Screening and application of CD38 molecular aptamer

Abstract

The invention discloses a CD38 molecular nucleic acid aptamer and application thereof, and belongs to the technical field of nucleic acid aptamers. The technical problem to be solved by the invention is how to realize the detection of the CD38 protein. Therefore, the invention provides a nucleic acid aptamer, the nucleic acid aptamer can be a nucleic acid aptamer A1, a nucleic acid aptamer A2, a nucleic acid aptamer A3, a nucleic acid aptamer A4 and / or a nucleic acid aptamer A5, and the nucleic acid aptamer A1 is a nucleic acid molecule containing a nucleotide sequence shown as SEQ ID NO: 1. According to the present invention, the nucleic acid aptamer is obtained through screening, wherein the binding epitope of the nucleic acid aptamer and the CD38 protein is different from the binding epitope of the existing CD38 monoclonal antibody Dartumumab and / or Istuximab; in addition, in-vitro binding tests and cell tests both show that the aptamer provided by the invention has high affinity with the CD38 protein. The nucleic acid aptamer provided by the invention can be used for effectively realizing the detection of the CD38 protein.

Description

Technical Field

[0001] The present application belongs to the field of nucleic acid technology, and specifically to a CD38 molecule nucleic acid aptamer and its application. Background Art

[0002] Multiple myeloma (MM) is a malignant disease characterized by abnormal proliferation of clonal plasma cells, and its incidence ranks second among hematological malignancies. Although the development of new therapies such as immunomodulatory drugs, proteasome inhibitors, and monoclonal antibodies in recent years has significantly improved the treatment of MM, the disease is highly heterogeneous and difficult to cure, and most patients will still relapse with a poor prognosis. CD38 is a transmembrane glycoprotein that plays a vital role in various physiological processes, including immune response regulation, calcium signaling, and nicotinamide adenine dinucleotide (NAD) metabolism. CD38 is significantly overexpressed in many types of malignant tumors, especially multiple myeloma, and is therefore an important biodiagnostic marker and therapeutic intervention target. Currently, CD38 monoclonal antibodies such as Daratumumab and Isatuximab can induce tumor cell apoptosis through multiple immune mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), and have shown good results in the treatment of MM.

[0003] However, CD38 monoclonal antibodies also pose new challenges to flow cytometry-based minimal residual disease (MRD) detection during treatment. Studies have shown that in patients treated with daratumumab, CD38 flow cytometry detection on the surface of plasma cells will be interfered with. This is because the CD38 site on the surface of plasma cells is competitively bound by the daratumumab drug, resulting in the conventional CD38 flow antibody binding site being masked and unable to bind to plasma cells, resulting in false negative results. This interference may last for 4 to 6 months, significantly increasing the risk of false negatives in MRD detection. In order to solve these problems, researchers previously considered using some alternative antibodies such as CD319, VS38 or CD38 multi-phenotype antibodies to reduce the impact of CD38 deficiency. However, traditional antibodies have a large molecular weight, and the production of antibodies usually requires complex biotechnology processes, including animal immunization and cell culture, which makes them relatively expensive. Therefore, a new recognition molecule is needed to solve this problem.

[0004] Aptamers are single-stranded DNA or RNA oligonucleotides that can specifically bind to specific target molecules. They usually contain 15-100 nucleotides and can fold into stable spatial structures, such as stem-loops, hairpins, and G-quadruplexes, and bind to target molecules through weak interactions such as van der Waals forces, hydrogen bonds, hydrophobic interactions, and electrostatic interactions. Compared with traditional antibodies, aptamers can be obtained by in vitro screening and chemical synthesis, and have the advantages of small molecular weight, simple synthesis, low cost, high stability, and strong specificity. In addition, the small size and flexibility of aptamers enable them to bind to smaller targets, or hidden domains that some antibodies cannot bind to, reducing the impact of steric hindrance and becoming an ideal substitute for traditional antibodies. The screening of aptamers relies on SELEX technology, which obtains high-affinity binding molecules in a short time through positive and negative screening processes, providing an important tool for targeted diagnosis and treatment of tumors. Summary of the invention

[0005] The technical problem to be solved by the present application is: how to detect CD38 protein. Furthermore, the technical problem to be solved by the present application is how to detect CD38 protein that may have been in contact with Daratumumab and / or Isatuximab, such as CD38 protein detection in multiple myeloma patients treated with Daratumumab and / or Isatuximab.

[0006] In order to solve the above technical problems, the present application provides a nucleic acid aptamer, which can be nucleic acid aptamer A1, nucleic acid aptamer A2, nucleic acid aptamer A3, nucleic acid aptamer A4 and / or nucleic acid aptamer A5.

[0007] The nucleic acid aptamer A1 may be any of the following:

[0008] A11) comprising a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1;

[0009] A12) a nucleic acid molecule comprising the nucleotide sequence of positions 24 to 57 of SEQ ID NO: 1;

[0010] A13) a nucleic acid molecule having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A11) or A12);

[0011] The nucleic acid aptamer A2 may be any of the following:

[0012] A21) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 2;

[0013] A22) A nucleic acid molecule comprising the nucleotide sequence of positions 4 to 55 of SEQ ID NO:2.

[0014] A23) comprising a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 6;

[0015] A24) a nucleic acid molecule comprising the nucleotide sequence of positions 4 to 50 of SEQ ID NO: 6;

[0016] A25) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A21), A22), A23) or A24);

[0017] The nucleic acid aptamer A3 may be any of the following:

[0018] A31) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 3;

[0019] A32) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A31);

[0020] The nucleic acid aptamer A4 may be any of the following:

[0021] A41) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 4;

[0022] A42) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A41);

[0023] The nucleic acid aptamer A5 may be any of the following:

[0024] A51) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 5;

[0025] A52) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A51).

[0026] The nucleic acid aptamer may be a nucleic acid aptamer that binds to CD38 protein.

[0027] The nucleic acid aptamer may be a nucleic acid aptamer that binds to CD38 protein but does not bind to EGF protein.

[0028] The nucleic acid aptamer may be a nucleic acid aptamer that binds to CD38 protein, the extracellular domain of CD38 protein, and / or cells or tissues that express or contain CD38 protein.

[0029] The reference sequence number of the CD38 protein in NCBI is NP_001766.2 (PRI 08-OCT-2024).

[0030] The reference sequence number of the EGF protein in NCBI is NP_001954.2 (PRI 29-OCT-2024).

[0031] The nucleic acid aptamer does not affect the binding of the monoclonal antibody Daratumumab and / or Isatuximab to the CD38 protein, that is, the binding epitope of the nucleic acid aptamer to the CD38 protein is different from that of Daratumumab and / or Isatuximab.

