An anti-human 4-1BB monoclonal antibody and its application

Abstract

This invention relates to the field of biomedicine and discloses an anti-human 4-1BB monoclonal antibody and its applications. This invention utilizes phage display technology, which, compared to hybridoma technology, can present the entire antibody library of an immunized animal, eliminating the cell fusion step and avoiding the tedious process of repeated subcloning due to hybridoma instability. This significantly increases the library capacity from several thousand clones in hybridomas to 10. 9 The anti-human 4-1BB monoclonal antibody obtained by screening in this invention significantly increased the production of the cytokine IFN-γ and increased the proportion of CD8+ cells in TILs in in vitro cellular validation, demonstrating good activation properties.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-human 4-1BB monoclonal antibody and its applications. Background Technology

[0002] 4-1BB, also known as CD137, is a member of the tumor necrosis factor (TNF) receptor family. Encoded by the TNFRSF9 gene, a member of the TNF receptor superfamily, it is expressed on various immune cells after activation, including T cells, dendritic cells, and natural killer cells. 4-1BB signaling can induce cytokine induction, prevent activation-induced cell death, and upregulate cytotoxic T cell activity. 4-1BB may also reduce regulatory T cell infiltration into tumors.

[0003] There are currently no marketed drugs for 4-1BB antibodies. Most are still in the clinical and preclinical research stages, and there are still many shortcomings in terms of antigen binding activity and activation of downstream T cell signals. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an anti-human 4-1BB monoclonal antibody that can specifically bind to 4-1BB molecules and exhibit activation properties on immune cells. It has potential applications in the treatment of various cancers through immunomodulatory effects.

[0005] The first objective of this invention is to provide an anti-human 4-1BB monoclonal antibody.

[0006] (1) The amino acid sequences of the three CDR regions of the heavy chain variable region of the anti-human 4-1BB monoclonal antibody: CDR1 (GDSITSGY), CDR2 (KSYSGST), and CDR3 (ARSLLWLGAMDY) are shown in SEQ ID No. 1, 2, and 3, respectively; and

[0007] (2) The amino acid sequences of the three CDR regions of the light chain variable region of the anti-human 4-1BB monoclonal antibody, namely CDR1 (QDVGTA), CDR2 (WAS) and CDR3 (QQYSSYPYT), are shown in SEQ ID No. 4, 5 and 6, respectively.

[0008] Furthermore, the aforementioned anti-human 4-1BB monoclonal antibody possesses:

[0009] (3) Its heavy chain has the amino acid sequence shown in SEQ ID No. 7 (QMQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWTWIRKFPGNRLEYMGFKSYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARSLLWLGAMDYWGQGTSVTVSS), and

[0010] (4) Its light chain has the amino acid sequence shown in SEQ ID No. 8 (MADIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEMKR).

[0011] A second object of the present invention is to provide a nucleic acid molecule encoding the aforementioned anti-human 4-1BB monoclonal antibody.

[0012] A third objective of this invention is to provide an expression vector containing the aforementioned nucleic acid molecule.

[0013] A fourth object of the present invention is to provide a recombinant cell containing the aforementioned nucleic acid molecule, or containing the aforementioned expression vector.

[0014] Furthermore, the host cell of the recombinant cells is a CHO cell, a HEK293 cell, a yeast cell, or a plant cell.

[0015] Host cells are used for the transient expression of antibodies and the construction of stable cell lines, enabling the research and industrial production of the antibodies.

[0016] A fifth object of the present invention is to provide the use of the monoclonal antibody in the preparation of medicaments for the treatment and prevention of 4-1BB-mediated diseases.

[0017] Furthermore, the disease in question is a tumor.

[0018] Furthermore, the tumors mentioned are lung cancer, stomach cancer, intestinal cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, and bladder cancer.

[0019] A sixth object of the present invention is to provide a pharmaceutical composition comprising:

[0020] (1) The anti-human 4-1BB monoclonal antibody described above; and,

[0021] (2) Pharmaceutically acceptable carrier.

