Specific mgt polypeptides, antibodies thereto and use in the clinic

Abstract

The present application relates to the field of biological medical technology, and more particularly to specific MGMT polypeptide, its antibody and application in clinic. The present application obtains polypeptide combination for malignant glioma prediction by screening after imbricate cutting, and then uses MGMT-02 and MGMT-04 in the polypeptide combination to prepare monoclonal antibody; the monoclonal antibody is coupled with TAT cell-penetrating peptide, and then is complexed with cell nanometer iron oxide particles, so that the complex formed can be used for reversing TMZ drug resistance by targeting intranuclear MGMT of TMZ drug-resistant GBM cell line under the premise of solving brain targeting delivery, and provides a new solution for developing targeted synergistic therapy for TMZ drug-resistant GBM in clinic, and has important translational medicine value and great industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to specific MGMT peptides, their antibodies, and their clinical applications. Background Technology

[0002] Glioblastoma (GBM) is a primary malignant brain tumor—glioma. Currently, the standardized treatment regimens for GBM both domestically and internationally mainly include: (1) maximally and safely resecting the tumor. (2) Postoperative routine intensity-modulated radiotherapy (IMRT) of the tumor target area combined with concurrent chemoradiotherapy with temozolomide (TMZ). (3) Subsequent adjuvant chemotherapy using a standardized 6-cycle regimen with TMZ (Stupp regimen). (4) Electric field therapy, targeted drug therapy, and immunotherapy, which have been developed in recent years. Although standardized therapies and promising new therapies are used, the overall efficacy is still very disappointing.

[0003] With the support of advanced surgical equipment and sophisticated surgical techniques, neurosurgeons have achieved near-total and total tumor resection as seen in imaging, reaching a level of surgical precision. The efficacy of radiotherapy is often limited by dosage due to radiation damage. Therefore, in the traditional cancer treatment paradigm, chemotherapy is extremely important for glioblastoma. However, the blood-brain barrier, the toxicity of chemotherapy drugs, and drug resistance significantly limit the true effectiveness of chemotherapy.

[0004] TMZ is currently a first-line chemotherapy drug in the STUPP regimen recommended for treating GBM both domestically and internationally. However, resistance to TMZ in GBM patients is a significant reason for poor chemotherapy outcomes. Studies have found that O 6 -Methylguanine-DNAtransferase (O 6 2-Methylguanine-DNA methyltransferase (MGMT)-mediated acquired resistance to TMZ is primarily caused by the repair of DNA damage within the nucleus to resist alkylating agent-induced DNA nucleotide methylation. Therefore, overcoming resistance to TMZ chemotherapy in glioblastoma has become a challenging problem that urgently needs to be solved in clinical treatment.

[0005] In their study, "Framework nucleic acid-based nanoparticles enhance temozolomide sensitivity in glioblastoma," Guo Hongbo et al. from Zhujiang Hospital of Southern Medical University developed a framework nucleic acid nanoparticle (FNN) based on a six-helix DNA bundle for loading and delivering siMGMT. The sidewalls of the DNA bundle were designed with a double-lock switch structure, while the vascular-targeting peptide Angiopep-2 (ANG) was conjugated to both ends of the DNA bundle. Before entering tumor tissue, the FNN effectively protected siMGMT from in vivo degradation. Guided by the ANG-targeting peptide, the siMGMT-loaded FNN penetrated the blood-brain barrier and targeted GBM. Upon reaching the tumor site, the overexpressed nucleotide-binding protein (NCL) in the tumor unlocked the double-lock switch, exposing the encapsulated siMGMT. This unique switching mechanism enabled siMGMT to be targeted and delivered to GBM cells, successfully silencing MGMT, enhancing TMZ sensitivity, and thus improving the therapeutic effect of TMZ. However, this study has some limitations: the siRNA strategy is a relatively "crude" intervention, aiming to knock down the entire MGMT protein. While effective, siRNA lacks fine-grained differentiation of specific functional domains of proteins. Furthermore, siRNA carries the risk of off-target effects, potentially silencing other genes with homologous sequences and causing unpredictable side effects. Additionally, siRNA needs to enter the cytoplasm, integrate into the RNA-induced silencing complex, and degrade mRNA; this process is time-delayed and cannot directly and rapidly neutralize existing MGMT proteins like antibodies.

[0006] Therefore, it is particularly necessary to develop targeted therapeutic drugs for glioblastoma. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a specific MGMT peptide, its antibody and its clinical application.

[0008] The present invention provides a polypeptide combination for the diagnosis of glioma patients, the polypeptide combination comprising: MGMT-02 polypeptide with amino acid sequence as shown in SEQ ID NO:2, MGMT-04 polypeptide with amino acid sequence as shown in SEQ ID NO:4, MGMT-07 polypeptide with amino acid sequence as shown in SEQ ID NO:7, MGMT-10 polypeptide with amino acid sequence as shown in SEQ ID NO:10, and / or MGMT-18 polypeptide with amino acid sequence as shown in SEQ ID NO:18.

[0009] The polypeptide combination can also be referred to as a biomarker combination.

[0010] This invention involves slicing the MGMT protein into 20 peptides using a shingled method. The coverage of these peptides was then assessed in subjects using the response of each peptide's autoantibody to malignant glioma (glioma) and healthy serum samples. The results showed that, compared with healthy controls, the peptides MGMT-02 (anti-MGMT-02), MGMT-04 (anti-MGMT-04), MGMT-07 (anti-MGMT-07), MGMT-10 (anti-MGMT-10), and MGMT-18 (anti-MGMT-18) exhibited higher coverage among the 20 peptides.

