A CD38 monoclonal antibody-MOFs composite biomaterial and kit

Abstract

The present invention belongs to the field of biological detection technology, and provides a CD38 monoclonal antibody-MOFs composite biomaterial. The CD38 monoclonal antibody-MOFs composite biomaterial includes a core and a coating layer, wherein the core is a MOFs composite biomaterial, and the coating layer is a CD38 monoclonal antibody or an irregular blood type antibody. In the CD38 monoclonal antibody-MOFs composite biomaterial provided by the present invention, Zu-MOF as a host not only does not affect the antibody's ability to detect its corresponding antigen, but also can be stored for a long time, avoiding high temperature and biological damage. The use of the CD38 monoclonal antibody-MOFs composite biomaterial provided by the present invention helps to dissipate the agglutination caused by the interference of the CD38 monoclonal antibody and improve the accuracy of the blood matching results.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a CD38 monoclonal antibody-MOFs composite biomaterial and a kit. Background Art

[0002] Multiple myeloma (MM) is a mature B-cell tumor and a malignant tumor with abnormal proliferation of a single plasma cell line in the bone marrow. The vast majority of patients have varying degrees of anemia, and the degree of anemia in most patients progressively worsens with the progression of the disease. In recent years, antibody drugs have been increasingly used in clinical practice. Currently, the CD38 monoclonal antibody Daratum has been approved by many countries for the clinical treatment of multiple myeloma (MM). Since the target of CD38 monoclonal antibody is not only expressed on tumor cells, but also on the surface of red blood cells to varying degrees, it interferes with transfusion compatibility testing.

[0003] In recent years, antibody drugs have been gradually promoted and applied in clinical practice, opening up new ways to treat diseases. CD38 monoclonal antibody-daratumumab is a monoclonal antibody drug that targets CD38. In 2019, my country's Food and Drug Administration approved CD38 monoclonal antibody-daratumumab for the treatment of relapsed and refractory multiple myeloma. The "Guidelines for the Diagnosis and Treatment of Multiple Myeloma in China (Revised in 2020)" clearly states that CD38 monoclonal antibody-daratumumab is used to treat relapsed and refractory multiple myeloma. Since the CD38 molecule is a type 2 transmembrane glycoprotein with both receptor and exonuclease activity, it is not only highly expressed on MM tumor cells, but also on red blood cells. Therefore, the use of daratumumab will bind to red blood cell CD38 to sensitize the patient's red blood cells, which will inevitably interfere with clinical transfusion compatibility testing experiments.

[0004] Daratum has the effect of inducing the weakening of erythrocyte CD38 expression. Flow cytometry results showed that after the patient was injected with Daratum, the number of Daratum bound to the surface of erythrocytes increased significantly, but began to weaken 6 hours after the medication. One week later, the microcolumn agglutination method could not detect the CD38 antigen on the surface of erythrocytes; 6 months after the drug was discontinued, the expression of erythrocyte CD38 in the patient's erythrocytes could be restored to the level before the medication. Therefore, the direct antiglobulin test of the patient who took the drug is often negative. On the other hand, since CD38 is expressed on the surface of erythrocytes, when the samples are tested using antiglobulin-based test methods, they all show positive reaction results, showing a broad spectrum of reactivity, which can be easily misjudged as autoantibodies or high-frequency antibodies. The clearance of Daratum is related to the dosage and frequency of administration. With the increase of dosage and frequency, the half-life is prolonged and the clearance rate is reduced. The average detection time of Daratum after discontinuation of the drug is about 6 months, but it has also been reported that Daratum can still be detected within 9 months after discontinuation of the drug. Regarding the interference of CD38 monoclonal antibody drugs in blood transfusion compatibility testing, the current clinical practice is to select blood donors with higher transfusion standard matching as much as possible for cross-matching, which cannot eliminate the interference caused by CD38 monoclonal antibody drugs on cross-matching experiments. Therefore, it is urgent to find effective measures to block the binding of CD38 monoclonal antibody drugs in patient serum and CD38 molecules on the surface of red blood cells of blood donors, thereby eliminating the interference caused by CD38 monoclonal antibody drugs on cross-matching experiments, ensuring clinical safe and effective blood use, and ensuring blood transfusion safety. Serological testing is the use of immunological methods to detect antigens or antibodies. The basic principle of testing is to use known antigens to detect unknown antibodies, or to use known antibodies to detect unknown antigens, and to judge whether the corresponding antigens or antibodies exist in the sample based on the experimental results. Since most of the antibodies used in the initial experiments came from serum, it is called serological testing. The monoclonal or polyclonal antibody reagents used in traditional serological tests have obvious advantages in terms of detection specificity and economic practicality. However, they also have serious shortcomings in the use and storage process: 1) The transportation and storage conditions must be cold chain throughout the process. Once out of the cold chain temperature, the biological characteristics are significantly affected, which in turn affects the detection level; 2) The sensitivity is poor, and the detection ability for some weak antigens is poor, such as the weak antigen detection ability for ABO subtype patients or rare blood type donors is significantly limited. This is also the biggest challenge currently faced by the application of blood type serological detection reagents. Summary of the invention