[0022] By means of the above-described solution, the present invention has at least the following advantages:

[0023] This invention utilizes phage display technology. Compared to hybridoma technology, phage display can present the entire antibody library of an immunized animal, eliminating the cell fusion step and avoiding the tedious process of repeated subcloning due to hybridoma instability. This significantly increases the library capacity from several thousand clones in hybridomas to 10. 9 The anti-human 4-1BB monoclonal antibody obtained by screening in this invention significantly increased the production of the cytokine IFN-γ and increased the proportion of CD8+ cells in TILs in in vitro cellular validation, demonstrating good activation properties.

[0024] The above description is only an overview of the technical solution and some results of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings. Attached Figure Description

[0025] Figure 1 SDS-PAGE analysis of recombinant 4-1BB-huIgG1 Fc fusion protein;

[0026] Figure 2 The binding activity of recombinant 4-1BB-huIgG1 Fc fusion protein;

[0027] Figure 3 Detection of the titer of recombinant 4-1BB-huIgG1 Fc fusion protein in mice;

[0028] Figure 4 Amplification of antibody light and heavy chain variable region genes;

[0029] Figure 5 Amplification of the antibody scFv gene;

[0030] Figure 6 ELISA detection of 4-1BB antibody affinity;

[0031] Figure 7 , 4 IFN-γ secretion detection in the stimulatory effect of -1BB antibody on PBMCs;

[0032] Figure 8 , 4 -1BB antibody on the proliferation of CD8+ T cells in TIL cell culture. Detailed Implementation

[0033] This invention discloses an anti-human 4-1BB monoclonal antibody and its 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 is particularly important to note 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.

[0034] the term:

[0035] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel).

[0036] An antibody is a protein composed of one or more polypeptides that specifically bind to antigens. One form of antibody constitutes its basic structural unit. This form is a tetramer, which consists of two pairs of identical antibody chains, each pair containing a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains work together to bind the antigen, while the constant regions are responsible for the antibody's effector function.

[0037] The "variable region" of an antibody heavy or light chain is the N-terminal maturation region of that chain. Currently known antibody types include κ and λ light chains, as well as α, γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ heavy chains, or their other equivalent types.

[0038] "Antibody" includes any isotype of antibody or immunoglobulin, or antibody fragment that maintains specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins that contain the antigen-binding portion of an antibody and non-antibody proteins.

[0039] A "humanized antibody" is an antibody that contains a CDR region derived from a non-human antibody, and the rest of the antibody molecule is derived from one (or more) human antibodies. Furthermore, to preserve binding affinity, some residues in the backbone (called FR) segment can be modified.

[0040] The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0041] As used herein, “CDR region” or “CDR” refers to the hypervariable region of the heavy and light chains of the antibody. There are three heavy chain CDRs and three light chain CDRs. Depending on the context, the term CDR or CDRs may be used to indicate one, several, or even all of these regions, which contain the majority of the amino acid residues responsible for binding via the antibody’s affinity for the antigen or its recognition epitope.

[0042] Phage display technology is a biotechnology that involves inserting the DNA sequence of a foreign protein or polypeptide into an appropriate position in the structural gene of a phage coat protein. This allows the foreign gene to be expressed along with the coat protein, and the foreign protein is simultaneously displayed on the phage surface as the phage reassembles. Phage display technology can directly yield antibody genes, facilitating the further construction of various genetically engineered antibodies.

[0043] The raw materials and reagents used in the anti-human 4-1BB monoclonal antibody and its application provided by this invention are all commercially available.

[0044] The present invention will be further illustrated below with reference to the embodiments:

[0045] Example 1: Construction and eukaryotic expression of an expression vector for a fusion protein (4-1BB-huIgG1 Fc) of amino acid sequences 24 to 186 of Human 4-1BB with the human IgG1 Fc region.

[0046] 1. Synthesis of the gene sequence of Human 4-1BB from amino acid position 24 to 186 and construction of the expression vector for the 4-1BB-huIgG1 Fc fusion protein.

[0047] The human 4-1BB gene sequence from positions 24 to 186 was synthesized chemically. The human IgG1 heavy chain constant region sequence from proline (position 100) to lysine (position 330) was also synthesized chemically. The human 4-1BB gene fragment was spliced ​​with the human IgG1 Fc gene fragment using molecular cloning. The splice product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit.

