Anti-CD19 antibody and application thereof

Abstract

The invention discloses an anti-CD19 antibody or an antigen binding fragment thereof and application of the anti-CD19 antibody or the antigen binding fragment thereof. The anti-CD19 antibody or the antigen binding fragment thereof comprises a complementary determining region (CDR) sequence selected from SEQ ID NO: 1-31. The anti-CD19 antibody or the antigen binding fragment thereof has high affinity, can effectively recognize and bind human and monkey CD19 antigens, and provides a safer and more effective immunotherapy choice for treatment of related diseases.

Description

Technical Field

[0001] The present application belongs to the field of antibody technology, and specifically relates to an anti-CD19 antibody and its application. Background Art

[0002] Human CD19 is a 95kDa type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. Human CD19 has two extracellular C-type IgSF domains and a cytoplasmic tail containing 242 residues. CD19 is widely expressed at most stages of B cell differentiation, but its expression is downregulated when B cells terminally differentiate into plasma cells. On B cells, CD19 binds to CD21, CD81, and CD225 to form a signal transduction complex, which lowers the threshold of B cell activation, promotes B cell proliferation, differentiation, and antibody production, and is a key participant in B cell development, activation, and functional execution.

[0003] CD19 has always been the focus of immunotherapy development. CD19 is expressed in most B-cell derived malignancies and follicular dendritic cells. CD19 is an important cell surface marker for normal B cells and B-cell derived cancers. By developing anti-CD19 antibodies, CD19 molecules can be specifically recognized and bound, thereby activating other immune cells to kill CD19 positive cells, thereby achieving the effect of treating tumors and autoimmune diseases.

[0004] At present, a variety of tumor immunotherapy methods based on anti-CD19 antibodies have been developed, such as ADC, CAR-T, and TCE. These therapies achieve precise killing of B cell tumors by specifically recognizing the CD19 antigen. However, existing anti-CD19 antibodies and immunotherapy methods still have some problems, such as high cost, efficacy and safety need to be further improved, etc. Summary of the invention

[0005] Based on the above problems, the present application provides a new antibody targeting CD19, which can effectively recognize and bind to CD19 molecules while not cross-reacting with other cell surface molecules, thereby specifically activating other immune cells to kill tumor cells or B cells.

[0006] The technical solution of this application is as follows:

[0007] 1. An anti-CD19 antibody or an antigen-binding fragment thereof, comprising a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 1-31.

[0008] 2. The anti-CD19 antibody or antigen-binding fragment thereof according to item 1, wherein:

[0009] The complementary determining regions include a heavy chain complementary determining region and a light chain complementary determining region;

[0010] The heavy chain complementarity determining region is:

[0011] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; or

[0012] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:7, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:8; or

[0013] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 12; or

[0014] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 16; or

[0015] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 18; or

[0016] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:21; or

[0017] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:23, a CDR2 having an amino acid sequence as shown in SEQ ID NO:24, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:25; or

[0018] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 29; and

[0019] The light chain complementarity determining region is:

[0020] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or

[0021] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:9; or

[0022] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; or

[0023] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or

[0024] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 19, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; or

[0025] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:22; or

[0026] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:26, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:27; or

[0027] It comprises a CDR1 with the amino acid sequence shown in SEQ ID NO:30, a CDR2 with the amino acid sequence shown in SEQ ID NO:5, and a CDR3 with the amino acid sequence shown in SEQ ID NO:31.

[0028] 3. The anti-CD19 antibody or antigen-binding fragment thereof according to item 2, wherein the complementarity determining regions comprise the following heavy chain complementarity determining regions and light chain complementarity determining regions:

[0029] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; or

[0030] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:8, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:9; or

[0031] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15; or

[0032] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 16, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; or

[0033] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or

[0034] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:20, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:22; or

[0035] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:24, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:25, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:27; or

[0036] The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:28, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:29, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:30, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:31.

[0037] 4. An anti-CD19 antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region sequence selected from the following: SEQ ID NO: 32-47 or an amino acid sequence that has at least 80% identity with the heavy chain variable region and the light chain variable region sequence, respectively.

[0038] 5. The anti-CD19 antibody or antigen-binding fragment thereof according to item 1, wherein:

[0039] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, or the amino acid sequence shown in SEQ ID NO: 34, or the amino acid sequence shown in SEQ ID NO: 36, or the amino acid sequence shown in SEQ ID NO: 38, or the amino acid sequence shown in SEQ ID NO: 40, or the amino acid sequence shown in SEQ ID NO: 42, or the amino acid sequence shown in SEQ ID NO: 44, or the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences; and

[0040] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, or the amino acid sequence shown in SEQ ID NO:35, or the amino acid sequence shown in SEQ ID NO:37, or the amino acid sequence shown in SEQ ID NO:39, or the amino acid sequence shown in SEQ ID NO:41, or the amino acid sequence shown in SEQ ID NO:43, or the amino acid sequence shown in SEQ ID NO:45, or the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences.

[0041] 6. The anti-CD19 antibody or antigen-binding fragment thereof according to item 4, wherein:

[0042] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; or

[0043] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35; or

[0044] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37; or

[0045] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39; or

[0046] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; or

[0047] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43; or

[0048] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:45; or

[0049] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:46 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47.

[0050] 7. An anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof is an antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies.

[0051] 8. The anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

[0052] 9. An anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises the constant domain of human IgG1, IgG2, IgG3 or IgG4.

[0053] 10. A nucleic acid encoding the anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

[0054] 11. A host cell comprising the polynucleotide according to item 10.

