A single-chain antibody against human cd19 and use thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an anti-human CD19 single-chain antibody and application. The anti-human CD19 single-chain antibody can be specifically combined with human CD19 protein and is stable in combination. By coupling the single-chain antibody with an adsorption column, B cells in blood can be adsorbed in a targeted manner, the level of pathogenic factors can be quickly reduced, and the abnormal activated or pathogenic B cells in circulation and the pathogenic factors such as antibodies, cytokines and the like generated by the B cells can be quickly reduced, so that the symptoms can be quickly relieved. The B cells can be relatively specifically removed, and the influence on other cells can be reduced. Time for other treatments is obtained, and in the acute attack or severe stage of the disease, the disease state can be stabilized, and favorable conditions for subsequent immune regulation treatment or other targeted treatment are created. The immune imbalance can be improved, and the imbalance state of the immune system can be adjusted, and the immune homeostasis can be recovered. The adsorption can be repeatedly performed as necessary to maintain the treatment effect, and the safety is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a single-chain antibody against human CD19 and application thereof. BACKGROUND

[0002] Autoimmune diseases refer to the immune system mistakenly attacking and destroying its own tissues and organs, leading to the body producing an autoimmune response. B cells are an important cell type in the immune system, which can help the body resist pathogens and foreign substances by producing antibodies. However, in autoimmune diseases, B cells will 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 self-tissues and organs, which can cause autoimmune reactions. For example, in type 1 diabetes, B cells produce islet cell antibodies, which attack and destroy islet cells, leading to abnormally high blood sugar levels. Systemic Lupus Erythematosus (SLE) patients have multiple antibodies against their own nuclear components, such as anti-double-stranded DNA antibodies, anti-Sm antibodies, etc. Rheumatoid Arthritis (RA): Produces rheumatoid factor (RF), anti-cyclic citrullinated peptide antibodies (anti-CCP), and other autoantibodies. b. Activating other immune cells: B cells can activate other immune cells, such as macrophages and T cells, to promote autoimmune reactions. For example, in systemic lupus erythematosus, B cells can activate T cells, leading to the immune system attacking multiple tissues and organs of the body. c. Promoting inflammatory reactions: B cells can secrete inflammatory factors to promote inflammatory reactions and exacerbate autoimmune reactions. For example, in arthritis, B cells can produce pro-inflammatory factors to cause joint inflammation. d. As antigen-presenting cells: For example, in multiple sclerosis (MS), B cells present self-antigens to activate T cells, leading to damage to the nerve myelin sheath. In myasthenia gravis (MG), B cells present acetylcholine receptor-related antigens to trigger autoimmune reactions.

[0003] B cells, as one of the main components of the human immune system, mainly participate in immune reactions by secreting antibodies, and can also interact with T cells to release 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, clearing B cells and inhibiting abnormal immune reactions have become a new strategy for treating autoimmune diseases, which has clinical significance and necessity.

[0004] Current B cell depletion therapy (BCDT) mainly includes: a. Targeting specific antigens of B cells (the specific surface molecules of B cells mainly include CD34, CD38, CD10, IGH, CD19, CD20, CD21, CD22 and CD23, etc.) by using antibody drugs, and mediating B cell lysis through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC); b. Targeting B cell survival required cytokines such as B cell activating factor (BAFF), thereby inducing B cell apoptosis.

[0005] According to targeted elimination and non-targeted elimination, it can be divided into: targeted drugs (targeting CD20, targeting CD19 (including CAR-T therapy), targeting B cell activation factor (BAFF), etc.); extracorporeal plasma exchange, immunosuppressants. Among them, targeting CD20 can be divided into: single antibody, double antibody; targeting CD19 is divided into single antibody, double antibody, ADC therapy, CAR-T therapy.