[0011] Furthermore, the individual effects of the above five peptides are not accurate enough for predicting malignant gliomas. When the five peptides are combined as a multi-indicator diagnostic model, the area under the curve increases to 0.810 (95% confidence interval is 0.752~0.869). This diagnostic combination has a specificity of 83% and a corresponding sensitivity of 75%. This diagnostic model achieves moderate diagnostic efficacy and can be used as a biomarker in the diagnosis of gliomas.

[0012] This invention provides the application of polypeptide combinations as biomarkers in the preparation of products for predicting malignant tumors.

[0013] The present invention provides an antibody obtained by immunizing mice with any polypeptide from the polypeptide combination described in the present invention as an immunogen.

[0014] Furthermore, in a specific embodiment of the present invention, the antibody includes antibody 1 (mAb1, or mAb1#scfV), antibody 2 (mAb2, mAb2#scfV) and antibody 3 (mAb3, or mAb3#scfV).

[0015] The amino acid sequence of CDR1 of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:26, the amino acid sequence of CDR2 is shown in SEQ ID NO:27, and the amino acid sequence of CDR3 is shown in SEQ ID NO:28.

[0016] The amino acid sequence of CDR1 of the light chain variable region of antibody 1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:33.

[0017] The amino acid sequence of CDR1 of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:38, the amino acid sequence of CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is shown in SEQ ID NO:40.

[0018] The amino acid sequence of CDR1 of the light chain variable region of antibody 2 is shown in SEQ ID NO:42, the amino acid sequence of CDR2 is shown in SEQ ID NO:43, and the amino acid sequence of CDR3 is shown in SEQ ID NO:44.

[0019] The amino acid sequence of CDR1 of the heavy chain variable region of antibody 3 is shown in SEQ ID NO:47, the amino acid sequence of CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of CDR3 is shown in SEQ ID NO:49.

[0020] The amino acid sequence of CDR1 of the light chain variable region of antibody 3 is shown in SEQ ID NO:52, the amino acid sequence of CDR2 is shown in SEQ ID NO:53, and the amino acid sequence of CDR3 is shown in SEQ ID NO:54.

[0021] Furthermore, in the antibody described in this invention,

[0022] The amino acid sequence of the light chain variable region of antibody 1 is shown in SEQ ID NO:25; the amino acid sequence of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:30.

[0023] The amino acid sequence of the light chain variable region of antibody 2 is shown in SEQ ID NO:37; the amino acid sequence of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:41.

[0024] The amino acid sequence of the light chain variable region of antibody 3 is shown in SEQ ID NO:51; the amino acid sequence of the heavy chain variable region of antibody 3 is shown in SEQ ID NO:46.

[0025] The present invention provides a conjugate (MGMT-04mAb-TAT) comprising the antibody and membrane-penetrating peptide described herein.

[0026] Furthermore, the membrane-penetrating peptide is a polypeptide rich in arginine, lysine, and / or glycine; and its amino acid count is 10 to 14.

[0027] Furthermore, the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID NO:34 or SEQ ID NO:55.

[0028] In this invention, MGMT-04mAb-TAT can combine with other targets to enhance the efficacy of TMZ, providing a new molecular tool and theoretical basis for overcoming TMZ resistance and improving the prognosis of GBM treatment.

[0029] This invention provides nanoparticle complexes comprising iron oxide nanoparticles and the coupling compounds described herein.

[0030] Furthermore, the iron oxide nanoparticles are extremely small iron oxide nanoparticles.

[0031] Furthermore, the extremely small iron oxide nanoparticles (ESIONPs) were purchased from CD Bioparticles, WNM-C003.

[0032] The nanoparticle complex described in this invention can precisely target gliomas in the brain, achieving effective delivery.

[0033] This invention provides the use of at least one of the following (I) to (III) in the preparation of a medicament for the prevention and treatment of tumors:

[0034] I) The antibody described in this invention;

[0035] II) The coupling compound described in this invention;

[0036] III) The nanoparticle complex described in this invention.

[0037] Furthermore, the tumors include brain tumors; the brain tumors include: glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, etc., derived from neuroepithelial tissue; meningioma derived from meninges; schwannomas such as acoustic neuroma derived from nerve sheaths; pituitary adenomas derived from pituitary glands; germ cell tumors and teratomas derived from germ cells; hemangioblastomas derived from blood vessels; metastatic tumors (brain metastases from lung cancer, breast cancer, etc.); and craniopharyngiomas, epidermoid cysts, dermoid cysts, chordomas, etc., derived from congenital / embryonic remnant tissues.

[0038] This invention provides a drug for the prevention and treatment of tumors, which uses at least one of the following ingredients (A) to (C) as raw materials:

[0039] A) The antibody described in this invention;

[0040] B) The coupling described in this invention;

[0041] C) The nanoparticle complex described in this invention.

[0042] This invention provides a drug combination comprising the drug described herein and other drugs for the prevention and treatment of tumors.

[0043] Other drugs used for cancer prevention and treatment include, but are not limited to, temozolomide, platinum-based drugs, antimetabolites, plant alkaloids, alkylating agents, targeted drugs, and / or immune checkpoint inhibitors.

[0044] The platinum-based drugs include cisplatin, carboplatin, and / or oxaliplatin, etc.; the antimetabolites include 5-fluorouracil, gemcitabine, and / or methotrexate, etc.; the alkaloids include paclitaxel, vincristine, and / or irinotecan, etc.; the alkylating agents include cyclophosphamide and / or carmustine, etc.; the targeted drugs include trastuzumab, bevacizumab, and / or imatinib, etc.; and the immune checkpoint inhibitors include pembrolizumab and / or nivolumab, etc.

