Differential anti-cd73 monoclonal antibodies with high enzymatic activity inhibiting ability and use thereof

Abstract

The application relates to the field of biological medicine, and discloses a differential anti-CD73 monoclonal antibody with high enzyme activity inhibition capacity and application thereof. The anti-CD73 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO:1 or has at least 95% homology with the amino acid sequence, and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO:2 or has at least 95% homology with the amino acid sequence. The anti-CD73 monoclonal antibody of the application recognizes a novel antigen epitope, has a unique mechanism of action, has high enzyme activity inhibition capacity on different forms of CD73 proteins, and can fully exert the curative effect potential of the CD73 target point and improve the treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition and its application. Background Technology

[0002] CD73, short for extracellular 5′-nucleotidase, is highly expressed in various cancers and is closely associated with poor prognosis, including breast cancer, lung cancer, pancreatic cancer, gastric cancer, and colorectal cancer. In the tumor microenvironment, CD73 hydrolyzes adenosine monophosphate (AMP) to produce large amounts of adenosine (ADO). The accumulated adenosine binds to adenosine receptors on immune cells, inhibiting the body's anti-tumor immune response. Specifically, it blocks the immune function of T cells, natural killer cells, and dendritic cells, while simultaneously enhancing the function of regulatory T cells and myeloid-derived suppressor cells, and promoting M2 macrophage polarization, leading to tumor immune escape and thus promoting tumor growth and metastasis. In the tumor microenvironment, adenosine can also act on adenosine receptors on endothelial cells, promoting intratumoral angiogenesis, directly promoting tumor cell growth, and enhancing the immunosuppressive function of tumor-associated fibroblasts. CD73 also directly promotes tumor cell invasion and metastasis through a mechanism independent of its enzymatic activity. Therefore, CD73 plays a crucial role in tumor progression, making it an attractive target for tumor immunotherapy.

[0003] Current positive results from preclinical studies indicate that CD73-targeted interventions, whether through gene editing or drug therapy, can effectively inhibit tumor growth and enhance the efficacy of traditional radiotherapy, chemotherapy, and immune checkpoint inhibitors (such as PD-(L)1 and CTLA-4). These findings have driven the development of CD73-targeted therapeutic antibodies and propelled them into clinical trials. Current Phase I and II clinical trial data show that the combined use of CD73 antibodies with other immune checkpoint inhibitors and radiotherapy not only enhances therapeutic efficacy but also demonstrates good safety. Most treatment-related adverse events were grade 3 or lower, and high expression of CD73 in tumors was found to be closely related to treatment response, suggesting its potential as a biomarker. These results highlight the significant value of CD73-targeted therapy in clinical translation and its anti-tumor potential.

[0004] Although CD73 antibodies have shown potential in clinical studies, the mechanisms of action of currently available CD73 antibodies in terms of antigen epitope binding and enzyme activity inhibition differ. These differences lead to incomplete inhibition of different forms of CD73 (including free and cell membrane surface forms). For example, TJ004309 significantly inhibits free CD73 activity but has a weaker inhibitory effect on cell membrane surface CD73; CPI-006 almost completely inhibits cell membrane surface CD73 activity but has limited inhibitory effect on free CD73; MEDI9447 exhibits a "hook effect" on free activity but incompletely inhibits cell membrane CD73 activity. This incomplete inhibition may weaken the antibody's blocking effect on the AMP-CD73-ADO axis, thus affecting its therapeutic efficacy. Therefore, antibodies that can completely inhibit CD73 activity may have superior therapeutic effects in clinical applications.

[0005] In summary, CD73, as an emerging therapeutic target for cancer, offers new insights into cancer treatment with its preliminary efficacy in clinical trials. However, the differences in antigen epitope binding and enzyme activity inhibition among currently clinically developed anti-CD73 antibodies may limit the full realization of the therapeutic potential of the CD73 target and affect its clinical therapeutic value. Future research needs to develop more potent anti-CD73 antibodies with differentiated mechanisms of action to realize the clinical translational value of this target. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition and its applications. The anti-CD73 monoclonal antibody of this invention has a novel antigenic epitope recognition mechanism and a unique action mechanism. It exhibits high enzyme activity inhibition against different forms of CD73 protein, thus fully realizing the therapeutic potential of the CD73 target and improving treatment efficacy.

[0007] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a differentiated anti-CD73 monoclonal antibody or its antigen-binding fragment with high enzyme activity inhibition capability, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or has at least 95% homology with it; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or has at least 95% homology with it.

[0008] Further, the anti-CD73 monoclonal antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein: the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 5; and the light chain comprises the amino acid sequence shown in SEQ ID NO: 6.

[0009] Secondly, the present invention provides a nucleotide encoding the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, comprising a portion encoding a heavy chain variable region and a portion encoding a light chain variable region. Wherein: the portion encoding the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 3; the portion encoding the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0010] Furthermore, the nucleotides include nucleotide sequences encoding the heavy chain as shown in SEQ ID NO: 7, and nucleotide sequences encoding the light chain as shown in SEQ ID NO: 8.

[0011] Thirdly, the present invention provides a carrier containing the above-mentioned nucleotides.

[0012] Fourthly, the present invention provides a host cell containing the above-mentioned nucleotides or vectors.

