Anti-human CD38 antibody and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an anti-human CD38 antibody and application thereof. The anti-human CD38 antibody disclosed by the invention can be specifically combined with human CD38 protein, and the combination is stable. The antibody is coupled with an adsorption column to pertinently adsorb B cells in blood, so that the level of pathogenic factors can be quickly reduced, and abnormally activated or pathogenic B cells in circulation and pathogenic factors such as antibodies, cell factors and the like generated by the abnormally activated or pathogenic B cells are quickly reduced, thereby quickly relieving symptoms. The B cells can be cleared relatively specifically, and the influence on other cells is reduced. The traditional Chinese medicine composition has the advantages that time is bought for other treatments, in the acute attack or serious stage of diseases, illness state stabilization is facilitated, favorable conditions are created for subsequent immunoregulation treatment or other targeted treatment, immune imbalance can be improved, the imbalance state of an immune system can be adjusted, and immune homeostasis can be recovered. When necessary, adsorption can be repeatedly carried out to maintain the treatment effect, and the safety is high.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antibody against human CD38 and its applications. Background Technology

[0002] Autoimmune diseases refer to the immune system mistakenly attacking and destroying its own tissues and organs, leading to an autoimmune response. B cells are an important cell type in the immune system; they help the body fight pathogens and foreign substances by producing antibodies. However, in autoimmune diseases, B cells mistakenly attack the body's own tissues and organs. B cells primarily affect autoimmune diseases through the following mechanisms: a. Producing autoantibodies: B cells can produce antibodies against their own tissues and organs, which can trigger an autoimmune response. For example, in type 1 diabetes, B cells produce islet cell antibodies, attacking and destroying islet cells, leading to abnormally high blood sugar levels. Systemic lupus erythematosus (SLE) patients have various antibodies against their own cell nuclear components, such as anti-double-stranded DNA antibodies and anti-Sm antibodies. Rheumatoid arthritis produces autoantibodies such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). b. Activating other immune cells: B cells can activate other immune cells, such as macrophages and T cells, thereby promoting autoimmune responses. For example, in systemic lupus erythematosus, B cells can activate T cells, causing the immune system to attack multiple tissues and organs in the body. c. Promoting inflammatory responses: B cells can secrete inflammatory factors, promoting inflammatory responses and exacerbating autoimmune responses. For example, in arthritis, B cells can produce pro-inflammatory factors, causing arthritis symptoms. d. As antigen-presenting cells: For example, in multiple sclerosis, B cells present autoantigens to activate T cells, leading to damage to the myelin sheath of nerve cells. In myasthenia gravis, B cells present acetylcholine receptor-associated antigens, triggering an autoimmune response.

[0003] B cells, as a major component of the human immune system, primarily participate in immune responses by secreting antibodies. They also interact with T cells, releasing cytokines to participate in the occurrence and development of autoimmune diseases. It has been confirmed that B cells and their effectors (such as antibodies and cytokines) are involved in the occurrence and development of multiple autoimmune diseases. Therefore, eliminating B cells and suppressing abnormal immune responses has become a novel strategy for treating autoimmune diseases, possessing clinical significance and necessity.

[0004] Currently, B cell elimination therapy (BCDT) mainly includes: a. using antibody drugs to target and bind to B cell-specific antigens (the main B cell surface molecules are CD34, CD38, CD10, IGH, CD19, CD20, CD21, CD22, and CD23, etc.), and mediating B cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC); b. inducing B cell apoptosis by targeting and binding to cytokines required for B cell survival, such as B cell activating factor (BAFF).

[0005] Based on targeted and non-targeted clearance, it can be divided into: targeted drugs (targeting CD20, targeting CD19 (including CAR-T therapy), targeting B cell activating factor (BAFF), etc.); in vitro plasma exchange; and immunosuppressants. Among them, targeting CD20 can be further subdivided into: monoclonal antibodies and bispecific antibodies; targeting CD19 can be further subdivided into monoclonal antibodies, bispecific antibodies, ADC therapy, and CAR-T therapy.