[0048] 2. Expression and purification of recombinant 4-1BB-huIgG1 Fc fusion protein

[0049] Five days after transfection of 293T cells (ATCC) with this expression vector, the culture supernatant was collected, and the recombinant 4-1BB-huIgG1 Fc fusion protein was purified using AKTA explorer 100 (GE). Due to glycosylation modifications and other reasons, the recombinant 4-1BB-huIgG1 Fc fusion protein, after reducing SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, showed a size of approximately 60 kDa. The results are as follows... Figure 1 As shown.

[0050] Example 2: ELISA detection of the binding of recombinant human 4-1BB (4-1BB-huIgG1 Fc) to biotinylated human 4-1BB Ligand

[0051] Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of recombinant human 4-1BB (4-1BB-huIgG1 Fc) to biotinylated human 4-1BB Ligand. The specific ELISA procedure was as follows: 100 ng / well of the prepared 4-1BB-huIgG1 Fc protein was added to a microplate and incubated overnight at 4°C. The plate was washed three times with PBS, and 200 μL / well of 1% BSA / PBS was added, and the plate was blocked at 37°C for 1 hour. After washing with 100 μL of PBS, serially diluted biotinylated human 4-1BB Ligand at an initial concentration of 50 ng / well was added, and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, and 100 μL of 1:5000 diluted HRP-Streptavidin was added, and the plate was incubated at 37°C for 1 hour. Wash three times with PBST, add 100 μL / well TMB chromogenic buffer, incubate at 37°C for 10 minutes, then add 100 μL / well ELISA stop buffer. Read the OD450 value using a microplate reader. The OD450 value reflects the binding of biotinylated human 4-1BB ligand to recombinant human 4-1BB, thus determining whether the expressed recombinant human 4-1BB can be used for mouse immunization. Experimental results are as follows: Figure 2 .

[0052] Example 3: Preparation of anti-human 4-1BB mouse antibody

[0053] 1. Immunized animals

[0054] 2 mg / mL of 4-1BB-huIgG1 Fc fusion protein was mixed with an equal volume of complete Freund's adjuvant (Sigma-Aldrich) and emulsified. Five 6-week-old female Balb / c mice were used for subcutaneous immunization, with 100 μg of antigen per mouse. After the initial immunization, booster immunizations were performed every ten days for a total of four subcutaneous immunizations. For the fifth immunization, 4-1BB-huIgG1 Fc fusion protein was directly used for spleen pulse immunization.

[0055] 2. Serum titer testing

[0056] Before each booster immunization, 50 μL of blood was collected from the tail vein, centrifuged to remove cells, and serum was retained. 50 ng / well of 4-1BB-his (ACRO Biosystems) was added to each well of the ELISA microplate and incubated overnight at 4°C. The plates were washed three times with PBS, and 200 μL / well of 1% BSA / PBS was added, with blocking at 37°C for 1 hour. Serially diluted mouse serum was added, and binding was incubated at 37°C for 1 hour. The plates were washed three times with PBST, and 100 μL of 1:5000 diluted HRP-goat anti-mouse IgG (Shanghai Yisheng) was added, with binding incubated at 37°C for 1 hour. The plates were washed three times with PBST, and 100 μL / well of TMB chromogenic buffer was added, with incubation at 37°C for 10 minutes. 100 μL / well of ELISA stop solution was added, and the OD450 value was read using a microplate reader. Figure 3 As shown.

[0057] 3. Constructing an immune library

[0058] 3.1 Obtaining total cDNA from mouse spleen cells

[0059] Four days after intraperitoneal immunization with the 4-1BB-huIgG1 Fc fusion protein, mice were sacrificed, and spleens were harvested. The entire spleen was ground using a 70-micron cell sieve to obtain spleen cells. After washing twice with PBS, the cells were centrifuged at 500g for 5 minutes to obtain spleen cells. Total RNA was extracted using the Trizol RNA extraction kit. Using the RNA as a template, SuperScript was used... TM The IV First-Strand Synthesis System kit synthesizes first-strand cDNA.

