A Fab-BH3 fusion protein and its preparation method and application

Abstract

The application discloses a Fab-BH3 fusion protein and a preparation method and application thereof, belongs to the technical field of biological medicines, and particularly relates to a Fab-BH3 fusion protein, wherein the amino acid sequence of the Fab-BH3 fusion protein is shown as SEQ ID NO. 2. The application also discloses an application of the Fab-BH3 fusion protein in preparation of a drug for treating non-small cell lung cancer. The Fab-BH3 fusion protein is formed by coupling a cetuximab Fab fragment and a BH3 polypeptide, does not affect the activity of the cetuximab, inhibits the growth of non-small cell lung cancer cells with overexpressed EGFR, and improves the treatment effect on non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a Fab-BH3 fusion protein, its preparation method and application. Background Technology

[0002] Lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs. It is one of the leading causes of cancer-related deaths worldwide. The development of lung cancer is related to multiple factors, including smoking, air pollution, and occupational exposure. For early-stage non-small cell lung cancer, lobectomy is the most common surgical procedure, removing the lobe containing the tumor to clear as much tumor tissue as possible. Systematic lymph node dissection is also an important part of the surgery because 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 taxane-based drugs 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 within lung cancer cells. For example, in 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 tumor cell proliferation signaling pathways and thus inhibiting tumor growth. In many malignant tumors, EGFR is often overexpressed or mutated. This abnormal EGFR signaling leads to uncontrolled proliferation, invasion, and metastasis of cancer cells. For instance, in non-small cell lung cancer (NSCLC), approximately 10% to 30% of patients have EGFR gene mutations, which increase the dependence of cancer cells on EGFR signaling. In some tumors with high EGFR expression or mutations, EGFR antibodies can be used as monotherapy. For example, cetuximab has some application in the treatment of head and neck squamous cell carcinoma; it can control tumor growth by blocking EGFR signaling, inhibiting tumor cell proliferation, and inducing tumor cell apoptosis. More often, EGFR antibodies are used in combination with other treatment methods. When combined with chemotherapy, such as in the treatment of colorectal cancer, EGFR antibodies combined with chemotherapy drugs (such as the FOLFOX or FOLFIRI regimen) can improve treatment efficacy.

[0003] The Fab (fragment of antigen binding) segment is formed when papain cleaves the immunoglobulin (Ig) near the N-terminus of the inter-heavy chain disulfide bond in the hinge region, creating two identical monovalent antigen-binding fragments.

[0004] BH3 peptides are a class of relatively small peptide sequences that are often important domains of members of a family of intracellular apoptosis regulatory proteins. The Bcl-2 family of proteins plays a crucial role in the intrinsic pathway of apoptosis, and this family includes anti-apoptotic proteins (such as Bcl-2 and Bcl-xL) and pro-apoptotic proteins. The BH3 domain is primarily found in pro-apoptotic proteins, such as Bax and Bak. In the 1990s, multiple laboratories, through sequence alignment and functional analysis of Bcl-2-related proteins, determined the key role of the BH3 domain in apoptosis signaling. For example, studies of tumor cell lines with apoptosis defects have revealed that mutations in certain genes affect the function of proteins containing the BH3 domain, thus highlighting its importance in apoptosis regulation. BH3 peptides can mimic the BH3 domain of endogenous pro-apoptotic proteins, binding to anti-apoptotic Bcl-2 proteins (such as Bcl-2 and Bcl-xL), thereby releasing the pro-apoptotic proteins Bax and Bak, initiating mitochondrial-mediated apoptosis. BH3 peptides can be used in combination with other anticancer therapies, such as chemotherapy drugs. Chemotherapy drugs often cause damage to tumor cells and activate intracellular apoptosis signaling pathways. BH3 peptides can enhance the apoptosis signaling in these pathways and overcome the drug resistance of tumor cells. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a Fab-BH3 fusion protein, its preparation method, and its application. By coupling the cetuximab Fab fragment with the BH3 peptide to form the 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 overexpressing EGFR, thereby improving the therapeutic effect of non-small cell lung cancer.