[0032] Furthermore, the nucleic acid aptamer A1 may be any of the following:

[0033] A11) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 1;

[0034] A12) is a nucleic acid molecule whose nucleotide sequence is positions 24 to 57 of SEQ ID NO: 1;

[0035] A13) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A11) or A12).

[0036] Furthermore, the nucleic acid aptamer A2 may be any of the following:

[0037] A21) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 2;

[0038] A22) is a nucleic acid molecule whose nucleotide sequence is positions 4 to 55 of SEQ ID NO:2.

[0039] A23) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 6;

[0040] A24) a nucleic acid molecule comprising the nucleotide sequence of positions 4 to 50 of SEQ ID NO: 6;

[0041] A25) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A21), A22), A23) or A24).

[0042] Furthermore, the nucleic acid aptamer A3 may be any of the following:

[0043] A31) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 3;

[0044] A32) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A31).

[0045] Furthermore, the nucleic acid aptamer A4 may be any of the following:

[0046] A41) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 4;

[0047] A42) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A41).

[0048] Furthermore, the nucleic acid aptamer A5 may be any of the following:

[0049] A51) is a nucleic acid molecule whose nucleotide sequence is SEQ ID NO: 5;

[0050] A52) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A51).

[0051] In some embodiments of the present application, the nucleic acid aptamer A1 may be the nucleic acid aptamer CD38jd4a and its truncated or optimized nucleic acid aptamers.

[0052] In some embodiments of the present application, A11) is a nucleic acid aptamer CD38jd4a. The nucleic acid aptamer CD38jd4a may specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:1.

[0053] In some embodiments of the present application, A12) is a nucleic acid aptamer CD38jd4c. The nucleic acid aptamer CD38jd4c may specifically be a single-stranded DNA having a nucleotide sequence of positions 24 to 57 of SEQ ID NO:1.

[0054] In some embodiments of the present application, the nucleic acid aptamer A2 may be the nucleic acid aptamer CD38jd1a and its truncated or optimized nucleic acid aptamer.

[0055] In some embodiments of the present application, A21) is a nucleic acid aptamer CD38jd1a. The nucleic acid aptamer CD38jd1a may specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:2.

[0056] In some embodiments of the present application, A22) is a nucleic acid aptamer CD38jd1b. The nucleic acid aptamer CD38jd1b is a truncated and optimized nucleic acid aptamer of CD38jd1a obtained by removing three deoxyribonucleotides AGG at the 5' end and one thymine deoxyribonucleotide (T) at the 3' end of the nucleic acid aptamer CD38jd1a (nucleotide sequence is SEQ ID NO: 2). The nucleic acid aptamer CD38jd1b can specifically be a single-stranded DNA having a nucleotide sequence of positions 4 to 55 of SEQ ID NO: 2.

[0057] In some embodiments of the present application, A23) is a nucleic acid aptamer CD38jd1c. The nucleic acid aptamer CD38jd1c is obtained by removing the adenine deoxyribonucleotide (A) at the 48th position of the nucleic acid aptamer CD38jd1b and mutating a thymine deoxyribonucleotide (T) at the 52nd position to a cytosine deoxyribonucleotide (C), thereby obtaining a truncated and optimized nucleic acid aptamer. The nucleic acid aptamer CD38jd1c can specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:6.

[0058] In some embodiments of the present application, A24) is a nucleic acid aptamer CD38jd1d. The nucleic acid aptamer CD38jd1d is a truncated and optimized nucleic acid aptamer obtained by removing the three deoxyribonucleotides CGC at the 5' end and the three thymine deoxyribonucleotides GCG at the 3' end of the nucleic acid aptamer CD38jd1c (nucleotide sequence is SEQ ID NO: 6). The nucleic acid aptamer CD38jd1d can specifically be a single-stranded DNA having a nucleotide sequence of positions 4 to 50 of SEQ ID NO: 6.

[0059] In some embodiments of the present application, A31) is a nucleic acid aptamer CD38jd2a. The nucleic acid aptamer CD38jd2a may specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:3.

[0060] In some embodiments of the present application, A41) is a nucleic acid aptamer CD38jd3a. The nucleic acid aptamer CD38jd3a may specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:4.

[0061] In some embodiments of the present application, A51) is a nucleic acid aptamer CD38jd11a. The nucleic acid aptamer CD38jd11a may specifically be a single-stranded DNA having a nucleotide sequence of SEQ ID NO:5.

[0062] The present application also provides nucleic acid aptamer derivatives, which are obtained by performing the following operations on each single nucleic acid aptamer in the nucleic acid aptamer: connecting fluorescein, anti-tumor drugs, radioactive elements, biological enzymes, biotin and / or nanomaterials to one end or the middle of the nucleic acid sequence of the nucleic acid aptamer to obtain a nucleic acid aptamer derivative having the same function as the single nucleic acid aptamer.

[0063] The same function may be specific binding to CD38 protein.

[0064] Furthermore, in the nucleic acid aptamer derivative, the fluorescein is selected from fluorescein isothiocyanate (FITC) or AF647.

[0065] The present application also provides a composition for detecting CD38 protein, wherein the composition contains the nucleic acid aptamer and / or the nucleic acid aptamer derivative.

[0066] The present application also provides the use of the nucleic acid aptamer, the nucleic acid aptamer derivative and / or the composition in any of the following:

[0067] (B1) Application in the preparation of products for screening or assisting in screening CD38 protein;

[0068] (B2) Application in screening or auxiliary screening of CD38 protein;

[0069] (B3) Use in the preparation of products for identifying or assisting in identifying CD38 protein;

[0070] (B4) application in identifying or assisting in identifying CD38 protein;

[0071] (B5) Use in preparing a product for screening or assisting in screening CD38-positive test samples;

[0072] (B6) Application in screening or auxiliary screening of CD38 positive test samples;

[0073] (B7) Application in the preparation of products for detecting MRD (minimal residual disease) of CD38 target-related diseases and / or assisting MRD detection of CD38 target-related diseases;

[0074] (B8) Use as a detection reagent in MRD detection of CD38 target-related diseases and / or assisting MRD detection of CD38 target-related diseases;

[0075] (B9) Use in the preparation of products for CD38 target related disease prognosis assessment and / or assisting CD38 target related disease prognosis assessment;

[0076] (B10) Use of a detection reagent in the prognosis assessment of CD38 target-related diseases and / or in assisting the prognosis assessment of CD38 target-related diseases;

[0077] (B11) Use in preparing a product for imaging CD38-positive cells to be tested and / or CD38-positive samples to be tested;

[0078] (B12) Use as an imaging agent in imaging CD38-positive cells to be tested and / or CD38-positive samples to be tested;

[0079] Application of (B13) in the preparation of a drug for treating multiple myeloma

[0080] (B14) Use as a drug in the treatment of multiple myeloma;

[0081] (B15) Application in the preparation of multiple myeloma (MM) related physiological and pathological research products.