[0055] 12. A method for preparing an anti-CD19 antibody or an antigen-binding fragment thereof, comprising culturing the host cell described in item 11 to thereby prepare the anti-CD19 antibody or the antigen-binding fragment thereof as described in any one of items 1 to 9.

[0056] 13. A pharmaceutical composition comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

[0057] 14. A pharmaceutical composition according to claim 13, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0058] 15. A conjugate comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

[0059] 16. A fusion protein comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 9.

[0060] 17. Use of the anti-CD19 antibody or antigen-binding fragment thereof according to any one of items 1 to 9, the pharmaceutical composition according to item 13 or 14, the conjugate according to item 15 or the fusion protein according to item 16 in the preparation of drugs for treating and / or preventing CD19-mediated related diseases.

[0061] Beneficial effects: The anti-CD19 antibody of the present application can be used to treat CD19-positive malignant tumors, such as acute lymphocytic leukemia, etc.; and autoimmune diseases, such as systemic lupus erythematosus, etc. CD19-positive cells can be effectively eliminated through anti-CD19 monoclonal antibody therapy or therapies such as dual antibodies, ADC, CAR-T, CAR-NK, CAR-M, etc. using anti-CD19 antibodies, thereby treating CD19-positive malignant tumors and autoimmune diseases. The anti-CD19 antibody or its antigen-binding fragment has high affinity and can effectively recognize and bind to human and monkey CD19 antigens, so that the antibody or its fragment is also effective in animal models before clinical trials, which improves the reliability of the conversion of preclinical data to human trials, and provides a safer and more effective immunotherapy option for the treatment of related diseases. In addition, the therapy can also be used in combination with other drugs to enhance the therapeutic effect and reduce side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings are used to better understand the present application and do not constitute an improper limitation on the present application.

[0063] Figure 1 The binding curves of 8 antibodies to recombinant human CD19-His protein are shown in the figure above. The binding curves of antibodies 1#-4# are shown in the figure above, and the binding curves of antibodies 5#-8# are shown in the figure below.

[0064] Figure 2 The binding curves of 8 antibodies to recombinant monkey CD19-His protein are shown in the figure above. The binding curves of antibodies 1#-4# are shown in the figure above, and the binding curves of antibodies 5#-8# are shown in the figure below.

[0065] Figure 3 The binding curves of 8 antibodies to human CD19 / HEK293T cells.

[0066] Figure 4 The results show the detection of NK cell killing effect on Raji cells mediated by 8 antibodies. DETAILED DESCRIPTION

[0067] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0068] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the functional differences of components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" are open-ended terms and should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present application, but the description is based on the general principles of the specification and is not intended to limit the scope of the present application. The scope of protection of the present application shall be determined by the attached claims.

[0069] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0070] Generally speaking, the terms used in this specification have the following meanings.

[0071] As used herein, the term "antibody" is used in the broadest sense and covers various antibody structures, including not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies (scFV), VHH antibodies, multispecific antibodies (e.g., bispecific antibodies), multispecific single-chain antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen recognition site of desired specificity, including antibody glycosylation variants, antibody amino acid sequence variants, and covalently modified antibodies.

[0072] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second, and third constant regions (CH1, CH2, CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL), and the light chain constant region includes κ (Kappa) or λ (Lambda). A full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses of the above), but the antibody does not need to belong to any particular class. Based on the antibody amino acid sequence of the constant region of the heavy chain, immunoglobulins can be assigned to different classes. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0073] As used herein, the term "antigen binding fragment" refers to a region of a molecule that specifically binds to an antigen. An antigen binding domain can be an antigen binding portion of an antibody or an antigen binding fragment thereof. An antigen binding fragment can include, for example, Fab', F(ab')2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), single domain antibody, VHH, VNAR, sdAb or nanobody.

[0074] As used herein, the term "variable region" refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also referred to as hypervariable regions. The CDRs in each chain are kept close together by the FRs, and together with the CDRs from the other chains, contribute to the formation of the antigen binding site of the antibody.

[0075] As used herein, the term "monoclonal antibody (mAb)" or "mAb" or "Mab" refers to a homogeneous antibody population, that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, directed against a single antigen or epitope. "Monoclonal" indicates that the antibody is a feature obtained from a substantially homogeneous antibody population and should not be construed as limiting the structure, source, or preparation method of the antibody. In some embodiments, monoclonal antibodies are prepared by hybridoma methods, phage display methods, yeast display methods, recombinant DNA methods, single cell screening, or single cell sequencing methods.

[0076] As used in this article, "plasmid" refers to a DNA molecule other than chromosomes (or nucleoids) in organisms such as bacteria, yeast and actinomycetes, which exists in the cytoplasm or nucleus and has the ability to replicate autonomously, so that it can maintain a constant copy number in daughter cells and express the genetic information it carries.

[0077] The term "vector" refers to a vector by which a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, a "viral vector" is a vector transformed from a viral genome, which introduces foreign genes into host cells through viral infection.

[0078] As used herein, the term "specific binding" refers to a binding affinity (equilibrium dissociation constant KD for antigen-antibody binding) of at least 10 -6 In some aspects, the antibody is present in an amount of at least about 10 -7 M, preferably 10 -8 M, 10 - 9 M, 10 -10 M, 10 -11 M or 10 -12 Affinity binding of M.

[0079] In this application, the terms "polynucleotide" or "nucleic acid", "nucleic acid molecule" are used interchangeably and include but are not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), mi RNA (micro RNA), genomic DNA, synthetic DNA, synthetic RNA and / or tRNA.

[0080] As used herein, sequence "identity" refers to the degree of similarity between amino acid sequences or between nucleotide sequences as determined by sequence comparison software, such as BLAST.

