Fab-BH3 fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a Fab-BH3 fusion protein as well as a preparation method and application thereof, belongs to the technical field of biological medicines, and particularly relates to the Fab-BH3 fusion protein, and the amino acid sequence of the Fab-BH3 fusion protein is as shown in SEQ ID NO. 2. The invention further discloses application of the Fa-BH3 fusion protein in preparation of drugs for treating non-small cell lung cancer. According to the invention, the Fab fragment of the cetuximab and the BH3 polypeptide are coupled to form the Fa-BH3 fusion protein, the activity of the cetuximab is not influenced, and the Fa-BH3 fusion protein inhibits the growth of non-small cell lung cancer cells overexpressing EGFR, so that the treatment effect on the non-small cell lung cancer is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a Fab-BH3 fusion protein and a preparation method and application thereof. Background Art

[0002] Lung cancer is a malignant tumor that originates in the bronchial mucosa or glands of the lungs. It is one of the most common malignant tumors with the highest morbidity and mortality rates worldwide. The incidence of lung cancer is associated with multiple factors, including smoking, air pollution, and occupational exposure. For early-stage non-small cell lung cancer, lobectomy is the most common surgical procedure. This procedure involves removing the lobe of the lung containing the tumor to minimize the amount of tumor tissue. Systematic lymph node dissection is also a crucial component of surgery, as lung cancer is prone to lymph node metastasis. Lymph node dissection allows for accurate pathological staging and reduces the risk of tumor recurrence. Chemotherapy drugs work by interfering with the growth and division of tumor cells. Commonly used chemotherapy drugs include platinum-based drugs such as cisplatin and carboplatin, and taxanes such as paclitaxel and docetaxel. For advanced non-small cell lung cancer, chemotherapy can prolong patient survival and alleviate symptoms. Targeted therapy, on the other hand, targets specific molecular targets in lung cancer cells. For example, for patients with EGFR mutation-positive non-small cell lung cancer, EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors), such as gefitinib and erlotinib, can specifically inhibit EGFR activity, blocking the proliferation signaling pathways of tumor cells and thereby suppressing tumor growth. In many malignancies, EGFR is often overexpressed or mutated. This aberrant EGFR signaling can lead to uncontrolled cancer cell proliferation, invasion, and metastasis. For example, in non-small cell lung cancer (NSCLC), approximately 10% to 30% of patients harbor EGFR mutations, which increase cancer cells' dependence on EGFR signaling. In some tumors with high EGFR expression or mutations, EGFR antibodies can be used as a single agent. For example, cetuximab has limited application in the treatment of head and neck squamous cell carcinoma. By blocking EGFR signaling, it inhibits tumor cell proliferation and induces apoptosis, thereby controlling tumor growth. EGFR antibodies are often used in combination with other therapies. When combined with chemotherapy, for example in the treatment of colorectal cancer, EGFR antibodies combined with chemotherapy drugs (such as FOLFOX or FOLFIRI regimens) can improve the therapeutic effect.

[0003] The Fab (fragment of antigen binding) segment is formed by papain cleaving the disulfide bond between the heavy chains of immunoglobulin (Ig) near the N-terminus in the hinge region, forming two identical monovalent antigen-binding fragments.

[0004] BH3-binding peptides are relatively small peptide sequences that often serve as key domains within members of the intracellular apoptosis-regulating protein family. The Bcl-2 family of proteins plays a key role in the intrinsic apoptosis pathway. This family includes both anti-apoptotic proteins (such as Bcl-2 and Bcl-xL) and pro-apoptotic proteins. BH3-binding domains are primarily found in pro-apoptotic proteins, such as Bax and Bak. In the 1990s, sequence alignment and functional analysis of Bcl-2-related proteins by multiple laboratories confirmed the critical role of the BH3-binding domain in apoptotic signaling. For example, studies of apoptosis-deficient tumor cell lines revealed that mutations in certain genes impaired the function of proteins containing BH3-binding domains, highlighting their importance in apoptosis regulation. BH3-binding peptides can mimic the BH3 domains of endogenous pro-apoptotic proteins and bind to anti-apoptotic Bcl-2 proteins (such as Bcl-2 and Bcl-xL), thereby releasing the pro-apoptotic proteins Bax and Bak and initiating mitochondrial-mediated apoptosis. BH3 peptides can be used in combination with other anti-cancer therapies, such as chemotherapy drugs. Chemotherapy drugs often cause certain damage to tumor cells and activate apoptosis signaling pathways within the cells. BH3 peptides can enhance the apoptosis signaling in these pathways and overcome the drug resistance of tumor cells. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes a Fab-BH3 fusion protein, a preparation method, and an application thereof. By coupling the Fab fragment of cetuximab with a BH3 polypeptide to form a Fab-BH3 fusion protein, the activity of cetuximab is not affected. The Fab-BH3 fusion protein inhibits the growth of non-small cell lung cancer cells that overexpress EGFR, thereby improving the therapeutic effect of non-small cell lung cancer.