[0082] Furthermore, the application includes a step of coupling the nucleic acid aptamer with a detection label before contacting with the sample to be tested.

[0083] Furthermore, the detection label is selected from fluorescein, radioactive elements, biological enzymes, nanomaterials and / or biotin.

[0084] Furthermore, the fluorescein is selected from fluorescein isothiocyanate (FITC) or AF647.

[0085] Furthermore, in the application, it is characterized in that: the sample to be tested can be blood, blood components, tissue, cells, tissue samples (processed tissues), cells and / or exfoliated matter.

[0086] The CD38-positive cells or the CD38-positive cells to be detected may be cells expressing CD38 protein.

[0087] Furthermore, the products include but are not limited to: detection reagents, imaging reagents, drugs, and research models.

[0088] Specifically, in the present application, the products described in (B1), (B3), B5), (B7) and B9) may be biosensors and / or diagnostic reagents. The working principle of the biosensor is that when the nucleic acid aptamer specifically binds to the target substance, its configuration will change accordingly. This characteristic is used to develop an electrochemical sensor based on the configuration change of the nucleic acid aptamer, namely an E-AB (Electrochemical aptamer-based) sensor. It has broad application prospects in the fields of medical diagnosis and so on. The working principle of the diagnostic reagent is that the nucleic acid aptamer and / or the nucleic acid aptamer derivative specifically binds to the CD38-positive sample to be tested.

[0089] Specifically, in the present application, the product described in (B11) can be an imaging agent. The working principle of (B11) and (B12) is that after the nucleic acid aptamer binds to the target substance (i.e., CD38), real-time monitoring and imaging of the target molecule are achieved through signal transduction mechanisms such as fluorescence and chemiluminescence.

[0090] Specifically, in the present application, the drug described in (B13) can be a drug delivery carrier or a drug. The applications described in (B13) and (B14) can be achieved through drug delivery. The working principle of drug delivery utilizes nucleic acid aptamers to bind to specific target cells or tissues (such as cells or tissues expressing CD38) for drug delivery. By combining drugs with nucleic acid aptamers, drugs can be accurately delivered to diseased cells or tissues, thereby improving the efficacy of drugs and reducing side effects.

[0091] The present application also provides a kit for detecting CD38, wherein the kit contains the nucleic acid aptamer, the nucleic acid aptamer derivative and / or the composition.

[0092] The present application also provides a method for detecting CD38 protein, comprising contacting the nucleic acid aptamer and / or the nucleic acid aptamer derivative with a sample to be tested, and determining whether CD38 protein exists and / or is expressed in the sample to be tested by detecting whether the nucleic acid aptamer and / or the nucleic acid aptamer derivative binds to the sample to be tested.

[0093] Furthermore, the method comprises the step of coupling the nucleic acid aptamer with a detection label before contacting with the sample to be tested.

[0094] Furthermore, the detection label is selected from fluorescein, radioactive elements, biological enzymes and / or biotin.

[0095] Furthermore, the fluorescein is selected from fluorescein isothiocyanate (FITC) or AF647.

[0096] Furthermore, the sample to be tested may be CD38 protein, blood, blood components, tissue, tissue sample (processed tissue) and / or exfoliated material that has been contacted with one or more CD38-specific binding agents.

[0097] Furthermore, the CD38 specific binding agent may be the monoclonal antibody Daratumumab and / or Isatuximab.

[0098] In the present application, the direct purpose of the use or method is non-disease diagnosis and / or treatment.

[0099] In the application or method, whether the nucleic acid aptamer and / or the nucleic acid aptamer derivative binds to the CD38 protein can be determined by the detection label.

[0100] In the application or method, whether the nucleic acid aptamer and / or the nucleic acid aptamer derivative binds to the sample to be tested can be determined by the detection label.

[0101] Furthermore, the detection marker is fluorescein, biological enzyme, biotin and / or radioactive element.

[0102] In some embodiments of the present application, the detection label is fluorescein.

[0103] If there is binding between the nucleic acid aptamer and the sample to be tested, it means that the sample to be tested contains CD38 protein.

[0104] In the present application, the sample to be tested may be CD38 protein or contain CD38 protein.

[0105] In the present application, the CD38 target-related disease may be a disease in which CD38 is positive or abnormally expressed, including but not limited to: multiple myeloma (MM), AIDS, autoimmune diseases (such as systemic lupus erythematosus), type II diabetes, osteoporosis, chronic B-lymphocytic leukemia (B-CLL), macroglobulinemia ( macroglobulinemia, primary systemic amyloidosis, mantle cell lymphoma, acute myeloid leukemia, acute lymphoid leukemia, NK cell leukemia.

[0106] The beneficial technical effects achieved by this application are as follows:

[0107] The present application screened and obtained nucleic acid aptamers whose binding epitopes to CD38 protein are different from those of the existing CD38 monoclonal antibodies Daratumumab and / or Isatuximab. In addition, in vitro binding tests and cell tests all show that the nucleic acid aptamers provided by the present application have a high affinity to CD38 protein. The nucleic acid provided by the present application can effectively detect CD38 protein. Furthermore, the nucleic acid aptamers provided by the present application can be used to detect CD38 protein that has been in contact with Daratumumab and / or Isatuximab. It is particularly suitable for the prognosis evaluation of multiple myeloma patients treated with Daratumumab and / or Isatuximab or the detection of minimal residual lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 Screening and characterization of nucleic acid aptamers targeting CD38 protein. a: Screening principle diagram. b: Binding of enriched library with target protein CD38. c: Binding of enriched library with counter-screening protein EGF.

[0109] Figure 2 Identification and characterization of nucleic acid aptamers. a: Prediction of secondary structure. b: SPR determination of nucleic acid aptamer affinity.

[0110] Figure 3 Characterization of the binding ability of nucleic acid aptamers. a: The binding ability of nucleic acid aptamers to the target protein CD38. b: The binding ability of nucleic acid aptamers to the anti-screening protein EGF.

[0111] Figure 4 The truncation and mutation of nucleic acid aptamers. a: Secondary structure of truncated mutant sequence. b: Characterization of binding ability of CD38jd1a and its truncated mutant sequence. c: Characterization of binding ability of CD38jd4a and its truncated mutant sequence.

