An antibody targeting mutant histone h3 and uses thereof

Abstract

The application discloses an antibody targeting a mutant histone H3 and application thereof. The application takes a histone H3 K27M mutant as a target protein, designs and synthesizes a K-M mutant polypeptide 1 (CLATKAARMSAPS) and a non-mutant polypeptide 2 (CLATKAARKSAPS), couples the polypeptide 1 with KLH to serve as an immunogen to prepare hybridoma cells, and obtains one monoclonal antibody specifically targeting the histone H3 K27M mutant through screening, with a clone number of 21C5-A6. The monoclonal antibody provided by the application can specifically recognize the K27M mutant polypeptide 1 and cannot recognize the non-mutant polypeptide 2, has the advantages of high specificity and high sensitivity, can accurately detect the histone H3 K27M mutation in a sample, and has wide clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody targeting a mutated histone H3 and its application. Background Technology

[0002] Histones help package DNA into nucleosome chains. A nucleosome is an octamer with two copies each of histones H2A, H2B, H3, and H4 at its core, surrounded by approximately 146-147 bp of tightly wound DNA, serving as the primary structural unit of chromatin. Histones not only perform structural functions but also dynamically regulate the active and inactive states of chromatin through various post-translational modifications, thus playing a crucial regulatory role in DNA replication, transcription, and damage repair. Specifically, the H3K27M mutation in histone H3 (i.e., the substitution of lysine (K) at position 27 with methionine (M)) can inhibit the activity of the PRC2 complex, leading to a decrease in genome-wide H3K27me3 levels, thereby abnormally activating oncogenes and triggering significant epigenetic reprogramming.

[0003] In clinical practice, diffuse midline glioma (DMG) is a type of invasive central nervous system tumor that commonly occurs in midline structures such as the brainstem, thalamus, spinal cord, and cerebellum. The vast majority of cases involve histone H3K27M mutations; these tumors are termed H3K27M-mutant diffuse midline gliomas, classified as grade IV gliomas by the World Health Organization (WHO). They are highly malignant and pose a significant threat to patients' lives and health. In exploring the pathological mechanisms of H3K27M-mutant diffuse midline gliomas, screening relevant targeted drugs, and providing targeted diagnosis, antibodies specifically targeting H3K27M are crucial and remain a key research focus in this field. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a monoclonal antibody that can specifically bind to the histone H3 K27M mutation site and its applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides an antibody targeting histone H3 with K27M mutation, wherein the heavy chain variable region sequence of the antibody is shown in SEQ ID NO:1 and the light chain variable region sequence is shown in SEQ ID NO:5.

[0006] Furthermore, the heavy chain variable region CDR1-3 sequences of the antibody are shown in SEQ ID NO:2-4, and the light chain variable region CDR1-3 sequences are shown in SEQ ID NO:6-8, respectively.

[0007] In some embodiments, mutants of the antibodies described in this invention are also included within the scope of protection of this invention, wherein the "mutant" has an amino acid sequence that is significantly or substantially identical or similar to that of the parent antibody, and the mutant retains the biological activity of the parent antibody. Mutants encompass, for example, variants of the antibodies described herein that retain the ability to recognize target cells to a similar, equal, or greater extent than the parent antibody. Referring to the parent antibody, the mutant may, for example, have at least about 75%, 80%, 85%, 90%, 95%, or higher identity with the parent antibody in terms of amino acid sequence.

[0008] In some embodiments, the amino acid sequences corresponding to the heavy chain variable regions CDR1-3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:2-4 or SEQ ID NO:10-12, and the amino acid sequences corresponding to the light chain variable regions CDR1-3 of the present invention are not limited to the amino acid sequences shown in SEQ ID NO:6-8 or SEQ ID NO:14-16. The amino acid sequences or nucleotide sequences corresponding to the CDRs obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable regions shown in SEQ ID NO:1 or SEQ ID NO:9, or defining CDR1, CDR2, and CDR3 in the light chain variable regions shown in SEQ ID NO:5 or SEQ ID NO:13, using any CDR numbering scheme (existing CDR numbering schemes, such as IMGT, Chothia, Kabat, Martin (enhanced Chothia), AbM, Aho, or new CDR numbering schemes to be generated in the future) are all within the scope of protection of the present invention.