[0006] CD38, a type II transmembrane glycoprotein, has become a key target in the treatment of autoimmune diseases due to its specific high expression on the surface of plasma cells and specific B cell subsets. This molecule has dual functions as both a receptor and an enzyme: on the one hand, its interaction with B cell receptor signaling can positively regulate the early activation and proliferation of B cells; on the other hand, its extracellular enzyme activity consumes key metabolic substrates such as NAD+, leading to mitochondrial dysfunction in B cells. This metabolic reprogramming is abnormally aggravated in the context of autoimmunity, thereby promoting the production of pathological autoantibodies. Drugs targeting CD38 (such as monoclonal antibodies) mainly utilize mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent phagocytosis (ADCP) to precisely eliminate CD38-overexpressing plasma cells and pathogenic B cell subsets, thereby reducing the production of autoantibodies at the source and intervening in abnormal immune activation circuits.

[0007] Currently, several CD38-targeting therapies have shown great potential in the clinical development of autoimmune diseases. For example, Takeda Pharmaceutical's Mezagitamab (TAK-079) met its primary endpoint in a Phase IIb study for the treatment of immune thrombocytopenic purpura (ITP), demonstrating rapid and durable platelet responses. Cannoa's CM313 also showed high response rates and good safety in a Phase 1 / 2 clinical trial for the treatment of adult primary immune thrombocytopenic purpura. In the field of kidney disease, Biogen's Felzartamab, acquired through the acquisition of HI-Bio, has received Breakthrough Therapy Designation from the FDA due to its positive efficacy in primary membranous nephropathy (PMN) and antibody-mediated rejection (AMR) after kidney transplantation. Furthermore, early-stage clinical trials of these drugs for indications such as systemic lupus erythematosus (SLE) and IgA nephropathy are also underway. Compared to traditional anti-CD20 therapies that primarily deplete mature B cells, CD38-targeted therapy has the advantage of effectively eliminating long-lived plasma cells, a "continuous source" of autoantibodies, and may cover pathological B cell subsets that are insensitive to traditional therapies, providing a new direction for improving existing treatment strategies.

[0008] Blood adsorption therapy is based on the principles of porous adsorption, and / or hydrophobic interaction, and / or electrostatic adsorption, and / or bioaffinity (complement fixation, Fc fragment binding, and antigen-antibody binding). Through extracorporeal circulation technology, it can broadly, selectively, or specifically adsorb pathogenic factors in the blood without affecting the body's normal functions, thereby purifying the blood and alleviating the condition.

[0009] Commonly used blood purification techniques in clinical practice include hemoperfusion, hemofiltration, and hemodialysis. Currently, blood purification has become the third treatment method after surgery and medication, and has been successfully applied to the treatment of autoimmune diseases, liver failure, kidney failure, and drug poisoning. In recent years, blood purification has also been widely used in the treatment of inflammatory factors, sepsis, and severe pancreatitis.

[0010] In the field of blood purification therapy for immune diseases, the Compibrane Protein A Immunosorbent Column successfully treated a 20-year-old girl who had suffered from a rare and severe disease, neuromyelitis optica spectrum disorder, for 10 years and was blind in both eyes. The patient recovered and was discharged from the hospital in just 18 days. The column infuses plasma from patients with immune diseases into the column, where genetically engineered recombinant staphylococcal protein A molecules quickly "capture" and remove pathogenic antibodies from the patient's plasma. The purified blood is then returned to the body, and this cycle is repeated continuously to remove pathogenic substances from the blood, thus achieving the therapeutic goal. This technology can be widely used in the treatment of renal failure and uremia, systemic lupus erythematosus, rheumatic immune diseases, myasthenia gravis and other neurological diseases, as well as organ transplant rejection, among other areas.

[0011] Therefore, complexes composed of immunoadsorbents and antibody drugs also have adsorption effects, and can be used as an adjunct therapy for autoimmune diseases with B-cell abnormalities. Summary of the Invention

[0012] The purpose of this invention is to overcome at least one deficiency of the prior art and to provide an antibody against human CD38 and its application.

[0013] The technical solution adopted in this invention is: The first aspect of the present invention provides: An antibody against human CD38 includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the complementarity-determining regions CDR1 to CDR3 of the heavy chain variable region are DGRCQ, TIYPQGDGDTGYAQEFQG and GDRYTSNGLDY, respectively. The amino acid sequences of the complementary determinant regions CDR1 to CDR3 of the light chain variable region are KARQGVVTKVA, SGSDREYQ, and QGHYEESGPRT, respectively.