[0112] Figure 5 Affinity characterization of truncated sequences obtained for SPR assays.

[0113] Figure 6 Characterization of the binding sites of monoclonal antibodies and nucleic acid aptamers. a: Schematic diagram of protein fixation. b: Flow cytometry characterization of the binding sites of nucleic acid aptamer CD38jd4a and Daratumumab. c: Flow cytometry characterization of the binding sites of nucleic acid aptamer CD38jd4a and Isatuximab.

[0114] Figure 7Figure 2 is the characterization of nucleic acid aptamers on cells. a: Flow cytometry characterization of the binding ability of nucleic acid aptamers to K562, CCRF-CEM, RPMI8226, and Ramos. bd is the binding of AF647-labeled CD38jd4a (b) and FITC-labeled Daratumumab (c) or Isatuximab (d) to Ramos cells treated with siCD38 or siCtrRNA. e: Bar graph, statistical analysis of 3 separate experiments. f: Co-staining of nucleic acid aptamer CD38jd4a and Daratumumab in CD38+ cell line Ramos. g: Co-staining of nucleic acid aptamer CD38jd4a and Isatuximab in CD38+ cell line Ramos.

[0115] Figure 8 Detection of nucleic acid aptamers in clinical samples. a: Nucleic acid aptamer CD38jd4a detects positive cell populations in a mixture of 90% K562 and 10% Ramos. bc: Co-staining of nucleic acid aptamer CD38jd4a and Daratumumab (b) / Isatuximab (c) in clinical blood samples. DETAILED DESCRIPTION

[0116] In this application, nucleic acid aptamers were screened for CD38 protein, and high-affinity nucleic acid aptamers CD38jd4a and CD38jd1a that specifically bind to CD38 were obtained. Functional verification found that the binding site of the aptamer CD38jd4a to CD38 is different from that of the monoclonal antibodies Daratumumab and / or Isatuximab, so the detection based on CD38jd4a will not be interfered by the CD38 monoclonal antibody drugs Daratumumab and / or Isatuximab, avoiding the risk of MRD missed detection. As a verification, we used flow cytometry to prove that CD38jd4a can be used for positive cell detection in the blood at the same time as CD38 monoclonal antibodies. These results show that CD38jd4a can overcome the shortcomings of current molecular tools, improve existing diagnostic methods, and provide a new solution for the accurate diagnosis and treatment of multiple myeloma.

[0117] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0118] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0119] CO2 cell culture incubator (Thermo Fisher Scientific); PCR instrument (Bio-Rad Bio-Medical Products Co., Ltd.); flow cytometer (Thermo Fisher Scientific); desktop low-speed centrifuge (Hunan Xiangyi Centrifuge Instrument Co., Ltd.); gel electrophoresis instrument (Bio-Rad Bio-Medical Products Co., Ltd.); vacuum centrifuge concentrator (Beijing Zhaoshengxing Instrument Equipment Co., Ltd.); cell counter (Thermo Fisher Scientific); fluorescence inverted microscope (Olympus Corporation).

[0120] CD38 (Sino Biological Technology Co., Ltd. 10818-H02H), EGF protein (Sino Biological Technology Co., Ltd. 10605-H01H); DPBS buffer (Langeco Technology Co., Ltd. BL310A); D-glucose (Sinopharm Chemical Reagent Co., Ltd. 63005518); magnesium chloride (Sinopharm Chemical Reagent Co., Ltd. 10012818); RPMI8226 (CL-0564), CCRF-CEM, Ramos, K562 (from Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences); NHS sugar ball: Cytiva product, catalog number 17071601; HsDNA: herring sperm DNA (Beijing Solebold Technology Co., Ltd., catalog number D8050).

[0121] In the following examples, the formula of Washing buffer is: DPBS+5mM MgCl2+4.5g / L D-glucose; in the following examples, the formula of Binding Buffer is: Washing buffer+1mg / mL BSA+0.1mg / mL HsDNA.

[0122] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0123] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test. P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) indicated a very significant difference.

[0124] Example 1. Screening and truncation optimization of nucleic acid aptamers

[0125] 1.1. Screening of nucleic acid aptamers targeting CD38 protein

[0126] In order to obtain aptamers that specifically target CD38 protein, aptamer screening was performed using purified human recombinant CD38 extracellular domain protein as the target. The screening process is as follows: Figure 1As shown in a. The brief description is as follows: First, the target protein (human recombinant CD38 extracellular domain protein, the reference sequence number in NCBI is NP_001766.2 (PRI 08-OCT-2024); it is a product of Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 10818-H02H) was coupled to the microbeads to facilitate separation from the enriched library. In order to enhance the specificity of the aptamer to CD38, we started from the third round and performed negative screening with EGF (the reference sequence number in NCBI is NP_001954.2 (PRI 29-OCT-2024); it is a product of Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 10605-H01H) as a negative screening protein before screening to remove some sequences that bind to EGF or microbeads. During the screening process, flow cytometry was used to monitor the binding of the enriched library to CD38 by detecting changes in fluorescence intensity. The enriched library in the third round had a strong binding to CD38 ( Figure 1 b), and the enriched library did not bind to the negative screening protein EGF ( Figure 1 (c). Since the fluorescence intensity of the sixth round of enriched library binding to CD38 did not increase much, it means that the enrichment of ssDNA sequences binding to CD38 has reached saturation, so we terminated the screening. Next, we performed library construction and high-throughput sequencing for each round of screening libraries according to the previous method. We analyzed the sequencing data of the sixth round of enriched libraries and ranked the top 100 sequences by sequence abundance.

[0127] The specific screening process of nucleic acid aptamers is as follows:

[0128] Screening process: Dissolve the 2OD initial library in 100μL DPBS, denature at 95℃ for 5min, cool on ice for 5min, and at room temperature for 15min. Incubate with the anti-screening protein EGF at room temperature, centrifuge and take the supernatant, then add the target protein CD38 for incubation, wash and leave the sequence bound to the target protein for PCR amplification, and then prepare the single chain for the next round of amplification.

[0129] Multiple rounds of screening: The conditions for multiple rounds of screening are shown in Table 1.

[0130] Table 1 Changes in screening conditions

[0131]

[0132] Process monitoring: The enriched library and protein-beads (CD38 protein fixed by NHS sugar beads) after each round of screening were shaken and incubated at room temperature for 30 minutes. The final concentration of the library was 200nM. The counter-screening protein EGF was used as a control. After the incubation, the sample was washed and the supernatant was removed. Washing buffer was added to resuspend, and the change in fluorescence intensity was detected by flow cytometry. Finally, the flow cytometry software FlowJo was used to process and analyze the test results.