[0013] Fifthly, the present invention provides a method for preparing the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, comprising the following steps: culturing host cells containing nucleotides encoding anti-CD73 monoclonal antibody or its antigen-binding fragment, causing them to express anti-CD73 monoclonal antibody or antigen-binding fragment, and collecting the expressed antibody or its antigen-binding fragment.

[0014] In a sixth aspect, the present invention provides the use of an anti-CD73 monoclonal antibody or its antigen-binding fragment, or the aforementioned nucleotide, or the aforementioned vector, or the aforementioned host cell in the preparation of a medicament. The medicament is a drug that inhibits CD73 enzyme activity and has antitumor effects.

[0015] Furthermore, the tumor is a tumor that highly expresses, or moderately / lowly expresses, CD73.

[0016] Further preferred, the tumor is triple-negative breast cancer or colorectal cancer.

[0017] Preferably, the drug also includes a pharmaceutically acceptable carrier.

[0018] Further preferably, the dosage form of the drug is an injectable preparation, existing in the form of an injection solution, which may be a solution or lyophilized powder, etc.

[0019] Compared with the prior art, the beneficial effects of the present invention are: (1) The anti-CD73 monoclonal antibody of the present invention has better enzyme activity inhibition ability against different forms of CD73 protein (including free form and cell membrane surface form) compared with the current clinical investigation anti-CD73 antibody.

[0020] (2) The anti-CD73 monoclonal antibody of the present invention has shown in animal experiments that it can exert a significant anti-tumor effect in vivo. No significant weight loss or abnormal blood routine and biochemical indicators were observed in animals within the effective dose range.

[0021] (3) The anti-CD73 monoclonal antibody of the present invention recognizes antigen epitopes and has a different mechanism of action than the current anti-CD73 antibodies under clinical investigation, and is a potential new clinical option.

[0022] (4) The anti-CD73 monoclonal antibody of the present invention has a unique mechanism of action and can exert enzyme activity inhibition through a monovalent antigen-binding fragment (Fab), and can be used as a choice for bispecific antibodies.

[0023] (5) The anti-CD73 monoclonal antibody of the present invention can play a co-stimulatory role and assist in the activation of T immune cells. Attached Figure Description

[0024] Figure 1 The inhibition rate of humanized antibody #12 and the currently clinically investigated anti-CD73 antibody on the activity of free CD73 enzyme.

[0025] Figure 2 The inhibition rate of CD73 enzyme activity on the surface of tumor cell membrane (MDA-MB-231, a triple-negative breast cancer cell line that highly expresses CD73) by humanized antibody #12 and the currently clinically investigated anti-CD73 antibody when the substrate incubation time is 2h.

[0026] Figure 3 The inhibition rate of CD73 enzyme activity on the surface of tumor cell membrane (MDA-MB-231, a triple-negative breast cancer cell line that highly expresses CD73) by humanized antibody #12 and the currently clinically investigated anti-CD73 antibody when the substrate incubation time is 8h.

[0027] Figure 4 The results show the affinity of the murine #12 antibody (#12(m)) before humanization and the modified humanized antibody #12 (#12(h)) for binding to free CD73.

[0028] Figure 5 Results of the affinity of humanized antibody #12 for CD73 on the surface membrane of CD73-positive T cells in peripheral blood of normal individuals.

[0029] Figure 6 Results of the affinity of humanized antibody #12 for CD73 on the surface membrane of CD73-positive B cells in peripheral blood of normal individuals.

[0030] Figure 7 The results show the affinity of humanized antibody #12 for CD73 on the surface membrane of human colorectal cancer cell line HCT-116.

[0031] Figure 8 The results show the affinity of humanized antibody #12 for CD73 on the surface membrane of human triple-negative breast cancer cell line MDA-MB-231.

[0032] Figure 9 This is a graph showing the results of a competitive binding experiment between humanized antibody #12 and currently clinically investigated antibodies.

[0033] Figure 10 The binding of humanized antibody #12 to CD73 mutants with different site mutations.

[0034] Figure 11 Experiments on the in vitro inhibition of the proliferation of human triple-negative breast cancer cell line MDA-MB-231 by humanized antibody #12.

[0035] Figure 12 Experiments on the in vitro inhibition of human colorectal cancer cell line HCT-116 by humanized antibody #12.

[0036] Figure 13 Experiments on the inhibition of humanized antibody #12 on the growth of human triple-negative breast cancer cell line MDA-MB-231 xenografts in nude mice.

[0037] Figure 14 The size of the tumor in mice was photographed at the endpoint of the in vivo experiment on humanized antibody #12 inhibiting the growth of human triple-negative breast cancer cell line MDA-MB-231 xenografts in nude mice.

[0038] Figure 15 Experiments on the inhibition of humanized antibody #12 on the growth of human colorectal cancer cell line HCT-116 xenografts in nude mice.

[0039] Figure 16 The results of tumor dissection photography and tumor weight were obtained at the endpoint of the in vivo experiment on the inhibition of human colorectal cancer cell line HCT-116 xenograft tumor in nude mice by humanized antibody #12.

[0040] Figure 17 Experiments on the in vitro induction of CD73 downregulation on the surface of human triple-negative breast cancer cell line MDA-MB-231 by humanized antibodies #12 and BR101.

