Anti-human CD19 single-chain antibody and application

Abstract

The invention belongs to the technical field of biology, and discloses an anti-human CD19 single-chain antibody and application. The anti-human CD19 single-chain antibody disclosed by the invention can be specifically combined with human CD19 protein, and the combination is stable. The single-chain antibody is coupled with an adsorption column, B cells in blood are adsorbed in a targeted manner, the pathogenic factor level can be rapidly reduced, abnormally activated or pathogenic B cells in circulation and pathogenic factors such as antibodies and cell factors generated by the abnormally activated or pathogenic B cells are rapidly reduced, and therefore symptoms are rapidly relieved. The B cells can be cleared relatively specifically, and the influence on other cells is reduced. The traditional Chinese medicine composition strives for time for other treatments, is helpful for stabilizing the illness state in the acute attack or serious stage of the disease, and creates favorable conditions for subsequent immunoregulation treatment or other targeted treatments. The immune imbalance can be improved, the imbalance state of the immune system can be adjusted, and the 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] The present invention belongs to the field of biotechnology, and in particular relates to an anti-human CD19 single-chain antibody and an application thereof. Background Art

[0002] Autoimmune diseases are when the immune system mistakenly attacks and destroys its own tissues and organs, causing the body to produce an autoimmune response. B cells are an important type of cell in the immune system, and they can 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 mainly affect autoimmune diseases through the following mechanisms: a. Producing autoantibodies: B cells can produce antibodies against their own tissues and organs, which can cause autoimmune reactions. For example, in type 1 diabetes, B cells produce islet cell antibodies that attack and destroy islet cells, leading to abnormally high blood sugar levels. In systemic lupus erythematosus, patients have a variety of antibodies against their own cell nuclear components, such as anti-double-stranded DNA antibodies, anti-Sm antibodies, etc. Rheumatoid arthritis: Produces autoantibodies such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP). b. Activate other immune cells: B cells can activate other immune cells, such as macrophages and T cells, thereby promoting autoimmune reactions. 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. Promote inflammatory responses: B cells can secrete inflammatory factors, promote inflammatory responses, and aggravate autoimmune responses. For example, in arthritis, B cells can produce proinflammatory factors that cause arthritis symptoms. d. Act as antigen presenting cells: For example, in multiple sclerosis, B cells present self-antigens to activate T cells, leading to nerve myelin damage. In myasthenia gravis, B cells present acetylcholine receptor-related antigens, triggering autoimmune reactions.

[0003] As one of the main components of the human immune system, B cells participate in immune responses mainly by secreting antibodies. They can also interact with T cells and release cytokines to participate in the occurrence and development of autoimmunity. 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 inhibiting abnormal immune responses have become new strategies for treating autoimmune diseases, which are clinically significant and necessary.

[0004] At present, the main B cell depletion therapies (BCDT) are: a. Using antibody drugs to target and bind to B cell-specific antigens (the specific surface molecules of B cells mainly include CD34, CD38, CD10, IGH, CD19, CD20, CD21, CD22 and CD23, etc.), 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 clearance and non-targeted clearance, it can be divided into: targeted drugs (targeted CD20, targeted CD19 (including CAR-T therapy), targeted B cell activating factor (BAFF), etc.); in vitro plasma exchange, immunosuppressants. Among them, targeted CD20 can be subdivided into: monoclonal antibody, bispecific antibody; targeted CD19 can be subdivided into monoclonal antibody, bispecific antibody, ADC therapy, and CAR-T therapy.