[0006] To achieve the above object, the present invention provides a Fab-BH3 fusion protein, the amino acid sequence of the Fab-BH3 fusion protein is shown in SEQ ID NO.2.

[0007] Preferably, the specific composition of the Fab-BH3 fusion protein is: the heavy chain variable region and the light chain variable region of the cetuximab Fab fragment are connected by a linker, a His tag is added to the N-terminus, and the C-terminus is connected to the BH3 polypeptide through a linker to form a fusion protein Fab-BH3.

[0008] The present invention also provides a method for preparing the Fab-BH3 fusion protein, comprising the following steps: amplifying a nucleotide sequence encoding the Fab-BH3 fusion protein and connecting it to a plasmid to obtain a recombinant plasmid vector; transferring the recombinant plasmid vector into competent Escherichia coli cells of an expression system, expressing the obtained positive transformants in prokaryotes, and isolating and purifying the expression products to obtain the Fab-BH3 fusion protein.

[0009] Preferably, the nucleotide sequence encoding the Fab-BH3 fusion protein is shown as SEQ ID NO.1.

[0010] The present invention also provides the use of the Fab-BH3 fusion protein in preparing a medicine for treating non-small cell lung cancer.

[0011] Preferably, the Fab-BH3 fusion protein has stronger cytotoxicity to EGFR non-small cell lung cancer cells and stronger ability to induce apoptosis of EGFR non-small cell lung cancer cells.

[0012] The present invention also provides a recombinant plasmid vector, which comprises a nucleotide sequence encoding a Fab-BH3 fusion protein. The nucleotide sequence encoding the Fab-BH3 fusion protein is shown in SEQ ID NO.1.

[0013] The present invention also provides the use of the recombinant plasmid vector in preparing medicine for treating non-small cell lung cancer.

[0014] The present invention also provides a recombinant expression strain, which can express Fab-BH3 fusion protein in prokaryotes. The amino acid sequence of the Fab-BH3 fusion protein is shown in SEQ ID NO.2.

