CN115960228A

CN115960228A - Monoclonal antibody specifically binding to CD19 and application thereof

Info

Publication number: CN115960228A
Application number: CN202211073657.1A
Authority: CN
Inventors: 张青; 郑骏年; 孙世硕; 高晓鸽
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2022-09-02
Filing date: 2022-09-02
Publication date: 2023-04-14

Abstract

The invention discloses a monoclonal antibody specifically binding to CD19 and application thereof, and also discloses a nucleic acid molecule for encoding the monoclonal antibody, an expression vector containing the nucleic acid molecule, a host cell and a host cell population containing the expression vector, a pharmaceutical composition, an antibody-drug conjugate and a kit.

Description

Monoclonal antibody specifically binding to CD19 and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, relates to an anti-CD19 monoclonal antibody, and particularly relates to a monoclonal antibody specifically binding to CD19 and application thereof.

Background

CD19 is a 95kDa transmembrane glycoprotein, widely expressed on the surface of B lymphocytes, and is an important membrane antigen involved in the processes of B lymphocyte activation, proliferation, differentiation, and antibody production, and in addition, CD19 has the following properties: CD19 is only expressed on the surface of normal or malignant B lymphocytes, and other tissues hardly express or only slightly express; CD19 is not lost during malignant transformation of B lymphocytes, meaning that it can still play a role even in patients who relapse, or in refractory cases; CD19 is not expressed on the surface of stem cells and B-lymphoid progenitor cells, so B-lymphoid cells can be effectively replenished by stem cells, progenitor cells, after treatment is stopped. Due to the above characteristics of CD19, CD19 has attracted considerable attention in recent years as a molecular target for immunotherapy of B lymphocyte malignancies, and both preclinical studies and clinical studies have shown the potential value of CD19 protein in immunotherapy of B lymphocyte malignancies, including immunotherapy of Acute Lymphocytic Leukemia (ALL), chronic Lymphocytic Leukemia (CLL), non-hodgkin's lymphoma, and the like. The CD 19-targeted therapeutic regimen is widely applied to the treatment of B lymphocyte malignant tumors clinically, such as monoclonal antibodies, bispecific antibodies and the like. At present, CD19 has become an ideal target for targeted therapy of B lymphocyte malignancies.

With the increasing clinical application of monoclonal antibodies, relevant researchers find that the monoclonal antibodies show good treatment effects in malignant tumors, organ transplantation and treatment of connective tissue diseases through research. The monoclonal antibody medicine has the characteristics of high targeting property, good purity and the like, and in recent years, the monoclonal antibody medicine is developed rapidly, and some monoclonal antibody medicines have been applied to clinical treatment of various diseases. However, the monoclonal antibody drug is difficult to develop in the early stage, needs to comprehensively consider various factors, and often needs a large amount of screening work; moreover, some monoclonal antibodies have the problems of unsatisfactory specificity or side effects, and the like, so that the application of the monoclonal antibodies in clinic is hindered. Although there are reports related to monoclonal antibodies targeting CD19 in the prior art, the prior art still has the defects of poor specificity and the like with CD19. In the face of the demand of B lymphocyte malignant tumor patients for therapeutic drugs, especially for monoclonal antibody drugs, at present, a monoclonal antibody with better specificity to CD19 still needs to be provided clinically.

Disclosure of Invention

In view of the above, in order to overcome the technical problems in the prior art, the present invention provides a monoclonal antibody specifically binding to CD19 and applications thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a monoclonal antibody against CD19.