[0009] A second aspect of the present invention provides a nucleic acid molecule that encodes the antibody described in the first aspect of the present invention.

[0010] The third aspect of the present invention provides any of the following products: 1) A recombinant vector comprising the antibody described in the first aspect of the present invention or the nucleic acid molecule described in the second aspect of the present invention.

[0011] 2) A recombinant cell comprising the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, or the recombinant vector.

[0012] 3) An antibody derivative, wherein the antibody derivative is a complex formed by directly or indirectly coupling the antibody described in the first aspect of the present invention to a detectable marker.

[0013] 4) A detection product comprising the antibody or antibody derivative described in the first aspect of the present invention.

[0014] In some embodiments, the recombinant vector includes plasmids, phage particles, granules, artificial chromosomes, or virus-derived vectors. The virus-derived vectors are lentiviral vectors, retroviral vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpesvirus vectors, or baculovirus vectors.

[0015] Furthermore, the recombinant cells include prokaryotic cells and eukaryotic cells.

[0016] In some embodiments, the prokaryotic cells include bacteria, actinomycetes, cyanobacteria, mycoplasma, chlamydia, and rickettsiae. The bacteria include *Escherichia coli*, *Bacillus subtilis*, *Salmonella typhimurium*, *Pseudomonas*, *Streptomyces*, and *Staphylococcus*. The eukaryotic cells include mammalian cells, insect cells, plant cells, and yeast cells.

[0017] Furthermore, the detectable markers include tags, bioluminescent agents, chemiluminescent agents, enzymes, photosensitizing diagnostic agents, paramagnetic ions, or radionuclides.

[0018] In some embodiments, the tags include fluorescent protein tags (such as GFP, RFP, YFP / CFP), bioluminescent tags (such as luciferase), chemiluminescent tags (such as APEX), purification tags (such as His tags, GST tags, FLAG tags, Strep tags), localization tags (such as nuclear localization signals NLS, membrane localization tags: palmitoylated sequences or cardamomylated sequences, secretion signal peptides: IgG signal peptides), and functional research tags (such as HA tags, Myc tags, SBP tags, SUMO tags). The bioluminescent agents include firefly luciferase, renin luciferase, luciferin, and jellyfish luminescent proteins. The chemiluminescent agents include luminol, isoluminol, acridinium esters, imidazoles, and oxalates. The enzymes include horseradish peroxidase and alkaline phosphatase. The photosensitive diagnostic agents include photosensitive biotin, rhodamine-based markers (such as rhodamine B, rhodamine 6G, rhodamine 101, tetramethylrhodamine, and dansyl chloride), cyanine dye markers (such as Cy3, Cy5, Cy5.5, Cy7, and Cy7.5), and the BODIPY (boron dipyrrole fluoride) series dyes. The paramagnetic ions include gadolinium ions, manganese ions, iron ions, cobalt ions, and nickel ions. The radionuclides include radioactive iodine, radioactive cesium, radioactive iridium, and radioactive cobalt.

[0019] Furthermore, the testing products include reagents, kits, chips, and test strips.

[0020] The fourth aspect of the present invention provides any of the following methods: 1) A method for preparing the recombinant cells according to the third aspect of the present invention, the method comprising introducing the recombinant vector according to the third aspect of the present invention into cells to obtain the recombinant cells.

[0021] 2) A method for producing the antibody according to the first aspect of the present invention, the method comprising culturing the recombinant cells according to the third aspect of the present invention, and isolating the antibody from the culture of the recombinant cells.

[0022] 3) A method for detecting histone H3K27M mutations in a sample in vitro for non-diagnostic, non-therapeutic purposes, the method comprising contacting the sample with the antibody described in the first aspect of the present invention, the antibody derivative described in the third aspect of the present invention, or a detection product, and detecting the formation of immune complexes.