[0133] High-throughput sequencing: The ssDNA obtained from the 1st to 6th rounds of screening was used to build a library and sent to Shanghai Sangon Biotechnology Co., Ltd. for high-throughput sequencing. The sequencing results were sorted from high to low by repetition rate, and the abundance of the top 100 ssDNA sequences was listed.

[0134] 1.2 Identification and characterization of nucleic acid aptamers

[0135] In order to identify the aptamers that bind to CD38 in the enriched library, we carefully analyzed the top 100 aptamers and finally selected 5 aptamers CD38jd1a, CD38jd2a, CD38jd3a, CD38jd4a, and CD38jd11a for preliminary sequence truncation and synthesis. The structures of the 5 aptamers were further predicted using the mfold website, and one of the possible secondary structures is as follows: Figure 2 As shown in a.

[0136] 1.2.1. Detection of affinity of nucleic acid aptamers by flow cytometry

[0137] The fluorescent molecule FITC (fluorescein isothiocyanate) was labeled at the 5' end of the five nucleic acid aptamers. Next, the binding of the five nucleic acid aptamers to the target protein CD38 and EGF protein was detected by flow cytometry. The specific detection steps were to dilute the five FITC-labeled nucleic acid aptamer sequences to 10 μM with DPBS and then put them on ice for standby. The nucleic acid aptamers CD38jd1a, CD38jd2a, CD38jd3a, CD38jd4a, CD38jd11a or the control aptamer ctrl were mixed with CD38 protein or EGF protein in Binding Buffer, and the final concentration of the nucleic acid aptamer was 200nM. The cells were incubated at room temperature in the dark for 30 minutes, and then washed twice with 200μL of Washing buffer, and the supernatant was removed. Finally, 150μL of Washing buffer was added to resuspend, and the fluorescence intensity changes of the nucleic acid aptamers were detected by flow cytometry. Finally, the flow software FlowJo was used to process and analyze the detection results.

[0138] The results showed that all five aptamers showed significant binding to CD38 protein ( Figure 3a, but not bound to EGF protein ( Figure 3 (b)

[0139] 1.2.2 Surface Plasmon Resonance (SPR) Detection of Aptamer Affinity

[0140] The equilibrium dissociation constants (Kd) of the five aptamers and CD38 were further detected by surface plasmon resonance (SPR) to evaluate the affinity of the five aptamers for binding to CD38. The specific detection steps are as follows: protein coupling is first performed: equal volumes of 0.1M NHS and 0.4M EDC are pre-mixed and added to a 96-well microplate to activate the carboxyl groups on the CM5 chip, with a flow rate of 10μL / min. CD38 protein is diluted to 20μg / mL with 10mM sodium acetate, pH=4.5, and added to a 96-well plate for coupling. The coupling time is set to 900s, the flow rate is 10μL / min, and then ethanolamine is used to block the chip at 10μL / min. Then the affinity of the nucleic acid aptamer to the CD38 protein was detected: the nucleic acid aptamer was gradiently diluted with SPR buffer (DPBS+5mM MgCl2) to concentrations of 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, and 1.5625nM, respectively. The samples were injected sequentially, and the binding of the nucleic acid aptamer to the CD38 protein was detected by surface plasmon resonance. Finally, data analysis and drawing were performed.

[0141] The results are as follows Figure 2 As shown in middle b, all five aptamers showed high affinity, and Kd reached the nanomolar (nM) level. Among them, CD38jd1a had the highest binding affinity to CD38, and its equilibrium dissociation constant (Kd) was only 4.8±0.2nM, which was consistent with the high affinity results detected by flow cytometry. Unexpectedly, although CD38jd4a was the weakest binding among the five sequences in the flow cytometry results, the Kd detected by SPR was 5.4n±0.6M, showing a very high affinity. The Kd of aptamers CD38jd2a, CD38jd3a and CD38jd11a were 12.2±0.3nM, 11.9±0.2nM and 37.2±0.4nM, respectively. Since CD38jd1a and CD38jd4a had the best affinity, they were further studied.

[0142] 1.3. Optimization of nucleic acid aptamer truncation

[0143] The more bases a nucleic acid aptamer has, the higher the synthesis cost will be, which further limits the application of nucleic acid aptamers. From the perspective of practical application, truncation and optimization of nucleic acid aptamers can improve their application potential. In addition, longer aptamers may contain some non-essential nucleotides, which are not helpful for the binding of the aptamer to the target, but may reduce the affinity. Therefore, we then tried to truncate and mutate the aptamers CD38jd1a and CD38jd4a to improve affinity. The truncation and mutation methods are as follows: Figure 4 As shown in a. Based on the secondary structure, we designed a series of truncated and mutant aptamers (Table 2).

[0144] The details of the truncation optimization are as follows:

[0145] (1)CD38jd1a

[0146] ① The three deoxyribonucleotides AGG at the 5' end and one thymine deoxyribonucleotide (T) at the 3' end of the nucleic acid aptamer CD38jd1a (nucleotide sequence is SEQ ID NO: 2) were removed to obtain a truncated and optimized nucleic acid aptamer of CD38jd1a, named CD38jd1b. The nucleotide sequence of CD38jd1b is positions 4 to 55 of SEQ ID NO: 2.

[0147] ② The adenine deoxyribonucleotide (A) at position 48 of the nucleic acid aptamer CD38jd1b was removed, and a thymine deoxyribonucleotide (T) at position 52 was mutated to a cytosine deoxyribonucleotide (C) to obtain a truncated and optimized nucleic acid aptamer CD38jd1c. The nucleotide sequence of the nucleic acid aptamer CD38jd1c is SEQ ID NO:6.

[0148] ② Remove the three deoxyribonucleotides CGC at the 5' end and the three thymine deoxyribonucleotides GCG at the 3' end of the nucleic acid aptamer CD38jd1c (nucleotide sequence is SEQ ID NO: 6) to obtain a truncated and optimized nucleic acid aptamer of CD38jd1a, named CD38jd1d. The nucleotide sequence of CD38jd1d is positions 4 to 50 of SEQ ID NO: 6.

[0149] (2)CD38jd4a

[0150] ① Remove the deoxyribonucleotides at positions 45 to 60 of the nucleic acid aptamer CD38jd4a (nucleotide sequence is SEQ ID NO: 1) to obtain a truncated and optimized nucleic acid aptamer of CD38jd4a, named CD38jd4b. The nucleotide sequence of CD38jd1b is positions 1 to 44 of SEQ ID NO: 1.