[0041] Figure 18 Experiments on the in vitro induction of CD73 downregulation on the surface of human colorectal cancer cell line HCT-116 by humanized antibody #12 and BR101.

[0042] Figure 19 The results show the effects of humanized antibody #12 on the T cell activation marker CD25 in vitro.

[0043] Figure 20These are the experimental results of humanized antibody #12 blocking the effect of high AMP on T cell cytokine release in vitro.

[0044] Figure 21 The results of in vitro experiments using humanized antibody #12 to block the effect of high AMP on T cell proliferation.

[0045] Figure 22 Results of the experiment on the activation of humanized antibody #12 to help T cells.

[0046] Figure 23 The results of an in vivo experiment on the inhibition of tumor growth in CD73 humanized mice (immunoblasts) using humanized antibody #12 on MC38 cell line-immunoblastic mice were obtained.

[0047] Figure 24 The effect of humanized antibody #12 on body weight in an in vivo experiment on the inhibition of tumor growth in immune-healthy mice (CD73 humanized mice) using mouse MC38 cell line.

[0048] Figure 25 The effect of humanized antibody #12 on blood routine indicators in an in vivo experiment on the inhibition of tumor growth in CD73 humanized mice (immunoblastic mice) with MC38 cell line.

[0049] Figure 26 The effect of humanized antibody #12 on blood biochemical parameters in an in vivo experiment on the inhibition of tumor growth in CD73 humanized mice (immunoblastic mice) with MC38 cell line. Detailed Implementation

[0050] The present invention will be further described below with reference to embodiments.

[0051] General Implementation Examples In a first aspect, the present invention provides a differentiated anti-CD73 monoclonal antibody or its antigen-binding fragment with high enzyme activity inhibition capability, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 95% homology with it; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 95% homology with it. Further, the amino acid sequence of the heavy chain of the anti-CD73 monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain is shown in SEQ ID NO: 6.

[0052] Secondly, the present invention provides a nucleotide encoding the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, comprising a portion encoding a variable region of the heavy chain and a portion encoding a variable region of the light chain. Wherein: the portion encoding the variable region of the heavy chain comprises the nucleotide sequence shown in SEQ ID NO: 3; the portion encoding the variable region of the light chain comprises the nucleotide sequence shown in SEQ ID NO: 4. Further, the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 8.

[0053] Thirdly, the present invention provides a carrier containing the above-mentioned nucleotides.

[0054] Fourthly, the present invention provides a host cell containing the above-mentioned nucleotides or vectors.

[0055] Fifthly, the present invention provides a method for preparing the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, comprising the following steps: culturing host cells containing nucleotides encoding anti-CD73 monoclonal antibody or its antigen-binding fragment, causing them to express anti-CD73 monoclonal antibody or antigen-binding fragment, and collecting the expressed antibody or its antigen-binding fragment.

[0056] Furthermore, the present invention can also prepare the above-mentioned anti-CD73 humanized monoclonal antibody by the following method, including the following steps: S1: Hybridoma monoclonal antibody screening was conducted using the hybridoma method: mice were immunized with recombinant CD73 protein, and hybridoma cells that produced anti-human CD73 monoclonal antibodies were screened through cell fusion. Based on affinity screening, hybridoma cells expressing the required antibodies were identified.

[0057] S2: Screening candidate antibodies with high enzyme activity inhibition ability using high-throughput methods: A screening system capable of detecting CD73 enzyme activity was established using enzyme-coupled assay. Based on this, lead antibodies can be screened in high-throughput to identify candidate antibodies that meet the requirements.

[0058] S3: By constructing a mutant antibody library from the heavy and light chains of monoclonal antibodies using CDR1, CDR2, and CDR3, antibodies are screened based on affinity to obtain successfully humanized monoclonal antibodies with unaffected affinity.

[0059] Sixthly, the present invention provides the use of an anti-CD73 monoclonal antibody or its antigen-binding fragment, or the aforementioned nucleotide, or the aforementioned vector, or the aforementioned host cell in the preparation of a drug. The drug is an anti-tumor drug that inhibits CD73 enzyme activity. Further, the tumor is a tumor that highly expresses, moderately / lowly expresses CD73; more preferably, it is triple-negative breast cancer or colorectal cancer.

[0060] Preferably, the drug also includes a pharmaceutically acceptable carrier.

[0061] More preferably, the dosage form of the drug is an injectable preparation, which is usually in the form of an injection solution, such as a solution or lyophilized powder. Specific Implementation Example 1: Comparison of the ability of humanized antibody #12 (the amino acid sequence of the heavy chain is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain is shown in SEQ ID NO: 6) to inhibit free CD73 enzyme activity with clinically investigated CD73 antibody (1) HCT-116 cells in logarithmic growth phase were taken, digested with trypsin, and centrifuged to collect the HCT-116 cell pellet.

[0063] (2) Add lysis buffer to the HCT-116 cell pellet to extract free CD73. Lyse on ice for 15 min, then centrifuge at 145000g for 10 min. Take the supernatant for free CD73 enzyme activity detection experiment.

[0064] (3) Add different final concentrations of antibody to the supernatant. Add isotype (human igg1) to the control group at a final concentration of 500ug / ml. Two replicates were set for each treatment. Incubate at room temperature for 30min.

[0065] (4) Use a biochemical analyzer to detect the enzyme activity of free CD73 in the supernatant after different treatments.