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

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

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

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

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

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

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

[0013] The present invention also provides the application of the recombinant plasmid vector in the preparation of a drug for treating non-small cell lung cancer.

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

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

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] This invention provides a Fab-BH3 fusion protein, formed by conjugating a cetuximab Fab fragment to a BH3 peptide. The heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment are linked by a linker, a His tag is added to the N-terminus, and the C-terminus is linked to the BH3 peptide via a linker. By forming a fusion protein of BH3 peptide and cetuximab Fab fragment without affecting Fab activity, the antitumor activity of the antibody drug is enhanced. The Fab-BH3 fusion protein has the same affinity as the antibody but exhibits stronger cytotoxicity against EGFR-overexpressing non-small cell lung cancer (NSCLC), while having no effect on the growth of EGFR-negative cells, thus reducing adverse reactions. The Fab-BH3 fusion protein inhibits the growth of NSCLC cells overexpressing EGFR, improving the therapeutic effect of NSCLC. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The images show the enzyme digestion diagrams of the recombinant plasmid vector of Example 1 and the recombinant plasmid vector of Comparative Example 1, where 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.

[0020] Figure 2 The images are of the recombinant plasmid vector of Example 1 and the recombinant plasmid vector of Comparative Example 1, where A is the image of the recombinant plasmid vector of Example 1 and B is the image of the recombinant plasmid vector of Comparative Example 1.

[0021] Figure 3 Figure A shows the expression, identification, and purification analysis of the Fab-BH3 fusion protein. In Figure B, M represents the marker, 1 represents the uninduced sample, and 2-6 represent the induced samples. Figure B shows the optimization and solubility analysis results of the Fab-BH3 fusion protein. In Figure B, M represents the marker, 1 represents the sample induced at 15℃ with 0.2mM IPTG, 2 represents the sample induced at 15℃ with 1.0mM IPTG, 3 represents the sample induced at 37℃ with 0.2mM IPTG, 4 represents the sample induced at 37℃ with 1.0mM IPTG, 5 represents the uninduced sample, 6 represents the precipitate after induction at 37℃ with 1.0mM IPTG, 7 represents the supernatant after induction at 37℃ with 1.0mM IPTG, 8 represents the precipitate after induction at 37℃ with 0.2mM IPTG, 9 represents the supernatant after induction at 37℃ with 0.2mM IPTG, and 10 represents the sample induced at 15℃ with 1.0mM IPTG. IPTG-induced precipitate, 11 represents the supernatant after 1.0 mM IPTG induction at 15℃, 12 represents the precipitate after 0.2 mM IPTG induction at 15℃, 13 represents the supernatant after 0.2 mM IPTG induction at 15℃, C is the SDS-PAGE analysis diagram of Fab-BH3 fusion protein purified by nickel agarose affinity chromatography, where M represents Marker, 1 represents precipitate after disruption, 2 represents supernatant after disruption, 3 represents eluent, 4 and 5 represent washing samples, 6 represents elution sample, D is the SDS-PAGE analysis diagram of Fab-BH3 fusion protein purified by gel filtration chromatography, where M represents Marker, S represents sample before gel filtration chromatography purification, B4, B2, C1, C5, C9, C12, D10, D7, D4, D1, E2, E5 and E8 represent protein collection samples;

