M NaCl solution to obtain thirty pheromones. The SDS-PAGE electrophoresis results of three of the pheromones are shown in Figure 3. The yield of pheromones reached 5-12 mg / ml.

[0085] Thirty pheromone molecules prepared were tested using liquid chromatography-mass spectrometry (LC-MS), confirming that the amino acid residues of the antibody mimics were located at the carboxyl terminus of the pheromone molecules. The thirty pheromone molecules tested by LC-MS were mixed in equal mass to obtain the pheromone array of this invention.

[0086] Example 2

[0087] In vitro inhibition experiment of pheromone array on small cell lung cancer cells

[0088] 1. Experimental Objective

[0089] The in vitro inhibitory effect of the pheromone array of the present invention on small cell lung cancer cells was investigated.

[0090] 2. Experimental Materials

[0091] Test drugs: (1) The pheromone array of the present invention prepared in Example 1, which is composed of 30 pheromones with amino acid sequences as shown in SEQ ID NO:32-61, mixed in equal mass, was provided by Chengdu FinoLixin Biotechnology Co., Ltd. (2) HB8627-pheromone, provided by Chengdu FinoLixin 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.

[0092] The small cell lung cancer cell line NCI-H446, cell culture medium RMPI 1640, and microscope LeicaDMi8 were provided by the Institute of Basic Medical Sciences, Peking Union Medical College.

[0093] 3. Experimental Location

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

[0095] 4. Experimental Methods

[0096] 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.

[0097] The experiment included a blank control group, a positive control group, and a pheromone array treatment group, as detailed below:

[0098] (1) Blank control group: Normal cultured small cell lung cancer cells NCI-H446, without any treatment.

[0099] (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.

[0100] (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.

[0101] 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.

[0102] 5. Experimental Results

[0103] After co-incubating small cell lung cancer cells 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). Small cell lung cancer cells in the blank control group had a polygonal morphology (Figure 4a). Small cell lung cancer cells co-incubated with the pheromone array for 48-72 hours had round, oval, or elongated morphologies, and most could no longer adhere to the cell wall, with all subcellular structures altered. Propidium iodide staining results indicated that the cell membranes of dead cells were destroyed, allowing propidium iodide to enter the cell and stain it red. Under the continuous attack of the pheromone array, all small cell lung cancer cells observed under the microscope had lost their viability (Figure 4b).

[0104] 6. Experimental Conclusions

[0105] The pheromone array of the present invention has a powerful effect in inhibiting and killing small cell lung cancer cells.

[0106] Example 3

[0107] Pharmacodynamic experiments of pheromone arrays on a mouse tumor-bearing model

[0108] 1. Experimental Objective

[0109] The pheromone array of the present invention was used to verify its killing effect on a mouse tumor-bearing model.

[0110] 2. Experimental Materials

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

[0112] 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 pheromone array of the present invention prepared in Example 1, which is composed of 30 pheromones with amino acid sequences as shown in SEQ ID NO:32-61, mixed in equal mass, was provided by Chengdu FinoLixin Biotechnology Co., Ltd. (2) HB8627-pheromone, provided by Chengdu FinoLixin 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.

[0114] 3. Experimental Methods

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

[0116] Balb / C nude mice were injected axillarily with cultured small cell lung cancer cells at a dose of 0.1 ml (5 × 10⁶ cells / ml). 7 Cells per mouse. 3–14 days after inoculation, the mouse tumor-bearing models were randomly assigned to groups and treatment was initiated.

[0117] (2) Administration method and dosage

[0118] The first experimental group of Balb / C nude mice was 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 physiological saline, 1 ml / mouse / day. Positive control group: intraperitoneal injection of HB8627-pheromone, 1 mg daily, twice daily, 0.5 mg / time. Pheromonomone array treatment group: intraperitoneal injection of the pheromone array of the present invention, 1 mg daily, twice daily, 0.5 mg / time. Dissection was performed after 28 days of continuous administration.

[0119] The second experimental group consisted of Balb / C nude mice inoculated with the small cell lung cancer cell line NCI-H446. Intraperitoneal administration began 14 days after inoculation. A blank control group (n=10) and a pheromone array treatment group (n=10) were established, where n represents the number of nude mice. Blank control group: Intraperitoneal injection of physiological saline, 1 ml / mouse / day. Pheromones array treatment group: Intraperitoneal injection of the pheromone array of the present invention, 2 mg / day, three times daily, approximately 0.7 mg / time. Dissection was performed after 21 days of continuous administration.

[0120] The third experimental group of Balb / C nude mice was 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 a pheromone array treatment group (n=10) were set up, where n represents the number of nude mice. Blank control group: intraperitoneal injection of physiological saline, 1 ml / mouse / day. Pheromones array treatment group: intraperitoneal injection of the pheromone array of the present invention, 2 mg / day, three times a day, approximately 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.