[0006] CD19 is a 95kpa glycoprotein on the surface of B cells. It is expressed from the early stage of B cell development until it differentiates into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily. As one of the components of the B cell surface signal transduction complex, it participates in regulating the signal transduction process of the B cell receptor. In the CD19-deficient mouse model, the number of B cells in the outer shield lymphoid tissue will be significantly reduced, and the response to vaccines and mitogens will also decrease, accompanied by a decrease in serum Ig levels. It is generally believed that the expression of CD19 is limited to the B-cell lineage, and not expressed on the surface of pluripotent hematopoietic stem cells. CD19 is also expressed on the surface of most B cell lymphomas, mantle cell lymphomas, ALLS, CLLS, hairy cell leukemia, and some acute myeloid leukemia cells. Therefore, in the treatment of leukemia / lymphoma. CD19 is a very valuable immunotherapy target. Importantly, CD19 is not expressed on the surface of most normal cells except B cells, including pluripotent hematopoietic stem cells. This feature makes CD19 a safe therapeutic target that can minimize the risk of autoimmune diseases or irreversible bone marrow toxicity in patients. Currently, anti-CD19 antibodies or scFv fragments have been developed, and their application prospects have been demonstrated in mouse models and humans / primates. In recent years, there has been fierce competition in the field of CD19CART cells, and some large pharmaceutical companies have also established partnerships with research institutions. After receiving CD19CAR T cells expressing CD28 or 4-1BB, children and adults with relapsed or refractory acute B-cell lymphoma have a complete remission rate of approximately 90%. However, CAR-T cell therapy is cumbersome to operate, and CAR-T cells require lymphocyte depletion chemotherapy with fludarabine and cyclophosphamide before administration. Moreover, the higher efficacy of CAR-T cells is accompanied by toxicity. The T cells in CAR-T cell therapy have intrinsic functional activity, which makes their toxicity more complex and severe, such as fatal cytokine release syndrome (CRS).

[0007] Blood adsorption therapy is based on the principles of pore adsorption, and / or hydrophobic effect, and / or electrostatic adsorption, and / or bioaffinity (complement binding, Fc segment binding and antigen-antibody binding). Through in vitro circulation technology, it can broadly, selectively or specifically adsorb pathogenic factors in the blood without affecting the normal functions of the body, thereby achieving the purpose of purifying the blood and alleviating the condition.

[0008] Commonly used blood purification techniques in clinical practice include hemoperfusion, hemofiltration, hemodialysis, etc. Currently, blood purification has become the third treatment method after surgery and drugs, and has been successfully used in 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.

[0009] In the field of blood purification for the treatment of immune diseases, the Kangbier Protein A immunoadsorption column successfully treated a 20-year-old girl who had been suffering from the rare and severe "neuromyelitis optica spectrum disorder" for 10 years and was blind. The patient recovered and was discharged from the hospital in just 18 days. It inputs the plasma of patients with immune diseases into the adsorption column, allowing the genetically engineered recombinant Staphylococcus protein A molecules on the column to quickly "grasp" and remove pathogenic antibodies in the patient's plasma, and then returns the purified blood to the human body. This cycle repeats continuously to remove pathogenic substances in the blood, thereby achieving the purpose of treatment. It can be widely used in the treatment of renal failure, uremia, systemic lupus erythematosus, rheumatism, myasthenia gravis and other neurological diseases, as well as organ transplant rejection reactions and other fields.

[0010] Therefore, the complex composed of the immunoadsorbent and the antibody drug also has an adsorption effect, so it can be used as an adjuvant treatment for autoimmune diseases with abnormal B cells. Summary of the invention

[0011] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a single-chain antibody against human CD19 and its application.

[0012] The technical solution adopted by the present invention is: An anti-human CD19 single-chain antibody comprises 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 respectively shown as SEQ ID NO.1 to SEQ ID NO.3, and the amino acid sequences of the complementarity determining regions CDR1 to CDR3 of the light chain variable region are respectively shown as SEQ ID NO.4 to SEQ ID NO.6.

[0013] In some examples of single-chain antibodies, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8.

[0014] In some examples of single-chain antibodies, they are chimeric antibodies, humanized antibodies, antibodies fused to an Fc fragment.

[0015] In some examples of single-chain antibodies, the Fc fragment is selected from the Fc fragment of human IgG1, IgG2, IgG3 or IgG4 or variants and modifications thereof.

[0016] In some examples of single-chain antibodies, the antibody is coupled to other antibodies via a linker sequence to form a bivalent or multivalent antibody, or to form a dimer or multimer.