[0151] ② Remove the deoxyribonucleotides at positions 1 to 23 and 58 to 60 of the nucleic acid aptamer CD38jd4a (nucleotide sequence is SEQ ID NO: 1) to obtain a truncated and optimized nucleic acid aptamer of CD38jd4a, named CD38jd4c. The nucleotide sequence of CD38jd1c is positions 24 to 57 of SEQ ID NO: 1.

[0152] The binding ability of all sequences to the target protein CD38 was then tested based on flow cytometry. Figure 4 As shown in Figures b-c, compared with CD38jd1a, CD38jd1b binds weaker, CD38jd1c and CD38jd1d hardly bind; and compared with CD38jd4a, CD38jd4b does not bind, and CD38jd4c binds weaker. We further directly detected the affinity of CD38jd1b, CD38jd1c, CD38jd1d, and CD38jd4c for binding to CD38 by SPR ( Figure 5 The affinity of CD38jd1b was 21.3±3.8nM, the affinity of CD38jd1c was 780±28.6nM, the affinity of CD38jd1d was 624±40.5nM, and the affinity of CD38jd4c was 505±57.1nM, which were all decreased compared with the original aptamer.

[0153] Table 2 DNA sequences used in the experiment

[0154]

[0155] Example 2: Verification of the binding site between nucleic acid aptamer and CD38

[0156] Currently, CD38 monoclonal antibodies such as Daratumumab and Isatuximab have shown good results in the treatment of MM. However, they also cause new interference in the detection of clinical CD38 because they will mask the sites where antibody-based detection kits bind to CD38.

[0157] The purpose is to investigate whether the binding sites of aptamers CD38jd4a and CD38jd1a and monoclonal antibodies Daratumumab or Isatuximab that bind to CD38 molecules are the same. Figure 6 As shown in a, the CD38 protein is fixed with the antibody Daratumumab or Isatuximab, and then the aptamers CD38jd4a and CD38jd1a are detected by flow cytometry to see whether they can still bind to the CD38 protein. The specific steps are as follows:

[0158] 1) Conjugate Daratumumab or Isatuximab with sugar spheres: Wash 50μL NHS sugar spheres with 100μL 1mM ice HCl three times, and centrifuge to remove the supernatant. Add 100μL DPBS to wash three times, and centrifuge to remove the supernatant. Add 50μg Daratumumab or Isatuximab to the sugar spheres and incubate at room temperature for 60 minutes. After incubation, add 100μL DPBS to wash twice, and centrifuge to remove the supernatant. Then add 100μL Buffer A to wash three times, Buffer B to wash three times, and Buffer A to wash three times. Let it stand at room temperature again for 30 minutes, then wash three times with Buffer B, Buffer A to wash three times, and Buffer B to wash three times, and centrifuge to remove the supernatant. Add 100μL BD sheath fluid (BD FACSFlow TM Wash twice with BD Sheath Fluid (Catalog No.: 342003), centrifuge and remove the supernatant. Finally, add 50 μL BD Sheath Fluid and store at 4°C.

[0159] Buffer A (50 mL): 1.88 g glycine (MW = 75.067, final concentration 0.5 M) + 1.46 g sodium chloride (MW = 58.44, final concentration 0.5 M) + 50 mL ddH2O.

[0160] Buffer B: 0.68 g sodium acetate trihydrate (MW=82.03, final concentration 0.1 M) + 1.46 g sodium chloride (MW=58.44, final concentration 0.5 M) + 50 mL ddH2O.

[0161] 2) Capture of CD38 protein: The Daratumumab or Isatuximab coupled to the sugar spheres was washed once with Washing buffer, and then CD38 protein was added to Binding buffer and incubated at room temperature for 30 min. After the incubation, the mixture was washed once with Washing buffer, and finally, the mixture was resuspended with Washing buffer and stored at 4°C.

[0162] 3) Binding and detection of aptamers CD38jd4a and CD38jd1a:

[0163] Aptamers CD38jd4a, CD38jd1a and control aptamer Ctrl were labeled with fluorescent molecule FITC (fluorescein isothiocyanate) at the 5' end for the experiment. According to the different antibody types, the experiment was divided into two groups: Daratumumab group and Isatuximab group.

[0164] Daratumumab group: The above Daratumumab-fixed CD38 protein was washed once with Washing buffer, and then aptamer CD38jd4a or CD38jd1a was added to the Binding buffer and incubated at room temperature for 30 min. After the incubation, it was washed twice with Washing buffer, and finally resuspended with Washing buffer for flow cytometry detection.

[0165] Isatuximab group: The CD38 protein fixed with Isatuximab was washed once with Washing buffer, and then aptamer CD38jd4a or CD38jd1a was added and incubated in Binding buffer at room temperature for 30 min. After the incubation, the cells were washed twice with Washing buffer, and finally resuspended with Washing buffer for flow cytometry detection.

[0166] The experiment set up a Ctrl group, and the detection reference was the Daratumumab group or the Isatuximab group, the difference being that the Ctrl group used the control aptamer Ctrl.

[0167] The results are as follows Figure 6 As shown in bc, after CD38 protein was fixed with monoclonal antibody (i.e., CD38 protein bound to Daratumumab or Isatuximab), CD38jd4a and CD38jd1a still bound to CD38, indicating that the epitopes bound to CD38 by CD38jd4a and CD38jd1a are different from those of monoclonal antibody Daratumumab or Isatuximab.

[0168] The above results show that the detection of CD38 by CD38jd4a and CD38jd1a will not be interfered by CD38 monoclonal antibody drugs, and the risk of MRD missed detection can be avoided. This result shows that the aptamer we screened will be more suitable for the detection of residual lesions in MM patients after treatment with Daratumumab or Isatuximab. At the same time, CD38jd4a and CD38jd1a have high affinity for CD38 and have very high application potential in the detection of residual microlesions.

[0169] Example 3: Binding of nucleic acid aptamers to cell surface CD38

[0170] 3.1. Detection of the binding of nucleic acid aptamers to cell surface CD38

[0171] Since the purified human recombinant protein may have certain differences from the natural structure, we further tested whether the aptamer can bind to the naturally existing CD38 on the cell. According to previous research reports and searches on the www.proteinatlas.org website, CD38 is highly expressed in lymphoma, myeloma, leukemia and other related cancer cells. Three positive cell lines, CCRF-CEM, RPMI8226 and Ramos, and one negative cell line, K562, were selected and the binding of nucleic acid aptamers CD38jd1a and CD38jd4a to the four cell lines was verified based on flow cytometry.