[0006] CD19 is a 95 kpa glycoprotein on the surface of B cells, which is expressed from the early stage of B cell development until it is differentiated into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily, which is involved in the regulation of B cell receptor signal transduction as one of the elements of the B cell surface signal transduction complex. In CD19-deficient mouse models, the number of B cells in the outer mantle lymphoid tissue is significantly reduced, the response to vaccines and mitogens is also reduced, and the serum Ig level is also reduced. It is generally believed that the expression of CD19 is limited to the B cell lineage, and is 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 a portion of 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 other than B cells, including pluripotent hematopoietic stem cells, which makes CD19 a safe therapeutic target that minimizes the risk of autoimmune disease or irreversible bone marrow toxicity in patients. Currently, antibodies or scFv fragments against CD19 have been developed and their application prospects have been demonstrated in mouse models and humans / primates. In recent years, the field of CD19 CAR T cells has been highly competitive, and some large pharmaceutical companies have established partnerships with research institutions. After receiving CD19 CAR T cells expressing CD28 or 4-1BB, pediatric and adult patients with relapsed or refractory acute B cell lymphoma have a complete remission rate of about 90%. However, CAR-T cell therapy is complicated, and CAR-T cells require lymphocyte-depleting chemotherapy with fludarabine and cyclophosphamide before administration. Moreover, the higher efficacy of CAR-T cells is accompanied by toxicity, and the T cells in CAR-T cell therapy have inherent functional activity, leading to more complex and severe toxicity, such as the life-threatening cytokine release syndrome (CRS).

[0007] Blood adsorption therapy is based on pore adsorption, and / or hydrophobic interaction, and / or electrostatic adsorption, and / or biological affinity (complement binding, Fc fragment binding and antigen-antibody binding) principles, through extracorporeal technology, to adsorb pathogenic factors in blood in a broad-spectrum, selective or specific manner without affecting normal body functions, thereby achieving the purpose of purifying blood and relieving the disease.

[0008] The commonly used blood purification technology in clinical practice includes blood perfusion, hemofiltration, hemodialysis, etc. At present, blood purification has become the third treatment method after surgery and drugs, and has been successfully applied to the treatment of autoimmune diseases and 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 blood purification treatment of immune diseases, Kangbi protein A immunoadsorption column successfully rescued a 20-year-old girl who had suffered from a rare severe "opticospinal myelitis spectrum disease" for 10 years and was blind in both eyes. In only 18 days, the patient was discharged from hospital. It inputs the plasma of patients with immune diseases into the adsorption column, and the genetically engineered recombinant staphylococcal protein A molecules on the column quickly "grasp" and remove the pathogenic antibodies in the plasma of the patients, and then the purified blood is returned to the human body, so as to continuously remove the pathogenic substances in the blood, so as to achieve the purpose of rescue. It can be widely used in the treatment of renal failure, uremia, systemic lupus erythematosus, immune diseases, myasthenia gravis and other nervous system diseases, organ transplant rejection and other fields.

[0010] Therefore, the complex composed of the immunoadsorbent and the antibody drug also has adsorption effect, so it can be used as an auxiliary treatment method for treating autoimmune diseases with B cell abnormalities. SUMMARY

[0011] The purpose of the present application is to overcome at least one deficiency of the prior art, and to provide an anti-human CD19 single-chain antibody and application.

[0012] The technical solution adopted by the present application is:

[0013] An anti-human CD19 single-chain antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of the complementarity determining regions CDR1-CDR3 of the heavy chain variable region are respectively shown in SEQ ID NO. 1-SEQ ID NO. 3, and the amino acid sequences of the complementarity determining regions CDR1-CDR3 of the light chain variable region are respectively shown in SEQ ID NO. 4-SEQ ID NO. 6.

[0014] In some examples of the single-chain antibody, 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.

[0015] In some examples of the single-chain antibody, it is a chimeric antibody, a humanized antibody, or an antibody fused with an Fc fragment.

[0016] In some examples of the single-chain antibody, the Fc fragment is selected from the Fc segment of human IgG1, IgG2, IgG3 or IgG4 or a variant or modification thereof.