[0017] The second aspect of the present invention provides: A nucleic acid molecule encoding the single-chain antibody according to the first aspect of the present invention.

[0018] The third aspect of the present invention provides: An expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0019] A fourth aspect of the present invention provides: The application of the single-chain antibody according to the first aspect of the invention comprises: preparing B cell immunoadsorbent; preparing B cell detection reagents; Preparing CD19 detection reagent; preparing an immunoconjugate targeting CD19; Preparation of antibody drugs for treating B cell related diseases.

[0020] In some application examples, the conjugate is a drug, and the B cell-related disease is selected from tumors and autoimmune diseases.

[0021] In some application examples, tumors include but are not limited to multiple myeloma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, glioblastoma; autoimmune diseases include but are not limited to lupus erythematosus, myasthenia gravis, rheumatoid arthritis, idiopathic inflammatory myositis, systemic sclerosis, and primary Sjögren's syndrome.

[0022] In some application examples, the immunoconjugate is composed of a nanobody and a conjugate connected to the nanobody, such as an ADC drug composed of an antibody and a small molecule.

[0023] In some application examples, the conjugate is a drug.

[0024] A fifth aspect of the present invention provides: An immunosorbent comprises a solid phase carrier, to which the single-chain antibody described in the first aspect of the present invention is coupled.

[0025] In some examples of immunosorbents, the solid phase carrier is selected from at least one of chitosan, agarose, cellulose, dextran, resin, and cellulose.

[0026] The sixth aspect of the present invention provides: a blood purification device, comprising the immunosorbent described in the fifth aspect of the present invention.

[0027] The beneficial effects of the present invention are: The anti-human CD19 single-chain antibodies of some embodiments of the present invention can specifically bind to the human CD19 protein and the binding is stable.

[0028] The single-chain antibodies against human CD19 in some examples of the present invention can be used to prepare CD19 protein detection reagents and adsorption reagents; and to prepare treatments or adjuvant treatments for related diseases caused by B cells.

[0029] In some examples of the present invention, by coupling the single-chain antibody of anti-human CD19 to an adsorption column, through extracorporeal blood circulation, the B cells in the blood are adsorbed in a targeted manner, which can quickly reduce the level of pathogenic factors, and quickly reduce the abnormally activated or pathogenic B cells in the circulation and the antibodies, cytokines and other pathogenic factors produced by them, thereby quickly alleviating symptoms. Secondly, it is highly targeted and can be relatively specifically targeted at B cells to remove, reducing the impact on other cell types. Moreover, it can buy time for other treatments, and in the acute onset or severe stage of the disease, it helps to stabilize the condition and create favorable conditions for subsequent immunomodulatory treatment or other targeted treatments. Not only that, it can also improve immune imbalance, adjust the imbalanced state of the immune system, and help restore immune homeostasis. Finally, it can be repeated when necessary to maintain the therapeutic effect, with high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the absorbance curve of anti-CD19 protein single-chain antibody with different concentrations.

[0031] Figure 2 It is a flow cytometer to detect the adsorption effect. DETAILED DESCRIPTION

[0032] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0033] In the present invention, the term "chimeric antigen receptor" (CAR) is the 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 (ChimericAntigenReceptorT) cell immunotherapy is considered to be one of the most promising means of conquering tumors. CAR-T cells use genetic modification methods to make T cells express CAR proteins. This CAR protein has the ability to recognize intact proteins on the membrane surface without relying on antigen presentation, thereby causing T cell activation and functional effects.