[0081] In this specification, "conjugate" refers to an antibody or antigen-binding fragment thereof connected to another agent such as a chemotherapeutic agent, a toxin, an immunotherapeutic agent, an imaging probe, etc. The connection can be a covalent bond or a non-covalent interaction, such as by electrostatic forces. A variety of linkers known in the art can be used to form a conjugate. In addition, the conjugate can be provided in the form of a fusion protein expressed by a polynucleotide encoding the conjugate.

[0082] In this specification, "enzyme-linked immunosorbent assay (ELISA)" refers to a detection method that utilizes the ability of antibody molecules to specifically bind to antigen molecules, combines free impurities with target proteins bound to a solid phase carrier, and uses special markers to conduct qualitative or quantitative analysis. The principle is: antigens or antibodies can be physically adsorbed on the solid surface and maintain their immune activity; antigens or antibodies can form enzyme conjugates with enzymes through covalent bonds while maintaining their respective immune activity or enzyme activity; after the enzyme conjugates bind to the corresponding antigens or antibodies, the occurrence of the immune reaction can be determined by the color reaction of the added substrate, and the depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double antibody sandwich method is the most commonly used method for detecting antigens. The method is to adsorb the antiserum containing known antibodies into the small holes of the microtiter plate and wash it once; add the antigen to be tested. If the two are specific, they will bind to each other, and then wash away the excess antibody; add an enzyme-linked antibody that reacts specifically with the antigen to be tested to form a "sandwich"; add the substrate of the enzyme, and if a colored enzymatic hydrolysis product is seen, it indicates the presence of the corresponding antigen.

[0083] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0084] Antibody

[0085] The present application provides an anti-CD19 antibody or an antigen-binding fragment thereof, which comprises a complementary determining region (CDR) sequence selected from the following: SEQ ID NO: 1-31. In some embodiments, the anti-CD19 antibody or an antigen-binding fragment thereof comprises a human universal framework region.

[0086] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0087] CDRH1:GYTFTNYW;

[0088] The amino acid sequence of SEQ ID NO:2 is as follows: CDRH2: IYPGDGDT;

[0089] The amino acid sequence of SEQ ID NO:3 is as follows: CDRH3: AYFGSSFENFDY;

[0090] The amino acid sequence of SEQ ID NO:4 is as follows: CDRL1: QDVGTA;

[0091] The amino acid sequence of SEQ ID NO:5 is as follows: CDRL2: WAS;

[0092] The amino acid sequence of SEQ ID NO:6 is as follows: CDRL3: QRYSIYPYT;

[0093] The amino acid sequence of SEQ ID NO:7 is as follows: CDRH1: GYTFTSYW;

[0094] The amino acid sequence of SEQ ID NO:8 is as follows: CDRH3:AREGFLLTTSAYYAMDS; the amino acid sequence of SEQ ID NO:9 is as follows: CDRL3:QQYSTYPLT;

[0095] The amino acid sequence of SEQ ID NO: 10 is as follows: CDRH1: GYSITSGYY;

[0096] The amino acid sequence of SEQ ID NO: 11 is as follows: CDRH2: IRYDGTN;

[0097] The amino acid sequence of SEQ ID NO: 12 is as follows: CDRH3: AREASYGPYYFDY; the amino acid sequence of SEQ ID NO: 13 is as follows: CDRL1: ESVDNYGISF; the amino acid sequence of SEQ ID NO: 14 is as follows: CDRL2: IAS;

[0098] The amino acid sequence of SEQ ID NO: 15 is as follows: CDRL3: QQSKEVP;

[0099] The amino acid sequence of SEQ ID NO: 16 is as follows: CDRH3: AYFGSSYEDFDY;

[0100] The amino acid sequence of SEQ ID NO: 17 is as follows: CDRH1: GYTFTTYW;

[0101] The amino acid sequence of SEQ ID NO: 18 is as follows:

[0102] CDRH3:AREGVLLTTSLYYAMDY;

[0103] The amino acid sequence of SEQ ID NO: 19 is as follows:

[0104] CDRL1:QDVGTG;

[0105] The amino acid sequence of SEQ ID NO:20 is as follows:

[0106] CDRH1:GYTFSNYW;

[0107] The amino acid sequence of SEQ ID NO:21 is as follows:

[0108] CDRH3:AYFSSSYEDFDY;

[0109] The amino acid sequence of SEQ ID NO:22 is as follows:

[0110] CDRL3:QRYSTYPYT;

[0111] The amino acid sequence of SEQ ID NO:23 is as follows:

[0112] CDRH1:GNSFTTYW;

[0113] The amino acid sequence of SEQ ID NO:24 is as follows:

[0114] CDRH2: IHPSDSET;

[0115] The amino acid sequence of SEQ ID NO:25 is as follows:

[0116] CDRH3:TTPGAYYIYEDSY;

[0117] The amino acid sequence of SEQ ID NO:26 is as follows:

[0118] CDRL2: WTS;

[0119] The amino acid sequence of SEQ ID NO:27 is as follows:

[0120] CDRL3:QQYSRYPYT;

[0121] The amino acid sequence of SEQ ID NO:28 is as follows:

[0122] CDRH1:GYTFTTYW;

[0123] The amino acid sequence of SEQ ID NO:29 is as follows:

[0124] CDRH2: IYPGDGYT;

[0125] The amino acid sequence of SEQ ID NO:30 is as follows:

[0126] CDRH3:AREGLLLTTSVYYAMDY;

[0127] The amino acid sequence of SEQ ID NO:31 is as follows:

[0128] CDRL3:QQYSTFPLK;

[0129] In some embodiments, the complementarity determining regions comprise a heavy chain complementarity determining region and a light chain complementarity determining region;

[0130] The heavy chain complementarity determining region is:

[0131] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; or

[0132] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:7, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:8; or

[0133] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 12; or

[0134] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 16; or

[0135] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 18; or

[0136] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:21; or

[0137] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:23, a CDR2 having an amino acid sequence as shown in SEQ ID NO:24, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:25; or

[0138] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 29; and

[0139] The light chain complementarity determining region is:

[0140] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or

[0141] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:9; or

[0142] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; or

[0143] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or

[0144] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 19, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; or

[0145] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:22; or

[0146] comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:26, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:27; or

[0147] It comprises a CDR1 with the amino acid sequence shown in SEQ ID NO:30, a CDR2 with the amino acid sequence shown in SEQ ID NO:5, and a CDR3 with the amino acid sequence shown in SEQ ID NO:31.

