Preparation and application of recombinant anti-human CDK4 antibodies

Abstract

This invention relates to the field of antibody technology, specifically to the preparation and application of recombinant anti-human CDK4 antibodies. The CDK4 recombinant antibody provided by this invention is prepared by constructing a human CDK4 antibody expression vector and using a CHO expression system. Simultaneously, the specificity of the antibody is verified by Western blotting and immunohistochemistry, demonstrating that the recombinant antibody provided by this invention has high specificity, stable properties, meets the requirements of experimental reproducibility, and is suitable for industrial production and application.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to the preparation and application of recombinant anti-human CDK4 antibodies. Background Technology

[0002] CDK4 (cyclin-dependent kinase 4) is an enzyme encoded by the CDK4 gene in the human body, belonging to the CDK family of cyclin-dependent protein kinases. This protein is encoded by the human CDK4 gene, located on chromosome 12q13-14. The full-length mRNA sequence is 2.14 kb, and the full-length cDNA is 912 bp, encoding 303 amino acids with a molecular weight of approximately 34 kb, very similar to CDK2. Because CDK4 lacks the typical PSTAIR sequence, its activity depends on the binding of cyclin D.

[0003] The malignant proliferation of tumor cells is mainly due to abnormal cell cycle regulation, and the core of this regulation mechanism is the phase-specific activation of CDKs. CDK4 binds to Cyclin D1 to form a complex that regulates the G1 / S phase of the cell cycle. Studies have shown that CDK4 is closely related to carcinogenesis. CDK4 can initiate DNA replication and mitosis; increased expression leads to increased DNA synthesis, which in turn causes uncontrolled cell division and proliferation, resulting in carcinogenesis. Overexpression and overactivation of CDK4 have been found in various tumor cells and cancerous tissues, making CDK4 an effective target for cancer treatment.

[0004] Reports indicate that CDK4 is almost universally positive in tumor tissues such as myxoid / round cell lipoma and rhabdomyosarcoma (≥95% of cases are positive); its positive expression rate is ≥75% in dedifferentiated liposarcoma, atypical liposarcoma, and low-grade osteosarcoma; its positive expression rate is ≥55% in pancreatic adenocarcinoma, low-grade central osteosarcoma, and pancreatic ductal adenocarcinoma; and it is also positively expressed in endometrial adenocarcinoma and low-grade fibromyxoid sarcoma. Furthermore, studies have found high expression of CDK4 in oral squamous cell carcinoma, lung cancer, breast cancer, and colon cancer. Therefore, CDK4 plays a significant role in the diagnosis, treatment, and prognosis of tumors.

[0005] Currently, traditional anti-CDK4 monoclonal antibodies on the market are still obtained by traditional animal immunization methods. This method cannot overcome batch-to-batch variations caused by individual animal differences, and the yield is low. The antibody stability is poor, and the problem of hybridoma cell loss cannot be avoided, which limits its further industrial production and application. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide the preparation and application of anti-human CDK4 recombinant antibody. The anti-human CDK4 recombinant antibody provided by the present invention has high specificity and stable properties, which helps to improve the reproducibility of experiments.

[0007] The CDK4 recombinant antibody provided by this invention comprises the following amino acid sequence:

[0008] (1) The CDR region of its heavy chain contains at least one of the amino acid sequences shown in SEQ ID NO:1, 2 or 3, and the CDR region of its light chain contains at least one of the amino acid sequences shown in SEQ ID NO:4, 5 or 6; or

[0009] (2) An amino acid sequence that has undergone substitution, addition, or deletion of one or more amino acids as shown in (1), but has the same or similar function as the amino acid sequence shown in I; or

[0010] (3) An amino acid sequence that has at least 80% homology with the amino acid sequence shown in (1) or (2).

[0011] In this invention, the CDK4 recombinant antibody contains:

[0012] I. At least one of the four FR regions of its heavy chain has an amino acid sequence as shown in SEQ ID NO:7,8,9 and10; at least one of the four FR regions of its light chain has an amino acid sequence as shown in SEQ ID NO:11,12,13 and14.

[0013] II. An amino acid sequence that has undergone substitution, addition, or deletion of one or more amino acids compared to the amino acid sequence shown in I, but has the same or similar function as the amino acid sequence shown in I; or

[0014] III. An amino acid sequence that has at least 80% homology with the amino acid sequence shown in I or II.