[0014] In some instances, the amino acid sequence of the heavy chain variable region is: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDGRCQWVKQRPGQGLEWIGTIYPQGDGDTGYAQEFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDRYTSNGLDYWGQGTSVTVSS.

[0015] In some instances, the amino acid sequence of the light chain variable region is: DIVMTQSHLSMSTSLGDPVSITCKARQGVVTKVAWYQQKPGQSPRRLIYSGSDREYQGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQGHYEESGPRTFGGGTKLEIK.

[0016] In some instances, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.1.

[0017] In some instances, the amino acid sequence of the light chain is as shown in SEQ ID NO.2.

[0018] In some instances, it is a single-chain antibody, a chimeric antibody, a humanized antibody, a scFv fused with an Fc fragment, or a homologous or heterologous bivalent or multivalent antibody.

[0019] A second aspect of the present invention provides: Genes encoding the antibodies described in the first aspect of this invention.

[0020] A third aspect of the present invention provides: A protein expression system containing the gene described in the second aspect of the present invention.

[0021] A fourth aspect of the present invention provides: An immunoadsorbent comprising a solid support on which the antibody described in the first aspect of the invention is coupled.

[0022] A fifth aspect of the present invention provides: The application of the antibody according to the first aspect of the present invention includes: Preparation of CD38-based B-cell immunosorbents; Preparation of CD38-based B cell detection reagent; Preparation of CD38 detection reagents; Prepare antibody-drug conjugates targeting CD38.

[0023] The beneficial effects of this invention are: Some examples of the anti-human CD38 antibodies of the present invention can specifically bind to human CD38 protein, and the binding is stable.

[0024] The antibodies against human CD38 in some examples of this invention can be used to prepare CD38 protein detection reagents and adsorption reagents; and to prepare treatments or adjuvant therapies for diseases caused by B cells.

[0025] In some embodiments of this invention, by conjugating the anti-human CD38 antibody to an adsorption column and circulating it in vitro through blood, the antibody specifically adsorbs B cells in the blood, rapidly reducing the level of pathogenic factors and quickly decreasing abnormally activated or pathogenic B cells in circulation, along with their produced antibodies, cytokines, and other pathogenic factors, thereby quickly alleviating symptoms. Secondly, it is highly targeted, able to relatively specifically clear B cells, reducing the impact on other cell types. Furthermore, it buys time for other treatments, helping to stabilize the condition during acute or severe stages of the disease and creating favorable conditions for subsequent immunomodulatory therapy or other targeted treatments. Moreover, it can improve immune imbalance, adjusting the imbalanced state of the immune system and helping to restore immune homeostasis. Finally, it can be repeated as needed to maintain the therapeutic effect, and has a high safety profile. Attached Figure Description

[0026] Figure 1 These are absorbance curves of anti-CD38 protein antibodies at different concentrations.

[0027] Figure 2 The adsorption effect is detected by flow cytometry. Detailed Implementation

[0028] The first aspect of the present invention provides: An antibody against human CD38 includes a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the complementarity-determining regions CDR1 to CDR3 of the heavy chain variable region are DGRCQ, TIYPQGDGDTGYAQEFQG and GDRYTSNGLDY, respectively. The amino acid sequences of the complementary determinant regions CDR1 to CDR3 of the light chain variable region are KARQGVVTKVA, SGSDREYQ, and QGHYEESGPRT, respectively.

[0029] In some instances, the amino acid sequence of the heavy chain variable region is: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDGRCQWVKQRPGQGLEWIGTIYPQGDGDTGYAQEFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDRYTSNGLDYWGQGTSVTVSS.

[0030] In some instances, the amino acid sequence of the light chain variable region is: DIVMTQSHLSMSTSLGDPVSITCKARQGVVTKVAWYQQKPGQSPRRLIYSGSDREYQGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQGHYEESGPRTFGGGTKLEIK.

[0031] In some instances, the amino acid sequence of the heavy chain is as shown in SEQ ID NO.1.

[0032] In some instances, the amino acid sequence of the light chain is as shown in SEQ ID NO.2.

[0033] In some instances, these are single-chain antibodies, chimeric antibodies, humanized antibodies, scFvs fused with Fc fragments, and homologous or heterologous bivalent or multivalent antibodies. These antibodies can be prepared using existing technologies. Different types of antibodies can be used depending on the application. For the preparation of immunoadsorbents, single-chain antibodies are a better choice because of their smaller molecular weight and because they do not affect the affinity for the target protein.

