Differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition capability and application thereof

Abstract

The invention relates to the field of biological medicine, and discloses a differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition capability 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 the heavy chain variable region has at least 95% homology with the amino acid sequence as shown in SEQ ID NO: 1; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or has at least 95% of homology with the amino acid sequence as shown in SEQ ID NO: 2. The anti-CD73 monoclonal antibody disclosed by the invention is novel in recognition epitope and unique in action mechanism, and has high enzyme activity inhibition capability on different forms of CD73 proteins, so that the curative effect potential of a CD73 target spot can be fully exerted, and the treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition ability and application thereof. Background Art

[0002] CD73, the full name of which is extracellular-5′-nucleotidase, is highly expressed in a variety of 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 can hydrolyze adenosine monophosphate (AMP) to produce a large amount of adenosine (ADO). The accumulated adenosine binds to adenosine receptors on immune cells to inhibit the anti-tumor immune response in the body, specifically blocking the immune function of T cells, natural killer cells, and dendritic cells, while enhancing the function of regulatory T cells and myeloid-derived suppressor cells, promoting the polarization of M2 macrophages, etc., leading to tumor immune escape, thereby promoting tumor growth and metastasis. Adenosine in the tumor microenvironment can also act on adenosine receptors on endothelial cells to promote the survival of intratumoral blood vessels, act on tumor cells to directly promote their growth, and act on tumor-associated fibroblasts to promote their immunosuppressive function. CD73 also directly promotes the invasion and metastasis of tumor cells through mechanisms that are independent of its enzymatic activity. Therefore, CD73 plays an important role in tumor progression, which makes it an attractive target for tumor immunotherapy.

[0003] At present, the positive results of preclinical studies have shown that interventions targeting CD73, whether through gene editing technology or drug therapy, can effectively inhibit tumor growth and enhance the therapeutic effects of traditional chemotherapy and immune checkpoint inhibitors (such as PD-(L)1, CTLA-4, etc.). These findings have promoted the development of therapeutic antibodies targeting CD73 and pushed it to the clinical trial stage. Current clinical phase I and II study data show that the combination of CD73 antibodies with other treatments such as immune checkpoint inhibitors and radiotherapy not only enhances the therapeutic effect, but also shows good safety. Most treatment-related adverse events were grade 3 or below, and it was found that high expression of CD73 in tumors was closely related to treatment response, suggesting its possibility as a potential biomarker. These results highlight the important value of targeted CD73 therapy in clinical transformation and its anti-tumor potential.

[0004] Although CD73 antibodies have shown potential in clinical studies, the existing CD73 antibodies have different mechanisms of action in terms of antigen epitope binding and enzyme activity inhibition. These differences lead to incomplete inhibition of the enzyme activity of different forms of CD73 (including free and cell membrane surface types). For example, TJ004309 has a significant inhibitory effect on the free CD73 enzyme activity, but the inhibitory effect on the cell membrane surface CD73 is weak; CPI-006 can almost completely inhibit the cell membrane surface CD73 enzyme activity, but the inhibitory effect on free CD73 is limited; MEDI9447 has a "hook effect" on free enzyme activity, and the enzyme activity of cell membrane CD73 is not completely inhibited. This incomplete inhibition may weaken the blocking effect of the antibody on the AMP-CD73-ADO axis, thereby affecting its therapeutic effect. Therefore, antibodies that can completely inhibit the CD73 enzyme activity may have better therapeutic effects in clinical applications.

[0005] In summary, as an emerging tumor treatment target, CD73's initial success in clinical trials provides new ideas for tumor treatment. However, the anti-CD73 antibodies currently under clinical development may limit the full potential of the efficacy of the CD73 target due to differences in antigen epitope binding and enzyme activity inhibition, affecting the realization of its clinical therapeutic value. Future research needs to develop more powerful anti-CD73 antibodies with differentiated mechanisms of action to realize the clinical transformation value of this target. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a differentiated anti-CD73 monoclonal antibody with high enzyme activity inhibition ability and its application. The anti-CD73 monoclonal antibody of the present invention has a novel antigen epitope recognition and a unique mechanism of action. It has a high enzyme activity inhibition ability against different forms of CD73 protein, so it can give full play to the therapeutic potential of the CD73 target and improve the therapeutic effect.