[0066] (5) The control group obtained the maximum enzyme activity value. The inhibition rate of the antibody against CD73 enzyme activity at each concentration was calculated using the formula: (control group - antibody groups of different concentrations) / control group * 100%.

[0067] The results are as follows Figure 1 The image shows a comparison of the inhibitory capacity of humanized antibody #12 on free CD73 enzyme activity with currently clinically investigated CD73 antibodies. BR101 is an anti-human CD73 therapeutic antibody developed by Zhejiang Borui Biopharmaceutical Co., Ltd., with its highest development stage in the field of tumor treatment being Phase I clinical trials; MEDI9447 is an anti-human CD73 therapeutic antibody developed by AstraZeneca, with its highest development stage in the field of tumor treatment being Phase III clinical trials; TJ004309 is an anti-human CD73 therapeutic antibody developed by I-Mab Biopharma, with its highest development stage in the field of tumor treatment being Phase III clinical trials; and CPI-006 is an anti-human CD73 therapeutic antibody developed by Corvus Pharmaceuticals, with its highest development stage in the field of tumor treatment being Phase I clinical trials.

[0068] Compared with these clinically investigated anti-CD73 antibodies, the humanized antibody #12 of this invention has superior enzyme activity inhibition ability (IC50: 0.69nM (#12) vs 1.263nM (BR101) vs 40.13nM (CPI-006) vs 67.03nM (TJ004309) vs 0.8573nM (MEDI9447)). The maximum inhibition rate of #12 is similar to that of TJ004309 and far superior to other antibodies. MEDI9447 and BR101 also exhibit a hook effect, which means that the enzyme activity inhibition effect decreases significantly under high concentrations of antibody.

[0069] Example 2: Comparison of the ability of humanized antibody #12 and clinically investigated CD73 antibody to inhibit CD73 enzyme activity on membrane surface (1) MDA-MB-231 breast cancer cell line with high expression of CD73 in log phase growth was taken and seeded into 96-well cell culture plates at 1w per well and cultured overnight at 37°C.

[0070] (2) On the second day, the culture supernatant was aspirated and serum-free culture medium containing different concentrations of antibodies was added. The control group was given isotype (human igg1) at a final concentration of 50ug / ml, and the blank group was given serum-free culture medium. Each treatment was set up in 2 replicates and incubated in an incubator for 2 hours.

[0071] (3) After incubation, serum-free medium containing AMP was added to the control group and antibody treatment group, with a final concentration of 500 uM. Serum-free medium was added to the blank group, and the incubator was incubated for 2-8 hours.

[0072] (4) At the designated time point, aspirate the cell supernatant, centrifuge at 1000g for 10min, aspirate 100ul of supernatant into a new 1.5ml EP tube, add an equal amount of methanol, mix well, and place at -20℃.

[0073] (5) The adenosine content in the supernatant after rewarming was detected by high performance liquid chromatography (HPLC). The control group yielded the maximum adenosine production, and the blank group yielded the minimum adenosine production. The inhibition rate of antibody on CD73 enzyme activity on cell membrane surface at each concentration was calculated using the formula: ((control group - blank group) - (different concentration antibody groups - blank group)) / (control group - blank group) * 100% (In the actual experiment, no adenosine production was detected in the blank group, so this formula can be used directly for calculation: (control group - different concentration antibody groups) / control group * 100%)

[0074] The results are as follows Figure 2 , 3The image shows a comparison of the inhibitory capacity of humanized antibody #12 and currently clinically investigated CD73 antibodies on the cell membrane surface CD73 enzyme activity. BR101 is an anti-human CD73 therapeutic antibody developed by Zhejiang Borui Biopharmaceutical Co., Ltd., with its highest development stage in the field of tumor treatment being Phase I clinical trials; MEDI9447 is an anti-human CD73 therapeutic antibody developed by AstraZeneca, with its highest development stage in the field of tumor treatment being Phase III clinical trials; TJ004309 is an anti-human CD73 therapeutic antibody developed by I-Mab Biopharma, with its highest development stage in the field of tumor treatment being Phase III clinical trials; and CPI-006 is an anti-human CD73 therapeutic antibody developed by Corvus Pharmaceuticals, with its highest development stage in the field of tumor treatment being Phase I clinical trials. The inhibitory activity of the humanized antibody #12 of this invention is similar to that of CPI-006, which has the best inhibitory activity (2h: same maximum inhibition rate, IC50: 0.4917nM (#12) vs 0.3637nM (CPI-006)), but it is least affected by incubation time compared to CPI-006 (8h: maximum inhibition rate of #12 is better than that of CPI-006, IC50: 1.420nM (#12) vs 1.219nM (CPI-006)).

[0075] Example 3: Affinity of humanized antibody #12 to bind free CD73 (1) The affinity between humanized antibody #12 and free CD73 protein was detected using biolayer interferometry (BLI).

[0076] (2) Using the Protein A / MFC Sensor, insert the sensor into the slot and equilibrate it in Q buffer [PBS (10mM PH7.4) + 0.02% Tween 20 + 0.2% BSA] for 30 seconds, and then determine the signal baseline.

[0077] (3) Immerse the sensor in Q buffer containing humanized #12 antibody for 120 seconds to allow humanized antibody #12 to be adsorbed onto the sensor surface.