[0045] This invention employs a swathenostomosis-based screening process to obtain a polypeptide combination suitable for predicting malignant gliomas. Then, monoclonal antibodies are prepared using MGMT-02 and MGMT-04 from this polypeptide combination. These monoclonal antibodies are conjugated with a TAT transmembrane peptide and then complexed with cellular iron oxide nanoparticles. The resulting complex, having addressed brain-targeted delivery, can be used to target intranuclear MGMT in TMZ-resistant GBM cell lines to reverse TMZ resistance. This provides a novel solution for the clinical development of targeted synergistic therapies against TMZ-resistant GBM, possessing significant translational medical value and enormous industrialization potential. Attached Figure Description

[0046] Figure 1 A schematic diagram illustrating the use of linear peptide microarray technology to monitor serum MGMT autoantibodies in glioma patients;

[0047] Figure 2 The response coverage of MGMT autoantibodies in the serum of glioma patients was shown.

[0048] Figure 3 The ROC curves of five dominant peptides in the glioma group are shown; where A is the ROC curve of MGMT-02 peptide; B is the ROC curve of MGMT-04 peptide; C is the ROC curve of MGMT-07 peptide; D is the ROC curve of MGMT-10 peptide; E is the ROC curve of MGMT-18 peptide; and F is the combined ROC curve of MGMT-02, MGMT-04, MGMT-07, MGMT-10 and MGMT-18 peptides.

[0049] Figure 4 A schematic diagram showing the synthesis and preparation of the MGMT-02 / 04 polypeptide antibody;

[0050] Figure 5 Parameter diagram of nano-iron oxide complex;

[0051] Figure 6 Preparation of ANG-IO@SS-M04Ab-TAT;

[0052] Figure 7The effect of ANG-IO@SS-M04Ab-TAT on GBM-R cell proliferation is shown; where A is a representative graph of the colony formation experiment results for each group of GBM-R cells; B is a quantitative analysis of the colony formation experiment results.

[0053] Figure 8 The effect of ANG-IO@SS-M04Ab-TAT combined with temozolomide on GBM-R apoptosis is shown; where A is a representative flow cytometry chromatogram of apoptosis markers in each group of GBM-R; B is the flow cytometry quantitative analysis of apoptosis markers.

[0054] Figure 9 This study investigated the effect of ANG-IO@SS-M04Ab-TAT combined with temozolomide on GBM-R cell apoptosis. In Figure A, Western blot analysis was used to detect the expression of apoptosis markers in GBM-R cells of each group; Figure B showed the quantitative analysis of apoptosis marker expression.

[0055] Figure 10 The effect of ANG-IO@SS-M04Ab-TAT on the tumorigenicity of GBM-R in nude mice is shown; where AB represents the distribution of ANG-IO@SS-M04Ab-TAT in tumor-bearing nude mice; CD represents the bioluminescence images in vivo and the quantitative analysis of the bioluminescence images; E represents the Kaplan-Meier survival curves of mice in the control group, TMZ, ANG-IO@SS-M04Ab-TAT group and TMZ+ANG-IO@SS-M04Ab-TAT group; F represents the trend of body weight change of nude mice in each group. Detailed Implementation

[0056] This invention provides a specific MGMT peptide, its antibody, and its clinical applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0057] MGMT-04mAb-TAT consists of three structures: 6His-SUMO-mAb1#scfV-TAT-C (Target MGMT-02), 6His-SUMO-mAb2#scfV-TAT-C (Target MGMT-04), and 6His-SUMO-mAb3#scfV-TAT-C (Target MGMT-04).

[0058] The amino acid sequence of 6His-SUMO-mAb1#scfV-TAT-C is: MGSSHHHHHHSSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGQVQLQQPGAELVRPGTSVKLSCKASGYTFTTFWMHWVKQRPGQGLEWIAMIDPSDSETHYNQMFRDK ATLTVDRSSSTAYIQLSSLTSADSAVYYCARADYWGQGTTLTVSSGGGGGSGGGGSGGGGSGGGGSDVMTQTPLTLSVSIGQPASISCKSSQSLLNNSGKTYLNWL LQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTKLEIKGSAAGAPKKKRKVGAPVPYPDPLEPRGGGGSC (SEQ IN NO:21);

[0059] The specific structure of 6His-SUMO-mAb1#scfV-TAT-C is as follows:

[0060] Initiating methionine-flexible linker-6H (amino acids at positions 1-10): MGSSHHHHHH (SEQ ID NO:22);

[0061] Linker peptide (amino acids 11-20): SSGMSDSEVN (SEQ ID NO:23);

[0062] SUMO tag (enhances protein expression and solubility, amino acids 21-111): QEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:24).

[0063] mAb1#scfV (single-chain antibody) VH (heavy chain variable region) (amino acids 112-223): QVQLQQPGAELVRPGTSVKLSCKASGYTFTTFWMHWVKQRPGQGLEWIAMIDPSDSETHYNQMFRDKATLTVDRSSSTAYIQLSSLTSADSAVYYCARADYWGQGTTLTVSS (SEQ ID NO:25);

[0064] mAb1#scfV of VH of CDR1:GYTFTTF (SEQ ID NO:26);

[0065] mAb1#scfV of VH of CDR2: AMIDPSDSETHYNQMF (SEQ ID NO:27);

[0066] mAb1#scfV of VH of CDR3:ARADY (SEQ ID NO:28);

[0067] Linker peptide (amino acids 224-243): GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:29);

[0068] mAb1#scfV (single-chain antibody) VL (light chain variable region, amino acids 244-355): DVVMTQTPLTLSVSIGQPASISCKSSQSLLNNSGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGIYYCWQGTHFPYTFGGGTKLEIK (SEQ ID NO:30);