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

[0008] Furthermore, 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] In a second aspect, the present invention provides a nucleotide encoding the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, which includes 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 includes the nucleotide sequence shown in SEQ ID NO: 3; the portion encoding the light chain variable region includes the nucleotide sequence shown in SEQ ID NO: 4.

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

[0011] In a third aspect, the present invention provides a vector containing the above-mentioned nucleotides.

[0012] In a fourth aspect, the present invention provides a host cell containing the above-mentioned nucleotide or vector.

[0013] In a fifth aspect, 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 a host cell containing nucleotides encoding the anti-CD73 monoclonal antibody or its antigen-binding fragment to express the 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 the above anti-CD73 monoclonal antibody or its antigen-binding fragment or the above nucleotide or the above vector or the above host cell in the preparation of a drug. Wherein, the drug is a drug that inhibits CD73 enzyme activity and is anti-tumor.

[0015] Furthermore, the tumor is a tumor with high, medium / low expression of CD73.

[0016] More preferably, the tumor is triple-negative breast cancer or colorectal cancer.

[0017] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0018] More preferably, the drug is in the form of an injection preparation, in the form of an injection solution, which may be a solution or a lyophilized powder.

[0019] Compared with the prior art, the beneficial effects of the present invention are: (1) Compared with the anti-CD73 antibodies currently under clinical development, the anti-CD73 monoclonal antibodies of the present invention have better enzyme activity inhibition ability against different forms of CD73 proteins (including free form and cell membrane surface form).

[0020] (2) Animal experiments show that the anti-CD73 monoclonal antibody of the present invention can exert significant anti-tumor effects in vivo. No obvious weight loss of animals and abnormalities in blood routine and biochemical indicators were observed 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 from the anti-CD73 antibodies currently under clinical development, and is a potential new clinical option.

[0022] (4) The anti-CD73 monoclonal antibody encoded by the present invention has a unique mechanism of action and can exert enzyme activity inhibition in the form of a monovalent antigen-binding fragment (Fab), and can be used as a choice of 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the inhibition rate of humanized antibody #12 and the anti-CD73 antibody currently under clinical development on free CD73 enzymatic activity.

[0025] Figure 2 The inhibition rate of humanized antibody #12 and the anti-CD73 antibody currently under clinical development on the surface CD73 enzyme activity of tumor cell membrane (MDA-MB-231, a triple-negative breast cancer cell line with high expression of CD73) when the substrate incubation time is 2 hours.

[0026] Figure 3 The inhibition rate of humanized antibody #12 and the anti-CD73 antibody currently under clinical development on the surface CD73 enzyme activity of tumor cell membrane (MDA-MB-231, a triple-negative breast cancer cell line with high expression of CD73) when the substrate incubation time is 8 hours.

[0027] Figure 4 Affinity results of the murine antibody #12 before humanization (#12(m)) and the humanized antibody #12 after modification (#12(h)) binding to free CD73.

[0028] Figure 5 The affinity results of humanized antibody #12 binding to CD73 on the surface membrane of CD73-positive T cells in normal human peripheral blood.

[0029] Figure 6 The affinity results of humanized antibody #12 binding to CD73 on the surface membrane of CD73-positive B cells in normal human peripheral blood.

[0030] Figure 7 Affinity results of humanized antibody #12 binding to CD73 on the surface membrane of human colorectal cancer cell line HCT-116.

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

[0032] Fig. 9 A graph showing the results of a competitive binding experiment between humanized antibody #12 and the current clinical development antibody.

[0033] Fig.10 Binding of humanized antibody #12 to CD73 mutants with mutations at different sites.

[0034] Fig.11 This is an experiment in which humanized antibody #12 inhibits the proliferation of human triple-negative breast cancer cell line MDA-MB-231 in vitro.

[0035] Fig.12 This is an experiment in which humanized antibody #12 inhibits the proliferation of human colorectal cancer cell line HCT-116 in vitro.

[0036] Fig.13 This is an experiment in which humanized antibody #12 inhibits the growth of nude mouse xenografts of the human triple-negative breast cancer cell line MDA-MB-231.

[0037] Fig.14 The tumor size of mice was photographed at the end point of the experiment in which humanized antibody #12 inhibited the growth of nude mouse xenografts of the human triple-negative breast cancer cell line MDA-MB-231 in vivo.