[0017] In some examples of the single-chain antibody, the antibody is coupled to other antibodies through a connecting sequence to form a bivalent or multivalent antibody, or to form a dimer or a multimer.

[0018] The second aspect of the present application provides:

[0019] A nucleic acid molecule encoding the single chain antibody of the first aspect of the application.

[0020] In a third aspect of the application, there is provided:

[0021] An expression vector comprising the nucleic acid molecule of the second aspect of the application.

[0022] In a fourth aspect of the application, there is provided:

[0023] Use of the single chain antibody of the first aspect of the application, the use comprising:

[0024] Preparation of a B cell immunoadsorbent;

[0025] Preparation of a B cell detection reagent;

[0026] Preparation of a CD19 detection reagent;

[0027] Preparation of an immunoconjugate targeting CD19;

[0028] Preparation of an antibody drug for treating a B cell related disease.

[0029] In some examples of use, the conjugate is a drug and the B cell related disease is selected from the group consisting of a tumor and an autoimmune disease.

[0030] In some examples of use, the tumor includes but is not limited to multiple myeloma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, glioblastoma; the autoimmune disease includes but is not limited to lupus erythematosus, myasthenia gravis, rheumatoid arthritis, idiopathic inflammatory myositis, systemic sclerosis, primary Sjogren's syndrome.

[0031] In some examples of use, the immunoconjugate is composed of a nanobody and a conjugate linked to the nanobody, such as an ADC drug composed of an antibody and a small molecule.

[0032] In some examples of use, the conjugate is a drug.

[0033] In a fifth aspect of the application, there is provided:

[0034] An immunoadsorbent comprising a solid phase carrier, the solid phase carrier having coupled thereto the single chain antibody of the first aspect of the application.

[0035] In some examples of the immunoadsorbent, the solid phase carrier is selected from the group consisting of at least one of chitosan, agarose, cellulose, dextran, resin, cellulose.

[0036] In a sixth aspect of the application, there is provided: a blood purification apparatus comprising the immunoadsorbent of the fifth aspect of the application.

[0037] The beneficial effects of the present application are:

[0038] The single-chain antibody against human CD19 of some examples of the present application can specifically bind to human CD19 protein and the binding is stable.

[0039] The single-chain antibody against human CD19 of some examples of the present application can be used for preparing CD19 protein detection reagents, adsorption reagents; preparing treatment or adjuvant treatment of B cell induced related diseases.

[0040] In some examples of the present application, by coupling the single-chain antibody against human CD19 to an adsorption column, through extracorporeal blood circulation, B cells in the blood are adsorbed specifically, the level of pathogenic factors is rapidly reduced, the abnormal activated or pathogenic B cells in the circulation and the pathogenic factors such as antibodies, cytokines produced by the B cells are rapidly reduced, thereby the symptoms are rapidly relieved. Secondly, it is specific and can relatively specifically remove B cells, reducing the impact on other cell types. Moreover, it can gain time for other treatments, which is helpful for stabilizing the disease condition, creating favorable conditions for subsequent immune regulation treatment or other targeted treatment. Furthermore, it can improve immune imbalance and adjust the imbalance of the immune system, which is helpful for restoring immune homeostasis. Finally, it can be repeated as necessary to maintain the therapeutic effect, which is safe. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is the absorbance curve of anti-CD19 protein single-chain antibody with different concentrations.

[0042] Figure 2 is the flow chart for detecting adsorption effect. DETAILED DESCRIPTION

[0043] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation steps used herein are widely used terms and conventional steps in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided as follows.

[0044] In the present application, the term "Chimeric Antigen Receptor" (CAR) is the core component of CAR cell treatment drugs, which can 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 means to conquer tumors. CAR-T cells are T cells that express CAR proteins using genetic modification methods. 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.