[0078] (4) Immerse the sensor in Q buffer to remove unadsorbed humanized antibody #12, rebalance for 60s, and measure the signal baseline.

[0079] (5) Immerse the sensor loaded with humanized antibody #12 into Qbuffer containing different concentrations of free CD73 protein for 120s and detect the antigen-antibody binding signal.

[0080] (6) Immerse the sensor in Q buffer for 180s to detect antigen-antibody dissociation signal.

[0081] (7) Use BLI software to analyze the data, determine the binding and dissociation kinetic parameters, and calculate the binding constant (KD).

[0082] The results are as follows Figure 4 As shown in Table 1, the affinity of humanized antibody #12 for binding to free CD73 is 9.55 × 10⁻⁶. -10 .

[0083] Table 1 Example 4: Affinity of humanized antibody #12 to CD73 on the membrane surface (1) Take MDA-MB-231 and HCT-116 cell lines in the logarithmic growth phase, digest them and leave the precipitate, resuspend them with cell staining buffer (2% FBS, PBS), and adjust the density of the single cell suspension to 100w / ml; take peripheral blood from normal people, obtain peripheral blood mononuclear cells (PBMCs) with lymphocyte separation solution, wash repeatedly with PBS and leave the cell precipitate, reselect with cell staining buffer, and adjust the density to 200w / ml.

[0084] (2) Prepare cell staining buffers containing different concentrations of antibodies, in a concentration gradient. The initial antibody concentration is 15ug / ml, diluted 3 times, for a total of 10 concentrations.

[0085] (3) Spread the above-treated cells evenly into the wells of a 96-well plate at a rate of 100 μL per well. Centrifuge at 350 g for 5 min, discard the supernatant, add 100 μL of staining buffer containing antibody, resuspend the cells, and incubate at 4 °C for 1 h.

[0086] (4) After incubation, wash twice with ice-cold PBS buffer.

[0087] (5) Add 100 μL of anti-human igg secondary antibody containing PE fluorescent labeling to the precipitate, resuspend the cells, and incubate at 4°C for 1 h.

[0088] (6) After incubation, wash twice with ice-cold PBS buffer and resuspend in 250 μL of PBS buffer.

[0089] (7) Antibody binding was detected using a BD fortessa flow cytometer and a BD HTS workstation.

[0090] (8) Calculate the MFI value of samples treated with different concentrations of antibody, and calculate the EC50 and KD value of the antibody using a nonlinear fitting method.

[0091] The results are as follows Figure 5-8As shown, the affinity of humanized antibody #12 for CD73 on the membrane surface of MDA-MB-231, HCT-116, and human peripheral blood CD3+ T and B cells was detected by flow cytometry. The affinity of #12 for CD73 on the membrane surface of MDA-MB-231 was 0.05829 ug / ml; for CD73 on the membrane surface of HCT-116 it was 0.03275 ug / ml; for CD73 on the membrane surface of CD3+ T cells it was 0.05881 ug / ml; and for CD73 on the membrane surface of CD19+ B cells it was 0.006406 ug / ml.

[0092] Example 5: Competitive binding assay between humanized antibody #12 and current clinical investigation antibodies (1) Take MDA-MB-231 in the logarithmic growth phase, digest it and leave the precipitate. Resuspend it with cell staining buffer (2% FBS, PBS) and adjust the density of the single cell suspension to 200w / ml.

[0093] (2) Prepare cell staining buffer containing 10ug / ml of unfluorescently labeled antibody, 5 groups, isotype (humanigg1), #12, BR101, CPI-006, TJ004309.

[0094] (3) Spread the treated cells evenly into the wells of a 96-well plate at a rate of 200 μL per well. Centrifuge at 350 g for 5 min, discard the supernatant, add 100 μL of staining buffer containing antibody, resuspend the cells, and incubate at 4°C for 1 h.

[0095] (4) Prepare a buffer containing 4ug / ml of FITC fluorescently labeled antibody, 4 types: #12-FITC, BR101-FITC, CPI-006-FITC, and TJ004309-FITC.

[0096] (5) After incubation, add 200 μL of pre-chilled PBS buffer to each well and wash twice. Resuspend the precipitate in 210 μL of buffer to each well, then divide it into 4 wells, 50 μL per well. Add 50 μL of each of the four fluorescently labeled antibodies to the 4 wells, to a final concentration of 2 μg / mL, and incubate at 4°C for 1 h.

[0097] (6) After incubation, wash twice with pre-cooled PBS buffer and resuspend in 250 μL of PBS buffer.

[0098] (7) The binding of fluorescently labeled antibodies was detected using a BD fortessa flow cytometer and a BD HTS workstation to determine the similarity of the binding epitopes between the antibodies.

[0099] See results Figure 9As shown, the binding mode and epitope of humanized antibody #12 are different from those of the current clinical investigation antibody, and it does not interfere with BR101.

[0100] Example 6: Humanized antibody #12 recognizes the antigenic epitope of CD73. (1) In the protein interaction structure prediction tool DMfold, the amino acid sequences of the heavy chain and light chain variable regions of antibody #12 and the amino acid sequence of mature CD73 protein were input in FASTA form to predict the interaction mode between humanized antibody #12 and CD73.