[0015] In this invention, the sequence having at least 80% homology is an amino acid sequence obtained by substituting, adding, or deleting one or more amino acids based on the original sequence, wherein the plurality of amino acids is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

[0016] In some embodiments, the three CDR regions of the CDK4 recombinant antibody heavy chain have amino acid sequences as shown in SEQ ID NO:1, 2 and 3, respectively;

[0017] In some embodiments, the three CDR regions of the CDK4 recombinant antibody light chain have amino acid sequences as shown in SEQ ID NO:4, 5 and 6, respectively.

[0018] In some embodiments, the four FR regions of the CDK4 recombinant antibody heavy chain have amino acid sequences as shown in SEQ ID NO:7, 8, 9 and 10, respectively;

[0019] In some embodiments, the four FRs of the CDK4 recombinant antibody light chain have amino acid sequences as shown in SEQ ID NO:11, 12, 13 and 14, respectively.

[0020] In some specific embodiments, the heavy chain variable region of the CDK4 recombinant antibody includes the CDR region shown in SEQ ID NO:1, 2 or 3 and the FR region shown in SEQ ID NO:7, 8, 9 and 10.

[0021] In this embodiment of the invention, the light chain variable region of the monoclonal antibody includes the CDR region shown in SEQ ID NO:4, 5 or 6 and the FR region shown in SEQ ID NO:11, 12, 13 and 14.

[0022] In some specific embodiments, the heavy chain variable region of the CDK4 recombinant antibody has an amino acid sequence as shown in SEQ ID NO:15; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:16.

[0023] This invention provides a biomaterial comprising at least one of the following:

[0024] 1) The nucleic acid encoding the CDK4 recombinant antibody;

[0025] 2) An expression vector containing the nucleic acid;

[0026] 3) Transform or transfect the host cells containing the expression vector;

[0027] 4) The conjugate prepared by coupling the CDK4 recombinant antibody with a solid or semi-solid medium;

[0028] 5) The aforementioned CDK4 recombinant antibody, which has been chemically or biologically labeled;

[0029] 6) The conjugate prepared by coupling the chemically or biologically labeled CDK4 recombinant antibody with a solid or semi-solid medium.

[0030] Specifically, the nucleic acid includes: a nucleic acid encoding the heavy chain variable region having a nucleotide sequence as shown in SEQ ID NO:17; and a nucleic acid encoding the light chain variable region having a nucleotide sequence as shown in SEQ ID NO:18.

[0031] The expression vector of the present invention can express and produce the CDK4 recombinant antibody in a host cell, wherein the host cell is selected from Escherichia coli, yeast, insect cells or mammalian cells.

[0032] The chemical marker is an isotope, immunotoxin, and / or chemical drug; the biomarker is biotin, avidin, or enzyme labeling. The enzyme labeling is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abscisicin, mistletoe lectin, sucralose root toxin, PAP, phytotoxicin, white tree toxin, or loofah toxin.

[0033] The solid or semi-solid medium refers to any support onto which the recombinant antibody or labeled recombinant antibody described in this invention can attach, including but not limited to nitrocellulose membranes, polyvinylidene fluoride (PVDF) membranes, iPDMS chips, microplates, polystyrene plates, microparticles, microcarriers, gels, etc.

[0034] The application of the CDK4 recombinant antibody, biomaterials, and CDK4 recombinant antibody expressed in host cells described in this invention in the preparation of tumor detection reagents or kits.

[0035] The tumor cells include any one or more of the following: myxoid / round cell lipoma, rhabdomyosarcoma, differentiated liposarcoma, atypical liposarcoma, low-grade osteosarcoma, pancreatic adenocarcinoma, low-grade central osteosarcoma, pancreatic ductal adenocarcinoma, endometrial adenocarcinoma, low-grade fibromyxosarcoma, oral squamous cell carcinoma, lung cancer, breast cancer, or colon cancer.

[0036] The present invention provides a detection reagent or detection kit, which includes the CDK4 recombinant antibody described in the present invention, the biological material, and the CDK4 recombinant antibody expressed by the host cell.

[0037] The detection reagent or detection kit may also include acceptable adjuvants, buffer solutions, excipients or carriers.