[0060] 3.2 Antibody gene amplification and light / heavy chain splicing

[0061] Using the cDNA as a template, and referring to the antibody amplification primers described in the literature (Schaefer JV, Honegger A., ​​Plückthun A. (2010) Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. In: Kontermann R., Dübel S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg), the heavy chain variable domain gene was amplified by PCR using upstream and downstream primers for the heavy chain variable domain, and the kappa chain variable domain gene was amplified by primer PCR using upstream and downstream primers for the light chain variable domain. In a 50 μL reaction system, 25 μL of phusion mastermix (NEB), 2.5 μL (25 pmol) of upstream primer, 2.5 μL (25 pmol) of downstream primer, 1.5 μL of DMSO, 0.5 μL of cDNA, and 18 μL of ddH2O were added, respectively. Perform the PCR reaction according to the following procedure: pre-denaturation at 98℃ for 1 minute, followed by temperature cycling: denaturation at 98℃ for 30 seconds, annealing at 58℃ for 30 seconds, extension at 72℃ for 1 minute, cycled 30 times, and finally extension at 72℃ for 10 minutes. Figure 4 The amplification of the variable regions of the antibody light and heavy chains is shown.

[0062] The amplified VH and VL genes were recovered using a DNA gel extraction kit. Equal amounts of the VH and VL genes were mixed and used as a template. The scFv gene was amplified by overlap PCR using the upstream primer scFv-F and the downstream primer scFv-R. In a 50 μL reaction mixture, 25 μL of Pharma Master Mix, 2.5 μL (25 pmol) of the upstream primer, 2.5 μL (25 pmol) of the downstream primer, 1.5 μL of DMSO, 0.5 μL of cDNA, and 18 μL of ddH2O were added. The PCR reaction was performed according to the following program: 98°C pre-denaturation for 1 minute, followed by temperature cycling: 98°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 1 minute, for 30 cycles, and a final extension at 72°C for 10 minutes. The amplified scFv gene fragment was recovered using a DNA gel extraction kit. Figure 5 The amplification of the antibody scFv gene is shown.

[0063] 3.3 Construction of immune libraries

[0064] The scFv gene fragment and the pcomb3XTT vector (Scripps Research, USA) were digested separately using SfiI DNA endonuclease. In a 50 μL reaction system, 2 μL of SfiI, 5 μL of 10x buffer, and 3 μg of DNA were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 50°C for 3 hours.

[0065] The digested scFv gene fragment and pcom3XTT vector were recovered using a DNA gel extraction kit. The digested scFv gene fragment and pcom3XTT vector were then cyclized using T4 ligase. In a 50 μL reaction mixture, 1 μL of T4 ligase, 5 μL of 10x buffer, 100 ng of scFv gene, and 500 ng of pcom3XTT vector were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 4°C for 16 hours. A small amount of the product was taken and agarose gel electrophoresis was performed to verify the ligation efficiency.

[0066] 10 μL of the above-mentioned ligation and cyclization product was added to self-made TG1 electroporation competent cells, and then electroporated. 10 μL of the electroporated bacteria were taken, diluted appropriately, and streaked onto plates containing ampicillin to count the bacteria and determine the size of the phage antibody library. The remaining electroporated bacteria were added to 2xYT medium containing 100 μg / mL ampicillin and 2% glucose and incubated in a heated incubator. After incubation, the culture was centrifuged at 4000G for 10 minutes at 4°C, and an appropriate amount of glycerol was added to the precipitate. The culture was then stored at -80°C as an antibody culture library. Through repeated electroporation, an scFv immunoglobulin library with a capacity exceeding 2E9 was obtained.

[0067] 4. Screening and identification of murine immune antibody phage libraries

[0068] 4.1 Biological Screening

[0069] Take Biotinylated Human 4-1BB Protein,His,Avitag TM(ACROBiosystems) Biopanning was used to select fusion proteins as target proteins in the above-mentioned murine immune antibody library to obtain antibodies that bind to 4-1BB. 100 OD bacteria were taken from the antibody library, recovered at an initial OD600 of 0.1, and grown to the logarithmic growth phase. The antibody library was then rescued using M13KO7 helper phage, centrifuged, resuspended in 2xYT medium containing ampicillin and kanamycin, and amplified overnight at 30°C. Phages were precipitated with PEG / NaCl, and the precipitate was dissolved in glycerol / PBST to obtain the immune library phage suspension. The collected phage suspension was added to a co-incubation system of casein-blocked Biotinylated Human 4-1BB Protein and casein-blocked Dynabeads M-270 streptavidin. Magnetic beads were washed with PBST to remove phages that could not bind to 4-1BB. Elution was performed under appropriate elution conditions. 10 μL of the eluted phage solution was used to determine the total amount of exported phage. The remaining phage solution was used to infect logarithmically growing TG1 cells, and the amplified cells overnight were used as the antibody library for the next round of panning. The biological panning process was performed in three rounds.