[0034] In the present invention, the term "antibody" has the conventional meaning in the art and is used in the present invention in the broadest sense. In the field of biological sciences, by analyzing the amino acid sequences of different antibody heavy chains and light chains, it is found that the amino acid sequences of the heavy chains and light chains near the N-terminus vary greatly, while the amino acid sequences of other parts are relatively constant. Therefore, the region near the N-terminus of the antibody light chain and heavy chain where the amino acid sequence varies greatly is called the variable region (variable region, V), and the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (constant region, C). The V region of the heavy chain and light chain is referred to as VH and VL, respectively, and the C region of the heavy chain and light chain is referred to as CH and CL, respectively. There is a small part of the amino acid residues in the variable region of the antibody that vary particularly strongly, and the residue composition and arrangement order of these amino acids are more prone to variation, which is called the hypervariable region (HVR); there are three hypervariable regions in the V region of the L chain and the H chain, and this part can form precise complementarity with the antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementarity determining region (CDR). In antibodies, common CDR division rules include Kabat, AbM, Chothia, Contact, and IMGT, which are well known to those skilled in the art. When a website that implements these rules is used, as long as the VH and VL sequences are input and the corresponding rules are selected, CDR sequences according to different rules can be obtained. It should be understood by those skilled in the art that the scope of protection of the present invention covers the combination of CDR sequences obtained by analyzing using different rules. The six CDR regions of an antibody jointly determine the recognition ability and specificity of the antibody to the corresponding antigen. It should be understood by those skilled in the art that when the present invention defines the amino acid sequences of the six CDR regions, the recognition ability and specificity of the antibody to the corresponding antigen are predictable.

[0035] In the present invention, the term "antigen binding site" has the conventional meaning in the art, and refers to the key site on an antibody that can specifically recognize and bind to an antigen, including the VH and / or VL regions.

[0036] In the present invention, the term "linker" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which connects any structure / region of the antibody or chimeric antigen receptor of the present invention together. The linker can be composed of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. When it is desired to ensure that two adjacent domains do not interfere with each other in space, a longer linker can be used.

[0037] The term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. A vector can transform, transduce or transfect a host cell so that the genetic material elements it carries can be expressed in the host cell. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication start site. The vector may also include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not only these substances.

[0038] Example 1: Preparation of CD19 scFv as target antigen and animal immunization The screening process of anti-CD19 antibodies in the present invention is as follows.

[0039] The DNA sequence encoding the amino acid sequence of CD19 scFv was constructed into a recombinant expression vector with a His tag, and the target antigen CD19scFv was obtained by purification after expression using mammalian cells HEK293; similarly, the same expression vector and expression system were used to prepare scFv against another target CD33 as a negative control antigen, and the molecular weight of the amino acid sequence of the negative control antigen CD33scFv was close to that of the aforementioned CD19scFv, and the same connecting sequence was used in the target antigen CD19 scFv and the negative control antigen CD33 scFv to connect VL and VH.

[0040] The target antigen CD19 scFv and negative control antigen CD33 scFv obtained above were used to immunize one New Zealand rabbit respectively, and each rabbit was immunized three times in total.

[0041] Example 2: ELISA detection of rabbit immune serum After three immunizations, rabbit immune serum was collected for enzyme-linked immunosorbent assay (ELISA assay), where ELISA assay was used to detect the specific binding and titer of the test sample with the target antigen at the protein level.

[0042] The test steps are as follows: S1) 7 days after the third immunization of New Zealand rabbits, rabbit immune serum was collected from the ear vein and diluted at different ratios as test samples; S2) using the target antigen CD19scFv at a concentration of 0.5 mg / mL and the negative control antigen at a concentration of 0.5 mg / ml to coat different 96-well plates, and blocking overnight; S3) Add the sample to be tested into the coated 96-well plate, and incubate the 96-well plate in a 37°C incubator for 1 hour; S4) washing the 96-well plate 4 times with a washing solution to wash away unbound test samples; S5) Add horseradish peroxidase-labeled goat anti-rabbit secondary antibody and incubate in a 37°C incubator for 1 hour; S6) Wash the 96-well plate four times with washing buffer and then add 3,3',5,5'-tetramethylbenzidine (TMB) for color development. Then measure the absorbance reading at 370 nm and perform data processing.

[0043] Antibody 1#, antibody 2# and antibody 3# with better adsorption effect were screened out.