[0038] The present invention also provides a diagnostic method for tumor-related diseases, including: testing a sample using the detection reagent or detection kit of the present invention.

[0039] This invention provides the preparation and application of recombinant anti-human CDK4 antibodies. A human CDK4 antibody expression vector is constructed, and the recombinant antibody is prepared using a CHO expression system. Compared with existing technologies, the recombinant antibody provided by this invention has high specificity, stable antibody properties, and meets the requirements for experimental reproducibility, making it suitable for industrial production and application. Immunohistochemical staining of colon cancer tissue using 0.08 μg / mL recombinant CDK4 antibody and 0.4 μg / mL commercially available CDK4 monoclonal antibody showed that the recombinant CDK4 antibody had a stronger staining effect. Attached Figure Description

[0040] Figure 1 Western blot pattern of CDK4 monoclonal antibody (M: molecular weight marker; 1: sp2 / 0 cell lysis buffer; 2: MAD109 cell lysis buffer);

[0041] Figure 2 Western blot image of CDK4 recombinant antibody (M: molecular weight marker; 1: mouse lung cancer tissue lysate; 2: MAD109 cell lysate);

[0042] Figure 3 The results of immunohistochemistry of CDK4 recombinant antibody in colon cancer tissue;

[0043] Figure 4 The results of immunohistochemistry of commercially available CDK4 mouse monoclonal antibody in colon cancer tissue are presented. Detailed Implementation

[0044] This invention provides the preparation and application of recombinant anti-human CDK4 antibodies. 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 realize and apply the technology of this invention.

[0045] The sequences involved in this invention are as follows:

[0046] The sequence shown in SEQ ID NO:1 is DFSLTRYA;

[0047] The sequence shown in SEQ ID NO:2 is IWRGGNT;

[0048] The sequence shown in SEQ ID NO:3 is AKDGYGTMDY;

[0049] The sequence shown in SEQ ID NO:4 is SVSY;

[0050] The sequence shown in SEQ ID NO:5 is STS;

[0051] The sequence shown in SEQ ID NO:6 is QQRSSYPPT.

[0052] The sequence shown in SEQ ID NO:7 is QVQLKQSGPGLVQPSQSLSITCTVS;

[0053] The sequence shown in SEQ ID NO:8 is IHWVRQSPGKGLEWLGV;

[0054] The sequence shown in SEQ ID NO:9 is DYNAVFMSRLSITKDTSKSQVFFKMNSLQADDTAIYYC;

[0055] The sequence shown in SEQ ID NO:10 is WGQGTSVTVSS;

[0056] The sequence shown in SEQ ID NO:11 is QIVLTQSPAIMSASPGEKVTITCSAS;

[0057] The sequence shown in SEQ ID NO:12 is MHWFQQKPGTSPKLWIY;

[0058] The sequence shown in SEQ ID NO:13 is NLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC;

[0059] The sequence shown in SEQ ID NO:14 is FGGGTKLEIK.

[0060] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0061] The present invention will be further illustrated below with reference to the embodiments:

[0062] Example 1: Design of CDK4 antigen

[0063] Based on the CDK4 protein sequence and structure published by Uniprot, amino acids 130-303 were selected for the preparation of recombinant CDK4 protein. The Uniprot accession number for CDK4 is P11802, and it contains 303 amino acids, of which 12-20 amino acids are nucleic acid binding sites. Based on bioinformatics analysis and literature review, amino acids 130-303 were selected as the preferred immunogen design for CDK4. The nucleotide and amino acid sequences of the recombinant CDK4 protein are shown below:

[0064] Nucleotide sequence:

[0065] (SEQ ID NO:23);

[0066] amino acid sequence:

[0067] FLHANCIVHRDLKPENILLVTSGGTVKLADFGLARIYSYQMALTPVVVTLWYRAPEVLLQSTYATPVDMWSVGCIFAEMFRRKPLFCGNSEADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLEMLTFNPHKRISAFRALQHSYLHKDEGNPE(SEQ ID NO:24)

[0068] Example 2: Fusion Expression and Purification

[0069] 1. The nucleic acid sequence at the tail of CDK4 was artificially synthesized, and the restriction enzyme sites BamHI and XhoI were introduced at both ends of the sequence. After synthesis, it was cloned into the pET-32a expression vector to construct the pET-32a-CDK4 expression vector.