[0034] A second aspect of the present invention provides: Genes encoding the antibodies described in the first aspect of this invention.

[0035] Genes can be constructed using existing technologies. Furthermore, genes can be codon-optimized based on differences in protein expression systems to achieve better expression.

[0036] A third aspect of the present invention provides: A protein expression system containing the gene described in the second aspect of the present invention.

[0037] A fourth aspect of the present invention provides: An immunoadsorbent comprising a solid support on which the antibody described in the first aspect of the invention is coupled.

[0038] A fifth aspect of the present invention provides: The application of the antibody according to the first aspect of the present invention includes: Preparation of CD38-based B-cell immunosorbents; Preparation of CD38-based B cell detection reagent; Preparation of CD38 detection reagents; Prepare antibody-drug conjugates targeting CD38.

[0039] Specifically, anti-CD38 antibodies can be conjugated to a solid-phase support, utilizing the affinity between the anti-CD38 antibody and the CD38 protein on the surface of B cells to achieve immunoadsorption of B cells. In particular, B cells that highly express CD38 have more CD38 protein and are more easily adsorbed. Similarly, by conjugating detectable markers, such as fluorescent groups, to anti-CD38 antibodies, the specific affinity between the anti-CD38 antibody and CD38 can be utilized to achieve CD38-based B cell detection, CD38 detection, and the preparation of antibody-conjugates targeting CD38.

[0040] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0041] In this invention, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising methods for conquering tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0042] In this invention, the term "antibody" has its conventional meaning in the art and is used in its broadest sense. In the field of bioscience, analysis of the amino acid sequences of different antibody heavy and light chains reveals that the amino acid sequences near the N-terminus of both heavy and light chains vary considerably, while the amino acid sequences in other parts remain relatively constant. Therefore, the regions in the antibody light and heavy chains with significant amino acid sequence variation near the N-terminus are called variable regions (V), and the regions with relatively stable amino acid sequences near the C-terminus are called constant regions (C). The V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and the C regions of the heavy and light chains are abbreviated as CH and CL, respectively. Within the antibody variable regions, a small number of amino acid residues exhibit particularly strong variations. These regions, where the composition and sequence of amino acid residues are more prone to variation, are called hypervariable regions (HVRs). There are three hypervariable regions in the V regions of both the L and H chains. Because these regions can form precise complementarity with the antigenic determinants in their spatial structure, they are also called complementarity determining regions (CDRs). In antibodies, common CDR (Cellular Recognition Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When using websites that apply these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of this invention covers combinations of CDR sequences obtained through analysis using different rules. The six CDR regions of an antibody collectively determine its recognition ability and specificity against the corresponding antigen. Those skilled in the art should understand that when this invention defines the amino acid sequences of the six CDR regions, the antibody's recognition ability and specificity against the corresponding antigen are predictable.

[0043] In this invention, the term "antigen binding site" has the conventional meaning in the art, referring to a key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0044] In this invention, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure / region of the antibody or chimeric antigen receptor of this invention. Linking sequences may consist of different amino acid residues (such as glycine and serine) to allow adjacent protein domains to move freely relative to each other. Longer linking sequences may be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0045] The term "vector" generally refers to a nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into it, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances.

[0046] Example 1: Preparation of target antigen and animal immunization The screening process for anti-CD38 antibodies in this invention is as follows.

[0047] The DNA sequence encoding the CD38 amino acid sequence was constructed into a recombinant expression vector with a His tag, and the target antigen CD38 was obtained after expression and purification in mammalian cells HEK293. Similarly, an amino acid sequence targeting another target CD33 was prepared as a negative control antigen using the same expression vector and expression system. The molecular weight of the amino acid sequence of the negative control antigen CD33 was close to that of the aforementioned CD38 amino acid sequence.

[0048] The target antigen CD38 and negative control antigen CD33 obtained above were used to immunize one New Zealand rabbit each, and each rabbit was immunized three times.

[0049] Example 2: ELISA detection of rabbit immune serum After three immunizations, rabbit immune serum was collected for enzyme-linked immunosorbent assay (ELISA). ELISA was used to detect the specific binding and titer of the test sample to the target antigen at the protein level.