[0172] The aptamer detection group was divided into CD38jd4a group, CD38jd1a group and Ctrl group. Taking CD38jd4a group as an example, the specific steps were as follows: (1) Cell preparation: 100 μL CCRF-CEM, RPMI8226, Ramos or K562 cells were taken in 96-well plates respectively; (2) The aptamer CD38jd4a labeled with the fluorescent molecule FITC (fluorescein isothiocyanate) at the 5' end was incubated with CD38+ cell lines CCRF-CEM, RPMI8226, Ramos and CD38-negative cell line K562 at 4°C for 30 minutes, and the final concentration of CD38jd4a in the system was 200 nM. After incubation, the cells were washed twice with Washing Buffer, and then suspended with 200 μL Washing Buffer. Finally, the cells were loaded and tested for binding by flow cytometry.

[0173] The detection of CD38jd1a and Ctrl groups referred to CD38jd4a group, the difference being that different nucleic acid aptamers were used: CD38jd1a group used CD38jd1a, and Ctrl group used control aptamer.

[0174] The results are as follows Figure 7 As shown in a, the two aptamers showed obvious binding to the three positive cell lines but not to the negative cell K562, which is consistent with the binding of two verified antibodies Daratumumab and Isatuximab.

[0175] To confirm that CD38jd4a indeed binds to CD38 on cells, co-staining and siRNA knockdown experiments were performed next.

[0176] 3.2 Co-staining experimental verification

[0177] Co-staining of nucleic acid aptamer and Daratumumab or Isatuximab in CD38+ cell line Ramos: Ramos cells were centrifuged and supernatant was removed, and the cells were washed once with Washing buffer and supernatant was removed. Daratumumab or Isatuximab (FITC-labeled secondary antibody) and nucleic acid aptamer labeled with AF647 were added respectively, and the final concentration of antibody and nucleic acid aptamer was kept at 200nM. The cells were incubated in Binding Buffer at 4℃ for 30min, washed with Washing buffer, and resuspended for processing.

[0178] Aptamer, Daratumumab or Isatuximab single staining group and blank group (Blank) were set as controls. The detection steps were referred to the co-staining group, with the only difference being that in the single staining group, only aptamer, Daratumumab or Isatuximab were added to the system at a final concentration of 200 nM, and in the blank group, blank cells were used for flow cytometry voltage adjustment.

[0179] The results are as follows Figure 7 In fg, CD38jd4a and Daratumumab or Isatuximab bind to CD38 protein on the cell surface at the same time. These results show that the aptamer CD38jd4a can specifically detect the expression of CD38 molecules on the cell membrane surface and is not interfered by CD38 antibodies Daratumumab or Isatuximab.

[0180] 3.3. Validation of CD38 knockdown in Ramos cells using siRNA

[0181] (1) CD38 knockdown in Ramos cells

[0182] Add 2 mL of culture medium without dual antibodies (GIBCO RPMI 1640 culture medium, catalog number: C11875500BT) to a six-well plate, add 200,000 CD38+ cell line Ramos, add the mixed transfection reagent and siRNA, transfer the cells to complete culture medium 6 hours after transfection and culture for 48 hours, and then detect the transfection efficiency by flow cytometry.

[0183] The formula of transfection reagent is 9 μL RNAiMAX Reagent (Thermo Fisher Scientific, 13778100) was used with 150 μL Medium (Thermo Fisher Scientific 31985062), 30 pmol siRNA was diluted with 150 μL Dilute with Medium, take 150μL Diluted siRNA and Diluted RNAiMAX Reagent was mixed evenly, and the content of siRNA in the transfection reagent was 25 pmol.

[0184] The sequence of (SiCD38) is as follows:

[0185] antisense:5'-AAGUGUUGAAUUCACCACACCTT-3';

[0186] sense: 5'-GGUGUGGUGAAUUCAACACUUTT-3'.

[0187] The sequence of (SiCtrl) is as follows:

[0188] sense: 5'-UUCUCCGAACGUGUCACGUTT-3';

[0189] antisense: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0190] SiCD38-transfected Ramos cells or SiCtrl-transfected Ramos cells were stained with antibodies and CD38jd4a according to the steps in 3.2, and analyzed by flow cytometry.

[0191] The results are as follows Figure 7 In the figure, compared with Ramos cells transfected with siCtrl, the fluorescence of CD38 antibody (Daratumumab or Isatuximab) and CD38jd4a in Ramos cells transfected with siCD38 was significantly weakened, indicating that the target of aptamer CD38jd4a on Ramos cells is indeed CD38.

[0192] 3.4. Evaluation of the detection ability of CD38jd4a for positive cells

[0193] The CD38+ cell line Ramos was centrifuged to remove the supernatant, washed with DPBS, stained with 10μM CFDA-SE staining solution, and washed twice with DPBS. Then, the CD38- cell line K562 was counted separately. The specific operation was as follows: 90% K562 and 10% Ramos were mixed evenly, CD38jd4a was added, and incubated at 4°C for 30 minutes. After washing twice, it was resuspended in washing buffer for flow cytometry detection.

[0194] The binding of CD38 antibody drugs to cell surface CD38 will hinder the application of flow cytometry to detect plasma cell diseases. Therefore, it is necessary to screen nucleic acid aptamers that bind to different sites of CD38 with monoclonal antibody drugs. First, the detection ability of CD38jd4a on positive cells was evaluated. The positive cells and negative cells were distinguished by staining Ramos. The aptamer CD38jd4a was added for incubation. It can be seen that even if there are only 10% positive cells, CD38jd4a can still identify the positive cell population from the cell mixture ( Figure 8 (a)

[0195] Example 4: Detection of CD38 by nucleic acid aptamers in clinical samples

[0196] To further evaluate the detection ability and potential application value of aptamer CD38jd4a in clinical samples, we used flow cytometry to verify its binding to clinical blood samples.

[0197] According to a peripheral blood sample obtained from Zhejiang Provincial Cancer Hospital (derived from a patient with a personal history of malignant lymphoma), AF647-labeled nucleic acid aptamer and Daratumumab or Isatuximab (FITC-labeled secondary antibody) were added with reference to the co-staining experimental operation steps in Example 3.2, with a final concentration of 200 nM, and incubated at room temperature in the dark for 30 min. After the incubation, red blood cell lysis buffer was added, lysed at 4°C in the dark for 10 min, and then centrifuged at 4°C 500g for 5 min. After removing the supernatant, DPBS was added to wash twice, and the supernatant was removed by centrifugation at 4°C 500g for 3 min. Finally, DPBS was added to resuspend and the sample was loaded on the flow cytometer for flow cytometry.