[0022] Figure 4Figure A shows the expression, identification, and purification analysis of L11P prepared in Comparative Example 1. In Figure B, M represents the marker, 1 represents the uninduced sample, and 2-6 represent the induced samples. Figure B shows the optimization and solubility analysis results of L11P. In Figure B, M represents the marker, 1 represents the sample induced at 15℃ with 0.2mM IPTG, 2 represents the sample induced at 15℃ with 1.0mM IPTG, 3 represents the sample induced at 37℃ with 0.2mM IPTG, 4 represents the sample induced at 37℃ with 1.0mM IPTG, 5 represents the uninduced sample, 6 represents the precipitate after induction at 37℃ with 1.0mM IPTG, 7 represents the supernatant after induction at 37℃ with 1.0mM IPTG, 8 represents the precipitate after induction at 37℃ with 0.2mM IPTG, 9 represents the supernatant after induction at 37℃ with 0.2mM IPTG, and 10 represents the sample induced at 15℃ with 1.0mM IPTG. IPTG-induced precipitate sample, 11 represents the supernatant sample after 1.0mM IPTG induction at 15℃, 12 represents the precipitate sample after 0.2mM IPTG induction at 15℃, 13 represents the supernatant sample after 0.2mM IPTG induction at 15℃, C is the SDS-PAGE analysis diagram of L11P nickel agarose affinity chromatography purification, in which M represents the marker, 1 represents the precipitate after disruption, 2 represents the supernatant after disruption, 3 represents the eluent, 4 and 5 represent the washing sample, 6 represents the elution sample, D is the SDS-PAGE analysis diagram of L11P gel filtration chromatography purification, in which M represents the 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;

[0023] Figure 5 The images show SDS-PAGE analysis of the Fab-BH3 fusion protein and the purified L11P protein. In the images, A represents the Fab-BH3 fusion protein, M represents the marker, and S represents the target fusion protein. In the images, B represents L11P, M represents the marker, and S represents the target fusion protein.

[0024] Figure 6 The images show Western blot diagrams of the Fab-BH3 fusion protein and L11P, where A represents the Fab-BH3 fusion protein and B represents L11P.

[0025] Figure 7 The image shows fluorescence of eukaryotic plasmid transfection. In the image, A549-MUT represents A549 cells transfected with the L11P mutant plasmid of Comparative Example 1, and A549-pDNA represents A549 cells transfected with the recombinant plasmid Fab-BH3 of Example 1. The scale bar is 50 μm.

[0026] Figure 8The graph shows the results of the MTT cytotoxicity assay. In the graph, A represents the MTT results for the Fab-BH3 fusion protein, where anti-EGFR-Fab-BH3 represents the Fab-BH3 fusion protein. " represents p<0.01", "" indicates 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.

[0027] Figure 9 The images show Hoechst fluorescence assays. In the figure, A represents the killing effect of different concentrations of Fab-BH3 fusion protein on A549 cells, B is a comparison of the killing effects of different concentrations of Fab-BH3 fusion protein and L11P on A549 cells, aEFB represents Fab-BH3 fusion protein, aEFBM represents L11P, and C represents the killing effect of different concentrations of Fab-BH3 fusion protein on A549 cells and CHO 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. The scale bar is 50 μm.

[0028] Figure 10 The results are shown in the cell colony assay. Figure A shows the inhibition of A549 cell migration by different concentrations of Fab-BH3 fusion protein and L11P, where aEFB represents Fab-BH3 fusion protein and aEFBM represents L11P, with a scale bar of 50 μm. Figure B shows the statistical effect of different concentrations of Fab-BH3 fusion protein and L11P on A549 cell migration inhibition, where aEFB represents Fab-BH3 fusion protein and aEFBM represents L11P. " represents p<0.01", "" represents p < 0.001;

[0029] Figure 11 Here are the structural schematic diagram and molecular structure diagram of BH3, where A is the structural schematic diagram of BH3 and B is the molecular structure diagram of BH3. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] Example 1

[0036] Preparation of Fab-BH3 fusion protein:

[0037] The nucleotide sequence encoding the Fab-BH3 fusion protein (SEQ ID NO.1) was double-digested and ligated into plasmid PET22b to obtain a recombinant plasmid vector (e.g., Figure 1 China A and Figure 2 (A) The recombinant plasmid vector was transformed into E. coli competent cells BL21(DE3) of the expression system. The obtained positive transformants were expressed in prokaryotes. The expression products were isolated and purified to obtain the Fab-BH3 fusion protein (its amino acid sequence is shown in SEQ ID NO.2).