[0148] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;

[0149] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:8, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:9;

[0150] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15;

[0151] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 16, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;

[0152] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9;

[0153] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:20, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:22;

[0154] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:24, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:25, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:27;

[0155] In some embodiments, the heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:28, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:29, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:30, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:31.

[0156] In some embodiments, the anti-CD19 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region sequence selected from the following: SEQ ID NO: 32-47 or an amino acid sequence that has at least 80% identity with the heavy chain variable region and the light chain variable region sequence, respectively.

[0157] The amino acid sequence of SEQ ID NO:32 is as follows:

[0158] KVQLQQSGAELARPGASVKVSCKASGYTFTNYWMQWVKQRPGQG LEWIGAIYPGDGDTRYSKKFKGKATLTADKSSSTAYMQLSSLASEDSAVY YCAYFGSSFENFDYWGQGTTLTVSS;

[0159] The amino acid sequence of SEQ ID NO:33 is as follows:

[0160] DIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYT FGGGTKLEIK;

[0161] The amino acid sequence of SEQ ID NO:34 is as follows:

[0162] QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG LEWIGAIYPGDGDTRYTQKFKGRATLTADKSSSTAYMQLSSLASEDSAVY YCAREGFLLTTSAYYAMDSWGQGTSVTVSS;

[0163] The amino acid sequence of SEQ ID NO:35 is as follows:

[0164] GILMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKV LIYWASTRHTGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYSTYPL TFGSGTKLEIK;

[0165] The amino acid sequence of SEQ ID NO:36 is as follows:

[0166] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGIKLE WMGYIRYDGTNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCA REASYGPYYFDYWGQGTTLTVSS;

[0167] The amino acid sequence of SEQ ID NO:37 is as follows:

[0168] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPP KLLIYIASKRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVP RTFGGGTKLEIK;

[0169] The amino acid sequence of SEQ ID NO:38 is as follows:

[0170] QVQLQQSGAELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQG LEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVY YCAYFGSSYEDFDYWGQGTTLTVSS;

[0171] The amino acid sequence of SEQ ID NO:39 is as follows:

[0172] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYT FGGGTKLEIK;

[0173] The amino acid sequence of SEQ ID NO:40 is as follows:

[0174] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQG LEWIGAIYPGDGDTRYLQKFKGKATLTADKSSNTAYMQLSSLASEDSAVY YCAREGVLLTTSLYYAMDYWGQGTSVTVSS;

[0175] The amino acid sequence of SEQ ID NO:41 is as follows:

[0176] GIVMTQSHKFMSTSVGDRVSITCKASQDVGTGVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSTYPLT FGSGTRLEIN;

[0177] The amino acid sequence of SEQ ID NO:42 is as follows:

[0178] QVQLQQSGAELARPGASVKLSCKASGYTFSNYWMQWIKQRPGQGL EWIGAIYPGDGDTRYSQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYY CAYFSSSYEDFDYWGQGTTLTVSS;

[0179] The amino acid sequence of SEQ ID NO:43 is as follows:

[0180] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSTYPYT FGGGTKLEIT;

[0181] The amino acid sequence of SEQ ID NO:44 is as follows:

[0182] QVQLQQPGAELVRPGDSVKLSCKASGNSFTTYWMNWVKQRPGQGL EWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPPTSEDSAVYYC TTPGAYYIYEDSYWGQGTTLTVSS;

[0183] The amino acid sequence of SEQ ID NO:45 is as follows:

[0184] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKAGQSPKL LFYWTSTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRYPY TFGGGTKLEIK;

[0185] The amino acid sequence of SEQ ID NO:46 is as follows:

[0186] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQG LEWIGAIYPGDGYTRYTQKFKGKATLTADKSASTAYMELSSLASEDSAVY YCAREGLLLTTSVYYAMDYWGQGTSVTVSS;

[0187] The amino acid sequence of SEQ ID NO:47 is as follows:

[0188] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTYFTLTINNVQSEDLADYFCQQYSTFPLK FGSGTKLEMK.

[0189] For example, when the anti-CD19 antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80% identical to the heavy chain variable region and light chain variable region sequences, respectively, it may comprise an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the heavy chain variable region and light chain variable region sequences, respectively.

[0190] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32, or the amino acid sequence shown in SEQ ID NO:34, or the amino acid sequence shown in SEQ ID NO:36, or the amino acid sequence shown in SEQ ID NO:38, or the amino acid sequence shown in SEQ ID NO:40, or the amino acid sequence shown in SEQ ID NO:42, or the amino acid sequence shown in SEQ ID NO:44, or the amino acid sequence shown in SEQ ID NO:46, or an amino acid sequence that is at least 80% identical to said amino acid sequence, respectively; and

[0191] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, or the amino acid sequence shown in SEQ ID NO:35, or the amino acid sequence shown in SEQ ID NO:37, or the amino acid sequence shown in SEQ ID NO:39, or the amino acid sequence shown in SEQ ID NO:41, or the amino acid sequence shown in SEQ ID NO:43, or the amino acid sequence shown in SEQ ID NO:45, or the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences.