[0198] The results showed that: in CD38jd4a (labeled AF647) and Daratumumab ( Figure 8 Isatuximab( Figure 8 In the case of co-staining with (labeled FITC) in (c), the aptamer can still accurately recognize the target CD38. The above results show that CD38jd4a, as a new type of molecular probe, has the potential for application in clinical diagnosis and treatment, such as for the detection and monitoring of CD38 target-related diseases.

[0199] Conclusion: In this paper, nucleic acid aptamers were screened for CD38 protein by SELEX technology. The screened nucleic acid aptamers have high affinity and specifically bind to the target protein CD38. By fixing CD38 with Daratumumab / Isatuximab at the protein level and co-staining Daratumumab / Isatuximab and CD38jd4a at the cell level, it was proved that CD38jd4a and Daratumumab / Isatuximab bind to different epitopes of CD38 protein. CD38jd4a can detect positive cells in a mixture of negative cells and positive cells, and the nucleic acid aptamer can detect CD38 in clinical blood samples without interference from Daratumumab / Isatuximab, thereby reducing the risk of MRD missed detection and facilitating CD38 monoclonal antibody drug treatment.

[0200] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A nucleic acid aptamer, characterized in that: The nucleic acid aptamer is nucleic acid aptamer A1, nucleic acid aptamer A2, nucleic acid aptamer A3, nucleic acid aptamer A4 and / or nucleic acid aptamer A5, The nucleic acid aptamer A1 is any one of the following: A11) comprising a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1; A12) a nucleic acid molecule comprising the nucleotide sequence of positions 24 to 57 of SEQ ID NO: 1; A13) a nucleic acid molecule having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A11) or A12); The nucleic acid aptamer A2 is any one of the following: A21) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 2; A22) A nucleic acid molecule comprising the nucleotide sequence of positions 4 to 55 of SEQ ID NO:

2. A23) comprising a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 6; A24) a nucleic acid molecule comprising the nucleotide sequence of positions 4 to 50 of SEQ ID NO: 6; A25) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A21), A22), A23) or A24); The nucleic acid aptamer A3 is any one of the following: A31) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 3; A32) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A31); The nucleic acid aptamer A4 is any one of the following: A41) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 4; A42) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A41); The nucleic acid aptamer A5 is any one of the following: A51) comprises a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 5; A52) is a nucleic acid molecule that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence of the nucleic acid molecule shown in A51).

2. A nucleic acid aptamer derivative, characterized in that: The nucleic acid aptamer derivative is obtained by performing the following operation on each single nucleic acid aptamer in the nucleic acid aptamer described in claim 1: connecting fluorescein, anti-tumor drugs, radioactive elements, biological enzymes, biotin and / or nanomaterials to one end or the middle of the nucleic acid sequence of the nucleic acid aptamer to obtain a nucleic acid aptamer derivative having the same function as the single nucleic acid aptamer.

3. The nucleic acid aptamer derivative according to claim 2, characterized in that: The fluorescein is selected from fluorescein isothiocyanate or AF647.

4. A composition for detecting CD38 protein, characterized in that: The composition contains the nucleic acid aptamer according to claim 1 and / or the nucleic acid aptamer derivative according to claim 2 or 3.

5. Use of the nucleic acid aptamer according to claim 1, the nucleic acid aptamer derivative according to claim 2 or 3, and / or the composition according to claim 4 in any of the following: (B1) Application in the preparation of products for screening or assisting in screening CD38 protein; (B2) Application in screening or auxiliary screening of CD38 protein; (B3) Use in the preparation of products for identifying or assisting in identifying CD38 protein; (B4) application in identifying or assisting in identifying CD38 protein; (B5) Use in preparing a product for screening or assisting in screening CD38-positive test samples; (B6) Application in screening or auxiliary screening of CD38 positive test samples; (B7) Application in the preparation of products for MRD detection of CD38 target-related diseases and / or assisting MRD detection of CD38 target-related diseases; (B8) Use as a detection reagent in MRD detection of CD38 target-related diseases and / or assisting MRD detection of CD38 target-related diseases; (B9) Use in the preparation of products for CD38 target related disease prognosis assessment and / or assisting CD38 target related disease prognosis assessment; (B10) Use of a detection reagent in the prognosis assessment of CD38 target-related diseases and / or in assisting the prognosis assessment of CD38 target-related diseases; (B11) Use in preparing a product for imaging CD38-positive cells to be tested and / or CD38-positive samples to be tested; (B12) Use as an imaging agent in imaging CD38-positive cells to be tested and / or CD38-positive samples to be tested; Application of (B13) in the preparation of a drug for treating multiple myeloma (B14) Use as a drug in the treatment of multiple myeloma; (B15) Application in the preparation of multiple myeloma-related physiological and pathological research products.

6. The use according to claim 5, characterized in that: Before contacting with the sample to be tested, the method comprises the step of coupling the nucleic acid aptamer with a detection label; Further, the detection label is selected from fluorescein, radioactive elements, biological enzymes, nanomaterials and / or biotin; Furthermore, the fluorescein is selected from fluorescein isothiocyanate or AF647.

7. The use according to claim 5 or 6, characterized in that: The sample to be tested is blood, blood components, tissue, tissue sample, cell and / or exfoliated material.

8. A kit for detecting CD38, characterized in that: The kit contains the nucleic acid aptamer according to claim 1, the nucleic acid aptamer derivative according to claim 2 or 3, and / or the composition according to claim 4.

9. A method for detecting CD38 protein, comprising contacting the nucleic acid aptamer according to claim 1 and / or the nucleic acid aptamer derivative according to claim 2 or 3 with a sample to be tested, and determining whether CD38 protein exists and / or is expressed in the sample to be tested by detecting whether the nucleic acid aptamer and / or the nucleic acid aptamer derivative binds to the sample to be tested; Furthermore, before contacting with the sample to be tested, the step of coupling the nucleic acid aptamer with a detection label is included; Furthermore, the detection marker is selected from fluorescein, radioactive elements, biological enzymes and / or biotin, and nanomaterials; Furthermore, the fluorescein is selected from fluorescein isothiocyanate or AF647.

10. The method according to claim 9, characterized in that: The sample to be tested is a CD38 protein, blood, blood component, tissue, tissue sample, cell and / or exfoliated material that has been exposed to one or more CD38-specific binding agents; Furthermore, the CD38 specific binding agent is the monoclonal antibody Daratumumab and / or Isatuximab.

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