[0015] The present invention also provides the use of the recombinant expression strain in preparing medicine for treating non-small cell lung cancer.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides a Fab-BH3 fusion protein. The Fab fragment of cetuximab is conjugated to a BH3 polypeptide to form the Fab-BH3 fusion protein. The heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment are connected via a linker, a His tag is added to the N-terminus, and the C-terminus is connected to the BH3 polypeptide via a linker to form the fusion protein Fab-BH3. While ensuring that the Fab activity is not affected, the BH3 polypeptide is combined with the Fab fragment of cetuximab to form a fusion protein, thereby enhancing the anti-tumor activity of the antibody drug. The Fab-BH3 fusion protein has the same affinity as the antibody but is more cytotoxic against EGFR-positive non-small cell lung cancer cells. It also has no effect on the growth of EGFR-negative cells, reducing adverse reactions. The Fab-BH3 fusion protein inhibits the growth of non-small cell lung cancer cells that overexpress EGFR, thereby improving the therapeutic efficacy of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The enzyme digestion diagrams of the recombinant plasmid vector of Example 1 and the recombinant plasmid vector of Comparative Example 1 are shown, wherein A is the enzyme digestion diagram of the recombinant plasmid vector of Example 1, and B is the enzyme digestion diagram of the recombinant plasmid vector of Comparative Example 1; Figure 2 The recombinant plasmid vector of Example 1 and the recombinant plasmid vector of Comparative Example 1 are shown in Figures A and B, respectively. Figure 3 Figure 2 is the expression identification and purification analysis diagram of Fab-BH3 fusion protein, where A is the identification analysis diagram of Fab-BH3 fusion protein, in which M represents Marker, 1 represents the uninduced sample, and 2~6 represent the induced samples; B is the optimization and solubility analysis result diagram of Fab-BH3 fusion protein, in which M represents Marker, 1 represents the sample induced at 15℃ and 0.2mM IPTG, 2 represents the sample induced at 15℃ and 1.0mM IPTG, 3 represents the sample induced at 37℃ and 0.2mM IPTG, 4 represents the sample induced at 37℃ and 1.0mM IPTG, 5 represents the uninduced sample, 6 represents the precipitate sample induced at 37℃ and 1.0mM IPTG, 7 represents the supernatant sample induced at 37℃ and 1.0mM IPTG, 8 represents the precipitate sample induced at 37℃ and 0.2mM IPTG, 9 represents the supernatant sample induced at 37℃ and 0.2mM IPTG, 10 represents the supernatant sample induced at 15℃ and 1.0mM IPTG. 11 represents the precipitate sample after induction with IPTG, 1.0 mM IPTG at 15°C, 12 represents the precipitate sample after induction with 0.2 mM IPTG at 15°C, 13 represents the supernatant sample after induction with 0.2 mM IPTG at 15°C, C is the SDS-PAGE analysis of the Fab-BH3 fusion protein purified by nickel-agarose affinity chromatography, in which M represents Marker, 1 represents the precipitate after crushing, 2 represents the supernatant after crushing, 3 represents the flow-through, 4 and 5 represent the washing samples, and 6 represents the elution sample, D is the SDS-PAGE analysis of the Fab-BH3 fusion protein purified by gel filtration chromatography, in which M represents Marker, S represents the sample before gel filtration chromatography purification, B4, B2, C1, C5, C9, C12, D10, D7, D4, D1, E2, E5 and E8 represent the collected protein samples; Figure 4: This is an expression identification and purification analysis diagram of L11P prepared in Comparative Example 1, wherein A is an L11P identification analysis diagram, in which M represents Marker, 1 represents an uninduced sample, 2 to 6 represent induced samples, and B is an L11P optimization and solubility analysis result diagram, in which M represents Marker, 1 represents a sample induced at 15°C, 0.2 mM IPTG, 2 represents a sample induced at 15°C, 1.0 mM IPTG, 3 represents a sample induced at 37°C, 0.2 mM IPTG, 4 represents a sample induced at 37°C, 1.0 mM IPTG, 5 represents an uninduced sample, 6 represents a precipitate sample induced at 37°C, 1.0 mM IPTG, 7 represents a supernatant sample induced at 37°C, 1.0 mM IPTG, 8 represents a precipitate sample induced at 37°C, 0.2 mM IPTG, 9 represents a supernatant sample induced at 37°C, 0.2 mM IPTG, 10 represents a precipitate sample induced at 15°C, 1.0 mM IPTG 11 represents the precipitate sample after IPTG induction, 11 represents the supernatant sample after 15°C, 1.0 mM IPTG induction, 12 represents the precipitate sample after 15°C, 0.2 mM IPTG induction, 13 represents the supernatant sample after 15°C, 0.2 mM IPTG induction, C is the SDS-PAGE analysis of L11P nickel agarose affinity chromatography purification, in which M represents Marker, 1 represents the precipitate after crushing, 2 represents the supernatant after crushing, 3 represents the effluent, 4 and 5 represent the washing samples, and 6 represents the elution sample, D is the SDS-PAGE analysis of L11P gel filtration chromatography purification, in which M represents Marker, S represents the sample before gel filtration chromatography purification, B9, B4, B2, C1, C5, C9, D10, D7, D3, E2, E5, E8, E12 and F10 represent protein collection samples; Figure 5 Figure 1 is an SDS-PAGE analysis of Fab-BH3 fusion protein and L11P purified protein, wherein A is Fab-BH3 fusion protein, M represents Marker, S represents fusion target protein, and B is L11P, M represents Marker, S represents fusion target protein; Figure 6 Western-blot images of Fab-BH3 fusion protein and L11P, where A is Fab-BH3 fusion protein and B is L11P; Figure 7 This is a fluorescence image of eukaryotic plasmid transfection. In the image, A549-MUT represents A549 cells transformed with the L11P mutant plasmid of Comparative Example 1, and A549-pDNA represents A549 cells transformed with the Fab-BH3 recombinant plasmid of Example 1. The scale bar is 50 μm. Figure 8 The results of the MTT cytotoxicity test are shown in Figure 1, where A is the MTT result of the Fab-BH3 fusion protein. In the figure, anti-EGFR-Fab-BH3 represents the Fab-BH3 fusion protein. " represents p < 0.01," "" represents p < 0.001; B is a comparison of the MTT assay results of Fab-BH3 fusion protein and L11P. In the figure, anti-EGFR-Fab-BH3 represents Fab-BH3 fusion protein, and anti-EGFR-Fab-BH3 (L11P) represents L11P. C is a comparison of different cells. In the figure, A549 represents non-small cell lung cancer cells expressing EGFR, and CHO represents hamster ovary cells that do not express EGFR. Figure 9 Figures 2 and 3 are Hoechst fluorescence experimental diagrams, wherein A shows the killing effect of Fab-BH3 fusion protein at different doses on A549 cells, B shows the comparison of the killing effect of Fab-BH3 fusion protein and L11P at different doses on A549 cells, in which aEFB represents Fab-BH3 fusion protein, aEFBM represents L11P, and C shows the killing effect of Fab-BH3 fusion protein at different doses on A549 cells and CHO cells, in which A549 represents non-small cell lung cancer cells expressing EGFR, and CHO represents hamster ovary cells that do not express EGFR. The scale bar is 50 μm. Figure 10 The results of the cell colony assay are shown in Figure 1, where A is a graph showing the inhibitory effect of different concentrations of Fab-BH3 fusion protein and L11P on the migration of A549 cells. In the figure, aEFB represents Fab-BH3 fusion protein, aEFBM represents L11P, and the scale is 50 μm. B is a statistical graph showing the inhibitory effect of different concentrations of Fab-BH3 fusion protein and L11P on the migration of A549 cells. In the figure, aEFB represents Fab-BH3 fusion protein, aEFBM represents L11P, and the scale is 50 μm. " represents p < 0.01," ” represents p < 0.001; Figure 11 These are the structural model diagram and molecular structure diagram of BH3, where A is the structural model diagram of BH3 and B is the molecular structure diagram of BH3. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Preparation of Fab-BH3 fusion protein: The nucleotide sequence encoding the Fab-BH3 fusion protein (SEQ ID NO. 1) was double-digested and ligated to the plasmid PET22b to obtain a recombinant plasmid vector (eg Figure 1 China A and Figure 2 The recombinant plasmid vector was transformed into the expression system Escherichia coli competent BL21 (DE3) cells, the obtained positive transformants were subjected to prokaryotic expression, and the expression products were isolated and purified to obtain Fab-BH3 fusion protein (the amino acid sequence of which is shown in SEQ ID NO.2).