[0038] Fig.15 This is an experiment in which humanized antibody #12 inhibits the growth of human colorectal cancer cell line HCT-116 xenograft tumors in nude mice.

[0039] Fig.16 This is the tumor detachment photography and tumor weight results at the end point of the experiment in which humanized antibody #12 inhibited the growth of nude mouse transplanted tumors of the human colorectal cancer cell line HCT-116 in vivo.

[0040] Fig.17 This is an experiment to investigate the downregulation of CD73 on the surface of human triple-negative breast cancer cell line MDA-MB-231 induced by humanized antibodies #12 and BR101 in vitro.

[0041] Fig.18 This is an experiment in which humanized antibodies #12 and BR101 induced downregulation of CD73 on the surface of human colorectal cancer cell line HCT-116 in vitro.

[0042] Fig.19 These are the experimental results of humanized antibody #12 blocking the effect of high AMP on T cell activation indicator CD25 in vitro.

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

[0044] Fig.21 These are the experimental results of humanized antibody #12 blocking the effect of high AMP on T cell proliferation in vitro.

[0045] Fig. 22 Results of the humanized antibody #12 helper T cell activation experiment.

[0046] Fig.23 The results of the humanized antibody #12 inhibiting the growth of transplanted tumors in MC38 cell line immunocompetent mice (CD73 humanized mice) in vivo.

[0047] Fig.24 The effect of humanized antibody #12 on body weight in an in vivo inhibition experiment of tumor growth transplanted in immunocompetent mice (CD73 humanized mice) bearing the mouse MC38 cell line.

[0048] Fig.25 The effect of humanized antibody #12 on routine blood test parameters in an in vivo inhibition experiment of tumor growth transplanted in immunocompetent mice (CD73 humanized mice) bearing the mouse MC38 cell line.

[0049] Fig.26 The effect of humanized antibody #12 on blood biochemical indicators in the in vivo inhibition experiment of tumor growth transplanted in immunocompetent mice (CD73 humanized mice) with the mouse MC38 cell line. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments.

[0051] Overall embodiment In the first aspect, the present invention provides a differentiated anti-CD73 monoclonal antibody or an antigen-binding fragment thereof with high enzyme activity inhibition ability, comprising a heavy chain variable region and a light chain variable region. Wherein: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or has at least 95% homology thereto; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 2, or has at least 95% homology thereto. Furthermore, the amino acid sequence of the heavy chain of the anti-CD73 monoclonal antibody or its antigen-binding fragment is as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 6.

[0052] In a second aspect, the present invention provides a nucleotide encoding the above-mentioned anti-CD73 monoclonal antibody or its antigen-binding fragment, which includes 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 includes a nucleotide sequence as shown in SEQ ID NO: 3; the portion encoding the light chain variable region includes a nucleotide sequence as 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] In a third aspect, the present invention provides a vector containing the above-mentioned nucleotides.

[0054] In a fourth aspect, the present invention provides a host cell containing the above-mentioned nucleotide or vector.

[0055] In a fifth aspect, 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 a host cell containing nucleotides encoding the anti-CD73 monoclonal antibody or its antigen-binding fragment to express the 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, comprising the following steps: S1: Use the hybridoma monoclonal antibody method to screen lead antibodies: Immunize mice with CD73 recombinant protein, screen hybridoma cells that produce anti-human CD73 monoclonal antibodies through cell fusion, and identify hybridoma cells that express antibodies that meet the requirements based on affinity screening.

[0057] S2: Use high-throughput methods to screen candidate antibodies with high enzyme activity inhibition ability: Use enzyme-coupled analysis to establish a screening system that can detect CD73 enzyme activity. Based on this, high-throughput screening of lead antibodies can be performed to determine candidate antibodies that meet the requirements.

[0058] S3: A mutant antibody library is constructed by screening antibodies based on affinity through the CDR1, CDR2 and CDR3 of the heavy and light chains of monoclonal antibodies to obtain monoclonal antibodies with successful humanization and unaffected affinity.