Further, the monoclonal antibody comprises a heavy chain variable region HCVR, a light chain variable region LCVR;

preferably, the HCVR comprises HCDR1, HCDR2, HCDR3;

preferably, the LCVR comprises LCDR1, LCDR2, LCDR3;

more preferably, the HCDR1 comprises an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence having at least 90% identity with SEQ ID NO. 1;

more preferably, the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence having at least 90% identity with SEQ ID NO. 2;

more preferably, the HCDR3 comprises an amino acid sequence as shown in SEQ ID No. 3 or an amino acid sequence having at least 90% identity to SEQ ID No. 3;

more preferably, the LCDR1 comprises the amino acid sequence as shown in SEQ ID No. 4 or an amino acid sequence having at least 90% identity to SEQ ID No. 4;

more preferably, the LCDR2 comprises an amino acid sequence as shown in SEQ ID No. 5 or an amino acid sequence having at least 90% identity to SEQ ID No. 5;

more preferably, the LCDR3 comprises the amino acid sequence as shown in SEQ ID No. 6 or an amino acid sequence having at least 90% identity to SEQ ID No. 6;

most preferably, the amino acid sequence of the HCVR is as set forth in SEQ ID NO 7;

most preferably, the amino acid sequence of the LCVR is as shown in SEQ ID NO 8;

most preferably, the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 11.

In a specific embodiment of the invention, said amino acid sequence having at least 90% identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 11 refers to an amino acid sequence obtained after replacing an amino acid in any one or more positions with any other amino acid on the basis of an amino acid sequence corresponding to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 11, as long as the amino acids having at least 90% identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8 or SEQ ID NO 11 as described herein are within the scope of the present invention.

CD19 is a surface protein expressed on B lymphocytes and follicular dendritic cells, belongs to a member of the immunoglobulin (Ig) superfamily, is located on the short arm of chromosome 16 (1693.2), and encodes a type I transmembrane glycoprotein of 556 amino acids with a molecular weight of 95KD. CD19 is closely related to B cell activation, signal transduction and growth regulation, is a functional receptor molecule on the surface of B lymphocytes, forms a B cell dual antigen binding model when B cell antigen receptors recognize antigens, and participates in Ca in B cells ²⁺ Regulate the activation and proliferation of B cells. CD19 is widely used as an important marker in diagnosis, prognosis and treatment of leukemia, lymphoma and immune system diseases.

In a second aspect of the invention, a pharmaceutical composition is provided.

Further, the pharmaceutical composition comprises an effective amount of the monoclonal antibody of the first aspect of the invention;

preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;

preferably, the pharmaceutical composition is for use in the treatment of a CD 19-associated hematological malignancy;

more preferably, the CD 19-associated hematological malignancy comprises multiple myeloma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-hodgkin's lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, burkitt's lymphoma, follicular lymphoma, anaplastic large cell lymphoma, diffuse large B cell lymphoma.

In a third aspect of the invention, an antibody-drug conjugate is provided.

Further, the antibody-drug conjugate comprises:

(1) A monoclonal antibody according to the first aspect of the invention;

(2) A small molecule substance conjugated to the monoclonal antibody;

preferably, the small molecule substance comprises a cytotoxin, a therapeutic agent;

more preferably, the cytotoxin comprises MMAE, DM1, PE-T20-KDEL, PE4E, PE40, PE38, PE25, PE38QQR, PE35, PE38KDEL, PE38DKEL, PE38RDEL, PE38KNEL, TLR8 agonist, doxorubicin, methotrexate, mitomycin, fluorouracil, vinblastine, calicheamicin, duocarmycin, pyrrolobenzodiazepine, camptothecin analogs, irinotecan;

more preferably, the therapeutic agent comprises an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, an inhibitor of chromatin function, an antiangiogenic agent, an antiestrogen, an antiandrogen, an immunomodulator.

In a fourth aspect, the invention provides a nucleic acid molecule.

Further, the nucleic acid molecule comprises a nucleotide sequence encoding the HCVR of the monoclonal antibody of the first aspect of the invention, a nucleotide sequence encoding the LCVR of the monoclonal antibody of the first aspect of the invention, or a nucleotide sequence encoding the monoclonal antibody of the first aspect of the invention;

preferably, the nucleotide sequence encoding HCVR is as shown in SEQ ID NO 9;

preferably, the nucleotide sequence encoding the LCVR is shown in SEQ ID NO. 10;

preferably, the nucleotide sequence encoding the monoclonal antibody is shown as SEQ ID NO. 15.