[0025] The recombinant plasmid vector was constructed as follows: the plasmid with the nucleotide sequence of Fab-BH3 fusion protein and the PET22b plasmid were respectively used Nde I and Xho After double digestion with restriction endonucleases, perform agarose gel electrophoresis, cut out the 5000 bp and 1500 bp bands, and use a gel recovery kit to recover DNA. EcoliE DNA ligase was used to connect the target gene and the vector to obtain a recombinant plasmid vector for subsequent transformation experiments.

[0026] The transformation was performed as follows: the recombinant plasmid vector (10 μL) was added to BL21 (DE3) competent cells (100 μL), ice-bathed for 30 min, heat-shocked at 42°C for 60 s, plated into ampicillin-resistant LB liquid medium (100 μg / mL ampicillin), cultured at 37°C overnight, and then single clones were picked and inoculated into fresh culture medium for overnight culture at 37°C.

[0027] The specific steps of prokaryotic expression are as follows: transfer the overnight cultured bacteria into new ampicillin-resistant LB liquid medium (50µg / mL ampicillin) at a volume ratio of 1:100, and culture at 37°C until the OD 600 When the pH value was within the range of 0.6-0.8, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, induced at 15°C for 16 h, centrifuged at 4000 rpm for 10 min, discarded the supernatant, and collected the bacteria.

[0028] The specific separation and purification steps are as follows: first, the bacteria obtained by prokaryotic expression are resuspended in a buffer solution, then ultrasonically disrupted for 15 minutes at a power of 130 W, centrifuged at 12,000 rpm for 20 minutes, and the supernatant is collected. The supernatant is purified by nickel column affinity chromatography using binding buffer and elution buffer to obtain a high-purity Fab-BH3 fusion protein.

[0029] Nickel column affinity chromatography purification: Load the column with Ni-NTA filler and wash and equilibrate the column with binding buffer; incubate the crude protein with the equilibrated column filler and collect the flow-through; wash the equilibrated column with binding buffer; wash the column with wash buffer and collect the flow-through; elute with elution buffer and collect the flow-through; treat the crude protein and the flow-through fraction separately, prepare samples for purification analysis, and use dialysate (PBS, 8M / 4M / 1M / 0M urea, pH 8) to renature the target protein to obtain the Fab-BH3 fusion protein.