[0069] mAb1#scfV of VL of CDR1:KSSQSLLNNSGKTY (SEQ ID NO:31);

[0070] mAb1#scfV of VL of CDR2:LVSKLDS (SEQ ID NO:32);

[0071] mAb1#scfV of VL of CDR3:WQGTHFPYT (SEQ ID NO:33);

[0072] TAT membrane-penetrating peptide (amino acids 356-369): GSAAGAPKKKRKVG (SEQ ID NO:34);

[0073] The C-terminal structure facilitates chemical coupling or immobilization (amino acids at positions 370-387): APVPYPDPLEPRGGGGSC (SEQ ID NO: 35);

[0074] The amino acid sequence of 6His-SUMO-mAb2#scfV-TAT-C is: MGSSHHHHHHSSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDS LRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMQWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSD KSSSTAYMDLTSLTSEDSAVYYCARRPLITVNSLYSMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQAAPSVPVTPGESVSISCRSNKSLLHSNGNTYLY WFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIKGSAAGAPKKKRKVGAPVPYPDPLEPRGGGGSC (SEQ ID NO:36);

[0075] The specific structure of 6His-SUMO-mAb2#scfV-TAT-C is as follows:

[0076] Initiating methionine-flexible linker-6H (amino acids at positions 1-10): MGSSHHHHHH (SEQ ID NO:22);

[0077] Linker peptide (amino acids 11-20): SSGMSDSEVN (SEQ ID NO:23);

[0078] SUMO tag (enhances protein expression and solubility, amino acids 21-111): QEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:24).

[0079] VH (heavy chain variable region, amino acids 112-234) of mAb2#scfV (single-chain antibody): EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMQWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMDLTSLTSEDSAVYYCARRPLITVNSLYSMDYWGQGTSVTVSS (SEQ ID NO:37).

[0080] mAb2#scfV of VH of CDR1:GYTFTSY (SEQ ID NO:38);

[0081] mAb2#scfV of VH of CDR2: YINPYNDGTKYNEKFK (SEQ ID NO:39);

[0082] mAb2#scfV of VH of CDR3: ARRPLITVNSLYSMDY (SEQ ID NO:40);

[0083] Linker peptide (amino acids 235-254): GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:9);

[0084] VL (light chain variable region, amino acids 255-366) of mAb2#scfV (single-chain antibody): DIVMTQAAPSVPVTPGESVSISCRSNKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK (SEQ ID NO:41);

[0085] mAb2#scfV of VL of CDR1: RSNKSLLHSNGNTYLY (SEQ ID NO:42);

[0086] mAb2#scfV of VL of CDR2: RMSNLAS (SEQ ID NO:43);

[0087] mAb2#scfV of VL of CDR3: MQHLEYPYT (SEQ ID NO:44);

[0088] TAT membrane-penetrating peptide (amino acids 367-380): GSAAGAPKKKRKVG (SEQ ID NO:34);

[0089] The C-terminal structure facilitates chemical coupling or fixation (381~398): APVPYPDPLEPRGGGGSC (SEQ ID NO:35);

[0090] The amino acid sequence of 6His-SUMO-mAb3#scfV-TAT-C is: MGSSHHHHHHSSGMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGK EMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGEVQLLETGGGLVQPKGSLKLSCAVSGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADS VKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVRFTLYYYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQSPASLAVSLGQRATISCRASKSVSTSGY SYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELKAAGAPKKKRKVGAPVPYPDPLEPR (SEQ ID NO:45);

[0091] Initiating methionine-flexible linker-6H (amino acids at positions 1-10): MGSSHHHHHH (SEQ ID NO:22);

[0092] Linker peptide (amino acids 11-20): SSGMSDSEVN (SEQ ID NO:23);

[0093] SUMO tag (enhances protein expression and solubility, amino acids 21-111): QEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (SEQ ID NO:24).

[0094] VH (heavy chain variable region, amino acids 112-232) of mAb3#scfV (single-chain antibody): EVQLLETGGGLVQPKGSLKLSCAVSGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQTMLYLQMNNLKTEDTAMYYCVRFTLYYYAMDYWGQGTSVTVSS (SEQ ID NO:46);

[0095] mAb3#scfV of VH of CDR1:VS (SEQ ID NO:47);

[0096] CDR2 of VH of mAb3#scfV: ARIRSKSNNYATYYA (SEQ ID NO:48);

[0097] mAb3#scfV of VH of CDR3:VRFTLYYYAMDY (SEQ ID NO:49);

[0098] Linker peptide (amino acids 233-247): GGGGSGGGGSGGGGS (SEQ ID NO:50);

[0099] VL (light chain variable region, amino acids 248-360) of mAb3#scfV (single-chain antibody): DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPLTFGAGTKLELKAA (SEQ ID NO:51);

[0100] mAb3#scfV's VL CDR1:RASKSVSTSGYSYM (SEQ ID NO:52);

[0101] mAb3#scfV of VL of CDR2:LASNLESG (SEQ ID NO:53);

[0102] mAb3#scfV of VL of CDR3:QHSRELPLT (SEQ ID NO:54);

[0103] TAT membrane-penetrating peptide (amino acids 361-370): GAPKKKRKVG (SEQ ID NO:55);

[0104] The C-terminal structure facilitates chemical coupling or immobilization (amino acids at positions 371-382): APVPYPDPLEPRGGGGSC (SEQ ID NO: 56);

[0105] ANG peptide: TFFYGGSRGKRNNFKTEEY (SEQ ID NO:57);

[0106] Transmembrane-penetrating peptide (TAT) is a transactivator of transmembrane-penetrating peptide.