Further, the nucleic acid molecule is isolated or purified. The sequence of the DNA molecule can be obtained by conventional techniques, or by using hybridoma technology. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Typically, long fragments are obtained by first synthesizing a plurality of small fragments and then ligating them together.

At present, the DNA sequence encoding the monoclonal antibody (or fragment thereof, or derivative thereof) of the present invention can be obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the sequence of the monoclonal antibody of the invention by chemical synthesis.

In a fifth aspect of the invention, an expression vector is provided.

Further, the expression vector comprises the nucleic acid molecule according to the fourth aspect of the invention;

preferably, the vector comprises a plasmid vector, a virus-derived vector, a phage vector;

more preferably, the virus-derived vector includes a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a poxvirus vector, a herpesvirus vector.

Further, the expression vector may further comprise a vector of a suitable promoter or control sequence. These expression vectors may be used to transform an appropriate host cell so that it can express the protein. The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: bacterial cells such as E.coli, streptomyces; salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO cells, COS7 cells, 293 cells, NSO cells or Bowes melanoma cells.

If desired, the recombinant monoclonal antibody can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

In a sixth aspect the invention provides an engineered host cell.

Further, the engineered host cell comprises a nucleic acid molecule according to the fourth aspect of the invention or an expression vector according to the fifth aspect of the invention;

preferably, the host cell includes prokaryotic cells, eukaryotic cells;

more preferably, the prokaryotic cell comprises a bacterium, actinomycete, cyanobacterium, mycoplasma, chlamydia, rickettsia;

most preferably, the bacteria include escherichia coli, bacillus subtilis, salmonella typhimurium, pseudomonas, streptomyces, staphylococcus;

more preferably, the eukaryotic cell comprises a mammalian cell, an insect cell, a plant cell, a yeast cell.

A seventh aspect of the invention provides a population of host cells.

Further, the population of host cells comprises engineered host cells according to the sixth aspect of the invention;

preferably, the population of host cells further comprises host cells that do not comprise a nucleic acid molecule according to the fourth aspect of the invention or an expression vector according to the fifth aspect of the invention;

more preferably, the host cell includes prokaryotic cells, eukaryotic cells;

most preferably, the prokaryotic cell comprises a bacterium, actinomycete, cyanobacterium, mycoplasma, chlamydia, rickettsia;

most preferably, the eukaryotic cell comprises a mammalian cell, an insect cell, a plant cell, a yeast cell.

An eighth aspect of the invention provides a kit.

Further, the kit comprises a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention.

For convenience in detection, the kit provided by the present invention may further comprise other detection reagents or auxiliary reagents, such as reagents conventionally used in ELISA kits, besides the monoclonal antibody according to the first aspect of the present invention, the nucleic acid molecule according to the fourth aspect of the present invention, the expression vector according to the fifth aspect of the present invention, the engineered host cell according to the sixth aspect of the present invention, and the host cell population according to the seventh aspect of the present invention, the properties of these reagents and their preparation methods are well known to those skilled in the art, such as chromogenic reagents, markers, secondary antibodies, sensitizers, etc. It will be understood by those skilled in the art that various variations of the detection kit are encompassed by the present invention, as long as the monoclonal antibody provided by the present invention is utilized as a reagent for recognizing CD19 therein. In addition, instructions for use may be included in the kit to specify the method of use of the reagents loaded therein. After obtaining the monoclonal antibody and/or the kit provided by the present invention, various immunology related methods can be used to detect whether CD19 is present or not in a sample, or a specific amount thereof.