[0070] 4.2 Initial screening and identification of clones

[0071] The antibody library obtained after the third round of biopanning was diluted and plated on plates containing ampicillin to obtain single clones. Single clones were selected and cultured overnight in deep-well plates. The next day, the deep-well plates were subjected to three freeze-thaw cycles, and the supernatant was used for subsequent two types of ELISA reactions.

[0072] ELISA reaction for detecting binding activity: Coating was performed overnight with 100 ng 4-1BB-his, followed by three washes with PBS. 200 μL of 1% BSA / PBS was added to each well, and the mixture was blocked at 37°C for 1 hour. After three washes with PBS, 100 μL of the supernatant was added, and the mixture was incubated at 37°C for 1 hour. After three washes with PBST, 100 μL of 1:5000 diluted HRP-conjugated goat anti-mouse IgG (Fab-specific) (Thermo) was added. After three washes with PBST, 100 μL of TMB chromogenic buffer was added to each well, and the mixture was incubated at 37°C for 10 minutes. 100 μL of ELISA stop buffer was added to each well, and the OD450 value was read using a microplate reader. Candidate clones were sequenced to obtain the antibody light chain variable domain and heavy chain variable domain sequences, named GT1C8. The results are shown in Table 1.

[0073] Table 1

[0074] OD450 1 2 3 4 5 6 7 8 9 10 11 12 A 0.1329 0.2328 0.0227 0.0231 0.0339 0.0213 0.0232 0.0191 0.0239 0.028 0.0228 0.0618 B 0.0341 0.3204 0.0218 0.0143 0.0211 0.0152 0.0223 0.0607 0.2248 0.0169 0.0156 1.4981 C 0.0485 0.2199 0.2726 0.2216 1.0348 0.2136 0.2129 1.1248 0.016 0.4981 0.0162 0.0874 D 0.0332 0.0159 0.0171 0.0198 0.0207 0.017 0.0165 0.1248 0.0152 0.1248 0.0178 0.0902 E 0.2021 0.0202 0.0156 0.0176 0.0253 0.0396 0.0328 0.024 0.0179 0.9018 0.0158 0.08 F 0.0295 0.0199 1.0837 1.2021 0.0564 0.0428 0.0349 0.0196 0.0428 0.0169 0.0271 0.092 G 0.028 0.0327 0.0837 0.0187 0.2221 0.0446 0.0168 0.2251 0.2187 0.0172 0.0195 0.0348 H 0.0491 0.0262 0.049 0.2295 0.0265 0.2295 0.0313 0.0308 0.0311 0.026 0.0275 0.0772

[0075] 4.3 Expression of GT1C8 clone hIgG1 antibody

[0076] The heavy chain variable region sequence of GT1C8 was cloned into pFUSEss-CHIg-hG1, and the light chain variable region sequence of GT1C8 was cloned into pFUSE2ss-CLIg-hK. Both plasmids were co-transfected into 293T cells (ATCC) at a 1:1 ratio for 5 days. The culture supernatant was collected, and the 6H6 antibody was purified using AKTA explorer 100 (GE). The GT1C8 antibody was stained with Coomassie Brilliant Blue after non-reducing SDS-PAGE electrophoresis, revealing a size of approximately 150 kDa.

[0077] Example 4: Preliminary in vitro evaluation of anti-4-1BB mouse antibody

[0078] 1. In vitro binding assay of huIgG1 type 4-1BB antibody

[0079] The affinity of GT1C8 huIgG1 antibody for 4-1BB antibodies was verified by ELISA: Human 4-1BB / TNFRSF9 Protein and His Tag (ACRO Biosystems) 1ug / ml 100ul per well were coated overnight in four layers. After washing and drying, the plates were blocked by incubation at 37°C for 1 hour with 1% BSA in PBS 200ul / well. After washing three times with 0.1% PBST and drying, the plates were incubated at room temperature for 1 hour with GT1C8 huIgG1 antibody (starting from 10ug / ml, 2-fold serial dilutions, for a total of 15 dilutions). After washing three times with 0.1% PBST, Goat anti-Mouse IgG F(ab')2 Secondary Antibody, HRP (Thermo) (1:10000 dilution) was added and incubated at room temperature for 1 hour. After washing the plate 6 times with 0.1% PBST, add 100 μL of TMB chromogenic solution to each well and incubate at room temperature for 10 minutes. Then add 100 μL of stop solution to each well and read the OD450 using a microplate reader. Analyze the data and calculate the EC50 of GT1C8 huIgG1 antibody binding to 4-1BB to be 0.1018 μg / mL. Figure 6 As shown.