[0059] In a sixth aspect, the present invention provides the use of the above anti-CD73 monoclonal antibody or its antigen-binding fragment or the above nucleotide or the above vector or the above host cell in the preparation of a drug. Wherein, the drug is a drug that inhibits CD73 enzyme activity and is anti-tumor. Further, the tumor is a tumor with high expression, medium / low expression of CD73; more preferably, triple-negative breast cancer and colorectal cancer.

[0060] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0061] More preferably, the drug is in the form of an injection preparation, usually in the form of an injection solution, which may be a solution or a lyophilized powder. Specific embodiments 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) and the CD73 antibody under clinical development to inhibit the enzymatic activity of free CD73 (1) HCT-116 cells growing in the logarithmic phase were obtained, digested with trypsin, and the HCT-116 cell precipitate was collected by centrifugation.

[0063] (2) Lysis buffer was added to the HCT-116 cell pellet to extract free CD73. The cell pellet was lysed on ice for 15 min and then centrifuged at 145,000 g for 10 min. The supernatant was used for free CD73 enzyme activity detection experiment.

[0064] (3) Antibodies with different final concentrations were added to the supernatant. The control group was added with isotype (human IgG1) at a final concentration of 500 ug / ml. Two replicate wells were set up for each treatment and incubated at room temperature for 30 min.

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

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

[0067] The results are as follows Figure 1 As shown, the inhibitory ability of humanized antibody #12 and the CD73 antibody currently under clinical development in free CD73 enzyme activity is compared. BR101 is an anti-human CD73 therapeutic antibody developed by Zhejiang Borui Biopharmaceutical Co., Ltd. In the field of tumor treatment, the highest development status is clinical phase I; MEDI9447 is an anti-human CD73 therapeutic antibody developed by AstraZeneca Pharmaceuticals, and the highest development status is clinical phase III in the field of tumor treatment; TJ004309 is an anti-human CD73 therapeutic antibody developed by Tianjin Biopharma Co., Ltd., and the highest development status is clinical phase III in the field of tumor treatment; CPI-006 is an anti-human CD73 therapeutic antibody developed by Corvus Pharmaceuticals, and the highest development status is clinical phase I in the field of tumor treatment.

[0068] Compared with these anti-CD73 antibodies under clinical development, the humanized antibody #12 of the present invention has better 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 #12 is similar to that of TJ004309 and is far superior to other antibodies. MEDI9447 and BR101 also have a hook effect, which is manifested as a significant decrease in the enzyme activity inhibition effect under the action of high concentrations of antibodies.

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

[0070] (2) On the second day, the culture supernatant was removed and serum-free culture medium containing different concentrations of antibodies was added. The control group was added with isotype (human IgG1) at a final concentration of 50 ug / ml, and the blank group was added with serum-free culture medium. Two replicate wells were set for each treatment and incubated in the incubator for 2 h.

[0071] (3) After the incubation, serum-free medium containing AMP was added to the control group and the antibody-treated group at a final concentration of 500 uM, and serum-free medium was added to the blank group, and the cells were incubated in the incubator for 2-8 h.

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

[0073] (5) The supernatant after rewarming was tested for adenosine content by high performance liquid chromatography (HPLC). The control group obtained the maximum adenosine production, and the blank group obtained the minimum adenosine production. The inhibition rate of the antibody on the CD73 enzyme activity on the cell membrane surface at each concentration was calculated using the formula: ((control group - blank group) - (antibody groups with different concentrations - blank group)) / (control group - blank group) * 100% (In actual experiments, adenosine production was not measured in the blank group, and this formula can be used directly for calculation: (control group - antibody groups with different concentrations) / control group * 100%).

[0074] The results are as follows Figure 2 , 3As shown, the cell membrane surface CD73 enzyme activity inhibition ability of humanized antibody #12 and the CD73 antibodies currently under clinical development is compared. BR101 is an anti-human CD73 therapeutic antibody developed by Zhejiang Borui Biopharmaceutical Co., Ltd. In the field of tumor treatment, the highest development status is clinical phase I; MEDI9447 is an anti-human CD73 therapeutic antibody developed by AstraZeneca Pharmaceuticals, and the highest development status is clinical phase III in the field of tumor treatment; TJ004309 is an anti-human CD73 therapeutic antibody developed by Tianjin Biopharma Co., Ltd., and the highest development status is clinical phase III in the field of tumor treatment; CPI-006 is an anti-human CD73 therapeutic antibody developed by Corvus Pharmaceuticals, and the highest development status is clinical phase I in the field of tumor treatment. The inhibitory ability of the humanized antibody #12 of the present invention is similar to that of CPI-006 with the best inhibitory ability (2h: the same maximum inhibition rate, IC50: 0.4917nM (#12) vs 0.3637nM (CPI-006)), but it is least affected by the incubation time compared with CPI-006 (8h: the 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 binding to free CD73 (1) BioLayer Interferometry (BLI) was used to detect the affinity between humanized antibody #12 and free CD73 protein.