[0101] (2) Download model1, which has the highest score in the prediction results. Open the model1.pdb file with ChimeraX and analyze the key residues that interact between the two. The predicted antigenic epitopes are: T44, E46, S49, K50, C51, V52, S55, R56, F65, Q69, R72, R73, V78, H103, R109, D111, K133, E134, K136, D317, I320, D323, K326, W327.

[0102] (3) Point mutations or combination mutations were performed on the predicted key interaction residues. The mutated CD73 sequence was cloned using the pcDNA3.1 plasmid, and an additional HA tag was added to the N-terminus of the CD73 sequence to detect the expression of the protein on cells used to express the mutated CD73.

[0103] (4) The binding of humanized antibody #12 to CD73 with different point mutations or combination mutations was detected by flow cytometry.

[0104] (5) Detect the binding of humanized antibody #12 to different CD73 mutants to determine the key epitopes for identification.

[0105] The constructed point mutation overexpression plasmids are shown in Table 2.

[0106] Table 2 See results Figure 10 As shown, humanized antibody #12 does not bind to CD73-V4, but it binds to all other point mutations. The positive control (whose epitope does not overlap with #12) BR101, however, maintains its binding to CD73-V4. Therefore, the epitopes recognized by humanized antibody #12 include PEDPSIKADINKWR.

[0107] Example 7: In vitro antitumor cell effect of humanized antibody #12 (1) MDA-MB-231 and HCT-116 cells in the logarithmic growth phase were digested and the precipitate was collected. The cells were resuspended in complete medium at 1640 and seeded into 96-well cell culture plates at 3000 cells per well. The cells were cultured overnight at 37°C.

[0108] (2) On the second day, the culture supernatant was aspirated, and complete culture medium containing different concentrations of humanized antibody #12 was added. The control group was given isotype (human igg1) at a final concentration of 15ug / ml. Each treatment was set up in 3 replicates. An additional blank group was set up with an equal volume of complete culture medium without cells. The cells were incubated in an incubator for 72 hours.

[0109] (3) Prepare the proliferation detection solution. Using a multichannel pipette, carefully add 10 μL of CCK-8 solution to each well, avoiding the formation of air bubbles. Incubate in an incubator for 2 hours.

[0110] (4) After incubation, the absorbance (OD value) at 450 nm was measured using an ELISA reader. The proliferation index under different antibody concentrations was calculated as (OD value of antibody group - OD value of blank group) / (OD value of control group - OD value of blank group).

[0111] The results are as follows Figure 11 , 12 As shown, humanized antibody #12 can significantly inhibit the in vitro proliferation of human breast cancer and colorectal cancer cell lines.

[0112] Example 8: In vivo anti-tumor cell effect of humanized antibody #12 (1) The 1-week-old female Balb / c nude mice were housed in a barrier for 1 week.

[0113] (2) Logarithmic growth phase MDA-MB-231 cells were digested, washed twice with PBS, resuspended in serum-free RPMI 1640 medium, and the cell concentration was adjusted to 6 × 10⁻⁶. 7 Add an equal volume of matrix gel to each ml of water, mix well, and store on ice.

[0114] (3) Logarithmic growth phase: HCT-116 cells were digested, washed twice with PBS, resuspended in serum-free RPMI 1640 medium, and the cell concentration was adjusted to 2.5 × 10⁻⁶. 7 / ml, store on ice.

[0115] (4) MDA-MB-231 suspension was injected into the fourth mammary fat pad on the left side of the mouse abdomen at a dose of 100 μl / mouse. HCT-116 suspension was injected into the left axillary fat pad of the mouse at a dose of 100 μl / mouse.

[0116] (5) Five days after MDA-MB-231 tumor inoculation (average tumor diameter 40mm) 3) Start medication, once every 3 days, 250ug each time, for a total of 8 times, via intraperitoneal injection. Start medication on the day of HCT-116 tumor inoculation, once every 3 days, 250ug each time, for a total of 5 times, via intraperitoneal injection.

[0117] (6) Grouping: Homotype antibody (human-iggl) vs. humanized antibody #12, 5 tumor-bearing mice in each group.

[0118] (7) Monitor tumor growth three times a week, measure the long and short diameters of the tumor, and calculate the volume as long diameter × long diameter × short diameter / 2. At the end of the experiment, euthanize the mice by dislocation, completely remove the subcutaneous tumor, weigh it, and take a photograph.

[0119] (8) Use GraphPad Prism software to draw a tumor volume growth curve.

[0120] See results Figure 13-16 As shown, humanized antibody #12 significantly inhibited the growth of human breast cancer and colorectal cancer cell lines in nude mice.

[0121] Example 9: Humanized antibody #12 downregulates CD73 levels on the surface of tumor cells (1) Take MDA-MB-231 and HCT-116 cell lines in the logarithmic growth phase, digest them and leave the precipitate. Resuspend the precipitate with cell staining buffer (2% FBS, PBS) and adjust the density of the single cell suspension to 100w / ml.

[0122] (2) Spread the treated cells evenly into the wells of a 96-well plate at a rate of 100 μL per well. Centrifuge at 350 g for 5 min, discard the supernatant, add 100 μL of staining buffer containing 15 μg / ml unlabeled fluorescent antibody, resuspend the cells, and incubate at 37 °C for 0, 1, 2, and 3 h.

[0123] (3) Antibody grouping: control group: isotype (human igg1), antibody treatment group 1: #12, antibody treatment group 2: BR101.