[0192] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33.

[0193] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:35.

[0194] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:37.

[0195] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:39.

[0196] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41.

[0197] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43.

[0198] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:45.

[0199] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:46 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:47.

[0200] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment is an antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment of these antibodies.

[0201] In some embodiments, the antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment or a scFv fragment.

[0202] In some embodiments, the anti-CD19 antibody or antigen-binding fragment thereof comprises the constant domain of human IgG1, IgG2, IgG3 or IgG4.

[0203] Nucleic acid molecules, vectors, host cells

[0204] The present application provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the aforementioned anti-CD19 antibodies or antigen-binding fragments thereof.

[0205] In one embodiment of the present application, the nucleic acid is an RNA or DNA molecule.

[0206] In one embodiment of the present application, the nucleic acid is single-stranded or double-stranded.

[0207] In one embodiment of the present application, the nucleic acid is linear or circular. The nucleic acid can be synthesized by cells or chemically synthesized.

[0208] In one embodiment of the present application, the nucleic acid is a chemically modified nucleic acid molecule.

[0209] The present application also provides a vector comprising the above-mentioned nucleic acid.

[0210] In one embodiment of the present application, the vector is a plasmid.

[0211] The present application also provides a host cell, which comprises the above-mentioned nucleic acid or vector.

[0212] Pharmaceutical compositions, conjugates and methods of treatment

[0213] The present application provides a pharmaceutical composition, which comprises any one of the above-mentioned anti-CD19 antibodies or antigen-binding fragments thereof, or the antibody expressed by the above-mentioned nucleic acid, or the antibody prepared by the above-mentioned vector, or the antibody produced by the above-mentioned host cell, and a pharmaceutically acceptable carrier.

[0214] The present application also provides a method for preparing an anti-CD19 antibody or an antigen-binding fragment thereof, which comprises culturing the above-mentioned host cell to prepare the anti-CD19 antibody or the antigen-binding fragment thereof as described in any one of the above items.

[0215] The present application also provides a pharmaceutical composition for treating or preventing CD19-mediated related diseases. In some embodiments, the pharmaceutical composition comprises the aforementioned antibody. In some embodiments, the pharmaceutical composition comprises a complex of the antibody or its antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises a nucleic acid molecule encoding the aforementioned antibody or its antigen-binding fragment. In some embodiments, the pharmaceutical composition comprises the virus, bacteriophage or cell. In some specific embodiments, the virus, bacteriophage or cell comprises a nucleic acid encoding the antibody or its antigen-binding fragment, and after entering the animal body and reaching the target organ, the antibody is expressed.

[0216] The pharmaceutical composition can be used to treat or prevent CD19-mediated related diseases. For example, the related diseases include but are not limited to acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and B-cell lymphoma.

[0217] The present application also provides a conjugate, which comprises any one of the above-mentioned anti-CD19 antibodies or antigen-binding fragments thereof, or the antibody expressed by the above-mentioned nucleic acid, or the antibody prepared by the above-mentioned vector, or the antibody produced by the above-mentioned host cell.

[0218] Furthermore, the conjugate further comprises a drug, a cytokine, a radionuclide or an enzyme connected to the antibody or the antigen-binding fragment thereof.

[0219] The present application also provides a fusion protein comprising any of the above-mentioned anti-CD19 antibodies or antigen-binding fragments thereof.

[0220] The present application also provides a method for treating or preventing CD19-mediated related diseases, including administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a user who needs treatment or prevention. The pharmaceutical composition of the present application can be administered alone or in combination with one or more therapeutic agents (or second therapeutic agents). In the combined administration described herein, the pharmaceutical composition herein can be administered before, after, or simultaneously with one or more additional agents.

[0221] The present application also provides the use of any of the above-mentioned anti-CD19 antibodies or antigen-binding fragments thereof, the above-mentioned pharmaceutical composition, the above-mentioned conjugate or the above-mentioned fusion protein in the preparation of drugs for treating and / or preventing CD19-mediated related diseases.

[0222] Products, test kits

[0223] The present application also provides a product or kit for detecting CD19, which comprises the aforementioned antibody or antigen-binding fragment thereof for CD19. In some embodiments, the present application provides the use of the aforementioned antibody or antigen-binding fragment thereof in the preparation of a product or kit for detecting CD19.

[0224] Example

[0225] The present application content will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in this area and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the scheme and obtain corresponding results according to the embodiments.

[0226] Example 1. Antibody screening and preparation

[0227] 1 Animal immunization:

[0228] Ten Balb / c mice (Bai Ying Biotechnology) were used for immunization, and the specific immunization scheme was as follows: each mouse was immunized with 10 μg of human CD19 mRNA-LNP (#P15391-1) for the first and second immunizations, 10 μg of cynomolgus monkey CD19 mRNA-LNP (#F7F486-1) for the third immunization, and 10 μg of human CD19 mRNA-LNP was used again for the fourth immunization; the immunization interval was two weeks, and blood was collected after the second, third and fourth immunizations to determine the serum titer; each candidate mouse was immunized with 25 μg of Recombinant Human CD19 / His Protein (Bai Ying Biotechnology).

[0229] 2. Serum titer detection:

[0230] Serum titer was detected by ELISA and flow cytometry. In the ELISA test, when the mouse serum was diluted at 1:1000, the OD450 should be greater than 2, and when it was diluted at 1:16000, the OD450 should be greater than 1, which was considered to meet the sorting standard. The secondary antibody was goat anti-mouse IgG Fc secondary antibody (Invitrogen, 31437). In the flow cytometry test (serum titer cell binding test), when it was diluted at 1:24300, the mean fluorescence intensity (mean) value should be greater than the blank control group (mean value) × 10.