[0107] Preparation methods of 6His-SUMO-mAb1#scfV-TAT-C (Target MGMT-02), 6His-SUMO-mAb2#scfV-TAT-C (Target MGMT-04), or 6His-SUMO-mAb3#scfV-TAT-C (Target MGMT-04):

[0108] 1. Preparation and sequence acquisition of mAb1#scfV, mAb2#scfV, and mAb3#scfV monoclonal antibodies:

[0109] BALB / c mice were immunized with a peptide-KLH conjugate. After a series of immunization procedures, mice with the target titer were selected for cell fusion. Spleen cells were fused with SP2 / 0 myeloma cells and cultured in semi-solid medium to obtain hybridoma clones. After three rounds of ELISA screening using peptide-BSA as the coating antigen, a hybridoma cell line stably secreting specific antibodies was successfully obtained. Antibody subtypes were used to identify the cell line subtype. All animal experiments were approved by the Ethics Committee of Soochow University (Approval No.: SUDA20241113A04). Subsequently, total RNA was extracted from the hybridoma cells and cDNA was synthesized by reverse transcription. Hybridoma-specific primers were used to amplify the variable region genes of the antibody heavy chain (VH) and light chain (VL). The PCR products were cloned into a T vector via TA, and single clones were selected by blue-white screening and sent for Sanger sequencing to obtain the nucleotide sequences of VH and VL, which were then used to identify the complementarity-determining region (CDR) and frame region (FR).

[0110] 2. Construction of the 6His-SUMO-mAb1 / 2 / 3#scfV-TAT-C structure:

[0111] Based on the VH and VL sequences obtained from sequencing, scFv coding sequences with an N-terminal 6×His-SUMO tag and a C-terminal transmembrane peptide were designed and synthesized. After codon optimization of the sequences for *E. coli*, the whole gene was synthesized and cloned into the pET21a expression vector, and the correctness of the construction was verified by sequencing. The constructed expression plasmid was transformed into Rosetta (DE3) competent cells and expressed by IPTG induction. The expression product mainly existed in the form of inclusion bodies. After sonication and washing, the inclusion bodies were dissolved with 6M guanidine hydrochloride and refolded by serial dilution. The refolded protein was purified by nickel affinity chromatography, eluted, dialyzed in citrate buffer at pH 7.4, and finally concentrated by ultrafiltration to a concentration >0.5 mg / mL.

[0112] There are many types of GBM-R cells; some express MGMT, while others do not.

[0113] Preparation method of IONPs-OA (oleic acid modified iron oxide nanoparticles):

[0114] FeCl3·6H2O (ferric chloride hexahydrate) and FeCl2·4H2O (ferrous chloride tetrahydrate) were dissolved in water at a molar ratio of 1:1. While stirring, an alkaline solution (such as ammonia or NaOH) was added to adjust the pH to approximately 10, and oleic acid was added rapidly at the same time. After reacting at 60℃~80℃ for approximately 30~60 minutes, the product was separated and washed using a magnet to obtain IONPs-OA.

[0115] Preparation method of IONPs-mPEG:

[0116] ESIONPs-mPEG was prepared using a co-precipitation method combined with in-situ PEG modification: First, FeCl3·6H2O and FeCl2·4H2O were weighed in a 1:1 molar ratio and dissolved in deionized water to prepare mixed solutions containing 0.3 mol / L of iron salts, respectively. Separately, polyethylene glycol (PEG 5000) with a molecular weight of 5000 was dissolved in deionized water at a concentration of 50 g / L, and ammonia was added to adjust the pH to 10-11, resulting in an alkaline solution containing PEG. Under mechanical stirring and nitrogen protection, the iron salt mixed solution was slowly added dropwise to the above alkaline solution via back titration (dropping rate approximately 1 mL / min-2 mL / min). As the addition proceeded, black Fe3O4 nanoparticles gradually formed in the system, while PEG was adsorbed in situ onto the particle surface to control growth and prevent aggregation. After the addition is complete, continue stirring for 30 to 60 minutes to allow for crystallization and maturation. Then, collect the black precipitate using magnetic separation and wash it repeatedly with deionized water 3 to 5 times to remove unreacted iron ions and excess PEG. Finally, redisperse the product in deionized water or PBS buffer to obtain a stable IONPs-mPEG aqueous dispersion.

[0117] The binding of 6His-SUMO-mAb2#scFv-TAT-C (i.e., M04Ab-TAT) to ANG peptide and IONPs-mPEG:

[0118] Starting with oleic acid-modified iron oxide nanoparticles (IONPs-OA), PEG chains, antibodies, and ANG peptides were sequentially linked. The specific steps are as follows: First, 10 mg of IONPs-OA was taken and added with equal masses (50 mg each) of DOPA-PEG5000-SS-Py (Py is pyridine) and DOPA-PEG5000-DBCO. The mixture was reacted in chloroform at 40°C for 24 hours. The intermediate product IONPs-PEG5000-SS-Py / DBCO with disulfide pyridine (SS-Py) and DBCO groups on its surface was obtained through DOPA ligand exchange. Then, the intermediate product was reacted with a free thiol-containing MO4Ab-TAT antibody (the specific amount of MO4Ab-TAT is usually added at a ratio of 1~2 µg antibody per 50 µg Fe) in degassed PBS at room temperature for 24 hours. The antibody was linked to the nanoparticles via a thiol-disulfide bond exchange reaction. After magnetic separation and purification, IONPs-PEG5000-SS-MO4Ab-TAT / DBCO was obtained. Finally, an excess of azide-modified ANG peptide (N3-ANG) was added, and the mixture was reacted in PBS in the dark at room temperature for 48 hours. The ANG peptide was linked via a copper-free click chemistry reaction between DBCO and the azide group. After magnetic separation and purification again, ANG-IO@SS-MO4Ab-TAT was obtained. Here, N3-ANG needs to be in excess (e.g., DBCO:Azide molar ratio of 1:5~1:10) to ensure complete reaction.