[0044] Outsource sequencing to determine the sequence of the antibody, The amino acid sequence of antibody 2# is as follows: VH: QSVEESGGRLVTPGTPLTLTCTVSGASLSSYTMSWVRQAPGKGLEYIGIISYDGNTYYASWAKGRATVSKTSTTVDLKITSPTTEDTATYACARLWAYDDGSSYGVNLWGQGTLVTVSS (SEQ ID NO.7) VL: DVVMTOTPASVEAAVGGTVTIKCQASOSTGSYLAWYOOKPGQPPKLLIVRASTLESGVPSRAKGSGSGTEATLTISDLECADAATYYCQCTYDSSSSDAAAGGGT (SEQ ID NO. 8).

[0045] The amino acid sequence of the complementarity determining region determined based on the Kabat numbering system is as follows: VH_CDR1: GASLSSYT, SEQ ID NO.1 VH_CDR2:ISYDGNT,SEQ ID NO.2 VH_CDR3:LWAYDDGSSYGVNL,SEQ ID NO.3 VL_CDR1: QASOSTGSYLA, SEQ ID NO.4 VL_CDR2: RASTLES, SEQ ID NO.5 VL_CDR3: QCTYDSSSSDAA, SEQ ID NO.6.

[0046] The amino acid sequence of antibody 2# is as follows: VH: QSVEESGGRLVTPGTPLTLTCTVSGASLNTYTMTWVRQAPGKGLEWIGIITSSGGTYYATWAKGRATISKSSSTTVDLKMTRLTTEDTATYACARGGYVIYGYDMTRLDLWGQGTLVTVSS (SEQ ID NO.9) VL:AYDMTQTPASVEVVVGGTVTIKCQASEDIGTNLAWYQQKPGQPPKLLIYAAARLASGVSSRAKGSRSETEATLAISGVQCDDAATYYCQQGYSCIDVDNVAGGGTEVVVK (SEQ ID NO. 10).

[0047] The amino acid sequence of antibody 1# is as follows: VH: QSVEESGGRLVTPGTPLTLTCTVSGASLSNYAMSWVRQAPGKGLEYIGIISNSGSAYYASWAKGRATISKTSTTVDLKITSPTTEDTATYACARPRSYGDDSDVYSYYGMDLWGPGTLVTVSS (SEQ ID NO.11) VL: DVVMTQTPASVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRAKGSGAGTEYTLTISDLECADAATYYCQCTRGSSSSSSTYGNAAGGGTEVVVK (SEQ ID NO. 12).

[0048] Example 3: In vitro expression and purification of antibodies The gene sequence of the screened CD19 protein antibody was transferred into the PET28 plasmid through the restriction sites NcoI and XhoI, and expressed in Escherichia coli BL21 (DE3). After amplification with LB medium containing 70ug / m kanamycin, the bacteria were collected, ultrasonically disrupted (on 5s, stop 10s, working time 20min), centrifuged at 10000rpm for 10min, and the supernatant was collected. The his tag was purified with nickel ion chelating filler, and the elution peak was collected to obtain the anti-CD19 protein antibody.

[0049] SDS-PAGE results showed that the anti-CD19 protein antibody was successfully expressed and the molecular weight was consistent with expectations.

[0050] Example 4: Detection of antibody binding activity to CD19 protein S1) Coat the CD19 protein (used as an antigen) on the ELISA plate, dilute the protein concentration to 5ug / ml with coating solution (0.1M PBS, pH=7.4), then add 100uL / well and incubate at 4℃ overnight; S2) The next day, discard the buffer in the wells, pat dry with absorbent paper, add 250uL of ELISA washing solution (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 h, and wash the plate three times; S4) After washing the plate, anti-CD19 antibody protein was added to each well, and the antibody was diluted to different concentrations (1000pg / ml, 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.6pg / mL, 0pg / mL), incubated in a 37°C constant temperature incubator for 2h, and the plate was washed 3 times; S5) After washing the plate, add anti-His tag HRP-labeled antibody at a dilution ratio of 1:5000, add 100 μL / well, then incubate in a 37°C constant temperature incubator for 1 hour, and wash the plate 6 times; S6) After washing, add 100 μL TMB colorimetric solution to each well and react at 37°C for 10-15 min in the dark. Stop the reaction when the wells in the experimental group turn blue and there is no obvious color change in the blank and negative control wells. S7) Add 100uL of 1M H2SO4 and stop solution to terminate the color reaction, and use an enzyme reader to detect the absorbance value at a wavelength of 450nm.