[0070] 2. Transform the recombinant plasmid constructed above into competent BL21(DE3) Escherichia coli cells, pick a single colony and culture it in 4 mL of LB liquid medium containing ampicillin (100 μg / mL), add IPTG to a final concentration of 1 mM for expression analysis, and preserve the strain that can express the target protein.

[0071] 3. Inoculate the strains that can express the target protein into 350 mL of LB liquid medium containing ampicillin (100 μg / mL), and culture at 37°C until the OD600 reaches 0.6. Then add IPTG to a final concentration of 0.4 mM and induce expression at 37°C for 4 h.

[0072] 4. Centrifuge to collect the induced bacterial cells, resuspend in ice-cold 10mM PBS, sonicate (350W, 20min, 3s working, 3s rest), centrifuge at 12000g for 10min, and collect the supernatant for further purification.

[0073] 5. Filter the supernatant solution through a 0.45 μm filter, and purify the filtrate using nickel agarose gel electrophoresis. Load the filtrate into an equilibrated gel at a flow rate of 1 mL / min. Wash with 10 mM PBS to remove unadsorbed protein, and perform linear elution with PBS and PBS containing 0.5 M imidazole. Collect different elution peaks for SDS-PAGE protein identification. Ultrafilter samples with a purity greater than 90% to 10 mM PBS.

[0074] Example 3: Screening of CDK4 hybridoma cell lines and preparation of monoclonal antibodies

[0075] 1. Animal Immunization: The CDK4 protein obtained above was purified separately. This protein was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into 6-8 week old BalB / c mice at a dose of 100 μg / mouse. A second immunization was performed two weeks later using Freund's incomplete adjuvant emulsification at the same dose, via intraperitoneal injection. After the second immunization, tail blood was collected and serum titers were determined using a serial dilution method with indirect ELISA. Mice with the highest antibody titers were selected for tail vein pulse immunization at a dose of 100 μg / mouse.

[0076] 2. Cell fusion: Myeloma cells were sp2 / 0 derived from BalB / c and were in the logarithmic growth phase at the time of fusion. Spleens of mice after shock immunization were aseptically harvested and spleen cells were prepared into a single-cell suspension. Mouse spleen cells and myeloma cells were mixed at a ratio of 1:5, 1 mL of 50% PEG (pH 8.0) at 37°C was added, incomplete culture medium was added, the supernatant was discarded after centrifugation, HAT culture medium was added to suspend and mix well, the volume was adjusted to 50 mL, and the suspension was added to 96-well cell culture plates and cultured in a 37°C, 5% CO2 incubator.

[0077] 3. Screening and Cloning: Cell clones are selected within 7-10 days after fusion for screening tests (ELISA, IHC, WB, and other methods). Positive wells are subjected to limiting dilutions, with ELISA values ​​measured 5-6 days after each dilution. Wells with high OD450 positive values ​​are selected for further limiting dilutions until the entire 96-well plate shows a positive ELISA result. High-positive-value clones are then selected to confirm the cell line.

[0078] 4. Preparation and purification of ascites monoclonal antibodies: 8-10 week old BalB / c mice were intraperitoneally injected with 0.5 ml of liquid paraffin. One week later, each mouse was intraperitoneally injected with a 1 ml syringe containing a monoclonal cell suspension washed and resuspended in PBS. The cell volume was 5 × 10⁶ cells / mouse, and each cell line was injected into 3 mice. After ascites fluid accumulated, it was collected, centrifuged, and the supernatant was purified by Protein A column chromatography. The purified monoclonal antibody concentration was determined, aliquoted, and stored at -20℃.

[0079] Example 4: Western blotting to detect the specificity of CDK4 monoclonal antibody

[0080] 1. Sample preparation: Prepare sp2 / 0 cell lysis buffer and MAD109 cell lysis buffer. After lysis with RAPI containing 1mM PMSF, add 5×SDS-PAGE Loading buffer to prepare the sample.

[0081] 2. Electrophoresis and transfer: Load 30 μg of each sample and electrophore at 80V for 20 min, then electrophore at 120V until the bromophenol blue reaches the bottom of the gel. Then start the transfer. Soak the NC membrane and filter paper in transfer buffer beforehand, and place them in a sandwich configuration in the wet transfer apparatus. The sequence is: electrode (-) - sponge - filter paper - gel - NC membrane - filter paper - sponge - electrode (+), constant voltage 90V, 90 min.