[0005] In order to solve the problem in the prior art that CD38 molecules interfere with blood transfusion compatibility detection, the present invention provides a CD38 monoclonal antibody-MOFs composite biomaterial.

[0006] The CD38 monoclonal antibody-MOFs composite biomaterial comprises a core and a coating layer, wherein the core is the MOFs composite biomaterial, and the coating layer is the CD38 monoclonal antibody or the irregular blood type antibody.

[0007] Furthermore, the MOFs composite biomaterial includes a metal ligand that is zinc.

[0008] The invention also provides a kit.

[0009] The kit comprises a substrate, and the substrate is the CD38 monoclonal antibody-MOFs composite biomaterial as described in any one of the above items.

[0010] The invention also provides an artificial intelligence detection platform.

[0011] The artificial intelligence detection platform comprises a detection platform, an artificial intelligence interpretation terminal movably connected to one side of the detection platform, a bright field microscope, and the above-mentioned reagent kit, wherein the artificial intelligence interpretation terminal and the bright field microscope are connected via a data line.

[0012] The present invention also provides a method for preparing the CD38 monoclonal antibody-MOFs composite biomaterial as described above.

[0013] The preparation method of the CD38 monoclonal antibody-MOFs composite biomaterial comprises the following steps:

[0014] The CD38 monoclonal antibody or the irregular blood type antibody coats the MOFs composite biomaterial.

[0015] Furthermore, the method further comprises the following steps:

[0016] The MOFs composite biomaterial whose metal ligand is zinc is prepared.

[0017] In the CD38 monoclonal antibody-MOFs composite biomaterial provided by the present invention, Zn-MOF as a host not only does not affect the detection ability of the antibody to its corresponding antigen, but also can be stored for a long time and avoid high temperature and biological damage.

[0018] The use of the CD38 monoclonal antibody-MOFs composite biomaterial provided by the present invention helps to dissipate the agglutination caused by the interference of the CD38 monoclonal antibody and improve the accuracy of the blood matching result. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The flowchart of the preparation method of CD38 monoclonal antibody-MOFs composite biomaterials;

[0020] Figure 2 This is an electron microscope scan of the Zn-MOFs model;

[0021] Figure 3 This is an electron microscope scanning image of CD38 monoclonal antibody-MOFs composite biomaterial;

[0022] Figure 4 This is the potential test result of CD38 monoclonal antibody-MOFs composite biomaterial;

[0023] Figure 5 This is the Zeta potential bar graph of CD38 monoclonal antibody-MOFs composite biomaterial;

[0024] Figure 6 This is the X-ray diffraction pattern of CD38 monoclonal antibody-MOFs composite biomaterial;

[0025] Figure 7 Ultraviolet UV results for CD38 monoclonal antibody-MOFs composite biomaterials;

[0026] Figure 8 To observe the patient's cross-matching results under an optical microscope. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and understandable, the specific implementation modes of the present invention are described in detail below with reference to the accompanying drawings, but they should not be construed as limiting the applicable scope of the present invention.

[0028] Metal-organic frameworks (MOFs) use inorganic metal (e.g., transition metals and lanthanide metals) ions / clusters as nodes and organic ligands (e.g., carboxylates, phosphonates, imidazolates, and phenolates) as supports to form extended infinite 1D / 2D / 3D MOF networks. In recent years, the application of MOFs in the biomedical field has attracted increasing attention. When the size of MOF particles is reduced to the nanoscale, these nano-MOFs (NMOFs) can be used as drug delivery carriers for imaging, chemotherapy, photothermal therapy, or photodynamic therapy. Compared with other porous materials, MOFs have many outstanding advantages, such as: (1) high specific surface area and porosity, which can be used for high loading of therapeutic drugs; (2) easy modification of the physical (such as pore size and shape) and chemical properties of MOFs by inorganic clusters and / or organic ligands, in addition, the desired functional groups can be added to the organic ligands through pre-design or post-synthetic modification methods; (3) the open windows and pores of MOFs allow the diffusion matrix to interact with the binding molecules; (4) moderate strength coordination bonds make MOFs biodegradable; (5) well-defined structures are conducive to host-guest interaction studies. Due to these unique properties, MOFs are considered to be one of the best candidates for drug delivery and cancer treatment.