A ninth aspect of the present invention provides any one of the following methods:

(1) A method of producing a monoclonal antibody according to the first aspect of the invention, the method comprising the steps of: culturing the engineered host cell of the sixth aspect of the invention or the population of host cells of the seventh aspect of the invention and isolating the monoclonal antibody of the first aspect of the invention from the culture;

(2) A method for detecting CD19 in a test sample at a non-diagnostic and non-therapeutic destination, said method comprising the steps of: contacting a sample to be tested with the monoclonal antibody of the first aspect of the invention, and detecting the formation of a complex between the monoclonal antibody and CD 19;

preferably, the monoclonal antibody is a monoclonal antibody labeled with a marker useful for detection;

more preferably, the label for detection comprises a fluorescent dye, avidin, paramagnetic atom, radioisotope;

most preferably, the fluorescent pigment is fluorescein, rhodamine, texas Red, phycoerythrin, phycocyanin, allophycocyanin, and polyanemxanthin-chlorophyll protein;

most preferably, the avidin is biotin, avidin, streptavidin, vitellin, avidin;

most preferably, the radioisotope is radioactive iodine, radioactive cesium, radioactive iridium, radioactive cobalt;

(3) A method of making an engineered host cell according to the sixth aspect of the invention, the method comprising the steps of: introducing an expression vector according to the fifth aspect of the invention into a host cell;

preferably, the methods of introduction include physical methods, chemical methods, biological methods;

more preferably, the physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation;

more preferably, the chemical process comprises a colloidal dispersion system, a lipid-based system;

most preferably, the colloidal dispersion system comprises macromolecular complexes, nanocapsules, microspheres, beads;

most preferably, the lipid-based system comprises an oil-in-water emulsion, micelles, mixed micelles, liposomes;

more preferably, the biological method comprises DNA vectors, RNA vectors, lentiviral vectors, poxvirus vectors, herpes simplex virus vectors, adenoviral vectors, adeno-associated virus vectors;

(4) A method of specifically inhibiting CD19 activity in vitro, the method comprising the steps of: the nucleic acid molecule according to the fourth aspect of the present invention is introduced into a cell of an organism, and the activity of CD19 is inhibited by expressing the monoclonal antibody according to the first aspect of the present invention.

In addition, the invention also provides a method for diagnosing whether a subject has a CD19 related disease.

Further, the method comprises the steps of: detecting the amount of CD19 in a sample from the subject using a monoclonal antibody according to the first aspect of the invention;

preferably, the method further comprises: comparing the amount of said CD19 in a sample from said subject to its amount in a known standard or reference sample and determining whether the level of CD19 in the sample from said subject falls within the level of CD19 of a CD 19-associated disease;

further, the CD 19-associated diseases include hematological malignancies, and any diseases associated with CD19 are within the scope of the present invention.

In addition, the invention also provides a method for treating a subject suffering from a CD 19-associated disease.

Further, the method comprises administering to a subject in need thereof an effective amount of the pharmaceutical composition according to the second aspect of the invention, the antibody-drug conjugate according to the third aspect of the invention.

A tenth aspect of the invention provides the use of any one of the following aspects:

(1) Use of a monoclonal antibody according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, an antibody-drug conjugate according to the third aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention for the preparation of a medicament for the prevention and/or treatment of a CD 19-associated hematologic malignancy;

(2) Use of a monoclonal antibody according to the first aspect of the invention, a pharmaceutical composition according to the second aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention for the preparation of an antibody-drug conjugate for the prevention and/or treatment of a CD 19-associated hematological malignancy;

(3) Use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention, a kit according to the eighth aspect of the invention for the detection of a CD19 protein or antigenic fragment thereof;

(4) Use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention, or a kit according to the eighth aspect of the invention, for the manufacture of a product for the detection of a CD19 protein or antigenic fragment thereof;

(5) Use of a monoclonal antibody according to the first aspect of the invention, a nucleic acid molecule according to the fourth aspect of the invention, an expression vector according to the fifth aspect of the invention, an engineered host cell according to the sixth aspect of the invention, a population of host cells according to the seventh aspect of the invention, a kit according to the eighth aspect of the invention, in the manufacture of a product for the diagnosis of a CD 19-associated hematological malignancy;

(6) The use of a monoclonal antibody according to the first aspect of the invention in the preparation of a nucleic acid molecule, an expression vector, an engineered host cell, a population of host cells;

(7) Use of a nucleic acid molecule according to the fourth aspect of the invention in the preparation of an expression vector, an engineered host cell, a population of host cells;