[0045] In the present application, the term "antibody" has the conventional meaning in the art and is used in the broadest sense in the present application. In the field of biological sciences, it is found by analyzing the amino acid sequences of different antibody heavy and light chains that the amino acid sequences near the N-terminus of the heavy and light chains vary greatly, and the other parts of the amino acid sequences are relatively constant. Therefore, the region of the antibody light and heavy chains with a large variation in the amino acid sequence near the N-terminus is called the variable region (V), and the region with a relatively stable amino acid sequence near the C-terminus is called the constant region (C). The V regions of the heavy and light chains are simply referred to as VH and VL, respectively, and the C regions of the heavy and light chains are simply 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 composition and arrangement order of these amino acid residues are more likely to vary. This region is called the hypervariable region (HVR); there are three hypervariable regions in the V region of the L chain and the H chain, respectively. This part can form a precise complement with the antigenic determinant in the spatial structure, so the hypervariable region is also called the complementarity determining region (CDR). In the antibody, the common CDR division rules are Kabat, AbM, Chothia, Contact, IMGT, which are well known to those skilled in the art. When the website implementing these rules is applied, as long as the VH and VL sequences are input and the corresponding rule is selected, the CDR sequences according to different rules can be obtained. Those skilled in the art should understand that the protection scope of the present application covers the combination of CDR sequences obtained by using different rules. The six CDR regions of the antibody together determine the recognition ability and specificity of the antibody to the corresponding antigen. Those skilled in the art should understand that when the present application limits the amino acid sequences of the six CDR regions, the recognition ability and specificity of the antibody to the corresponding antigen are predictable.

[0046] In the present application, the term "antigen binding site" has the conventional meaning in the art and refers to the key site on the antibody that can specifically recognize and bind to the antigen, including the VH and / or VL regions.

[0047] In the present application, the term "linker sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length, which links any structure / region of the antibody of the present application, chimeric antigen receptor together. The linker sequence can be composed of different amino acid residues, such as glycine and serine, so that the adjacent protein domains are free to move 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 sequence can be used.

[0048] The term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein is expressed. The vector can transform, transduce or transfect a host cell, so that the genetic material elements carried by the vector are expressed in the host cell. For example, the vector includes: a plasmid; a phagemid; a cosmid; an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC); a bacteriophage such as a lambda phage or a M13 phage, and an animal virus, etc. The types of animal viruses used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), pox viruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector can also contain a replication initiation site. The vector can also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0049] Example 1: Preparation of CD19 scFv as target antigen and performance of animal immunization

[0050] The screening process of the anti-CD19 antibody in the present application is as follows.

[0051] The DNA sequence encoding the amino acid sequence of the CD19 scFv is constructed into a recombinant expression vector with a His tag, and the target antigen CD19 scFv is obtained after expression and purification using mammalian cells HEK293; similarly, using the same expression vector and expression system, an scFv against another target CD33 is prepared as a negative control antigen, the amino acid sequence of the negative control antigen CD33 scFv has a molecular weight close to that of the aforementioned CD19 scFv, and the same linker sequence is used in the target antigen CD19 scFv and the negative control antigen CD33 scFv for connecting VL and VH.

[0052] The target antigen CD19 scFv and the negative control antigen CD33 scFv obtained above were respectively used for immunizing one New Zealand rabbit each, and each rabbit was immunized for three times.

[0053] Example 2: ELISA detection of rabbit immune serum

[0054] After the three immunizations, the rabbit immune serum was collected for enzyme-linked immunosorbent assay (i.e., ELISA detection), wherein the ELISA detection was used for detecting the specific binding and titer of the sample to be tested to the target antigen at the protein level.