[0124] (4) After the first incubation, wash twice with pre-cooled PBS buffer.

[0125] (5) Second incubation: The cell pellet was resuspended in 100ul of unlabeled fluorescent antibody staining buffer containing 15ug / ml of the same as the first incubation. The control group was divided into two groups, which were incubated for the second time with #12 and BR101 respectively. The cells were incubated at 4°C for 1h.

[0126] (6) After incubation, wash twice with pre-cooled PBS buffer, add 100 μL of anti-human igg secondary antibody containing PE fluorescent labeling, resuspend the cells, and incubate at 4°C for 1 h.

[0127] (7) After incubation, wash twice with ice-cold PBS buffer and resuspend in 250 μL of PBS buffer.

[0128] (8) Antibody binding was detected using a BD fortessa flow cytometer and a BD HTS workstation.

[0129] (9) Record the MFI value of the sample and calculate the percentage decrease of CD73 on the cell surface relative to the control group after different antibody treatment times: (isotype treatment t hours - antibody treatment t hours) / isotype treatment t hours.

[0130] The results are as follows Figure 17 , 18 As shown, humanized antibody #12 has a stronger effect on downregulating CD73 levels on the surface of tumor cell lines compared to BR101.

[0131] Example 10: Humanized antibody #12 blocks the immunosuppressive effect of AMP-CD73-ADO (1) Take 40 ml of normal human peripheral blood, obtain peripheral blood mononuclear cells (PBMCs) using lymphocyte separation solution, wash repeatedly with PBS to retain cell pellet, resuspend with 90 μl cell sorting buffer (MACS: 2% FBS, 1 mM EDTA, PBS buffer), add 30 μl of CD8 cation magnetic beads, and incubate on ice for 20 minutes.

[0132] (2) After incubation, add 10ml MACS to wash once, resuspend the precipitate with 3ml MACS, and filter through a 70um filter.

[0133] (3) Assemble the sorting device according to the Miltenyi LS column instructions. Rinse the sorting column with 3 ml of MACS solution three times. After the last rinse, add 3 ml of single-cell suspension filtered through a 70 μm filter. When the cell suspension has completely entered the sorting column, add 3 ml of MACS solution to rinse, repeating three times. Carefully remove the sorting column and place the lower end in a 15 ml centrifuge tube. Add 3 ml of MACS solution and use the provided plunger to inject the MACS solution into the centrifuge tube. At this time, the CD8+ cells adsorbed in the sorting column will enter the centrifuge tube with the MACS solution. Repeat three times to improve the yield.

[0134] (4) Centrifuge at 370g for 7min to obtain cell pellet, then resuspend the pellet in complete culture medium and adjust the cell density to 100w / ml.

[0135] (5) Grouping: Activation group: α CD3 + α CD28 treatment; Control group: α CD3 + α CD28 + AMP + isotype treatment; Antibody group: α CD3 + α CD28 + AMP + different concentrations of humanized antibody #12, with the highest concentration being 135ug / ml, diluted 3 times, for a total of 5 concentrations; Positive control group: α CD3 + α CD28 + AMP + AB680 (10uM).

[0136] (6) Treatment sequence: Add isotype, #12 and AB680 to cells, incubate at 37℃ for 2h, then add AMP solution to a final concentration of 500uM, and incubate for 1h. Finally, add αCD3 and αCD28 to each well to a final concentration of 1ug / ml, and incubate for 24h.

[0137] (7) After culture, centrifuge and use the supernatant to detect cytokines. The cell pellet is resuspended in 50 μL of staining buffer containing anti-CD25BV605, anti-CD69 percp cy5.5, anti-CD71 BV421, anti-OX40 BV711, and anti-CD73 FITC (BR101), and incubated at 4°C for 30 minutes. After incubation, wash with PBS.

[0138] (8) The precipitate was resuspended and the activation level of CD8+T was detected by flow cytometry.

[0139] (9) The proliferation experiment is the same as the above procedure, except that CD8+ T cells are first labeled with CTV dye before antibody incubation, and the proliferation is detected by flow cytometry after 4 days of culture.

[0140] (10) The experiment was repeated using CD8+T from at least 3 different normal human sources.

[0141] The results are as follows Figure 19-21 As shown, humanized antibody #12 can completely block the immunosuppressive effect of AMP-CD73-ADO under high AMP conditions, which is reflected in the complete rescue of CD8+ T cell activation, cytokine release and proliferation capacity.

[0142] Example 11: Humanized antibody #12 can activate CD73 co-stimulatory molecule to assist in the activation of CD8+ T cell function (1) Add 50 μL of antibody-containing PBS buffer to a 96-well plate and incubate overnight at 4°C. Group settings: Antibody 1: α CD3, Antibody 2: α CD3 + α CD28, Antibody 3: α CD3 + #12, α CD3 final concentration 0.25 μg / ml, α CD28 and #12 final concentration 0.5 μg / ml.

[0143] (2) As mentioned above, CD8+ T cells were obtained from peripheral blood of normal individuals and resuspended in complete culture medium at a cell concentration of 100w / ml.

[0144] (3) On the second day, the liquid in the well was aspirated, the cells were washed three times with PBS buffer, 100 μL of cell suspension was added, and the cells were incubated for two days.