[0030] The lysis buffer consisted of 8 M urea, 50 mM Tris, 300 mM NaCl, 0.1% Triton X-100, the solvent was water, and the pH was 8; the binding buffer consisted of 8 M urea, 50 mM Tris, 300 mM NaCl, the solvent was water, and the pH was 8.0; the elution buffer consisted of 8 M urea, 50 / 100 / 200 / 500 mM Tris, 300 mM NaCl, 200 mM imidazole, the solvent was water, and the pH was 8.0.

[0031] The specific composition of the Fab-BH3 fusion protein is: the heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment are connected by a linker, a His tag is added to the N-terminus, and the C-terminus is connected to the BH3 polypeptide through a linker to form the fusion protein Fab-BH3.

[0032]

[0033] SEQ ID NO.2: MHHHHHHEFDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAGGGGSGGGGSGGGGSQVQLKQSGPG LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSGGGGSGGGGSGGGGSPLGLAGDASTKKLSECLRRIGDELDS.

[0034] As shown in SEQ ID NO. 2 above, the bold portion is the His tag, the single underlined portion is the light chain variable region of the cetuximab Fab fragment, the bold and underlined portion is the linker, the double underlined portion is the heavy chain variable region of the cetuximab Fab fragment, the wavy portion is the MMP-2 cleavage site, the shading portion is the BH3 polypeptide, and the shading and underlined portion is the mutation point of the BH3 polypeptide.

[0035] Comparative Example 1 Preparation of Fab-mutant BH3 fusion protein (L11P): The nucleotide sequence encoding L11P (SEQ ID NO. 3) was double-digested and ligated to plasmid PET22b to obtain a recombinant plasmid vector (eg Figure 1 Middle B and Figure 2 The recombinant plasmid vector was transformed into the expression system Escherichia coli BL21 (DE3) cells, the obtained positive transformants were subjected to prokaryotic expression, and the expression product was isolated and purified to obtain L11P (the amino acid sequence of which is shown in SEQ ID NO.4).

[0036] The recombinant plasmid vector was constructed as follows: the plasmid with the nucleotide sequence of L11P and the PET22b plasmid were respectively used Nde I and Xho After double digestion with restriction endonucleases, perform agarose gel electrophoresis, cut out the 5000 bp and 1500 bp bands, and use a gel recovery kit to recover DNA. Ecoli E DNA ligase was used to connect the target gene and the vector to obtain a recombinant plasmid vector for subsequent transformation experiments.

[0037] The transformation was performed as follows: the recombinant plasmid vector (10 μL) was added to BL21 (DE3) competent cells (100 μL), ice-bathed for 30 min, heat-shocked at 42°C for 60 s, plated into ampicillin-resistant LB liquid medium (100 μg / mL ampicillin), cultured at 37°C overnight, and then single clones were picked and inoculated into fresh culture medium for overnight culture at 37°C.

[0038] The specific steps of prokaryotic expression are as follows: transfer the overnight cultured bacteria into new ampicillin-resistant LB liquid medium (50µg / mL ampicillin) at a volume ratio of 1:100, and culture at 37°C until the OD 600 When the pH value was within the range of 0.6-0.8, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, induced at 15°C for 16 h, centrifuged at 4000 rpm for 10 min, discarded the supernatant, and collected the bacteria.

[0039] The specific separation and purification steps are as follows: first, the bacteria obtained by prokaryotic expression are resuspended in a buffer solution, then ultrasonically disrupted for 15 minutes at a power of 130 W, centrifuged at 12000 rpm for 20 minutes, the supernatant is collected, and the supernatant is purified by nickel column affinity chromatography using binding buffer and elution buffer to obtain high-purity L11P.

[0040] Nickel column affinity chromatography purification: Ni-NTA filler was loaded onto the column, and the column was washed and equilibrated with binding buffer; the crude protein was incubated with the equilibrated column filler and the flow-through was collected; the equilibrated column was washed with binding buffer; the column was washed with wash buffer and the flow-through was collected; the column was eluted with elution buffer and the flow-through was collected; the crude protein and the flow-through fractions were treated separately, and samples were prepared to analyze the purification effect. The target protein was renatured with dialysate (PBS, 8 / 4 / 2 / 1 / 0 M urea, pH 8) to obtain L11P.

[0041] The lysis buffer consisted of 8 M urea, 50 mM Tris, 300 mM NaCl, 0.1% Triton X-100, the solvent was water, and the pH was 8; the binding buffer consisted of 8 M urea, 50 mM Tris, 300 mM NaCl, the solvent was water, and the pH was 8.0; the elution buffer consisted of 8 M urea, 50 / 100 / 200 / 500 mM Tris, 300 mM NaCl, 200 mM imidazole, the solvent was water, and the pH was 8.0.