[0038] The recombinant plasmid vector construction specifically involves: using a plasmid containing the nucleotide sequence of the Fab-BH3 fusion protein and a PET22b plasmid respectively... Nde I and XhoAfter double digestion with restriction endonucleases, agarose gel electrophoresis was performed to extract the 5000bp and 1500bp bands. The DNA was recovered using a gel extraction kit. Ecoli E DNA ligase ligates the target gene and the vector to obtain a recombinant plasmid vector, which is then used for subsequent transformation experiments.

[0039] The transformation process was as follows: 10 μL of the recombinant plasmid vector was added to 100 μL of BL21 (DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 60 s, spread onto ampicillin-resistant LB liquid medium (100 µg / mL ampicillin), and cultured overnight at 37°C. Single clones were then picked and inoculated into fresh medium and cultured overnight at 37°C.

[0040] The prokaryotic expression process involves transferring the overnight cultured bacterial suspension to fresh ampicillin-resistant LB liquid medium (50 µg / mL ampicillin) at a 1:100 volume ratio and incubating at 37°C until OD500. 600 Within the range of 0.6~0.8, add 1mM isopropyl-β-D-thiogalactoside (IPTG), induce at 15℃ for 16h, centrifuge at 4000rpm for 10min, discard the supernatant, and collect the bacteria.

[0041] The separation and purification process was as follows: the prokaryotic expression cells were first resuspended in buffer, then sonicated for 15 min at 130 W, centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was purified by nickel column affinity chromatography using binding buffer and elution buffer to obtain high-purity Fab-BH3 fusion protein.

[0042] Nickel column affinity chromatography purification: Pack Ni-NTA packing material into a column and wash and equilibrate the column with binding buffer; incubate the crude protein with the equilibrated packing material and collect the eluent; wash and equilibrate the column with binding buffer; wash the column with washing buffer and collect the eluent; elute with elution buffer and collect the eluent; process the crude protein and eluent separately, prepare samples to analyze the purification effect, and refold the target protein using dialysis buffer (PBS, 8M / 4M / 1M / 0M urea, pH 8) to obtain the Fab-BH3 fusion protein.

[0043] The lysis buffer was prepared by 8M urea, 50mM Tris, 300mM NaCl, and 0.1% Triton X-100, with water as the solvent and pH 8; the binding buffer was prepared by 8M urea, 50mM Tris, and 300mM NaCl, with water as the solvent and pH 8.0; the elution buffer was prepared by 8M urea, 50 / 100 / 200 / 500mM Tris, 300mM NaCl, and 200mM imidazole, with water as the solvent and pH 8.0.

[0044] The Fab-BH3 fusion protein is composed of the heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment connected by a linker, a His tag added to the N-terminus, and the C-terminus connected to the BH3 peptide by a linker to form the fusion protein Fab-BH3.

[0045]

[0046] SEQ ID NO.2: MHHHHHHEFDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAGGGGSGGGGSGGGGSQVQLKQSGPG LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSGGGGSGGGGSGGGGSPLGLAGDASTKKLSECLRRIGDELDS.

[0047] As shown in SEQ ID NO.2 above, the bolded part is the His tag, the single underlined part is the light chain variable region of the cetuximab Fab fragment, the bolded and underlined part is the linker, the double underlined part is the heavy chain variable region of the cetuximab Fab fragment, the wavy line part is the MMP-2 restriction site, the background part is the BH3 polypeptide, and the background and underlined part is the mutation point of the BH3 polypeptide.