[0055] The test steps were as follows:

[0056] S1) Seven days after the third immunization of the New Zealand rabbit, the rabbit immune serum was collected from the marginal vein of the ear and diluted at different ratios to serve as the sample to be tested;

[0057] S2) The target antigen CD19 scFv at a concentration of 0.5 mg / mL and the negative control antigen at a concentration of 0.5 mg / mL were respectively used to coat different 96-well plates, and the plates were blocked overnight;

[0058] S3) The sample to be tested was added to the coated 96-well plate, and the 96-well plate was incubated in a 37°C incubator for 1 hour;

[0059] S4) The 96-well plate was washed with washing solution for 4 times to remove the unbound sample to be tested;

[0060] S5) Horseradish peroxidase-labeled goat anti-rabbit secondary antibody was added, and the plate was incubated in a 37°C incubator for 1 hour;

[0061] S6) After washing the 96-well plate with washing solution for 4 times, 3,3',5,5'-tetramethylbenzidine (TMB) was added for color development, and the absorbance reading was measured at 370 nm and the data was processed.

[0062] Antibodies 1#, 2# and 3# with better adsorption effect were screened out.

[0063] The sequences of the antibodies were determined by outsourcing sequencing, wherein,

[0064] The amino acid sequence of antibody 2# is as follows:

[0065] VH: QSVEESGGRLVTPGTPLTLTCTVSGASLSSYTMSWVRQAPGKGLEYIGIISYDGNTYYASWAKGRATVSKTSTTVDLKITSPTTEDTATYACARLWAYDDGSSYGVNLWGQGTLVTVSS (SEQ ID NO. 7)

[0066] VL: DVVMTOTPASVEAAVGGTVTIKCQASOSTGSYLAWYOOKPGQPPKLLIVRASTLESGVPSRAKGSGSGTEATLTISDLECADAATYYCQCTYDSSSSDAAAGGGT (SEQ ID NO. 8).

[0067] The amino acid sequences of the complementarity determining regions determined based on the Kabat numbering system are as follows:

[0068] VH_CDR1: GASLSSYT, SEQ ID NO. 1

[0069] VH_CDR2: ISYDGNT, SEQ ID NO. 2

[0070] VH_CDR3: LWAYDDGSSYGVNL, SEQ ID NO. 3

[0071] VL_CDR1: QASOSTGSYLA, SEQ ID NO. 4

[0072] VL_CDR2: RASTLES, SEQ ID NO. 5

[0073] VL_CDR3: QCTYDSSSSDAA, SEQ ID NO. 6.

[0074] The amino acid sequence of antibody 2# is as follows:

[0075] VH: QSVEESGGRLVTPGTPLTLTCTVSGASLNTYTMTWVRQAPGKGLEWIGIITSSGGTYYATWAKGRATISKSSSTTVDLKMTRLTTEDTATYACARGGYVIYGYDMTRLDLWGQGTLVTVSS (SEQ ID NO. 9)

[0076] VL: AYDMTQTPASVEVVVGGTVTIKCQASEDIGTNLAWYQQKPGQPPKLLIYAAARLASGVSSRAKGSRSETEATLAISGVQCDDAATYYCQQGYSCIDVDNVAGGGTEVVVK (SEQ ID NO. 10).

[0077] The amino acid sequence of antibody 1# is as follows:

[0078] VH: QSVEESGGRLVTPGTPLTLTCTVSGASLSNYAMSWVRQAPGKGLEYIGIISNSGSAYYASWAKGRATISKTSTTVDLKITSPTTEDTATYACARPRSYGDDSDVYSYYGMDLWGPGTLVTVSS (SEQ ID NO. 11)

[0079] VL: DVVMTQTPASVEAAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYRASTLESGVPSRAKGSGAGTEYTLTISDLECADAATYYCQCTRGSSSSSTYGNAAGGGTEVVVK (SEQ ID NO. 12).

[0080] Example 3: In vitro expression and purification of the antibody

[0081] The gene sequence of the selected CD19 protein antibody was transferred into PET28 plasmid through Ncol and Xhol enzyme cutting sites, and expressed in E. coli BL21 (DE3). After expanding culture in LB medium containing 70 ug / m kanamycin, the bacterial cells were collected, ultrasonically broken (5 s on, 10 s off, working time 20 min), and then centrifuged at 10,000 rpm for 10 min to collect the supernatant. The his tag was used for purification with nickel ion chelation filler, and the elution peak was collected to obtain the anti-CD19 protein antibody.