[0080] 2. In vitro validation of the agonistic activity of the GT1C8 antibody

[0081] Human whole blood was used to separate PBMCs using Ficoll density gradient centrifugation. The separated PBMCs were then activated and cultured for three days with anti-CD3 antibody (1 μg / mL). 4-1BB antibody was diluted with PBS to 20 μg / ml, 10 μg / ml, and 1 μg / ml at 50 μl / well and coated overnight. An equal volume of huIgG1 isotype control (of the same species, subtype, and dose as the 4-1BB antibody, and therefore not reacting with the 4-1BB target) was added to the control group. After activation, PBMCs were cultured overnight with different concentrations of 4-1BB antibody. IFN-γ secretion was measured by ELISA. The measurement of IFN-γ secretion indirectly reflects the activation activity of the antibody on PBMC cells. Results are as follows: Figure 7 As shown, GT1C8 increased IFN-γ secretion in PBMCs from different donors compared to the isotype control, demonstrating strong agonistic activity.

[0082] Example 5: Effect of 4-1BB antibody on the proliferation of CD8+ T cells in TIL cell culture

[0083] The preREP donor (0614 / 1003) was removed from liquid nitrogen or dry ice and thawed in a 37°C water bath. After thawing, the cells were quickly transferred to preheated culture medium and centrifuged at 500g for 5 min. After centrifugation, the supernatant was discarded, and the cells were mixed with 1 mL of culture medium by pipetting. The culture medium was then added to an appropriate volume for cell counting. Cell suspensions were sampled and counted. Based on the counting results, the culture medium was added to adjust the cell density to 1E6 cells / mL. 30 ng / mL of CD3 antibody was added per 24 wells for dissolution, 3000 IU / mL of IL-2 was added, along with different concentrations of 4-1BB antibody and isotype control antibody. After 48 h of culture, PBMC feeder was added at a ratio of 1:200 according to the cell count at activation, adjusting the feeder cell density to 1E7 / mL. Cell counting and flow cytometry were performed after two weeks of culture. The experimental results are shown below. Figure 8 As shown, the number of cells proliferated significantly higher than that of the control group after the addition of 4-1BB antibody, and the proportion of CD8+ T cells in the cells increased. The 10ug / ml group of 4-1BB antibody concentration was significantly better than that of the 1ug / ml group.

[0084] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-human 4-1BB monoclonal antibody, characterized in that, (1) the amino acid sequences of the three CDR regions of the heavy chain variable region of the anti-human 4-1BB monoclonal antibody are respectively shown as SEQ ID No. 1, 2 and 3; and (2) the amino acid sequences of the three CDR regions of the light chain variable region of the anti-human 4-1BB monoclonal antibody are respectively shown as SEQ ID No. 4, 5 and 6. 2.The anti-human 4-1BB monoclonal antibody of claim 1, characterized in that, The anti-human 4-1BB monoclonal antibody has: (3) the heavy chain has an amino acid sequence shown as SEQ ID No. 7, and (4) the light chain has an amino acid sequence shown as SEQ ID No.

8.

3. A nucleic acid molecule encoding the monoclonal antibody of claim 1 or 2.

4. An expression vector, characterized by, containing the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that, containing the nucleic acid molecule of claim 3, or containing the expression vector of claim 4.

6. The recombinant cell of claim 5, wherein, The host cell of the recombinant cell is a CHO cell, a HEK293 cell or a yeast cell.

7. The use of the monoclonal antibody of claim 1 or 2 in the preparation of a medicament for the treatment and prevention of 4-1BB-mediated diseases, wherein the disease is a tumor, and the tumor is lung cancer, gastric cancer, intestinal cancer, liver cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer.

8. A pharmaceutical composition, characterized by, comprising: (1) the anti-human 4-1BB monoclonal antibody of claim 1 or 2; and, (2) a pharmaceutically acceptable carrier.

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