[0050] The experimental steps are as follows: S1) Seven days after the third immunization of New Zealand rabbits, rabbit immune serum was collected from the ear vein and diluted at different ratios to serve as test samples. S2) Different 96-well plates were coated with the target antigen CD38scFv at a concentration of 0.5 mg / mL and the negative control antigen at a concentration of 0.5 mg / mL, and blocked overnight. S3) Add the sample to be tested into the coated 96-well plate and incubate the 96-well plate in a constant temperature oven at 37°C for 1 hour; S4) Wash the 96-well plate four times with washing solution to remove unbound test samples; S5) Add horseradish peroxidase-labeled goat anti-rabbit secondary antibody and incubate at 37°C for 1 hour; S6) After washing the 96-well plate four times with washing solution, add 3,3',5,5'-tetramethylbenzidine (TMB) for color development, measure the absorbance at 450 nm and perform data processing.

[0051] Example 3: Establishment of a CD38 rabbit phage antibody library Peripheral blood mononuclear cells (PBMCs) were obtained from rabbits after three immunizations. RNA was then extracted from these cells and reverse transcribed into cDNA. After PCR amplification, the product was cleaved using HindIII and NotI enzymes and ligated into the phage vector pHIAT-1. This recombinant vector was then transformed into E. coli TG1 to construct the initial phage library.

[0052] After bacterial strain TG1 reached the logarithmic growth phase, helper phage M13KO7 was introduced into the culture system. After overnight culture, the supernatant was collected by centrifugation. Subsequently, phage particles were precipitated using polyethylene glycol (PEG), washed with PBS, and filtered through a 0.45 μm filter for sterilization, thereby obtaining the CD38 antibody library. The antibody library had a capacity of 4.5 × 10⁻⁶. 12 This provides abundant antibody resources for subsequent experiments.

[0053] Example 4: Screening of monoclonal phage antibodies binding to CD38 protein The titer of helper phage M13KO7 was determined to be 1 × 10⁻⁶. 12 pfu / ml. The TG1 strain was infected using this helper phage M13KO7.

[0054] First round of screening: First, human CD38 protein was coated onto ELISA plates at a concentration of 2 µg per well, and then incubated overnight at 4°C. The plates were then washed and blocked with 3% BSA. 100 µL of phage library solution was added to each well, and the plates were incubated at 37°C for 2 hours, followed by 6 washes. Elution was then performed with glycine-buffered saline (gly-HCl), followed by neutralization with Tris-HCl buffer. 10 μL of the eluent was used for titer determination, and the remaining eluent was added to 5 mL of TG1 bacteria in logarithmic growth phase. These bacteria were then infected and incubated at 37°C for 30 min, followed by the addition of preheated 2×YT medium to a total volume of 10 mL. The plates were incubated at 37°C and 250 rpm for another 30 min, then ampicillin was added to a final concentration of 100 μg / mL, and the plates were incubated for another 2 hours. Subsequently, kanamycin was added to achieve a final concentration of 70 μg / mL, and the mixture was incubated overnight. After centrifugation at 4000 rpm for 15 minutes at 4°C, the supernatant was collected. The supernatant was mixed with 5 mL of PEG / NaCl and incubated on ice for 1 h. Then, it was centrifuged at 9000 rpm for 20 minutes at 4°C, and the supernatant was discarded. Finally, the mixture was resuspended in PBS buffer and centrifuged to remove cell debris, yielding phage antibody particles.

[0055] Second round of screening: CD38 protein was coated onto ELISA plates, 2µg / well; the remaining operations were the same as the first round of screening.

[0056] Third round of screening: CD38 protein was coated onto ELISA plates at 2 µg / well; the remaining procedures were the same as in the first round of screening.

[0057] After screening, the selected phages were used to infect TG1 bacteria, and these bacteria were plated in culture dishes. Single clones were randomly selected from the cultured plates, using the original library as a negative control. Preliminary positive clones with an OD450 value more than twice that of the original library were screened using ELISA. After sequencing, the sequencing results were deduplicated, yielding a series of different sequences. The best-performing anti-CD38 protein antibody sequence was selected, and its amino acid sequence is as follows: Anti-CD38 antibody heavy chain: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDGRCQWVKQRPGQGLEWIGTIYPQGDGDTGYAQEFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDRYTSNGLDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO.1).