[0042] The specific composition of L11P is: the heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment are connected by a linker, a His tag is added to the N-terminus, and the C-terminus is connected to the mutant BH3 polypeptide through a linker to form a fusion protein L11P.

[0043]

[0044] SEQ ID NO.4: MHHHHHHEFDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAGGGGSGGGGSGGGGSQVQLKQSGPG LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSGGGGSGGGGSGGGGSPLGLAGDASTKKLSECPRRIGDELDS.

[0045] As shown in SEQ ID NO. 4 above, the bold portion is a His tag, the single underlined portion is the light chain variable region of the cetuximab Fab fragment, the bold and underlined portion is a linker, the double underlined portion is the heavy chain variable region of the cetuximab Fab fragment, the wavy portion is the MMP-2 cleavage site, the shading portion is the mutated BH3 polypeptide, and the shading and underlined portion is the mutation point of the BH3 polypeptide.

[0046] Experimental Example 1 Expression and identification of Fab-BH3 fusion protein and L11P: 1. Small-scale expression of Fab-BH3 fusion protein: The recombinant plasmid vector was transformed into competent Escherichia coli BL21 (DE3) cells. After heat shock at 42°C, the cells were spread on plates containing 50µg / mL ampicillin and cultured at 37°C for 20h. Five monoclonal colonies were picked and cultured in five tubes of liquid culture medium containing antibiotics at 37°C. When the OD value reached 0.6, 0.5mM inducer IPTG was added and cultured at 37°C for 6h. The negative control was the one without inducer. A total of 6 tubes of samples were collected by centrifugation for sample preparation.

[0047] The culture was inoculated and cultured until the OD value reached 0.6-0.8. IPTG was then added to a final concentration of 0.2 mM and 1.0 mM. The cells were cultured at 37°C and 15°C for 6 and 20 hours, respectively, to induce fusion protein expression. The cells were centrifuged at 4000 rpm for 10 minutes, the supernatant discarded, and the cells collected. The collected cells were suspended in buffer and thoroughly dissolved using an ultrasonic disruptor. The supernatant and precipitate were collected by centrifugation and dissolved in buffer. The precipitate was then prepared for sample preparation and gel loading.

[0048] The expression and identification results of Fab-BH3 fusion protein are shown in the figure Figure 3 China A and Figure 3 As shown in B.

[0049] 2. Large-scale expression and affinity chromatography purification of Fab-BH3 fusion protein: Select the optimally expressing clone and culture it at 37°C in a medium containing the appropriate antibiotic. When the OD value reaches 0.6-0.8, add 1.0 mM IPTG. Cultivate overnight at 15°C for large-scale expression. Collect the cells by centrifugation. The collected sample is then affinity purified.

[0050] Cells were lysed with buffer, disrupted by sonication, and the supernatant crude protein was collected by centrifugation. Ni-NTA packing was loaded onto a column, and the column was washed and equilibrated with binding buffer. The crude protein was incubated with the equilibrated column packing, and the flow-through was collected. The column was washed with binding buffer, and the column was washed with wash buffer, and the flow-through was collected. The column was eluted with elution buffer, and the flow-through was collected. The crude protein and flow-through fractions were processed separately, and samples were prepared for SDS-PAGE analysis. Purification was then performed by gel filtration chromatography (Superdex75) (PBS, 8 M urea, pH 7.4), and samples were prepared for SDS-PAGE analysis.

[0051] The results of purification analysis of Fab-BH3 fusion protein are as follows Figure 3 Middle C and Figure 3 As shown in D.

[0052] 3. Small-scale and large-scale expression of L11P and affinity chromatography purification: L11P was formed in the same manner as the Fab-BH3 fusion protein, and the results were as follows Figure 4 shown.

[0053] like Figure 5 and Figure 6 As shown, after purification, the Fab-BH3 fusion protein and L11P fusion protein showed obvious bands near the theoretical molecular weight by SDS-PAGE electrophoresis analysis (as shown in Figure 5 China A and Figure 5In B), it can be preliminarily determined that the fusion protein has been successfully purified (e.g. Figure 6 China A and Figure 6 Middle B).

[0054] Experimental Example 2 Eukaryotic plasmid transfection experiment: The recombinant plasmid vector prepared in Example 1 and the recombinant plasmid vector prepared in Comparative Example 1 were transferred into A549 cells, starved for 4 h before transfection, and then transfected with PEI reagent. 24 h after transfection, the cells were stained with PI and Hoechst and observed under a microscope.