[0082] 3. Blocking: Immerse the NC membrane in blocking solution (TBST containing 5% skim milk) and incubate at 37°C for 2 hours.

[0083] 4. Primary antibody incubation: Place the NC membrane in a monoclonal antibody containing anti-CDK4 (dilute the antibody 1:2000 with blocking buffer), incubate at 37°C for 1 hour, and then wash the membrane 3 times with TBST for 5 minutes each time.

[0084] 5. Secondary antibody incubation: Place the NC membrane in HRP-labeled goat anti-mouse IgG secondary antibody (the antibody was diluted 1:5000 with blocking buffer), incubate at 37°C on a shaker for 30 min, and then wash the membrane 3 times with TBST for 5 min each time.

[0085] 6. Color development: Mix components A and B in the ECL luminescent solution at a ratio of 1:1 and add it to the NC film, then expose it in an exposure apparatus.

[0086] Color development results as follows Figure 1 As shown, the CDK4 monoclonal antibody exhibits a target band of approximately 34 kDa in MAD109 cell lysates.

[0087] Example 5: CDK4 antibody gene extraction

[0088] 1. Total RNA extraction: Resuscitate CDK4 hybridoma cells, then test cell line titer and collect cells; add 1 ml Trizol, mix well, and incubate at room temperature for 5-10 min until cells are completely lysed; centrifuge at 12000 rpm for 5 min, discard the precipitate, then add 200 μL chloroform, invert vigorously, and incubate for 15 min; centrifuge at 12000 rpm at 4℃ for 15 min and collect the upper aqueous phase; add an equal volume of isopropanol, mix gently, and incubate for 10 min; then centrifuge at 12000 rpm at 4℃ for 10 min and discard the supernatant; add 1 ml pre-chilled 75% ethanol, gently tap the bottom of the tube, centrifuge at 12000 rpm at 4℃ for 5 min, discard the supernatant, air dry at room temperature for several minutes, then add 30-50 μL pre-chilled DEPC water until dissolved to obtain the RNA product; store at -80℃ for long-term storage; masks and gloves must be worn throughout the extraction process to prevent RNase contamination.

[0089] 2. Obtaining CDK4 antibody gene via 5' RACE

[0090] The 5' sequence of the CDK4 antibody was isolated using the SMARTer RACE 5' / 3' Kit (Clontech, Cat. No. 634859). Using 1 μg of total RNA as a template, first-strand cDNA synthesis was performed according to the manufacturer's instructions using the 5'-CDS primer A, SMART II A oligo, and 3'-CDS primer A provided in the kit, yielding 5'-RACE-Ready cDNA. PCR (Rapid Amplification of cDNA Ends) was then performed using these cDNAs as templates. 5'-RACE PCR used UPM (Universal Primer) primers, and GSP (gene-specific primer) primers designed based on the gene-specific sequence. The first round of PCR reaction system consisted of 50 μL of the following components: 2.5 μL of 5'-RACE-Ready cDNA, 5 μL of 10×UPM, 1 μL of 10 μM GSP, 25 μL of 2×SeqAmp buffer, 1 μL of SeqAmp DNA polymerase, and 15.5 μL of ddH2O. The PCR reaction program was as follows: 5 cycles, each cycle 94℃ for 30 sec, 72℃ for 3 min; 5 cycles, each cycle 94℃ for 30 sec, 70℃ for 30 sec, 72℃ for 3 min; 25 cycles, each cycle 94℃ for 30 sec, 68℃ for 30 sec, 72℃ for 3 min; PCR products were stored at 4℃. 5 μL of the PCR product was subjected to 1.0% agarose gel electrophoresis for detection. If a single band was observed, the remaining product was excised, recovered, and reconstructed onto pRACE for further sequencing of positive clones. If the bands are not uniform, nested PCR is required for further amplification before constructing the amplified material onto pRACE and then selecting positive clones for sequencing.