[0058] Anti-CD38 antibody light chain: DIVMTQSHLSMSTSLGDPVSITCKARQGVVTKVAWYQQKPGQSPRRLIYSGSDREYQGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQGHYEESGPRTFGGGTKLEIK-RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.2).

[0059] Analysis revealed the following amino acid sequence for the antibody heavy chain variable region: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDGRCQWVKQRPGQGLEWIGTIYPQGDGDTGYAQEFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDRYTSNGLDYWGQGTSVTVSS (SEQ ID NO.3); the following amino acid sequence for the antibody light chain variable region: DIVMTQSHLSMSTSLGDPVSITCKARQGVVTKVAWYQQKPGQSPRRLIYSGSDREYQGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQGHYEESGPRTFGGGTKLEIK (SEQ ID NO.4); and the CDRs for the heavy and light chains are shown in Table 1.

[0060] Table 1. CDR analysis results of anti-CD38 antibody CDR Name amino acid sequence Serial Number CDR-H1 DGRCQ 5 CDR-H2 TIYPQGDGDTGYAQEFQG 6 CDR-H3 GDRYTSNGLDY 7 CDR-L1 KARQGVVTKVA 8 CDR-L2 SGSDREYQ 9 CDR-L3 QGHYEESGPRT 10 Example 5: In vitro expression and purification of antibodies The gene sequence of the selected CD38 protein antibody was transformed into the PET28 plasmid via NcoI and XhoI restriction sites and expressed in *E. coli* BL21(DE3). After expansion culture in LB medium containing 70 μg / ml kanamycin, the bacterial cells were collected, sonicated (5 s on, 10 s off, working time 20 min), and centrifuged at 10,000 rpm for 10 min. The supernatant was collected. Purification was performed using a His tag with a nickel ion chelating packing material, and the elution peak was collected to obtain the anti-CD38 protein antibody.

[0061] SDS-PAGE results showed that the anti-CD38 protein antibody was successfully expressed, and the molecular weight was consistent with expectations.

[0062] Example 6: Detection of antibody binding activity to CD38 protein S1) Coat CD38 protein (used as antigen) on an ELISA plate, dilute the protein concentration to 5ug / ml with coating buffer (0.1M PBS, pH=7.4), and then add 100uL / well. Incubate overnight at 4°C. S2) The next day, discard the buffer solution in the wells, pat dry with absorbent paper, and add 250uL of ELISA washing buffer (0.05% Tween 20 / 0.1M PBS) to each well again, and wash the plate 3 times; S3) Block with 5% BSA, incubate at 37°C for 2 hours, and wash the plate 3 times; S4) After washing the plate, add anti-CD38 antibody protein to each well. The antibody is diluted to different concentrations (1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL, 0 pg / mL). Incubate at 37°C for 2 hours and wash the plate 3 times. S5) After washing the plate, add anti-His tag HRP-labeled antibody at a ratio of 1:5000, add 100 μL / well, and then incubate at 37℃ for 1 hour. Wash the plate 6 times. S6) After washing the plate, add 100 μL of TMB colorimetric solution to each well and react at 37°C in the dark for 10-15 min. Terminate the reaction when the wells of the experimental group turn blue and there is no obvious color change in the wells of the blank and negative control groups. S7) Add 100uL of 1M H2SO4 to stop the colorimetric reaction, and use an ELISA reader to detect the absorbance at a wavelength of 450nm.

[0063] The results are shown in Table 2 and Figure 1 As shown.

[0064] Table 2. Absorbance values ​​of different concentrations of anti-CD38 protein single-chain antibodies Antibody concentration / pg / ml 0 15.6 31.25 62.5 125 250 500 1000 CD38 protein sample 0.205 0.247 0.295 0.46 0.68 0.978 1.176 1.383 Protein A control 0.17 0.183 0.19 0.197 0.203 0.207 0.213 0.221 ELISA results showed that the anti-CD38 protein antibody had binding activity against CD38 protein, but no binding activity against protein A. Figure 1 Antibodies exhibit better binding activity.