[0055] The results are as follows Figure 7 As shown, GFP is green fluorescence, proving the successful transfection of the plasmid, PI is live-dead cell staining and Hoechst staining was performed, proving the successful transfer of the two fusion proteins Fab-BH3 of Example 1 and L11P of Comparative Example 1, and that the mutant plasmid group containing L11P of Comparative Example 1 did not undergo cell apoptosis, while the recombinant plasmid group containing Fab-BH3 of Example 1 underwent cell apoptosis.

[0056] Experimental Example 3 Toxicity testing of Fab-BH3 fusion protein and L11P: A549 cells (human alveolar basal epithelial cells of lung cancer) in good logarithmic growth phase were selected for the experiment, and the cell concentration was adjusted to 1×10 5 Cells were seeded into a 96-well plate at a volume of 100 μL per well. During the addition process, the cells were mixed at all times to prevent changes in cell concentration. The cells were shaken horizontally several times, back and forth or left and right. The cells were cultured in a cell incubator overnight.

[0057] The Fab-BH3 fusion protein and the L11P fusion protein were dissolved in 2 mL of serum-free culture medium to form gradient concentrations for later use; after the cells adhered overnight, the 96-well plate was removed, the culture medium in the wells was discarded, 100 μL of the drug at the planned experimental concentration was added to each well, and the cells were cultured in the incubator for 24 hours; after 24 hours of drug action, 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C for another 4 hours; the cell samples were removed from the cell culture incubator, the liquid in the wells was carefully discarded, 200 μL of room temperature DMSO was added to each well, and the cells were shaken at low speed on a shaker for 20 minutes to fully dissolve the crystals; the plate absorbance was measured at 492 nm using a microplate reader.

[0058] MTT is one of the most commonly used methods for determining cell viability. A549 cells that are not killed by the drug delivery system produce blue-purple crystals. Cells that are killed by the drug delivery system lose mitochondrial activity and are unable to function, resulting in no blue-purple crystals. The blue-purple crystals produced in each experimental group were dissolved by thorough oscillation with DMSO. The absorbance of each cell group was measured at 492nm using a microplate reader. The percentage of viable cells in the sample was calculated and plotted.

[0059] The results are as follows Figure 8 Middle A, Figure 8 Middle B and Figure 8 As shown in C, it can be seen that the cell apoptosis rate in the Fab-BH3 fusion protein group gradually increased with the increase of the drug concentration, while the L11P group did not show apoptosis phenomenon with the increase of the drug concentration, which proves the pro-apoptotic effect of Fab-BH3 fusion protein.

[0060] Experimental Example 4 Evaluation of the ability of Fab-BH3 fusion protein and L11P to promote apoptosis of A549 cells: The effect of Fab-BH3 fusion protein and L11P on the apoptosis of A549 cells was evaluated by Hoechst staining. To ensure the reliability of the experiment, the experiment should be repeated at least three times. A549 cells in logarithmic growth state were selected for the experiment, and the cell suspension concentration was adjusted to 2×10 5 Inoculate cells per well, then add complete culture medium to a final volume of 1 mL per well. Shake horizontally several times to mix the cells and culture overnight. Dissolve the Fab-BH3 fusion protein and the L11P fusion protein in serum-free culture medium to the desired experimental concentration and label for later use. After the cells adhere, discard the waste liquid from the wells and add 1 mL of each solution to each well. Continue incubating for 24 hours. After 24 hours, incubate the A549 cells with Hoechst dye in the dark for 20 minutes. Wash twice with PBS. Observe and photograph at least three areas per group using a fluorescence microscope.

[0061] The Hoechst fluorescence assay is a commonly used test to determine the live or dead state of tumor cells. Hoechst fluorescent dye has good cell membrane permeability, so the nuclei of both living and apoptotic tumor cells can be stained with the dye. However, the nuclei of viable tumor cells in good condition appear as light blue circles after Hoechst staining; while the nuclei of apoptotic tumor cells are in poor condition, and due to apoptosis, the nuclear material concentrates and exhibits bright blue fluorescence. The morphology of the nuclei also often becomes abnormally fragmented or lobed. The difference in fluorescence intensity and morphological characteristics of the nuclei of normal cells and apoptotic cells after Hoechst fluorescence staining is very obvious, making them easy to distinguish. The Hoechst staining experiment was used to observe the nuclear morphology of A549 cells 24 hours after treatment with Fab-BH3 fusion protein to verify the sample's ability to promote apoptosis.