[0048] Comparative Example 1

[0049] Preparation of Fab-mutant BH3 fusion protein (L11P):

[0050] The nucleotide sequence encoding L11P was double-digested (SEQ ID NO.3) and ligated into plasmid PET22b to obtain a recombinant plasmid vector (e.g., Figure 1 China B and Figure 2 (B) The recombinant plasmid vector was transformed into E. coli competent cells BL21(DE3) of the expression system. The obtained positive transformants were expressed in prokaryotes. The expression products were isolated and purified to obtain L11P (the amino acid sequence of which is shown in SEQ ID NO.4).

[0051] The recombinant plasmid vector construction specifically involves: using a plasmid containing the L11P nucleotide sequence and a PET22b plasmid respectively... Nde I and Xho After double digestion with restriction endonucleases, agarose gel electrophoresis was performed to extract the 5000bp and 1500bp bands. The DNA was recovered using a gel extraction kit. Ecoli E DNA ligase ligates the target gene and the vector to obtain a recombinant plasmid vector, which is then used for subsequent transformation experiments.

[0052] The transformation process was as follows: 10 μL of the recombinant plasmid vector was added to 100 μL of BL21 (DE3) competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 60 s, spread onto ampicillin-resistant LB liquid medium (100 µg / mL ampicillin), and cultured overnight at 37°C. Single clones were then picked and inoculated into fresh medium and cultured overnight at 37°C.

[0053] The prokaryotic expression process involves transferring the overnight cultured bacterial suspension to fresh ampicillin-resistant LB liquid medium (50 µg / mL ampicillin) at a 1:100 volume ratio and incubating at 37°C until OD500. 600 Within the range of 0.6~0.8, add 1mM isopropyl-β-D-thiogalactoside (IPTG), induce at 15℃ for 16h, centrifuge at 4000rpm for 10min, discard the supernatant, and collect the bacteria.

[0054] The separation and purification process was as follows: the prokaryotic cells were first resuspended in buffer solution, then sonicated for 15 min at 130 W, centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was purified by nickel column affinity chromatography using binding buffer and elution buffer to obtain high-purity L11P.

[0055] Nickel column affinity chromatography purification: Pack Ni-NTA packing material into a column and wash and equilibrate the column with binding buffer; incubate crude protein with the equilibrated packing material and collect the eluent; wash and equilibrate the column with binding buffer; wash the column with washing buffer and collect the eluent; elute with elution buffer and collect the eluent; process the crude protein and eluent separately, prepare samples to analyze the purification effect, and refold the target protein using dialysis buffer (PBS, 8 / 4 / 2 / 1 / 0M urea, pH 8) to obtain L11P.

[0056] The lysis buffer was prepared by 8M urea, 50mM Tris, 300mM NaCl, and 0.1% Triton X-100, with water as the solvent and pH 8; the binding buffer was prepared by 8M urea, 50mM Tris, and 300mM NaCl, with water as the solvent and pH 8.0; the elution buffer was prepared by 8M urea, 50 / 100 / 200 / 500mM Tris, 300mM NaCl, and 200mM imidazole, with water as the solvent and pH 8.0.

[0057] L11P is specifically composed of the heavy chain variable region (VH) and light chain variable region (VL) of the cetuximab Fab fragment connected by a linker, with a His tag added to the N-terminus, and the C-terminus linked to a mutated BH3 peptide to form the fusion protein L11P.

[0058]

[0059] SEQ ID NO.4: MHHHHHHEFDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAGGGGSGGGGSGGGGSQVQLKQSGPG LVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSGGGGSGGGGSGGGGSPLGLAGDASTKKLSECPRRIGDELDS.

[0060] As shown in SEQ ID NO.4 above, the bolded part is the His tag, the single underlined part is the light chain variable region of the cetuximab Fab fragment, the bolded and underlined part is the linker, the double underlined part is the heavy chain variable region of the cetuximab Fab fragment, the wavy line part is the MMP-2 restriction site, the background part is the mutated BH3 polypeptide, and the background and underlined part is the mutation point of the BH3 polypeptide.