[0029] Due to the special pore structure of metal organic framework compounds (MOFs), antibodies can be encapsulated or antibodies can be infiltrated into the MOF structure through covalent or non-covalent processes. In this way, MOF materials and blood type antibodies are integrated to ensure that the biological properties of the antibodies are not affected. In addition to forming simple antibody / MOF composite biomaterials, other materials can also be introduced into the composite to promote the advanced application of blood type antibody-MOFs composite biomaterials. The present invention proposes to protect blood type antibodies through encapsulation, pore penetration and surface adsorption, and to give them a higher level of biocompatibility and functional diversity.

[0030] Based on the inventive concept of "constructing a CD38 model coated with a metal organic framework structure (MOFs) to remove the interference of cross-matching of multiple myeloma patients", the present invention provides a CD38 monoclonal antibody-MOFs composite biomaterial and a kit. The present invention selects non-biotoxic Zn as the metal ligand of MOFs, successfully constructs a Zn-MOFs model for subsequent experiments, and the blood type antibody-MOFs composite biomaterial can be used in animals or humans in the future.

[0031] The CD38 monoclonal antibody-MOFs composite biomaterial comprises a core and a coating layer, wherein the core is the MOFs composite biomaterial, and the coating layer is the CD38 monoclonal antibody or the irregular blood type antibody.

[0032] Furthermore, the metal ligand of the MOFs composite biomaterial is zinc.

[0033] The combination of antibodies and catalytic metal-organic frameworks (MOFs) formed a bifunctional MOF / antibody complex, and Zn-MOF as a host not only did not affect the detection ability of antibodies to their corresponding antigens, but also allowed long-term storage, avoiding high temperatures and biological damage.

[0034] The invention also provides a kit.

[0035] The kit comprises a substrate, wherein the substrate is the CD38 monoclonal antibody-MOFs composite biomaterial as described above.

[0036] The present invention utilizes the above-mentioned characteristics of CD38 monoclonal antibody-MOFs composite biomaterial to construct a blood type antigen detection kit for improving the existing blood type detection kit.

[0037] The invention also provides an artificial intelligence detection platform.

[0038] The artificial intelligence detection platform comprises the detection platform, an artificial intelligence interpretation terminal movably connected to one side of the detection platform, a bright field microscope, and the above-mentioned reagent kit, wherein the artificial intelligence interpretation terminal and the bright field microscope are connected via a data line.

[0039] The present invention provides a method for preparing the CD38 monoclonal antibody-MOFs composite biomaterial as described above.

[0040] The method for preparing the CD38 monoclonal antibody-MOFs composite biomaterial is characterized by comprising the steps of:

[0041] The CD38 monoclonal antibody or the irregular blood type antibody coats the MOFs composite biomaterial.

[0042] Furthermore, the method further comprises the following steps:

[0043] The MOFs composite biomaterial whose metal ligand is zinc is prepared.

[0044] See also Figure 1 In one embodiment, the preparation method of CD38 monoclonal antibody-MOFs composite biomaterial includes:

[0045] S1: With zinc as the metal ligand, α,α'-bipyridine is connected by self-assembly to form the Zn-MOFs model;

[0046] S2: The Zn-MOFs model was coated with CD38 monoclonal antibody or irregular blood type antibody to prepare CD38 monoclonal antibody-MOFs composite biomaterial.

[0047] Example 1 is provided according to the method for preparing the CD38 monoclonal antibody-MOFs composite biomaterial provided above.

[0048] Example 1

[0049] (1) Construction of Zn-MOFs model

[0050] The Zn(NO 3 ) 2 , terephthalic acid (or 1,4-benzenedicarboxylic acid, H2BDC) and 1,4-diazabicyclo[2.2.2]octane (dabco) were heated at 110°C for 2 days to obtain a crystalline product [Zn 2 (1,4-BDC) 2 (dabco)]·4DMF·1=2H 2 O(1·4DMF·1=2H 2 O), namely Zn-MOFs.