[0121] (3) Weight detection

[0122] Animals were weighed using an electronic digital scale 3 days before vaccination and daily after administration.

[0123] (4) Observe the condition of tumor-bearing lesions

[0124] After dissection, the tumor-bearing lesions of the mice were observed. The tumors were isolated, weighed, fixed, and sectioned for pathological examination. After reviewing the pathological slides, scores were assigned based on the pathological changes in the tumors of each group.

[0125] (5) Statistical methods for results

[0126] The pairedt test was used for statistical analysis to evaluate the changes in 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 results of the first experimental group showed that after 28 days of continuous administration, the average tumor weight in the blank control group (CK) was 129.33 mg, the average tumor weight in the positive control group (PMC-8627) was 89.25 mg, and the average tumor weight in the pheromone array treatment group (PMC-array) was 3.33 mg (Figure 5).

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

[0131] The results of the third experimental group showed that after 21 days of continuous administration, the average tumor weight in the blank control group (CK) was 123.83 mg (n=6), and the average tumor weight in the pheromone array treatment group (PMC-array) was 11 mg (n=6). After 21 days of continuous administration followed by a 21-day withdrawal period, the average tumor weight in the blank control group (CK) was 202.75 mg (n=4), and the average tumor weight in the pheromone array treatment group (PMC-array) was 7.25 mg (n=4) (Figure 7).

[0132] Table 2

[0133] *A tumor weight of 0 indicates that no tumor was found after dissecting the mouse.

[0134] 5. Experimental Conclusions

[0135] The pheromone array of this invention exhibited a strong killing effect on a nude mouse tumor-bearing model (human small cell lung cancer). Three weeks after drug withdrawal, no tumor growth was observed (unlike the continuous growth of tumors in the blank control group during the same period) (Figure 7). Tumor anatomy results (Figure 8) and pathological section observation results (Figure 9) also confirmed that the pheromone array effectively eliminated small cell lung cancer tumors induced by inoculation. The experimental mice gained weight, confirming that the pheromone array did not cause toxic damage to the tested animals.

[0136] Example 4

[0137] Toxicity test of pheromone array on normal guinea pigs

[0138] 1. Experimental materials

[0139] Laboratory animals: 10 guinea pigs (half male and half female) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0140] Test drug: The pheromone array of the present invention prepared in Example 1 is composed of an equal mass mixture of thirty pheromones with amino acid sequences as shown in SEQ ID NO:32-61, and was provided by Chengdu FinoLixin Biotechnology Co., Ltd.

[0141] 2. Experimental Grouping

[0142] Guinea pigs were divided into two groups: a blank control group (n=4) and a pheromone array treatment group (n=6), for a total of 10 guinea pigs.

[0143] 3. Experimental Methods

[0144] (1) Administration method and dosage

[0145] Blank control group: Intraperitoneal injection of physiological saline, 2 ml / mouse / day.

[0146] Information-producing bacteria array treatment group: The information-producing bacteria array of the present invention was injected intraperitoneally, 3 mg / mouse / day.

[0147] All groups were given the medication once daily for 30 consecutive days. After blood samples were collected for testing, the patients were euthanized.

[0148] (2) Weight measurement

[0149] Animals were weighed using an electronic digital scale 3 days before vaccination and every 7 days after administration.

[0150] (3) Blood biochemistry test

[0151] Blood samples were sent to the Animal Hospital of China Agricultural University for testing to compare changes in blood biochemical indicators.

[0152] 4. Experimental Results

[0153] Compared with the control group, the animals injected with the pheromone array showed no change in appetite and behavior, and there was no difference in the results of blood biochemical indicators (Figure 10) (no damage to liver, kidney, or immune functions was observed).

[0154] 5. Experimental Conclusions

[0155] No toxic side effects were observed in experimental animals after 30 days of treatment with the pheromone array (intraperitoneal injection).

Claims

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

2. Use of the polypeptide with the amino acid sequence shown in SEQ ID NO:1-30 in the preparation of a drug for 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 linking a channel domain of coliform to a polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30.

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

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

31.

7. The medicament according to any one of claims 4-6, wherein the polypeptide with the amino acid sequence shown in SEQ ID NO:1-30 is respectively linked to the carboxyl terminus and / or amino terminus of the channel domain of the coliformin.

8. The medicament of claim 7, wherein the polypeptides with amino acid sequences as shown in SEQ ID NO:1-30 are respectively linked to the carboxyl terminus of the channel domain of the coliform.

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

10. A method for preparing the anti-small cell lung cancer drug according to any one of claims 4-9, comprising linking a polypeptide with an amino acid sequence as shown in SEQ ID NO:1-30 to a channel domain of an escherichia coli to obtain a fusion protein.

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