[0231] 3. Antibody Screening:

[0232] The spleen and bone marrow of the mice were removed, and plasma cells were enriched using CD138 magnetic beads. Recombinant human CD19-His protein labeled with AF-488 was used to sort specific B lymphocytes using the FRET principle. A single-cell cDNA library was constructed and sequenced by NGS. After bioinformatics analysis, the base and amino acid sequences of the paired light and heavy chains were obtained.

[0233] 4 Expression and purification of candidate antibodies:

[0234] The variable region heavy chain obtained by screening was combined with human IgG1Fc, and the light chain was combined with the light chain Kappa constant region to obtain the full-length human-mouse chimeric antibody sequence; DNA was synthesized according to the antibody protein sequence, an expression vector was constructed, and the antibody was transferred into CHO cells for antibody expression. After protein A affinity purification, a recombinant antibody with an Fc region of hIgG1 subtype was finally prepared. The results are shown in Table 1. The expression level and purity of the screened antibodies (1#-8#) were very high.

[0235] Table 1 Expression results of candidate antibodies (1#-8#)

[0236] Antibody ID Yield (mg / L) purity 1# 173.25 96.285％ 2# 202.50 98.086％ 3# 194.00 98.62％ 4# 184.50 97.024％ 5# 188.00 97.966％ 6# 171.00 98.797％ 7# 188.50 96.997％ 8# 176.50 98.582％

[0237] The amino acid sequences of candidate antibodies (1#-8#) are as follows:

[0238] The heavy chain variable region of antibody 1# is:

[0239] KVQLQQSGAELARPGASVKVSCKASGYTFTNYWMQWVKQRPGQG LEWIGAIYPGDGDTRYSKKFKGKATLTADKSSSTAYMQLSSLASEDSAVY YCAYFGSSFENFDYWGQGTTLTVSS (SEQ ID NO: 32);

[0240] The light chain variable region of antibody 1# is:

[0241] DIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYT FGGGTKLEIK(SEQ ID NO:33);

[0242] The heavy chain variable region of antibody 2# is:

[0243] QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQG LEWIGAIYPGDGDTRYTQKFKGRATLTADKSSSTAYMQLSSLASEDSAVY YCAREGFLLTTSAYYAMDSWGQGTSVTVSS(SEQ ID NO:34);

[0244] The light chain variable region of antibody 2# is:

[0245] GILMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKV LIYWASTRHTGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYSTYPL TFGSGTKLEIK(SEQ ID NO:35);

[0246] The heavy chain variable region of antibody 3# is:

[0247] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGIKLE WMGYIRYDGTNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCA REASYGPYYFDYWGQGTTLTVSS(SEQ ID NO:36);

[0248] The light chain variable region of antibody 3# is:

[0249] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPP KLLIYIASKRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVP RTFGGGTKLEIK(SEQ ID NO:37);

[0250] The heavy chain variable region of antibody 4# is:

[0251] QVQLQQSGAELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQG LEWIGAIYPGDGDTRYTQKFKGKATLTADKSSSTTAYMQLSSLASEDSAVY YCAYFGSSYEDFDYWGQGTTLTVSS(SEQ ID NO:38);

[0252] The light chain variable region of antibody 4# is:

[0253] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYT FGGGTKLEIK(SEQ ID NO:39);

[0254] The heavy chain variable region of antibody 5# is:

[0255] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQG LEWIGAIYPGDGDTRYLQKFKGKATLTADKSSNTAYMQLSSLASEDSAVY YCAREGVLLTTSLYYAMDYWGQGTSVTVSS(SEQ ID NO:40);

[0256] The light chain variable region of antibody 5# is:

[0257] GIVMTQSHKFMSTSVGDRVSITCKASQDVGTGVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSTYPLT FGSGTRLEIN(SEQ ID NO:41);

[0258] The heavy chain variable region of antibody 6# is:

[0259] QVQLQQSGAELARPGASVKLSCKASGYTFSNYWMQWIKQRPGQGL EWIGAIYPGDGDTRYSQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYY CAYFSSSYEDFDYWGQGTTLTVSS(SEQ ID NO:42);

[0260] The light chain variable region of antibody 6# is:

[0261] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKL LIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSTYPYT FGGGTKLEIT(SEQ ID NO:43);

[0262] The heavy chain variable region of antibody 7# is:

[0263] QVQLQQPGAELVRPGDSVKLSCKASGNSFTTYWMNWVKQRPGQGL EWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPPTSEDSAVYYCTTPGAYYIYEDSYWGQGTTLTVSS(SEQ ID NO:44);

[0264] The light chain variable region of antibody 7# is:

[0265] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKAGQSPKL LFYWTSTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRYPY TFGGGTKLEIK(SEQ ID NO:45);

[0266] The heavy chain variable region of antibody 8# is:

[0267] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQGLEWIGAIYPGDGYTRYTQKFKGKATLTADKSASTAYMELSSLASEDSAVYYCAREGLLLTTSVYYAMDYWGQGTSVTVSS(SEQ ID NO:46);

[0268] The light chain variable region of antibody 8# is:

[0269] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTYFTLTINNVQSEDLADYFCQQYSTFPLKFGSGTKLEMK(SEQ ID NO:47).