[0062] The results are as follows Figure 9 Middle A, Figure 9 Middle B and Figure 9 As shown in middle C, it can be seen that with the increase in the concentration of Fab-BH3 fusion protein administration, the number of bright blue cell nuclei gradually increases, indicating the occurrence of cell apoptosis, and the morphology of the cell nuclei also appears fragmented.

[0063] The cell colony assay is an effective method for evaluating the ability of test samples to inhibit tumor cell growth. A549 cells are dispersed as close to single cells as possible, seeded in a well plate, and cultured for seven days to observe the effects of Fab-BH3 fusion protein or L11P on colony formation. This assay can assess the proliferative capacity of viable lung cancer cell progeny through colony formation, thus avoiding experimental errors in evaluating cell viability by miscounting dead or weakened A549 cells that are unable to divide as viable cells.

[0064] Depend on Figure 10 China A and Figure 10 As shown in B, it can be observed that the Fab-BH3 fusion protein drug group has a good ability to inhibit the growth of A549 cells, while L11P does not have this ability.

[0065] like Figure 11 A in the figure shows the structural model of BH3. Figure 11 In the figure, B represents the molecular structure of BH3. Fab-BH3 fusion proteins prepared based on the amino acid sequence of BH3 can inhibit the growth of A549 cells.

[0066] In summary, the Fab-BH3 fusion protein provided by the present invention ensures that it can be specifically taken up by A549 cells without affecting the activity of cetuximab Fab, thereby enhancing the anti-tumor activity of the antibody drug. Cetuximab Fab specifically binds to the EGFR receptor expressed on the surface of normal cells and various cancer cells, and competitively blocks the binding of EGFR to other ligands, such as transforming growth factor-α (TGF-α). It is an IgG1 monoclonal antibody targeting the EGFR receptor. After specific binding, it inhibits the tyrosine kinase (TK) bound to the EGFR receptor, blocking intracellular signal transduction pathways, thereby inhibiting cancer cell proliferation, inducing cancer cell apoptosis, and reducing the production of matrix metalloproteinases and vascular endothelial growth factor. When used as a drug for the treatment of EGFR-receptor non-small cell lung cancer, the Fab-BH3 fusion protein exhibits enhanced cytotoxicity against EGFR-receptor non-small cell lung cancer and a stronger ability to induce apoptosis in EGFR-receptor cells. It can be used in the treatment of EGFR-receptor non-small cell lung cancer, providing a new breakthrough in the research of targeted anticancer drugs for EGFR-receptor non-small cell lung cancer. When preparing Fab-BH3 fusion protein, the Fab-BH3 fusion protein was expressed using an E. coli expression system, which is cheap and efficient compared to eukaryotic expression systems and other prokaryotic expression systems.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A Fab-BH3 fusion protein, characterized in that The amino acid sequence of the Fab-BH3 fusion protein is shown in SEQ ID NO.

2.

2. The method for preparing the Fab-BH3 fusion protein according to claim 1, wherein: The following steps are involved: The nucleotide sequence encoding the Fab-BH3 fusion protein is amplified and connected to a plasmid to obtain a recombinant plasmid vector; the recombinant plasmid vector is transformed into competent Escherichia coli cells of the expression system, the obtained positive transformants are subjected to prokaryotic expression, and the expression products are separated and purified to obtain the Fab-BH3 fusion protein.

3. The preparation method according to claim 2, characterized in that The nucleotide sequence encoding the Fab-BH3 fusion protein is shown in SEQ ID NO.

1.

4. Use of the Fab-BH3 fusion protein according to claim 1 in the preparation of a drug for treating non-small cell lung cancer.

5. The application according to claim 4, characterized in that: The Fab-BH3 fusion protein has stronger cytotoxicity to EGFR non-small cell lung cancer cells and stronger ability to induce apoptosis of EGFR non-small cell lung cancer cells.

6. A recombinant plasmid vector, characterized in that: The recombinant plasmid vector contains a nucleotide sequence encoding a Fab-BH3 fusion protein, and the nucleotide sequence encoding the Fab-BH3 fusion protein is shown in SEQ ID NO.

1.

7. Use of the recombinant plasmid vector according to claim 6 in the preparation of a drug for treating non-small cell lung cancer.

8. A recombinant expression strain, characterized in that: The recombinant expression strain can express Fab-BH3 fusion protein in prokaryotes, and the amino acid sequence of the Fab-BH3 fusion protein is shown in SEQ ID NO.

2.

9. Use of the recombinant expression strain according to claim 8 in the preparation of a drug for treating non-small cell lung cancer.

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