[0051] The results are shown in Table 1 and Figure 1 shown.

[0052] Table 1. Absorbance values ​​of anti-CD19 protein single-chain antibody at different concentrations

[0053] ELISA results showed that anti-CD19 protein antibodies 1#, 2#, and 3# had binding activity to CD19 protein, but had no binding activity to protein A ( Figure 1 ), antibody 3# has the best binding activity.

[0054] Example 5: Synthesis of immunosorbent and evaluation of adsorption performance Synthesis of immunosorbents: S1) 1 mL of cyanogen bromide-activated agarose was placed in a disposable chromatography column, the filler was washed with about 30 mL of 1 mmol HCl solution, and then dried; S2) Add 0.5 mL of 1 mmol HCl solution and 0.5 mL of protein solution with a buffer system of 0.1 M Na2CO3, 0.5 M NaC1, pH = 8.0 (the amount of protein added to CD19 monoclonal antibody (antibody 3#) is 15 mg), place on a decolorizing shaker, 100 rpm, and mix at room temperature for 2 h; S3) Rinse the filler with 10 mL of pH 8.0, 0.1 M Tris-HCl solution, and then add 1 mL of pH 8.0, 0.1 M Tris-HCl solution to block at room temperature for 3 h; S4) Wash the filler with 0.2M CH3COOH solution and store for later use.

[0055] Adsorption performance evaluation: Use a 10mL EP tube to load 1ml of the above synthetic filler and 2ml of B cell suspension, respectively. After 1-2h contact at 28℃ and 100rpm, place the filler in a disposable affinity chromatography column and drain the filler. Use flow cytometry to detect the changes in B cell content before and after adsorption, and the results are shown in the figure.

[0056] Depend on Figure 2 It can be seen that after adsorption, the peak moved forward and the number of B cells decreased, indicating that the immunoadsorbent has an adsorption effect.

[0057] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A single-chain antibody against human CD19, comprising a heavy chain variable region and a light chain variable region, characterized in that: The amino acid sequences of the complementarity determining regions CDR1 to CDR3 of the heavy chain variable region are shown as SEQ ID NO.1 to SEQ ID NO.3, respectively, and the amino acid sequences of the complementarity determining regions CDR1 to CDR3 of the light chain variable region are shown as SEQ ID NO.4 to SEQ ID NO.6, respectively.

2. The single-chain antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The single-chain antibody according to claim 1 or 2, characterized in that It is a chimeric antibody, a humanized antibody, or an antibody fused to an Fc fragment.

4. The single-chain antibody according to claim 3, characterized in that The Fc fragment is selected from the Fc segment of human IgG1, IgG2, IgG3 or IgG4 or its variants and modifications.

5. A nucleic acid molecule, characterized in that Encoding the single-chain antibody according to any one of claims 1 to 4.

6. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

7. Use of the single-chain antibody according to claims 1 to 4, characterized in that: The applications include: preparing B cell immunoadsorbent; preparing B cell detection reagents; Preparing CD19 detection reagent; preparing an immunoconjugate targeting CD19; Preparation of antibody drugs for treating B cell related diseases.

8. The use according to claim 7, characterized in that: The conjugate is a drug, and the B cell-related disease is selected from tumors and autoimmune diseases.

9. An immunosorbent, comprising a solid phase carrier, characterized in that: The single-chain antibody according to claims 1 to 4 is coupled to the solid phase carrier.

10. A blood purification device, characterized in that: Comprising the immunoadsorbent according to claim 9.

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