[0270] The heavy chain of antibody 1# is:

[0271] KVQLQQSGAELARPGASVKVSCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDGDTRYSKKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCAYFGSSFENFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:48);

[0272] The light chain of antibody 1# is:

[0273] DIVMTQSHKFMSTSVGDRVTITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:49);

[0274] The heavy chain of Antibody 2# is:

[0275] QVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGRATLTADKSSSTAYMQLSSLASEDSAVYYCAREGFLLTTSAYYAMDSWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:50);

[0276] The light chain of Antibody 2# is:

[0277] GILMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKVLIYWASTRHTGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYSTYPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:51);

[0278] The heavy chain of antibody 3# is:

[0279] DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGIKLEWMGYIRYDGTNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTATYYCAREASYGPYYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:52);

[0280] The light chain of antibody 3# is:

[0281] DIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYIASKRGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:53);

[0282] The heavy chain of antibody 4# is:

[0283] QVQLQQSGAELARPGASVKLSCKASGYTFTNYWMQWVKQRPGQGLEWIGAIYPGDGDTRYTQKFKGKATLTADKSSTTAYMQLSSLASEDSAVYYCAYFGSSYEDFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:54);

[0284] The light chain of antibody 4# is:

[0285] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSIYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:55);

[0286] The heavy chain of antibody 5# is:

[0287] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQGLEWIGAIYPGDGDTRYLQKFKGKATLTADKSSNTAYMQLSSLASEDSAVYYCAREGVLLTTSLYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:56);

[0288] The light chain of antibody 5# is:

[0289] GIVMTQSHKFMSTSVGDRVSITCKASQDVGTGVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCQQYSTYPLTFGSGTRLEINRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:57);

[0290] The heavy chain of antibody 6# is:

[0291] QVQLQQSGAELARPGASVKLSCKASGYTFSNYWMQWIKQRPGQGLEWIGAIYPGDGDTRYSQKFKGKATLTADKSSSTAYMQLSSLASEDSAVYYCAYFSSSYEDFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:58);

[0292] The light chain of antibody 6# is:

[0293] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQRYSTYPYTFGGGTKLEITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:59);

[0294] The heavy chain of antibody 7# is:

[0295] QVQLQQPGAELVRPGDSVKLSCKASGNSFTTYWMNWVKQRPGQGLEWIGMIHPSDSETRLNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTTPGAYYIYEDSYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:60);

[0296] The light chain of antibody 7# is:

[0297] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKAGQSPKLLFYWTSTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSRYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:61);

[0298] The heavy chain of antibody 8# is:

[0299] QVQLQQSGAELARPGASVKLSCKASGYTFTTYWMQWVKQRPGQGLEWIGAIYPGDGYTRYTQKFKGKATLTADKSASTAYMELSSLASEDSAVYYCAREGLLLTTSVYYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:62);

[0300] The light chain of antibody 8# is:

[0301] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTYFTLTINNVQSEDLADYFCQQYSTFPLKFGSGTKLEMKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:63).

[0302] Example 2 Antibody Binding to Human or Cynomolgus Monkey CD19 Protein Levels

[0303] The ELISA method was used to verify the binding effect of candidate antibodies on human or cynomolgus monkey CD19 protein.

[0304] (1) Protein coating: Coat the plate with antigen protein at a concentration of 2 μg / mL, 100 μL / well, incubate at 37°C for 1 h, wash the plate 6 times, and pat dry.

[0305] (2) Blocking: Use 3% BSA-PBS blocking solution, 200 μL / well, incubate at 25°C for 1 h, wash the plate 6 times, and pat dry.

[0306] (3) Primary antibody incubation: The sample to be tested was diluted 3-fold with blocking solution at 100 nM or 15 μg / mL in the first well, and the sample was diluted 3-fold with blocking solution in 8 gradients. The blocking solution was added to the last well as a blank at 100 μL / well and incubated at 25°C for 1 h. The plate was washed 6 times and patted dry.

[0307] (4) Secondary antibody incubation: The secondary antibody (Invitrogen, A18817) was diluted with blocking buffer, 100 μL / well, reacted at 25°C for 1 h, washed 6 times, and patted dry.

[0308] (5) Color development: Add 100 μL / well TMB (Invitrogen, 002023) and develop color at room temperature in the dark for 3-5 min.

[0309] (6) Termination: Add 50 μL / well ELISA stop solution (BBI, E661066-0500).

[0310] (7) Reading: Read OD450 using a microplate reader.

[0311] (8) Data processing: The data were fitted using the log(agonist) vs. response--Variable slope(four parameters) equation of GraphPad Prism 8.

[0312] The results are as follows Figure 1-Figure 2 As shown, the results showed that antibodies 1#-8# could bind to recombinant human CD19-His protein and recombinant cynomolgus monkey CD19-His protein, presenting an obvious dose-dependent curve, and the binding to cynomolgus monkey CD19-His was stronger than that of the control antibody (Tafasitamab).

[0313] Example 3. Detection of antibody binding to human CD19 expressing cells

[0314] Flow cytometry was used to verify the binding activity of the antibody to HEK293T cells overexpressing human CD19.

[0315] (1) Collect overexpressing cells in the exponential growth phase and resuspend them at a density of 3×10 6 cells / mL, 50 μl / well was added to a 96-well plate.

[0316] (2) Antibodies, positive controls, and negative controls were diluted 3-fold with 0.5% BSA-PBS diluent, with a final concentration of 33 nM in the first well. The dilutions were repeated 8 times, with a blank in the last well. The dilutions were added to a 96-well plate at 50 μL / well and incubated at 4°C for 1 h.

[0317] (3) Centrifuge at 400 g and 4°C for 5 min, discard the supernatant, and wash twice with 150 μL of 0.5% BSA-PBS diluent.

[0318] (4) Add 100 μL of diluted fluorescent secondary antibody (Invitrogen, A18818).