[0091] Example 6: Preparation of Recombinant Antibody

[0092] 1. Based on sequencing results and the IMGT database, heavy chain primers and light chain primers were designed. The primers for amplifying the heavy chain variable region gene are as follows:

[0093] HF: TAAACGGATCTCTAGCGAATTCATGGCTGTCTTAGCGCTGCTC (SEQ ID NO: 19);

[0094] HR:CGAGCGGCCGCTAGCAAGCTTTCATTTACCAGGAGAGTGGGAG (SEQ ID NO: 20);

[0095] The primers for amplifying the light chain variable region gene are as follows:

[0096] LF: TAAACGGATCTCTAGCGAATTCATGGATTTACAGGTGCAGAT (SEQ ID NO: 21);

[0097] LR:CGAGCGGCCGCTAGCAAGCTTTTAACACTCATTCCTGTTGAAG (SEQ ID NO: 22);

[0098] Using cDNA as a template, amplification was performed using PrimeSTAR Max DNA Polymerase (TAKARA Code No. R045Q). The PCR reaction conditions were: 98℃ for 3 min; 98℃ for 10 s, 62℃ for 10 s, 72℃ for 10 s, for a total of 32 cycles; 72℃ for 5 min. The PCR-amplified fragments and pTT5 vector were digested with EcoRI / HindIII for 3 h, and then recovered by agarose gel electrophoresis. The antibody expression vector was then constructed via recombination, and plasmids were extracted from correctly sequenced positive clones for transfection.

[0099] 2. Identification of antibody heavy and light chain gene information and analysis of the IMGT database.

[0100] Heavy chain genes:

[0101]

[0102] Heavy chain amino acid sequence:

[0103] QVQLKQSGPGLVQPSQSLSITCTVSDFSLTRYAIHWVRQSPGKGLEWLGVIWRGGNTDYNAVFMSRLSITKDTSKSQVFFKMNSLQADDTAIYYCAKDGYGTMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:15).

[0104] IMGT analysis:

[0105] CDR-H1: DFSLTRYA(SEQ ID NO:1); CDR-H2: IWRGGNT(SEQ ID NO:2); CDR-H3: AKDGYGTMDY(SEQ ID NO:3).

[0106] FR-H1 QVQLKQSGPGLVQPSQSLSITCTVS(SEQ ID NO:7);

[0107] FR-H2 IHWVRQSPGKGLEWLGV(SEQ ID NO:8);

[0108] FR-H3 DYNAVFMSRLSITKDTSKSQVFFKMNSLQADDTAIYYC(SEQ ID NO:9);

[0109] FR-H4 WGQGTSVTVSS(SEQ ID NO:10).

[0110] Light chain gene:

[0111] ATGGATTTACAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTAGTTACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT(SEQ ID NO:18).

[0112] Light chain amino acid sequence:

[0113] QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPPTFGGGTKLEIKRAD AAAPVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECC(SEQ IDNO:16).

[0114] IMGT Analysis:

[0115] CDR-L1: SSVSY (SEQ ID NO: 4); CDR-L2: STS (SEQ ID NO: 5); CDR-L3: QQRSSYPPT (SEQ ID NO: 6).

[0116] FR-L1 QIVLTQSPAIMSASPGEKVTITCSAS(SEQ ID NO:11);

[0117] FR-L2MHWFQQKPGTSPKLWIY(SEQ ID NO:12);

[0118] FR-L3 NLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYC(SEQ ID NO:13);

[0119] FR-L4 FGGGTKLEIK (SEQ ID NO: 14).

[0120] 3. Expression and purification of anti-CDK4 recombinant antibody, including pre-transfection cell culture, preparation of transfected cells, transient transfection, and product expression and detection;

[0121] (1) The method of cell culture before transfection is as follows: place CHO cells in a 5% CO2 constant temperature shaker and culture at 37℃ and 120rpm. When passaged, cell count should be performed first. After confirming the density, there is no need to centrifuge the cells. The cell suspension can be directly added to the culture medium according to the required ratio. If too many dead cells occur during the culture process, the cells should be discarded and new cells should be used.