[0119] Preparation of ANG-IONPs (also written as ANG-ESIONPs): This refers to empty nanoparticles without MO4Ab-TAT antibody as a control. The preparation method is as follows: 10 mg IONPs-OA is reacted with 50 mg DOPA-PEG5000-SS-Py and 50 mg DOPA-PEG5000-DBCO, and purified to obtain the intermediate product IONPs-PEG5000-SS-Py / DBCO. This intermediate product is reacted with excess N3-ANG in PBS at room temperature in the dark for 48 hours. The ANG peptide is then linked via a click chemistry reaction between DBCO and the azide group. Finally, ANG-IONPs are obtained by magnetic separation and purification. This product does not contain MO4Ab-TAT antibody, retaining only the iron core, PEG spacer arm, and ANG targeting peptide, and is used as a negative control in subsequent experiments.

[0120] DOPA-PEG5000-DBCO was custom-made by Xi'an Qiyue Biotechnology.

[0121] Specific preparation steps for DOPA-PEG5000-SS-Py:

[0122] Step 1: Synthesize HO-PEG5000-SS-Py. The goal of this step is to selectively introduce an o-pyridine disulfide group at one end of the PEG chain. The specific steps are as follows:

[0123] Under an inert gas atmosphere (such as nitrogen or argon), dihydroxy polyethylene glycol (HO-PEG5000-OH), 2,2'-dipyridine disulfide (Aldrithiol-2), and triphenylphosphine (PPh3) were dissolved in anhydrous dichloromethane (DCM). The reaction was stirred overnight at room temperature. After the reaction was complete, the reaction solution was concentrated by rotary evaporation. The concentrate was added dropwise to a large amount of cold diethyl ether to precipitate the product. The precipitate was collected and dried under vacuum to give the product HO-PEG5000-SS-Py, which has an o-pyridine disulfide group at one end.

[0124] Step 2: Synthesize DOPA-PEG5000-SS-Py. The goal of this step is to convert the hydroxyl group at the other end of the product from the previous step into a DOPA group. The specific steps are as follows:

[0125] Dissolve HO-PEG5000-SS-Py in anhydrous DMF or DCM, and add DOPA (or dopamine hydrochloride). Add coupling agent EDC·HCl and NHS (as catalyst), and stir the reaction at room temperature (e.g., for 24-48 hours). After the reaction is complete, filter to remove insoluble matter. Dialyze the filtrate thoroughly in deionized water (using a dialysis bag with an appropriate molecular weight cutoff, such as MWCO 1000Da) to remove unreacted small molecules and solvent. Lyophilize after dialysis to obtain the final product DOPA-PEG5000-SS-Py.

[0126] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0127] Example 1: Application of specific MGMT peptides, their antibodies, and their conjugates in the prevention and treatment of glioma.

[0128] I. Preliminary Analysis of MGMT Autoantibodies and Their Diagnostic Value

[0129] The MGMT protein and its corresponding amino acid sequence information were obtained from the NCBI database. The MGMT protein was then cleaved into 20 polypeptides (as shown in Table 1) for detection using polypeptide microarray technology (the detection procedure is as follows). Figure 1 The peptide microarray was then incubated with serum from 378 subjects, including 67 patients with malignant gliomas (preoperative) and 311 healthy donors, for serological screening of MGMT autoantibodies.

[0130] Cluster analysis of samples from patients with malignant gliomas and healthy donors revealed that the expression of MGMT peptides in gliomas was significantly higher than in healthy individuals. The responses of each peptide's autoantibody to malignant glioma (glioma) and healthy serum samples were then analyzed. The results showed that, compared to healthy controls, MGMT-02 (anti-MGMT-02), MGMT-04 (anti-MGMT-04), MGMT-07 (anti-MGMT-07), MGMT-10 (anti-MGMT-10), and MGMT-18 (anti-MGMT-18) peptides exhibited higher coverage among the 20 peptides. Figure 2 ).

[0131] ROC analysis was then used to evaluate the predictive value of the five highly responsive peptides. The responses of anti-MGMT-02, anti-MGMT-04, anti-MGMT-07, anti-MGMT-10, and anti-MGMT-18 peptide autoantibodies were tested in samples from 67 glioma patients (preoperative). Compared with 311 healthy subjects, the area under the curve (AUC) of the five peptide responses was not a satisfactory predictor of malignant gliomas. Combining the five peptides as a multi-indicator diagnostic model increased the AUC to 0.810 (95% confidence interval 0.752–0.869). This diagnostic combination achieved a specificity of 83% and a sensitivity of 75%. Figure 3 This diagnostic model achieves moderate diagnostic efficacy and is helpful in the diagnosis of gliomas. When comparing the differences in autoantibody responses to five dominant peptides between high-grade and low-grade gliomas, it was found that only the autoantibody levels of MGMT-02 and MGMT-04 peptides were significantly higher in high-grade gliomas than in low-grade gliomas, and the differences were statistically significant.

[0132] Table 1

[0133]

[0134] II. Preparation of Specific MGMT Monoclonal Antibodies

[0135] A complete monoclonal antibody preparation system was established during the development of therapeutic antibodies. SPF-grade BALB / c mice were immunized with chemically synthesized specific peptides MGMT-02 and MGMT-04. Hybridoma cells were obtained using polyethylene glycol-mediated cell fusion technology. Advanced methods such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry were used for multiple rounds of screening and subcloning to ultimately obtain highly specific monoclonal antibodies. Figure 4 ).