[0061] Experimental Example 1

[0062] Expression and identification of Fab-BH3 fusion protein and L11P:

[0063] 1. Low-level expression of the Fab-BH3 fusion protein:

[0064] The recombinant plasmid vector was transformed into competent E. coli BL21(DE3) cells. After heat shock at 42°C, the cells were plated on plates containing 50 µg / mL ampicillin and cultured at 37°C for 20 h. Five single colonies were picked from each cell and cultured in five tubes of liquid culture medium containing antibiotics at 37°C. When the OD value reached 0.6, 0.5 mM IPTG was added as an inducer and cultured at 37°C for 6 h. The cells without an inducer served as a negative control. A total of six tubes of samples were collected by centrifugation.

[0065] When the bacterial culture reached an OD value of 0.6–0.8, IPTG was added to final concentrations of 0.2 mM and 1.0 mM, respectively, and incubated at 37°C and 15°C for 6 h and 20 h, respectively, to induce fusion protein expression. The cells were then centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected. Buffer was added to the collected bacterial cells for resuscitation, and the cells were thoroughly dissolved using an ultrasonic homogenizer. The supernatant and precipitate were collected by centrifugation, and the precipitate was dissolved in buffer. The supernatant and precipitated proteins were then prepared separately for gel electrophoresis.

[0066] The expression identification results of the Fab-BH3 fusion protein are shown in the figure below. Figure 3 China A and Figure 3 As shown in B.

[0067] 2. Large-scale expression and affinity chromatography purification of the Fab-BH3 fusion protein:

[0068] The optimal expression clone strain was selected and cultured at 37°C in a medium containing the corresponding antibiotic. When the OD value reached 0.6–0.8, 1.0 mM IPTG was added, and the culture was carried out overnight at 15°C for mass expression. The cells were collected by centrifugation. The collected samples were processed and then purified using affinity chromatography.

[0069] Cellular cells were dissolved in buffer, sonicated, and the supernatant crude protein was collected by centrifugation. Ni-NTA packing material was packed into a column, and the column was washed and equilibrated with binding buffer. The crude protein was incubated with the equilibrated column packing material, and the eluent was collected. The column was washed and equilibrated with binding buffer. The column was washed with washing buffer, and the eluent was collected. Elution was performed with elution buffer, and the eluent was collected. The crude protein and eluent fractions were processed separately, and samples were prepared for SDS-PAGE analysis. Then, gel filtration chromatography (Superdex 75) was performed for purification (PBS, 8M urea, pH 7.4), and samples were prepared for SDS-PAGE analysis.

[0070] The purification and analysis results of the Fab-BH3 fusion protein are as follows: Figure 3 C and Figure 3 As shown in D.

[0071] 3. Low-level and high-level expression of L11P and affinity chromatography purification:

[0072] The formation of L11P is similar to that of the Fab-BH3 fusion protein, and the results are as follows: Figure 4 As shown.

[0073] like Figure 5 and Figure 6 As shown, the Fab-BH3 fusion protein and the L11P fusion protein, after purification, showed distinct bands near their theoretical molecular weights according to SDS-PAGE electrophoresis analysis (e.g., Figure 5 China A and Figure 5 (e.g., B) can be used to preliminarily determine that the fusion protein has been successfully purified. Figure 6 China A and Figure 6 (B)

[0074] Experiment Example 2

[0075] Eukaryotic plasmid transfection experiment: The recombinant plasmid vector prepared in Example 1 and the recombinant plasmid vector prepared in Comparative Example 1 were transfected into A549 cells. The cells were starved for 4 hours before transfection, and then transfected with PEI transfection reagent. After 24 hours of transfection, the cells were stained with PI and Hoechst and observed under a microscope.