[0023] In some embodiments, the methods of introduction include calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, the TALEN method, the ZFN method, non-viral vector-mediated transfection (e.g., liposomes) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenovirus infection), and other physical, chemical, or biological means for transfer into cells, such as transposon technology, CRISPR-Cas9, etc. Those skilled in the art can introduce the nucleic acid molecules or expression vectors of the present invention into host cells in various suitable ways, and are not limited to the methods listed herein.

[0024] In some implementations, the method for detecting histone H3K27M mutations in samples for non-diagnostic and non-therapeutic purposes includes, but is not limited to, the following applications: as a highly specific probe, used for co-crystallization with mutant histone peptides or nucleosomes to elucidate the microconformational effects of H3K27M mutations on the higher-order structure of chromatin; as a FRET (fluorescence resonance energy transfer) or single-molecule fluorescent probe in an in vitro recombinant system to monitor the dynamic conformational changes of mutant histones on nucleosomes in real time; and by specifically binding to the mutant epitope, blocking or promoting the interaction between other chromatin modifying enzymes (such as histone deacetylases or demethylases) and nucleosomes, thereby elucidating the transcriptional regulatory mechanisms of downstream genes.

[0025] The fifth aspect of the present invention provides for any of the following applications: 1) The use of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector, recombinant cell, antibody derivative or detection product described in the third aspect of the present invention in the preparation of products for diagnosing diseases related to histone H3 K27M mutation.

[0026] 2) The application of the antibody described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the recombinant vector, recombinant cell, antibody derivative or detection product described in the third aspect of the present invention in the in vitro non-diagnostic, non-therapeutic target detection of histone H3K27M mutation in samples.

[0027] Furthermore, the histone H3 K27M mutation-related disease is diffuse midline glioma.

[0028] Furthermore, the diffuse midline glioma is an H3 K27M mutant diffuse midline glioma.

[0029] Advantages and beneficial effects of the present invention: This invention uses the histone H3 K27M mutant as the target protein, designs and synthesizes KM-mutated peptide 1 (CLATKAARMSAPS) and unmutated peptide 2 (CLATKAARKSAPS). Peptide 1 is conjugated with KLH as an immunogen to prepare hybridoma cells. Through screening, a monoclonal antibody specifically targeting the histone H3 K27M mutant was obtained, clone number 21C5-A6. The monoclonal antibody provided by this invention can specifically recognize the K27M-mutated peptide 1 but not the unmutated peptide 2, exhibiting high specificity and high sensitivity. It can accurately detect the histone H3 K27M mutation in samples and has broad clinical application value. Attached Figure Description

[0030] Figure 1 The results show the positive screening results for antibodies coating peptide 1.

[0031] Figure 2 The result is the negative screening result for antibodies coating peptide 2.

[0032] Figure 3 The results are from SDS-PAGE analysis of the 21C5-A6 antibody.

[0033] Figure 4 The results are from the immunohistochemical verification of the 21C5-A6 antibody. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents.

[0035] The materials and equipment used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions are typically performed under conventional conditions or as recommended by the manufacturer. Specifically, the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0036] Example 1: Preparation, Screening and Validation of Antibodies I. Experimental Methods 1. Experimental materials 1) Immunogen: Using Histone H3 (mutated K27M) as the target protein, KM-mutated and unmutated polypeptide sequences were designed and synthesized as follows: KM-mutated polypeptide 1, CLATKAARMSAPS; wild-type polypeptide 2, CLATKAARKSAPS. These two polypeptides were prepared chemically. Polypeptide 1 was conjugated with keyhole hemocyanin (KLH) to prepare the immunogen.

[0037] 2) Culture media: DMEM medium was purchased from Gibco (Hyclone); HAT and HT selective media and phytidine were purchased from Sigma.

[0038] 3) Experimental animals: Balb / c mice, 28-34 days old, female, SPF grade animal culture.

[0039] 4) Other materials: Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma; PEG4000 was purchased from Fluka; HRP-goat anti-mouse IgG antibody was purchased from Jackson Immune; the remaining reagents were domestically produced analytical grade products.