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

[0077] (3) The sensor was immersed in Q buffer containing humanized antibody #12 for 120 s to allow the humanized antibody #12 to adsorb to the sensor surface.

[0078] (4) The sensor was immersed in Q buffer to remove unadsorbed humanized antibody #12, equilibrated again for 60 s, and the signal baseline was measured.

[0079] (5) The sensor loaded with humanized antibody #12 was immersed in Qbuffer containing different concentrations of free CD73 protein for 120 s, and the antigen-antibody binding signal was detected.

[0080] (6) Immerse the sensor in Q buffer for 180 s to detect the 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, it can be seen that the affinity of humanized antibody #12 for binding to free CD73 is 9.55*10 -10 .

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

[0084] (2) Prepare cell staining buffer containing different concentrations of antibodies, with a concentration gradient, starting at 15 ug / ml antibody concentration, 3-fold dilution, for a total of 10 concentrations.

[0085] (3) Evenly plate 100ul of the treated cells in each well of a 96-well plate. Centrifuge at 350g for 5min, discard the supernatant, add 100ul of staining buffer containing antibodies, resuspend the cells, and incubate in a refrigerator at 4°C for 1h.

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

[0087] (5) Add 100 μL of PE fluorescently labeled anti-human IgG secondary antibody to the precipitate, resuspend the cells, and incubate in a 4°C refrigerator for 1 h.

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

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

[0090] (8) Calculate the MFI values ​​of samples treated with different antibody concentrations, and use the nonlinear fitting method to calculate the antibody EC50 and KD values.

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

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

[0093] (2) Prepare cell staining buffer containing 10ug / ml unfluorescently labeled antibody, 5 groups, isotype (human IgG1), #12, BR101, CPI-006, TJ004309.

[0094] (3) Evenly plate 200ul of the treated cells in each well of a 96-well plate. Centrifuge at 350g for 5min, discard the supernatant, add 100ul of staining buffer containing antibodies, resuspend the cells, and incubate in a refrigerator at 4°C for 1h.

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

[0096] (5) After incubation, add 200ul of pre-cooled PBS buffer to each well and wash twice. Add 210ul of buffer to each well to resuspend the precipitate, and then divide it into 4 wells, 50ul per well. Take 50ul of each of the 4 fluorescent labeled antibodies and add them to 4 wells respectively, with a final concentration of 2ug / ml, and incubate in a refrigerator at 4℃ for 1h.

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

[0098] (7) Use BD Fortessa flow cytometer and BD HTS workstation to detect the binding of fluorescently labeled antibodies and determine the similarity of the binding epitopes between antibodies.

[0099] Results Fig. 9As shown, the binding mode and epitope of humanized antibody #12 are different from those of the current clinical research antibodies and do 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 the mature CD73 protein (excluding the signal peptide required for protein sorting and maturation) were input in FASTA format to predict the interaction between humanized antibody #12 and CD73.

[0101] (2) Download model1 with the highest score in the prediction results, open the modell.pdb file with ChimeraX, and analyze the key residues for the interaction between the two. The predicted antigen 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, and W327.

[0102] (3) Point mutation or combined mutation was performed on the predicted key interaction residues, and the mutant CD73 sequence was cloned using the pcDNA3.1 plasmid. An HA tag was additionally added to the N-terminus of the CD73 sequence to detect the expression of the protein in cells expressing the mutant 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 recognized.

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

[0106] Table 2 Results Fig.10 As shown, humanized antibody #12 does not bind to CD73-V4, but binds to other point mutations. The positive control (which does not overlap with the epitope of #12) BR101 maintains binding to CD73-V4, so the epitope recognized by humanized antibody #12 includes PEDPSIKADINKWR.