[0076] The results are as follows Figure 7 As shown, GFP is green fluorescence, proving the successful transfection of the plasmid. PI is live and dead cell staining and Hoechst staining, proving the successful transfection of the two fusion proteins Fab-BH3 of Example 1 and L11P of Comparative Example 1. Furthermore, no apoptosis occurred in the mutant plasmid group containing L11P of Comparative Example 1, while apoptosis occurred in the recombinant plasmid group containing Fab-BH3 of Example 1.

[0077] Experimental Example 3

[0078] Toxicity testing of Fab-BH3 fusion protein and L11P:

[0079] A549 cells (human alveolar basal epithelial cells for lung cancer) in good logarithmic growth phase were selected for the experiment, and the cell concentration was adjusted to 1×10⁻⁶. 5 Cells / mL. Seed 100 μL of cell suspension into each well of a 96-well plate. During the sample addition process, be sure to mix the cells frequently to prevent changes in cell concentration. Shake the plate horizontally a few times back and forth or left and right. Incubate overnight in a cell culture incubator.

[0080] The Fab-BH3 fusion protein and the L11P fusion protein were dissolved in 2 mL of serum-free medium to prepare gradient concentrations for later use. After the cells adhered overnight, the 96-well plate was removed, the culture medium in the wells was discarded, and 100 μL of the planned concentration of the drug was added to each well. The cells were then cultured in an incubator for 24 h. After 24 h of drug treatment, 10 μL of MTT solution was added to each well, and the cells were incubated at 37 °C for another 4 h. The cell samples were removed from the cell culture incubator, the liquid in the wells was carefully discarded, and 200 μL of room temperature DMSO was added to each well. The cells were then shaken at low speed for 20 min on a shaker to fully dissolve and crystallize the cells. The absorbance of the plate was measured using a microplate reader at a wavelength of 492 nm.

[0081] MTT assay is one of the most commonly used methods for assessing cell viability. A549 cells that are not killed by the drug delivery system will produce blue-purple crystals. Cells that can be killed by the drug delivery system will have their mitochondria inactive and unable to function, thus failing to produce blue-purple crystals. The blue-purple crystals produced in each experimental group were dissolved by thorough shaking with DMSO. The absorbance of each group of cells was measured at 492 nm using a microplate reader. The percentage of viable cells in the sample was calculated and plotted.

[0082] The results are as follows Figure 8 China A Figure 8 China B and Figure 8 As shown in Figure C, it can be seen that the apoptosis rate of the Fab-BH3 fusion protein group gradually increased with the increase of the drug concentration, while no apoptosis phenomenon was observed in the L11P group with the increase of the drug concentration, which proves the apoptosis-promoting effect of the Fab-BH3 fusion protein.

[0083] Experiment Example 4

[0084] Evaluation of the ability of Fab-BH3 fusion protein and L11P to promote apoptosis in A549 cells:

[0085] The effects of Fab-BH3 fusion protein and L11P on the pro-apoptosis of A549 cells were evaluated using Hoechst staining assays. To ensure experimental reliability, the experiments were repeated at least three times. A549 cells in logarithmic growth phase were selected for the experiment, and the cell suspension concentration was adjusted to 2 × 10⁻⁶. 5 Seed cells per well, then add complete culture medium to a final volume of 1 mL per well. Gently shake horizontally a few times to mix the cells, and incubate overnight. Dissolve the Fab-BH3 and L11P fusion proteins in serum-free medium to the required concentrations, and label them for later use. After cell attachment, discard the waste liquid in the wells, add 1 mL of the solution to each well, and continue culturing for 24 h. After 24 h, incubate A549 cells with Hoechst dye in the dark for 20 min, and wash twice with PBS after incubation. Observe and photograph at least three areas in each group using a fluorescence microscope.