[0040] 2. Establishment of hybridoma cell lines 1) Animal immunization: Mix the antigen and Freund's complete adjuvant in equal volumes and emulsify thoroughly. Administer the mixture subcutaneously at multiple sites, with each Balb / c mouse receiving approximately 100 μg. For booster immunization, use an emulsion of the antigen and Freund's incomplete adjuvant.

[0041] 2) Preparation of hybridoma cells: Mouse spleen cells and myeloma cells SP2 / 0 were fused separately. (Well-grown SP2 / 0 cells were pipetted and centrifuged at 1500 rpm for 3 min. The supernatant was discarded, and the cells were resuspended in 30 mL of preheated DMEM medium. After centrifugation at 1500 rpm for 3 min, the supernatant was discarded, and an appropriate amount of preheated DMEM medium was added to resuspend the cells and count them. The appropriate cell amounts were taken at a ratio of 10:1 for mouse spleen cells and SP2 / 0 cells, mixed thoroughly in a 50 mL centrifuge tube, centrifuged at 1500 rpm for 3 min, and the supernatant was discarded. Then, 1 mL of preheated PEG was slowly added, and the mixture was incubated at 37℃ for 25 min. 5 mL of preheated DMEM medium containing 10% fetal bovine serum was slowly added dropwise along the tube wall, and then the speed was gradually increased, adding 15 mL of preheated DMEM medium containing 10% fetal bovine serum, finally adding to a total of 40 mL.) Centrifuge at rpm for 5 min, discard the supernatant, and add 25 mL of recovery medium. Continue culturing and passage the hybridoma cell lines in DMEM medium containing 10% fetal bovine serum. Then, use HAT medium for selective culture. After 10–15 days post-convergence, collect the supernatant and use indirect ELISA to screen for antibody-secreting hybridoma cell lines. After one passage, perform a second screening using indirect ELISA. Finally, perform subclonal selection on the confirmed cell lines using limiting dilution to obtain stable monoclonal antibody-secreting hybridoma cell lines.

[0042] 3. ELISA screening and affinity verification Coating with peptide 1 / 2 or peptide 1 / 2 conjugated with BSA, add 100 µL to each well and incubate overnight at 4°C. Wash the plate three times with PBS, then block with 5% milk-PBS at room temperature for 1 hour, followed by one wash with PBS. Add serially diluted hybridoma cell supernatant (for screening / validation) or monoclonal antibody (for affinity verification) to the ELISA plate and incubate for 1 hour at room temperature. Wash the plate three times with PBS, blot dry, add 1:2000 diluted HRP-labeled goat anti-mouse IgG (Fc) secondary antibody, and incubate for 1 hour at room temperature. Wash the plate five times with PBS, blot dry, add TMB for color development, and incubate for 20 min in the dark at room temperature. Finally, add 50 µL of stop solution, mix well, and read the OD on a microplate reader. 450 -OD 630 value.

[0043] 4. Antibody preparation Adult BALB / c mice were selected and intraperitoneally injected with Freund's incomplete adjuvant, 0.5 ml per mouse. 7-10 days later, 16th generation hybridoma cells were intraperitoneally injected, 1 × 10⁶ cells per mouse. 6-2×10 6 Cells were collected. Five days later, when the abdomen was noticeably distended and the skin felt taut to the touch, ascites fluid was collected. The ascites fluid was centrifuged at 13000 r / min for 30 min to remove cellular components and other precipitates, and the supernatant was collected. Purification was performed using a Protein G purification column with 20 mM PBS buffer as the loading buffer and 50 mM glycine buffer (pH 2.7) as the elution buffer to obtain purified monoclonal antibodies.