[0136] This invention discovered that MGMT-04 is the most specific polypeptide with the highest responsiveness among five polypeptides during antibody screening. Through the above antibody preparation and screening, 14 MGMT-04-specific monoclonal antibodies were first obtained, with a molecular weight of approximately 150-200 kDa. After high-level purification, high-titer MGMT-04-specific monoclonal antibodies MGMT-04mAb, abbreviated as M04 monoclonal antibody (its single-chain antibodies are mAb2#scfV and mAb3#scfV, respectively).

[0137] The single-chain antibody for the peptide MGMT-02 is mAb1#scfV.

[0138] III. Study on the effect of specific MGMT monoclonal antibody coupled with membrane-penetrating peptide TAT on TMZ-resistant glioblastoma cells

[0139] Through deep sequencing analysis of the antibody variable region, it was discovered that the third complementarity-determining region of the heavy chain variable region of the MGMT-04 specific monoclonal antibody has unique amino acid composition characteristics, which may be the key structural basis for the antibody to exhibit high affinity. Therefore, a fusion expression system of single-chain antibody fragment and membrane-penetrating peptide was developed, which significantly improved the delivery efficiency of antibody molecules in tumor cells and created favorable conditions for subsequent in vivo therapeutic research.

[0140] However, these monoclonal antibodies still face challenges as a treatment method: the monoclonal antibodies themselves cannot directly enter tumor cells to target the precise site of MGMT. Therefore, the MGMT-04 specific monoclonal antibody (M04 monoclonal antibody) is coupled with the transmembrane peptide trans-activator of transcription (TAT) with unique transmembrane ability to construct MGMT-04mAb-TAT (including 6His-SUMO-mAb2#scfV-TAT-C (Target MGMT-04) and 6His-SUMO-mAb3#scfV-TAT-C (Target MGMT-04)) that can be precisely located to the cell nucleus to complete the delivery of the monoclonal antibody into the cell.

[0141] IV. Research on Precision Targeted Therapy Using MGMT-04 Specific Monoclonal Antibody-Nanoic Iron Oxide Particles to Cross the Blood-Brain Barrier

[0142] However, how MGMT-04mAb-TAT or MGMT-02mAb-TAT can penetrate the blood-brain barrier (BBB) ​​in vivo to enter the brain and target GBM remains a challenge. Therefore, oleic acid-modified iron oxide nanoparticles (IONPs-OA) were chosen to achieve the purpose of monoclonal antibody delivery.

[0143] Then, the IONPs-OA was modified with PEG5000 to obtain IONPs-mPEG. Figure 5() is used for specific delivery.

[0144] Due to technical limitations, only the IONPs-mPEG-MGMT-04 antibody complex was synthesized; then, the IONPs-mPEG-MGMT-04 antibody complex was surface-modified with ANG peptide (preparation process as follows). Figure 6 ANG peptide is a 19-amino acid polypeptide commonly used as a drug delivery carrier, especially in cross-BBB targeted therapy, where it effectively recognizes and binds to low-density lipoprotein receptor-related protein 1 (LRP1) on brain capillary endothelial cells, significantly enhancing its cross-BBB capacity. Furthermore, MGMT-04mAb-TAT-ANG (6His-SUMO-mAb2#scfV-TAT-C) and ANG peptide are linked via disulfide bonds to IONPs-OA to construct ANG-IO@SS-M04Ab-TAT (6His-SUMO-mAb2#scfV-TAT-C). Figure 7 This refers to a specific MGMT-04 polypeptide monoclonal antibody modified with iron oxide nanoparticles and fused with a TAT membrane-penetrating peptide, specifically 6His-SUMO-mAb2#scfV-TAT-C (Target MGMT-04). The disulfide bonds can be cleaved in the high-concentration glutathione reducing environment within tumor cells, thereby achieving the specific release of MGMT-04mAb-TAT within the tumor. This design not only increases the local effective concentration of MGMT-04mAb-TAT but also reduces non-specific exposure and toxicity risks to normal tissues.

[0145] In in vitro experiments, cell cloning experiments demonstrated that when the temozolomide-resistant glioblastoma strain (GBM-R) expressed MGMT, ANG-IO@SS-M04Ab-TAT primarily enhanced the ability of TMZ (temozolomide) to inhibit GBM-R proliferation by specifically targeting and binding to nuclear MGMT. Apoptosis and Western blotting experiments further demonstrated that MGMT-04mAb-TAT primarily enhanced the ability of TMZ to promote GBM-R apoptosis by specifically targeting and binding to nuclear MGMT. Figures 8-10 ; Figures 8-10In the study, the control group consisted of PBS; ANG-IONPs (ANG-ESIONPs) were iron oxide nanoparticles conjugated with ANG peptide; ANG-IO@SS-M04Ab-TAT was an iron oxide nanoparticle-modified monoclonal antibody specifically targeting MGMT-04 peptide; IONPs were IONPs-OA (oleic acid-modified iron oxide nanoparticles); TMZ was temozolomide; TMZ+IONPs were temozolomide + iron oxide nanoparticles; TMZ+ANG-IO@SS-M04Ab-TAT was temozolomide + iron oxide nanoparticle-modified monoclonal antibody specifically targeting MGMT-04 peptide; T98G-R was the T98G temozolomide-resistant cell line; and SHG141-R was the SHG141 temozolomide-resistant cell line. Finally, in vivo tumorigenesis experiments showed that ANG-IO@SS-M04Ab-TAT, after tail vein injection, successfully penetrated the blood-brain barrier, precisely targeting gliomas in the brain, achieving effective delivery. Combined TMZ therapy can significantly inhibit the tumor progression of TMZ-resistant glioblastoma, improve the drug resistance status, and demonstrate good therapeutic potential. Figure 10 ).