[0051] (2) Construction of CD38 monoclonal antibody-MOFs composite biomaterials

[0052] 1. Dissolve 2g Zn-MOFs powder evenly in 20ml Tris-HCl buffer (PH=7.8), then add 100ug CD38 protein, stir overnight at 4°C to form a homogeneous solution;

[0053] 2. The homogeneous solution obtained in step 1 was centrifuged at 10000 rpm for 10 minutes, and washed three times with deionized water to remove unbound proteins. The precipitate was then freeze-dried to obtain a product, which was the CD38 monoclonal antibody-MOFs composite biomaterial.

[0054] test:

[0055] 1. Scan the surface morphology of the Zn-MOFs model with an electron microscope, and the results show that Figure 2 .

[0056] 2. The constructed CD38 monoclonal antibody-MOFs composite biomaterial was tested by electron microscopy and X-ray diffraction (XRD). The results showed that Figure 3 and Figure 6 .

[0057] 3. Use ultraviolet light to detect CD38 monoclonal antibody in CD38 monoclonal antibody-MOFs composite biomaterials. The results show that Figure 7 .

[0058] 4. The CD38 monoclonal antibody-MOFs composite biomaterial was tested using a Zeta potential instrument. The results showed Figure 4 and Figure 5 .

[0059] 5. The CD38 monoclonal antibody-MOFs composite biomaterial prepared in the example was used for cross-matching experiment. The results of cross-matching were observed under an optical microscope. Figure 8 .

[0060] Result analysis:

[0061] Figure 2 The scanning electron microscopy results show that the morphology of Zn-MOFs is similar to that in the literature. 1 Consistent with the report, the energy spectrum results also showed that the Zn element was uniformly incorporated into the Zn-MOFs model.

[0062] See also Figure 3 , Figure 6 The CD38 monoclonal antibody-MOFs composite biomaterial was successfully constructed by scanning electron microscopy and X-ray diffraction (XRD). Ultraviolet (UV) was also used to detect the coated CD38 monoclonal antibody. Figure 7, the results showed a peak at 280 nm, which confirmed the successful construction of CD38 monoclonal antibody-MOFs composite biomaterials.

[0063] See also Figure 4 and Figure 5 The Zeta potential results showed that due to the positive charge of the amino group of the protein, the Zeta potential of the CD38 monoclonal antibody-MOFs complex was significantly increased compared with that of Zn-MOFs alone.

[0064] Figure 8 A shows the cross-matching results of the patient and the screening blood after using CD38 monoclonal antibody drugs, showing 1+ agglutination, indicating that the blood is not compatible. However, after adding CD38 monoclonal antibody-MOFs composite biomaterials, the agglutination caused by the interference of CD38 monoclonal antibody dissipated, and the blood matching results showed compatibility ( Figure 8 B). Cell functional experiments confirmed that the CD38 monoclonal antibody-MOFs composite biomaterial successfully eliminated the interference of CD38 monoclonal antibody on patient cross-matching.

[0065] Preliminary experiments of the present invention successfully constructed a Zn-MOFs model coated with CD38 monoclonal antibody or irregular blood type antibody, and prepared antibody-MOFs composite biomaterials. The constructed blood type antibody-MOFs composite biomaterials were detected by scanning electron microscopy and infrared spectroscopy, and the accuracy and reliability of the experimental results have been verified.

[0066] References:

[0067] 1. Dybtsev DN, Chun H, Kim K. Rigid and flexible: a highly porous metal-organic framework with unusual guest-dependent dynamic behavior. Angew ChemInt Ed Engl. 2004 Sep 27; 43 (38): 5033-6. doi: 10.1002 / anie.200460712. PMID: 15384114.

Claims

1. A CD38 monoclonal antibody-MOFs composite biomaterial, characterized in that: It comprises a core and a coating layer, wherein the core is a MOFs composite biomaterial, and the coating layer is a CD38 monoclonal antibody; The metal ligand of the MOFs composite biomaterial is zinc.

2. A kit comprising a substrate, characterized in that The substrate is the CD38 monoclonal antibody-MOFs composite biomaterial as described in claim 1.

3. An artificial intelligence testing platform, comprising a testing platform, characterized in that: It also includes an artificial intelligence interpretation terminal, a bright field microscope, and a test kit as described in claim 2, which are movably connected to one side of the detection platform, and the artificial intelligence interpretation terminal and the bright field microscope are connected via a data cable.

4. A method for preparing the CD38 monoclonal antibody-MOFs composite biomaterial as claimed in claim 1, characterized in that: Includes steps: The CD38 monoclonal antibody coats the MOFs composite biomaterial.

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