[0044] 5. Immunohistochemistry Tissue sections were baked at 60℃ for 3 h; after dewaxing and rehydration with xylene and graded ethanol, they were placed in EDTA solution for microwave antigen retrieval (heated to boiling, incubated at room temperature for 6 min, repeated 4 times), and cooled to room temperature. Endogenous peroxidase inhibitor was added, and the sections were incubated at room temperature for 10 min, followed by washing with PBS. Blocking with histochemistry-specific blocking buffer was performed at 37℃ for 1 h; primary antibody was added, and the sections were incubated overnight at 4℃, equilibrated at room temperature for 1 h the next day, and washed with PBST. Reaction enhancement buffer was added, and the sections were incubated at room temperature for 20 min, followed by washing with PBST; the corresponding secondary antibody was added, and the sections were incubated at room temperature for 20 min, followed by washing with PBS. DAB staining was performed (time controlled under a microscope), followed by washing with PBST; hematoxylin staining of nuclei, hydrochloric acid differentiation, and ammonia blue reversion. The sections were dehydrated with graded ethanol and xylene, mounted with neutral resin, air-dried, and then scanned using a slide scanner.

[0045] 6. Immunoblotting detection The specificity of the antibody was detected using a conventional Western blotting method. 100 μg of mutant protein sample was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane by wet transfer, and then Western blotting was performed using purified antibody.

[0046] II. Experimental Results Indirect ELISA screening results are as follows Figure 1 , 2 As shown in Table 1, the positive clones obtained after positive and negative screening were re-screened and verified. The antibody with clone number 21C5 was selected for subsequent subclone screening, and the results are shown in Table 2. The antibody with clone number 21C5-A6 was selected for subsequent sequencing and affinity verification. The sequence information of the 21C5-A6 antibody is shown in Table 3. After SDS-PAGE analysis, the purity of the 21C5-A6 antibody was greater than 95%. Figure 3 Immunohistochemical results showed that the 21C5-A6 antibody specifically recognized the K27M mutation site ( Figure 4 The affinity results showed that the antibody and peptide 1 antigen had a detection sensitivity of 0.005 µg / ml, indicating good affinity (Table 4).

[0047] Table 1. Validation of positive clones through rescreening

[0048] Table 2 Subcloning Screening

[0049] Table 3 Antibody sequence information

[0050] Table 4 Affinity Results

[0051] The above results collectively demonstrate that the antibody 21C5-A6 prepared in this invention has high affinity and strong specificity, and can specifically recognize histone H3 K27M mutations.

[0052] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antibody targeting histone H3 with a K27M mutation, characterized in that, The heavy chain variable region sequence of the antibody is shown in SEQ ID NO:1, and the light chain variable region sequence is shown in SEQ ID NO:

5.

2. The antibody according to claim 1, characterized in that, The heavy chain variable region CDR1-3 sequences of the antibody are shown in SEQ ID NO:2-4, and the light chain variable region CDR1-3 sequences are shown in SEQ ID NO:6-8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody as described in claim 1 or 2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the antibody of claim 1 or 2 or the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that, The recombinant cells contain the antibody of claim 1 or 2, the nucleic acid molecule of claim 3, or the recombinant vector of claim 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells include prokaryotic cells and eukaryotic cells.

7. An antibody derivative, characterized in that, The antibody derivative is a complex formed by directly or indirectly coupling the antibody of claim 1 or 2 to a detectable marker.

8. The antibody derivative according to claim 7, characterized in that, The detectable markers include tags, bioluminescent agents, chemiluminescent agents, enzymes, photosensitizing diagnostic agents, paramagnetic ions, or radionuclides.

9. A testing product, characterized in that, The detection product contains the antibody as described in claim 1 or 2 or the antibody derivative as described in claim 7 or 8.

10. The testing product according to claim 9, characterized in that, The testing products include reagents, kits, chips, and test strips.

11. A method for preparing the recombinant cells according to claim 5 or 6, characterized in that, The method includes introducing the recombinant vector of claim 4 into cells to obtain the recombinant cells.

12. A method for producing the antibody according to claim 1 or 2, characterized in that, The method includes culturing the recombinant cells of claim 5 or 6 and isolating the antibody from the culture of the recombinant cells.

13. The use of the antibody of claim 1 or 2, the antibody derivative of claim 7 or 8, or the detection product of claim 9 or 10 in the preparation of a product for diagnosing histone H3 K27M mutant diffuse midline glioma.

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