[0086] The Hoechst fluorescence assay is a commonly used test to determine the viability of tumor cells. Hoechst fluorescent dye has good cell membrane permeability, so it can stain the nuclei of both viable and apoptotic tumor cells. However, the nuclei of healthy, viable tumor cells appear as pale blue and round after Hoechst staining; while the nuclei of apoptotic tumor cells are in poor condition, exhibiting bright blue fluorescence due to the concentration of intranuclear material caused by apoptosis, and their morphology often becomes abnormally fragmented or lobed. The difference in fluorescence intensity and morphological characteristics between normal and apoptotic cell nuclei after Hoechst staining is significant, making them easily distinguishable. This study used Hoechst staining to observe the nuclear morphology of A549 cells treated with Fab-BH3 fusion protein for 24 hours to verify the sample's ability to promote apoptosis.

[0087] The results are as follows Figure 9 China A Figure 9 China B and Figure 9 As shown in Figure C, it can be seen that as the concentration of Fab-BH3 fusion protein increases, the number of bright blue cell nuclei gradually increases, indicating the occurrence of apoptosis, and the cell nuclei also show a fragmented morphology.

[0088] Cell colony assays are an effective method for evaluating the ability of test samples to inhibit tumor cell growth. A549 cells are dispersed into single-cell states and seeded in wells of a plate. After seven days of continuous culture, the effects of the Fab-BH3 fusion protein or L11P on colony formation are observed. This experiment can examine the progeny proliferation capacity of viable lung cancer cells through cell colony formation, thus avoiding experimental errors in evaluating cell viability by mistakenly counting dead cells or A549 cells in poor condition that cannot continue dividing as viable cells.

[0089] Depend on Figure 10 China A and Figure 10 As shown in Figure 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.

[0090] like Figure 11 Figure A shows a structural schematic diagram of BH3. Figure 11 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.

[0091] In summary, the Fab-BH3 fusion protein provided by this invention ensures that the activity of cetuximab Fab is not affected, and that the Fab-BH3 fusion protein can be specifically taken up by A549 cells, enhancing the antitumor activity of the antibody drug. Cetuximab Fab can specifically bind to the EGFR receptor expressed on the surface of normal cells and various cancer cells, and competitively block the binding of EGFR and other ligands, such as α-transforming growth factor (TGF-α). It is an IgG1 monoclonal antibody targeting the EGFR receptor. After specific binding, it inhibits intracellular signal transduction pathways by inhibiting tyrosine kinase (TK) bound to the EGFR receptor, thereby inhibiting cancer cell proliferation, inducing cancer cell apoptosis, and reducing the production of matrix metalloproteinases and vascular endothelial growth factor. When the Fab-BH3 fusion protein is used as a drug to treat EGFR non-small cell lung cancer, it exhibits stronger cytotoxicity and apoptosis-inducing ability in EGFR non-small cell lung cancer cells, providing a new breakthrough for the research of targeted anticancer drugs for EGFR non-small cell lung cancer. When preparing the Fab-BH3 fusion protein, the E. coli expression system was used to express the Fab-BH3 fusion protein. Compared with eukaryotic expression systems and other prokaryotic expression systems, it has the characteristics of being inexpensive and efficient.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope 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 as described in claim 1, characterized in that, Includes the following steps: The nucleic acid encoding the Fab-BH3 fusion protein was amplified and ligated into a plasmid to obtain a recombinant plasmid vector. The recombinant plasmid vector was transformed into competent E. coli cells of the expression system, and the obtained positive transformants were expressed in prokaryotes. The expression products were isolated and purified to obtain the Fab-BH3 fusion protein.

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

1.

4. The use of the Fab-BH3 fusion protein as described in claim 1 in the preparation of a medicament for treating non-small cell lung cancer.

5. A recombinant plasmid vector, characterized in that, The recombinant plasmid vector contains a sequence of nucleic acid encoding the Fab-BH3 fusion protein, the nucleotide sequence of which is shown in SEQ ID NO.

1.

6. The use of the recombinant plasmid vector as described in claim 5 in the preparation of a medicament for treating non-small cell lung cancer.

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

2.

8. The use of the recombinant expression strain as described in claim 7 in the preparation of a medicament for treating non-small cell lung cancer.

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