[0145] (4) After the culture was completed, the well plate was centrifuged at 350g for 5 min, and the supernatant was collected to detect the IL-2 content. The precipitate was resuspended in 50 μL of staining buffer containing anti-CD25BV605, anti-CD69 percp cy5.5, anti-CD71 BV421, anti-OX40 BV711, and anti-CD73 FITC (BR101), incubated at 4°C for 30 min, washed once with PBS, and after resuspending, the CD8+T activation level was detected by flow cytometry.

[0146] (5) The experiment was repeated using CD8+T from at least 3 different normal human sources.

[0147] The results are as follows Figure 22 As shown, humanized antibody #12 can act as a co-stimulatory molecule to assist in the activation of CD8+ T cells.

[0148] Example 12: Antitumor effect of humanized antibody #12 in CD73-targeted humanized mice (1) Six-week-old CD73-targeted humanized mice (purchased from Southern Model Biotechnology, C57BL / 6Smoc-Nt5etm3(NT5E)Smoc) were housed in a barrier for one week.

[0149] (2) Logarithmic growth phase mouse colorectal cancer cell line MC38 was digested, washed twice with PBS, resuspended in serum-free RPMI 1640 medium, and the cell concentration was adjusted to 1×10⁻⁶ cells / cells. 7 / ml, administer the suspension subcutaneously into the right back of the mouse at a dose of 100μl / mouse.

[0150] (3) When the tumor diameter reaches 100mm 3 After being randomly assigned to groups, patients were given 200 μg every 3 days for a total of 8 times, via intraperitoneal injection.

[0151] (4) Grouping: Isotype antibody (human-igg1) vs. humanized antibody #12, 6 tumor-bearing mice in each group.

[0152] (5) Monitor tumor growth 3 times a week, measure the long diameter and short diameter of the tumor, and the volume = long diameter × long diameter × short diameter / 2.

[0153] (6) When the tumor volume reaches 2000 mm 3If tumor necrosis occurs, mice are euthanized by dislocation of the neck, the subcutaneous tumor is completely removed, weighed, and photographed for record-keeping.

[0154] (7) Use GraphPad Prism software to draw a tumor volume growth curve.

[0155] The results are as follows Figure 23 As shown, the humanized antibody #12 had limited efficacy in the MC38 xenograft model, but two out of six mice showed a good drug response, indicating a decent tumor control rate. Therefore, the single-drug response rate of humanized antibody #12 was 33%. Further evaluation of the efficacy of humanized antibody #12 in combination with αPD-1, chemotherapy, or radiotherapy in the MC38 model will be considered.

[0156] Example 13: Safety of humanized antibody #12 in CD73-targeted humanized mice (1) Using the same mouse xenograft model as described above, the body weight of mice was monitored during treatment with humanized antibody #12. At the same time, peripheral blood was collected by enucleating the eyeballs of the mice before they were sacrificed and aliquoted into anticoagulant tubes (inverted and shaken) and ordinary EP tubes. The anticoagulant tubes were directly used for blood routine testing. After centrifuging the EP tubes at 10,000 rpm for 5 min, the supernatant was transferred to a biochemical testing cup and used for testing liver function (alanine aminotransferase, aspartate aminotransferase), kidney function (blood urea nitrogen) and other biochemical indicators.

[0157] The results are as follows Figure 24-26 As shown, there was no significant difference in body weight, blood routine and blood biochemical indicators between the humanized antibody #12 treatment group and the isotype group, and the #12 antibody showed no significant systemic toxicity.

[0158] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0159] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An anti-CD73 monoclonal antibody or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:1; The light chain variable region includes an amino acid sequence as shown in SEQ ID NO:

2.

2. The anti-CD73 monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: It includes the amino acid sequence of the heavy chain as shown in SEQ ID NO:5, and the amino acid sequence of the light chain as shown in SEQ ID NO:

6.

3. A nucleotide encoding the anti-CD73 monoclonal antibody of claim 1 or 2 or an antigen-binding fragment thereof, comprising a portion encoding a heavy chain variable region and a portion encoding a light chain variable region, characterized in that: The portion encoding the variable region of the heavy chain includes a nucleotide sequence as shown in SEQ ID NO:3; The portion encoding the variable region of the light chain includes a nucleotide sequence as shown in SEQ ID NO:

4.

4. The nucleotide according to claim 3, characterized in that: This includes the nucleotide sequence encoding the heavy chain as shown in SEQ ID NO:7, and the nucleotide sequence encoding the light chain as shown in SEQ ID NO:

8.

5. A carrier containing the nucleotide of claim 3 or 4.

6. A host cell containing the nucleotide of claim 4 or the vector of claim 5.

7. A method for preparing an anti-CD73 monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that... The method includes the following steps: culturing host cells containing nucleotides that can encode the anti-CD73 monoclonal antibody or its antigen-binding fragment, enabling them to express the anti-CD73 monoclonal antibody or its antigen-binding fragment, and collecting the expressed antibody or its antigen-binding fragment.

8. The use of the anti-CD73 monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, or the nucleotide according to claim 3 or 4, or the vector according to claim 5, or the host cell according to claim 6, in the preparation of a drug, characterized in that: The drug is an anti-tumor drug that inhibits CD73 enzyme activity; The tumor is either triple-negative breast cancer or colorectal cancer.

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