[0146] in conclusion:

[0147] Compared with traditional MGMT inhibitors, the ANG-IO@SS-M04Ab-TAT of the present invention has the following advantages:

[0148] 1. High specificity and precise delivery: Through specific antibody recognition of MGMT-04 specific polypeptide epitopes, TAT peptide ensures that MGMT-04mAb-TAT can cross the membrane and enter the cell nucleus, effectively acting on MGMT in the nucleus;

[0149] 2. Target Diversity: When GBM-R does not express MGMT, MGMT-04mAb-TAT can bind to other targets to enhance the efficacy of TMZ. Even when the glioblastoma-resistant strain (GBM-R) does not express MGMT, MGMT-04mAb-TAT can still bind to other undefined key molecular targets within the GBM-R cell nucleus, interfering with DNA repair or survival-related pathways in tumor cells, thereby continuously enhancing the anti-tumor effect of TMZ. These advantages provide new molecular tools and theoretical foundations for overcoming TMZ resistance and improving the prognosis of GBM treatment.

[0150] In summary, this technology is the first to achieve MGMT targeting the nuclear MGMT in TMZ-resistant GBM cell lines to reverse TMZ resistance, and utilizes ANG peptide-modified nanocarriers to solve the challenge of brain-targeted delivery. This provides a novel solution for the clinical development of targeted synergistic therapies against TMZ-resistant GBM, possessing significant translational medical value and enormous industrialization potential.

[0151] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide combination for the diagnosis of glioma patients, characterized in that, The polypeptide combination includes: MGMT-02 polypeptide with amino acid sequence as shown in SEQ ID NO:2, MGMT-04 polypeptide with amino acid sequence as shown in SEQ ID NO:4, MGMT-07 polypeptide with amino acid sequence as shown in SEQ ID NO:7, MGMT-10 polypeptide with amino acid sequence as shown in SEQ ID NO:10, and / or MGMT-18 polypeptide with amino acid sequence as shown in SEQ ID NO:

18.

2. The use of the polypeptide combination of claim 1 as a biomarker in the preparation of products for predicting malignant tumors.

3. An antibody, characterized in that, The mice were obtained by immunizing them with any polypeptide from the polypeptide combination described in claim 1 or 2 as an immunogen.

4. The antibody according to claim 3, characterized in that, The antibodies include antibody 1, antibody 2 and antibody 3; The amino acid sequence of CDR1 of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:26, the amino acid sequence of CDR2 is shown in SEQ ID NO:27, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

28. The amino acid sequence of CDR1 of the light chain variable region of antibody 1 is shown in SEQ ID NO:31, the amino acid sequence of CDR2 is shown in SEQ ID NO:32, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

33. The amino acid sequence of CDR1 of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:38, the amino acid sequence of CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

40. The amino acid sequence of CDR1 of the light chain variable region of antibody 2 is shown in SEQ ID NO:42, the amino acid sequence of CDR2 is shown in SEQ ID NO:43, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

44. The amino acid sequence of CDR1 of the heavy chain variable region of antibody 3 is shown in SEQ ID NO:47, the amino acid sequence of CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

49. The amino acid sequence of CDR1 of the light chain variable region of antibody 3 is shown in SEQ ID NO:52, the amino acid sequence of CDR2 is shown in SEQ ID NO:53, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

54.

5. The antibody according to claim 4, characterized in that, The amino acid sequence of the light chain variable region of antibody 1 is shown in SEQ ID NO:25; the amino acid sequence of the heavy chain variable region of antibody 1 is shown in SEQ ID NO:

30. The amino acid sequence of the light chain variable region of antibody 2 is shown in SEQ ID NO:37; the amino acid sequence of the heavy chain variable region of antibody 2 is shown in SEQ ID NO:

41. The amino acid sequence of the light chain variable region of antibody 3 is shown in SEQ ID NO:51; the amino acid sequence of the heavy chain variable region of antibody 3 is shown in SEQ ID NO:

46.

6. A coupling, characterized in that, Includes the antibody and membrane-penetrating peptide as described in any one of claims 3 to 5.

7. The coupling according to claim 6, characterized in that, The membrane-penetrating peptide is a polypeptide rich in arginine, lysine and / or glycine; and its amino acid count is 10 to 14.

8. The coupling according to claim 7, characterized in that, The amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID NO:34 or SEQ ID NO:

55.

9. A nanoparticle complex, characterized in that, It includes iron oxide nanoparticles and the coupling compound as described in any one of claims 6 to 8.

10. The nanoparticle complex according to claim 9, characterized in that, The iron oxide nanoparticles are extremely small iron oxide nanoparticles.

11. Use of at least one of the following shown in I) to III) in the preparation of a drug for the prevention and treatment of tumors: I) The antibody according to any one of claims 3 to 5; II) The coupling compound according to any one of claims 6 to 8; III) The nanoparticle complex according to claim 9 or 10.

12. The application according to claim 11, characterized in that, The tumors include glioblastoma, astrocytoma, oligodendroglioma, ependymoma, medulloblastoma, meningioma, schwannoma, pituitary adenoma, germ cell tumor, teratoma, craniopharyngioma, epidermoid cyst, dermoid cyst, and / or chordoma.

13. A drug for the prevention and treatment of tumors, characterized in that, Use at least one of the following A) to C) as raw materials: A) The antibody according to any one of claims 3 to 5; B) The coupling compound according to any one of claims 6 to 8; C) The nanoparticle complex according to claim 9 or 10.

14. A drug combination, characterized in that, This includes the drug as described in claim 13 and other drugs used for the prevention and treatment of tumors.

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