[0319] (5) Incubate at 4°C for 30 min.

[0320] (6) Centrifuge at 400 g and 4°C for 5 min, discard the supernatant, and wash twice with 150 μL of 0.5% BSA-PBS diluent.

[0321] (7) Resuspend in 100 μL 0.5% BSA-PBS diluent and load onto an analyzer (Invitrogen, Attune NxT).

[0322] (8) Data processing: The data were fitted using the log(agonist) vs. response--Variable slope(four parameters) equation of GraphPad Prism 8.

[0323] The results are as follows Figure 3 As shown, the results showed that antibodies 1#-8# all bound to human CD19 / HEK293T cells, presenting an obvious dose-dependent curve, and the binding intensity to human CD19 / HEK293T cells was close to that of the control antibody (Tafasitamab).

[0324] Example 4. Detection of anti-CD19 antibody-mediated killing effect

[0325] The luciferase reporter gene expression method was used to detect the antibody-mediated killing effect of NK cells on CD19 positive cells.

[0326] (1) Collect Raji cells expressing luciferase reporter gene in the exponential growth phase and resuspend them at a density of 0.4×10 6 cells / mL, 50 μL / well was added to a 96-well plate.

[0327] (2) Antibodies, positive controls, and negative controls were diluted 3-fold with culture medium, with a final concentration of 30 nM in the first well. The dilutions were added into 8 gradients and added to a 96-well plate containing Raji cells at 50 μL / well. The plates were incubated at 37°C for 20 min.

[0328] (3) Resuspension of sorted PBNK cells at a density of 4 × 10 6 cells / mL, 50 μL / well was added to a 96-well plate.

[0329] (4) Centrifuge at 100 g for 30 seconds.

[0330] (5) Incubate at 37°C for 4 h.

[0331] (6) Take out and equilibrate at room temperature for 15 minutes, add 100 μl of luciferase detection substrate (Genomeditech, GM-040513B), and react for 10 minutes.

[0332] (7) Transfer 200 μl of supernatant to a white opaque 96-well plate and use an ELISA reader to detect the fluorescence intensity.

[0333] (8) Data processing: The data were fitted using the log(agonist) vs. response--Variable slope(four parameters) equation of GraphPad Prism 8.

[0334] The results are as follows Figure 4 As shown, the results showed that antibodies 1#-8# could mediate NK cell killing of CD19-positive cells, and the effect was stronger than or close to the positive control antibody (Tafasitamab).

[0335] The present application provides an anti-CD19 antibody and its application. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. An anti-CD19 antibody or an antigen-binding fragment thereof, comprising a complementarity determining region, CD19 The complementary determining regions include a heavy chain complementary determining region and a light chain complementary determining region; The heavy chain complementarity determining region is: comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 3; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:7, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:8; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 10, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 11, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 12; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 16; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 18; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:20, a CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:21; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:23, a CDR2 having an amino acid sequence as shown in SEQ ID NO:24, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:25; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 17, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 28, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 29; and Wherein the light chain complementary determining region is: comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:9; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 13, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 14, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 15; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:6; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO: 19, a CDR2 having an amino acid sequence as shown in SEQ ID NO: 5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 9; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:22; or comprising a CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a CDR2 having an amino acid sequence as shown in SEQ ID NO:26, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:27; or It comprises a CDR1 having an amino acid sequence as shown in SEQ ID NO:30, a CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a CDR3 having an amino acid sequence as shown in SEQ ID NO:

31.

2. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein the complementarity determining regions comprise the following heavy chain complementarity determining regions and light chain complementarity determining regions: The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:3, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:6; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:8, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:9; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 13, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 15; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 16, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:20, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:21, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:22; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:23, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:24, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:25, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:26, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:27; or The heavy chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:17, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:28, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:29, and the light chain complementary determining region is a CDR1 comprising the amino acid sequence shown in SEQ ID NO:30, a CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO:

31.

3. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, CD19 among them: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32, or the amino acid sequence shown in SEQ ID NO: 34, or the amino acid sequence shown in SEQ ID NO: 36, or the amino acid sequence shown in SEQ ID NO: 38, or the amino acid sequence shown in SEQ ID NO: 40, or the amino acid sequence shown in SEQ ID NO: 42, or the amino acid sequence shown in SEQ ID NO: 44, or the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences; and The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, or the amino acid sequence shown in SEQ ID NO:35, or the amino acid sequence shown in SEQ ID NO:37, or the amino acid sequence shown in SEQ ID NO:39, or the amino acid sequence shown in SEQ ID NO:41, or the amino acid sequence shown in SEQ ID NO:43, or the amino acid sequence shown in SEQ ID NO:45, or the amino acid sequence shown in SEQ ID NO:47, or an amino acid sequence that is at least 80% identical to each of the amino acid sequences.

4. The anti-CD19 antibody or antigen-binding fragment thereof according to claim 3, wherein: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 32 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 33; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 34 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 35; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 36 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 39; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:41; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:43; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:44 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:45; or The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:46 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

47.

5. The anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is an antibody, a recombinant antibody, a fully human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antigen-binding fragment thereof.

6. A nucleic acid encoding the anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. A host cell comprising the polynucleotide of claim 6.

8. A method for preparing an anti-CD19 antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to claim 7 to prepare the anti-CD19 antibody or the antigen-binding fragment thereof according to any one of claims 1 to 5.

9. A pharmaceutical composition comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

10. A conjugate comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

11. A fusion protein comprising the anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

12. Use of the anti-CD19 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the pharmaceutical composition according to claim 9, the conjugate according to claim 10 or the fusion protein according to claim 11 in the preparation of a medicament for treating and / or preventing CD19-mediated related diseases.

Images