[0122] (2) The method for preparing transfected cells is as follows:

[0123] Before transient transfection, cell density and viability need to be determined; cells do not need to be centrifuged, but can be directly added to CHO culture medium to dilute the cell density to 2×10⁻⁶.6 Cells / mL; place the shake flask in a 5% CO2 constant temperature shaker and incubate at 37℃ and 120 rpm for 10 min before transfection. For transient transfection, prepare two 15 mL sterile centrifuge tubes. Add 5 mL of KPM and sterile plasmid DNA to one tube at a heavy chain:light chain ratio of 1:1, and gently pipette to mix. Take the other centrifuge tube and add 5 mL of KPM and 500 μL of TA-293 transfection reagent, and gently pipette to mix. Transfer all liquid from the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently pipette to mix. Incubate at room temperature for 10 minutes to prepare the plasmid-vector complex. Remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO2 constant temperature shaker for incubation. After 3 hours, add an appropriate amount of antibiotic as needed.

[0124] Product expression and detection methods: 600 μL of 293 cell protein expression enhancer can be added 24 hours after transfection to increase the product expression level; transient transfection nutrient additive can be added 24 hours after transfection.

[0125] (3) Antibody expression and purification: Cells were adjusted to the logarithmic growth phase, transfected with plasmids, and cultured for 48–72 h before cell collection. The supernatant was centrifuged to remove cells, and then purified by affinity chromatography using a Protein G column. The antibodies obtained from the binding adsorption were eluted with citrate buffer at pH 3.0, and the eluent was collected and rapidly neutralized to pH 7.2–7.4 using Tris-HCl solution at pH 8.8. The purified antibodies were then concentrated to a concentration of 1 mg / mL or higher by dialysis.

[0126] Example 7: Western blotting to detect the specificity of CDK4 recombinant antibody

[0127] For specific experimental steps, please refer to Example 4. The specific samples used were mouse lung cancer tissue lysis buffer and MAD109 cell lysis buffer.

[0128] The results are as follows Figure 2 As shown, the CDK4 recombinant antibody exhibited the target band (approximately 34 KD) in both mouse lung cancer tissue lysate and MAD109 cell lysate.

[0129] Example 8: Preparation of CDK4 Immunohistochemical Detection Reagent

[0130] The immunohistochemical reagent in this embodiment is an immunohistochemical detection reagent, and a CDK4 recombinant antibody working solution is constructed based on the recombinant antibody in Example 6. The specific components of the reagent include: a recombinant antibody concentration of 0.08 μg / mL, a TBS buffer containing 10 mg / mL BSA (bovine serum albumin), and preservatives Proclin 300 and Proclin 950, all added at a ratio of 1:1000 (V / V).

[0131] Example 9: Immunohistochemical (IHC) detection of the specificity of CDK4 recombinant antibody

[0132] The anti-human CDK4 immunohistochemical reagent prepared in Example 8 was used for immunohistochemical staining. A parallel comparison experiment was conducted using a commercially available mouse monoclonal antibody with clone number EP180. The concentration of the recombinant CDK4 antibody was 0.08 μg / mL. The specific experimental procedure is as follows:

[0133] 1. Take a paraffin block of colon cancer and slice it using a Leica tissue slicer. The tissue thickness is 3μm. Dry bake at 65℃ for 2 hours.

[0134] 2. The antibodies of this invention were subjected to immunohistochemical staining using a manual immunohistochemical method. The specific steps were as follows: High-temperature repair was performed for 20 min using DAKO HIGH pH repair solution; after cooling, 100 μL of endogenous peroxidase blocking solution was used for incubation at room temperature for 5 min, followed by immersion in washing solution twice, 5 min each time; 100 μL of the above two antibody working solutions were added for the primary antibody, and incubation was performed at 37°C for 30 min, followed by immersion in washing solution twice, 5 min each time; 100 μL of enzyme-labeled polymer was used for incubation at room temperature for 20 min, followed by immersion in washing solution twice, 5 min each time; 100 μL of DAB chromogenic solution was used for incubation at room temperature for 5 min, followed by immersion in purified water twice, 5 min each time; 100 μL of hematoxylin was added for counterstaining, and incubation was performed at room temperature for 5 min, followed by rinsing with tap water to achieve blue staining.

[0135] 3. Dehydration, clearing and mounting: Wash with deionized water for 3 min; 85% ethanol for 1 min; 95% ethanol for 1 min; 100% ethanol for 1 min, twice; xylene for 1 min, twice; mount with neutral resin.