[0107] Example 7: In vitro anti-tumor cell effects of humanized antibody #12 (1) Digest the MDA-MB-231 and HCT-116 cells in the logarithmic growth phase and collect the pellets. Resuspend them in 1640% complete medium and seed 3000 cells per well in a 96-well cell culture plate and culture at 37°C overnight.

[0108] (2) On the second day, the culture supernatant was removed and complete medium containing different concentrations of humanized antibody #12 was added. The control group was added with isotype (human IgG1) at a final concentration of 15 ug / ml. Three replicate wells were set up for each treatment. An additional blank group was set up with an equal volume of complete medium without cells. The cells were incubated in an incubator for 72 h.

[0109] (3) Prepare proliferation detection solution. Use a multichannel pipette to carefully add 10 μl of CCK-8 solution to each well, avoiding bubbles. Incubate in an incubator for 2 h.

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

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

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

[0113] (2) Digestion of MDA-MB-231 cells in the logarithmic growth phase, wash twice with PBS, and resuspend in serum-free RPMI 1640 medium to adjust the cell concentration to 6×10 7 / ml, add an equal volume of matrix gel, mix well, and store on ice.

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

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

[0116] (5) Day 5 after MDA-MB-231 tumor inoculation (average tumor diameter 40 mm) 3) started to be administered, once every 3 days, 250ug each time, for a total of 8 times, by intraperitoneal injection. The day of HCT-116 tumor inoculation, the administration started, once every 3 days, 250ug each time, for a total of 5 times, by intraperitoneal injection.

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

[0118] (7) The tumor growth was monitored three times a week, and the long and short diameters of the tumor were measured. Volume = long diameter × long diameter × short diameter / 2. At the end of the experiment, the mice were killed by cervical dislocation, and the subcutaneous tumor was completely removed, weighed, and photographed for record.

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

[0120] Results Figure 13-16 As shown, humanized antibody #12 can significantly inhibit 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, keep the precipitate, resuspend them in cell staining buffer (2% FBS, PBS), and adjust the single cell suspension density to 100w / ml.

[0122] (2) Evenly plate 100ul of the treated cells in each well of a 96-well plate. Centrifuge at 350g for 5min, discard the supernatant, add 100ul of staining buffer containing 15ug / ml unlabeled fluorescent antibody, resuspend the cells, and incubate at 37°C for 0, 1, 2, and 3h.

[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 100 μl of the same unlabeled fluorescent antibody staining buffer as the first incubation containing 15 μg / ml. The control group was divided into two groups, which were incubated with #12 and BR101 for the second time, respectively, and incubated in a 4°C refrigerator for 1 h.

[0126] (6) After incubation, wash twice with pre-cooled PBS buffer, add 100 μl of PE fluorescently labeled anti-human IgG secondary antibody, resuspend the cells, and incubate in a 4°C refrigerator for 1 h.

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

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

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

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

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

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

[0133] (3) Assemble the sorting device according to the instructions of Miltenyi Biotec LS column, rinse the sorting column with 3ml MACS, and rinse three times. After the last rinse, add 3ml of single cell suspension filtered through a 70um filter. When the cell suspension completely enters the sorting column, add 3ml MACS solution to rinse, and repeat three times. Carefully remove the sorting column, place the lower part in a 15ml centrifuge tube, add 3ml MACS solution, and use the built-in 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 increase the yield.

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

[0135] (5) Group setting: 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, the highest concentration was 135ug / ml, 3-fold dilution, a total of 5 concentrations, positive control group: α CD3+α CD28+AMP+AB680 (10uM).

[0136] (6) Treatment sequence: isotype, #12 and AB680 were added to the cells and incubated at 37°C for 2 h, followed by the addition of AMP solution to a final concentration of 500 uM and incubation for 1 h. Finally, α CD3 and α CD28 were added to each well at a final concentration of 1 ug / ml and cultured for 24 h.

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

[0138] (8) Resuspend the pellet and detect the activation level of CD8+ T cells on a flow cytometer.