[0082] The SDS-PAGE results showed that the anti-CD19 protein antibody was successfully expressed, and the molecular weight was consistent with the expected value.

[0083] Example 4: Detection of the binding activity of the antibody to CD19 protein

[0084] S1) Coat CD19 protein (used as antigen) on ELISA plate, dilute the protein concentration to 5 ug / m1 with coating solution (0.1M PBS, pH=7.4), then add 100 uL / well, 4°C overnight;

[0085] S2) The next day, discard the buffer in the wells, use a blotting paper to dry, and add 250 uL of ELISA washing solution (0.05% Tween 20 / 0.1M PBS) to each well, wash the plate 3 times;

[0086] S3) Block with 5% BSA, incubate at 37°C for 2 h, and wash the plate 3 times;

[0087] S4) After washing the plate, anti-CD19 antibody protein was added to each well, and the antibody was 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), and incubated in a 37°C constant temperature incubator for 2h, and washed 3 times;

[0088] S5) After washing the plate, anti-His tag with HRP labeled antibody was added, diluted at a ratio of 1:5000, 100 μL / well, then placed in a 37°C constant temperature incubator for 1 hour, and washed 6 times;

[0089] S6) After washing the plate, 100 μL of TMB color developing solution was added to each well, protected from light, and reacted at 37°C for 10-15 min. When the experimental group wells turned blue and the blank and negative control group wells showed no obvious color change, the reaction was terminated;

[0090] S7) 100 uL of 1M H2SO4 was added to terminate the color developing reaction, and the absorbance value was detected using an enzyme label instrument at a wavelength of 450 nm.

[0091] The results are shown in Table 1 and Figure 1 .

[0092] Table 1, absorbance values of different concentrations of anti-CD19 protein single-chain antibodies

[0093]

[0094] ELISA results showed that anti-CD19 protein antibodies 1#, 2# and 3# had binding activity to CD19 protein, but no binding activity to protein A (Table 1) Figure 1 ), and antibody 3# had the best binding activity.

[0095] Example 5: Synthesis of immunoadsorbent and evaluation of adsorption performance

[0096] Synthesis of immunoadsorbent:

[0097] S1) Take 1 mL of cyanogen bromide activated agarose and place it in a disposable chromatography column, wash the packing with about 30 mL of 1 mmol HC1 solution, and dry it;

[0098] S2) Add 0.5 mL of 1 mmol HCl solution and 0.5 mL of buffer system 0.1M Na2CO3, 0.5M NaC1, pH=8.0 protein solution (take CD19 mAb (antibody 3#) and add 15 mg of protein), place on a decolorizing shaker, mix at 100 rpm at room temperature for 2h;

[0099] 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;

[0100] S4) Clean the packing material with 0.2M CH3COOH solution and store it for later use.

[0101] Adsorption performance evaluation:

[0102] 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.

[0103] 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.

[0104] 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. 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 in 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 in 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 includes chimeric antibodies, humanized antibodies, and antibodies fused with Fc fragments.

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 or modifiers.

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

6. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 5.

7. The application of single-chain antibodies, characterized in that, The single-chain antibody is as described in any one of claims 1 to 4, and the application includes: Preparation of B-cell immunosorbents; Preparation of B-cell detection reagents; Preparation of CD19 detection reagents; Prepare an immunoconjugate targeting CD19.

8. The application according to claim 7, characterized in that, The conjugate is a drug.

9. An immunoadsorbent comprising a solid-phase support, characterized in that, The solid-phase support is coupled with the single-chain antibody according to any one of claims 1 to 4.

10. A blood purification device, characterized in that, Includes the immunosorbent as described in claim 9.

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