[0065] Example 7: Synthesis and Adsorption Performance Evaluation of Immunosorbents Synthesis of immunoadsorbents: S1) Place 1 mL of agarose activated with cyanogen bromide into a disposable chromatography column, wash the packing material with about 30 mL of 1 mmol HCl solution, and dry it under vacuum; S2) Add 0.5 mL of 1 mmol HCl solution and 0.5 mL of a buffer system consisting of 0.1 M Na2CO3, 0.5 M NaCl, and a protein solution with pH=8.0 (the amount of protein added to the CD38 monoclonal antibody is 15 mg). Place the solution on a decolorizing shaker and mix at 100 rpm at room temperature for 2 h. S3) Rinse the packing material with 10 mL of pH 8.0, 0.1 M Tris-HCl solution, then add 1 mL of pH 8.0, 0.1 M Tris-HCl solution and seal at room temperature for 3 h; S4) Clean the packing material with 0.2M CH3COOH solution and store it for later use.

[0066] Adsorption performance evaluation: 1 ml of the above-mentioned synthetic packing material and 2 ml of B cell suspension were loaded into 10 mL EP tubes, respectively. After contacting at 28°C and 100 rpm for 1–2 h, the packing material was placed in a disposable affinity chromatography column and the packing material was dried. The change in B cell content before and after adsorption was detected by flow cytometry, and the results are shown in the figure.

[0067] Depend on Figure 2 It can be seen that after adsorption, the peak shifts forward and the number of B cells decreases, indicating that the immunoadsorbent has an adsorption effect.

[0068] Example 8: Cytotoxicity assay of CD38-ADC-conjugated drugs Maytansine was dissolved in 30% N,N-dimethylacetamide (DMA) to a concentration of 8 mg / ml. The antibody was prepared at a concentration of 10 mg / ml. 6 mg of the antibody was added to 3 mg of sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester (SulfoSMCC), and the mixture was reacted at room temperature for 3 hours. Then, while stirring, maytansine solution was slowly added until the molar amount of maytansine was 6 times the molar amount of antibody, and the reaction was continued at room temperature for 2 hours to obtain the conjugated drug. B cells (1×10⁻⁶) were then... 6 Cells were seeded into 6-well plates (cells / well). One group was treated with antibody-drug conjugate (10 μg / mL), and the other group was treated with 100 μL of the same single-chain antibody added to the cell suspension. Cells were incubated for 1 hour, followed by washing twice with PBS buffer to remove free drug. After 48 hours of further culture, cell viability was assessed using the Annexin V-FITC / PI apoptosis assay kit.

[0069] The cell survival rate was 96.26% in the control group and 28.53% in the drug-treated group. This indicates that the anti-CD38 antibody of the present invention can be used to prepare CD38-targeting conjugates.

[0070] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. An antibody against human CD38, comprising a heavy chain variable region and a light chain variable region, characterized in that, The amino acid sequences of the complementary determinant regions CDR1 to CDR3 of the heavy chain variable region are: DGRCQ, TIYPQGDGDTGYAQEFQG and GDRYTSNGLDY, respectively. The amino acid sequences of the complementary determinant regions CDR1 to CDR3 of the light chain variable region are KARQGVVTKVA, SGSDREYQ, and QGHYEESGPRT, respectively.

2. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDGRCQWVKQRPGQGLEWIGTIYPQGDGDTGYAQEFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDRYTSNGLDYWGQGTSVTVSS.

3. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region is: DIVMTQSHLSMSTSLGDPVSITCKARQGVVTKVAWYQQKPGQSPRRLIYSGSDREYQGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQGHYEESGPRTFGGGTKLEIK.

4. The antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO.

1.

5. The antibody according to claim 1, characterized in that, The amino acid sequence of the light chain is shown in SEQ ID NO.

2.

6. The antibody according to claim 1, characterized in that, These include single-chain antibodies, chimeric antibodies, humanized antibodies, scFvs fused with Fc fragments, and homologous or heterologous bivalent or multivalent antibodies.

7. A gene encoding the antibody according to any one of claims 1 to 6.

8. A protein expression system, characterized in that, It contains the gene described in claim 7.

9. An immunoadsorbent comprising a solid-phase support, characterized in that, The solid-phase support is coupled with the antibody according to any one of claims 1 to 6.

10. The application of the antibody according to any one of claims 1 to 6, characterized in that, The applications include: Preparation of CD38-based B-cell immunosorbents; Preparation of CD38-based B cell detection reagent; Preparation of CD38 detection reagents; Prepare antibody-drug conjugates targeting CD38.

Images