[0136] 4. Microscopic examination

[0137] The localization results of CDK4 recombinant antibody and CDK4 murine monoclonal antibody in colorectal cancer tissues are as follows: Figure 3 and Figure 4 As shown, by Figure 3 and Figure 4It can be seen that both the CDK4 recombinant antibody and the control antibody were positive in colon cancer tissue. By comparing the control antibody (concentration of 0.4 μg / mL), the staining of the CDK4 recombinant antibody was slightly stronger, and the concentration of the CDK4 recombinant antibody was 0.08 μg / mL. Therefore, this antibody has good affinity and stronger staining effect.

[0138] In the immunohistochemical evaluation of other tissues, the staining results of CDK4 recombinant antibody and CDK4 mouse monoclonal antibody products were compared as follows: The paraffin blocks used for evaluation included 28 types of tumors, such as mesenchymal tumors, normal colon, colon cancer, renal dedifferentiated liposarcoma, pancreatic cancer, ovarian serous carcinoma, urothelial carcinoma, synovial sarcoma, breast fat-derived tumors, solitary fibromas, serous carcinoma, pelvic myxoid fibrosarcoma, spindle cell tumors of the lower right femur, retroperitoneal tumors, and cervical squamous cell carcinoma.

[0139] The tissue evaluation results of CDK4 recombinant antibody and CDK4 mouse monoclonal antibody are shown in Table 1:

[0140]

[0141] The results in Table 1 show that the CDK4 recombinant antibody and the CDK4 mouse monoclonal antibody have the same positive and negative results in tissue evaluation.

[0142] 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 CDK4 recombinant antibody, characterized in that, The amino acid sequences of the three CDR regions of its heavy chain are shown in SEQ ID NO:1, 2 and 3, respectively; The amino acid sequences of the three CDR regions of its light chain are shown in SEQ ID NO:4, 5 and 6, respectively.

2. The CDK4 recombinant antibody as described in claim 1, characterized in that, The amino acid sequences of the four FR regions of its heavy chain are shown in SEQ ID NO:7, 8, 9 and 10, respectively; The amino acid sequences of the four FR regions of its light chain are shown in SEQ ID NO:11, 12, 13 and 14, respectively.

3. The CDK4 recombinant antibody as described in claim 1 or 2, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:15; The amino acid sequence of its light chain variable region is shown in SEQ ID NO:

16.

4. A biomaterial, characterized in that, Includes at least one of the following: 1) Nucleic acid encoding the CDK4 recombinant antibody as described in any one of claims 1 to 3; 2) An expression vector containing the nucleic acid described in 1); 3) Transform or transfect host cells with the expression vector described in 2); 4) A conjugate prepared by conjugating the CDK4 recombinant antibody according to any one of claims 1 to 3 with a solid or semi-solid medium; 5) The recombinant CDK4 antibody according to any one of claims 1 to 3, which has been chemically or biologically labeled; 6) The conjugate prepared by coupling the CDK4 recombinant antibody of any one of claims 1 to 3, which is chemically or biologically labeled, with a solid or semi-solid medium.

5. The biomaterial as described in claim 4, characterized in that, The nucleic acids include: The nucleic acid encoding the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO:17; The nucleic acid encoding the variable region of the light chain has a nucleotide sequence as shown in SEQ ID NO:

18.

6. The method for preparing the CDK4 recombinant antibody according to any one of claims 1 to 3, characterized in that, include: The host cells as described in claim 4 are cultured to induce the expression of the CDK4 recombinant antibody.

7. The use of at least one of the following (i) to (iii) in the preparation of tumor detection reagents or kits: i. The CDK4 recombinant antibody as described in any one of claims 1 to 3; ii. The biomaterial as described in claim 4; iii. The CDK4 recombinant antibody was prepared by the method described in claim 6; The tumor is any one or more of the following: myxoid / round cell lipoma, rhabdomyosarcoma, differentiated liposarcoma, atypical liposarcoma, low-grade osteosarcoma, pancreatic adenocarcinoma, endometrial adenocarcinoma, low-grade fibromyxoid sarcoma, oral squamous cell carcinoma, lung cancer, breast cancer, or colon cancer.

8. A detection reagent or detection kit, characterized in that, Includes at least one of the following ①~③: ① The CDK4 recombinant antibody as described in any one of claims 1 to 3; ② The biomaterial as described in claim 4 or 5; ③ The CDK4 recombinant antibody was prepared by the preparation method described in claim 6.

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