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

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

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

[0142] Example 11: Humanized antibody #12 can activate CD73 co-stimulatory molecules to assist in the activation of CD8+ T cell function (1) Add 50ul of PBS buffer containing antibodies to a 96-well plate and incubate overnight at 4°C. Group setting: antibody 1: αCD3, antibody 2: αCD3+αCD28, antibody 3: αCD3+#12, αCD3 final concentration 0.25ug / ml, αCD28 and #12 final concentration 0.5ug / ml.

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

[0144] (3) On the next day, remove the liquid from the wells, wash three times with PBS buffer, add 100 μl of cell suspension, and culture in an incubator for 2 days.

[0145] (4) After the culture, the plate was centrifuged at 350g for 5 minutes, the supernatant was collected, and the IL-2 content was detected. The precipitate was resuspended in 50ul of staining buffer containing anti-CD25BV605, anti-CD69 percp cy5.5, anti-CD71 BV421, anti-OX40 BV711, and anti-CD73FITC (BR101), incubated at 4°C for 30 minutes, washed once with PBS, and resuspended and tested on a flow cytometer for the activation level of CD8+ T cells.

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

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

[0148] Example 12: Anti-tumor effect of humanized antibody #12 in CD73 target humanized mice (1) Six-week-old CD73 target humanized mice (purchased from Southern Model Organisms, C57BL / 6Smoc-Nt5etm3(NT5E)Smoc) were taken and housed in a barrier cage for 1 week.

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

[0150] (3) When the tumor diameter reaches 100 mm 3 After randomization, the drug was administered once every 3 days, 200 μg each time, for a total of 8 times, by intraperitoneal injection.

[0151] (4) Group setting: isotype antibody (human-IgG1) vs humanized antibody #12, 6 tumor-bearing mice in each group.

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

[0153] (6) When the tumor volume reaches 2000mm 3Or when tumor necrosis occurred, the mice were killed by cervical dislocation, and the subcutaneous tumor was completely removed, weighed, and photographed for record.

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

[0155] The results are as follows Fig.23 As shown in the figure, humanized antibody #12 has limited efficacy in the MC38 transplant tumor model, but 2 out of 6 mice responded well to the drug, and the tumor control rate was acceptable, so the single-drug response rate of humanized antibody #12 was 33%. In the future, the efficacy of humanized antibody #12 will be evaluated in combination with αPD-1, chemotherapy or radiotherapy in the MC38 model.

[0156] Example 13: Safety of humanized antibody #12 in CD73 target humanized mice (1) Using the same mouse transplant tumor model as above, the body weight of mice was monitored during the treatment with humanized antibody #12. At the same time, the eyeballs of mice were removed to obtain peripheral blood before they were killed. The blood was divided into anticoagulant tubes for routine blood test (shaken upside down) and ordinary EP tubes. The anticoagulant tubes were directly tested on the machine for routine blood test; the EP tubes were centrifuged at 10,000 rpm for 5 minutes, and the supernatant was transferred to a cup for biochemical test, and other biochemical indicators such as liver function (alanine aminotransferase, aspartate aminotransferase) and kidney function (urea nitrogen) were tested on the machine.

[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. The #12 antibody had no obvious systemic toxicity.

[0158] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0159] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A differentiated anti-CD73 monoclonal antibody or an antigen-binding fragment thereof with high enzyme activity inhibition ability, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1, or has at least 95% homology thereto; The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or has at least 95% homology thereto.

2. The anti-CD73 monoclonal antibody or antigen-binding fragment thereof 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 or antigen-binding fragment thereof according to claim 1 or 2, 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 heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO: 3; The portion encoding the light chain variable region includes the nucleotide sequence shown in SEQ ID NO:

4.

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

8.

5. A vector comprising the nucleotide according to claim 3 or 4.

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

7. A method for preparing the anti-CD73 monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The method comprises the following steps: culturing a host cell containing a nucleotide encoding an anti-CD73 monoclonal antibody or an antigen-binding fragment thereof, causing the host cell to express the anti-CD73 monoclonal antibody or the antigen-binding fragment thereof, and collecting the expressed antibody or the antigen-binding fragment thereof.

8. Use of the anti-CD73 monoclonal antibody or antigen-binding fragment thereof 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.

9. The use according to claim 8, characterized in that: The tumor is a tumor expressing CD73.

10. The use according to claim 9, characterized in that: The tumor is triple-negative breast cancer or colorectal cancer.

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