(8) Use of an expression vector according to the fifth aspect of the invention in the preparation of an engineered host cell, population of host cells;

(9) Use of an engineered host cell according to the sixth aspect of the invention in the preparation of a population of host cells;

preferably, the CD 19-associated hematological malignancy comprises multiple myeloma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-hodgkin's lymphoma, mucosa-associated lymphoid tissue lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, burkitt's lymphoma, follicular lymphoma, anaplastic large cell lymphoma, diffuse large B cell lymphoma.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the invention provides a novel anti-CD19 monoclonal antibody, a nucleic acid molecule for encoding the monoclonal antibody, an expression vector containing the nucleic acid molecule, a host cell and a host cell population containing the expression vector, a pharmaceutical composition, an antibody-drug conjugate and a kit, lays a foundation for development of B lymphocyte malignant tumor drugs, treatment of B lymphocyte malignant tumor, mechanism research of B lymphocyte malignant tumor and the like, and has an important application prospect in treatment of CD19 related diseases.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a diagram showing the results of SDS-PAGE identification of a purified CD19 antibody;

FIG. 2 is a diagram showing the flow verification results of the CD19 antibody prepared according to the present invention;

FIG. 3 is a schematic structural diagram of Ctrl-CAR and CD19-CAR, wherein Anti-CD19 scFv is a newly screened Anti-human CD19 antibody (clone 6E1B 8), namely the antibody screened in example 1 of the present invention;

FIG. 4 is a flow chart of the preparation of CAR-T cells according to the present invention;

FIG. 5 is a graph showing the results of flow-based detection of the positive rate of CAR expression, on day 10 of in vitro cell culture and amplification, 2X 10 cells were collected ⁵ The individual cells were used for flow detection and CAR expression was detected with a primary anti-C-myc antibody and a fluorescent secondary antibody labeled with Alexa Fluor 647;

FIG. 6 is a graph showing the results of ELISA assay for detecting cytokine-releasing ability of CD19-CAR-T cells, taking 1X 10 ⁴ Co-incubating Ctrl-T or CD19-CAR-T cells and human leukemia cells Raji or Nalm-6 cells respectively in a U-shaped bottom 96-well plate for 24 hours according to the proportion of 2; data statistics adopts Independent sample T Test (Independent-Samples T Test); * P<0.001；****P<0.0001；

FIG. 7 is a graph showing the results of flow assay of the killing effect of CD19-CAR-T on leukemia cells, which is 2X 10 ⁵ Each target cell (Raji or Nalm-6) was compared with 4X 10 ⁵ After the Ctrl-T or CD19-CAR-T cells are co-incubated for 48h, the cells are centrifuged and stained, and the killing effect is detected by a flow cytometer.

Detailed Description

The present invention has been extensively and extensively studied to select a novel anti-CD19 monoclonal antibody that specifically binds to CD19, comprising HCVR comprising HCDR1, HCDR2 and HCDR3 and LCVR comprising LCDR1, LCDR2 and LCDR3; the amino acid sequence of the HCDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the HCDR2 is shown as SEQ ID NO. 2, the amino acid sequence of the HCDR3 is shown as SEQ ID NO. 3, the amino acid sequence of the LCDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the LCDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the LCDR3 is shown as SEQ ID NO. 6.

In the present invention, the term "monoclonal antibody", and "monoclonal antibody" and "antibody" are used interchangeably to refer to an antibody molecule having a single molecular composition, obtained from a population of substantially identical antibodies. The monoclonal antibody exhibits a single binding specificity and affinity for a particular epitope. Typically, an immunoglobulin has a heavy chain and a light chain. Each heavy and light chain includes a constant region and a variable region (regions are also referred to as "domains"). The Light Chain Variable Region (LCVR) and the Heavy Chain Variable Region (HCVR) comprise four Framework Regions (FR) that are interrupted by three hypervariable regions (HVRs), also known as "Complementarity Determining Regions (CDRs)". The CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs for each chain are typically CDR1, CDR2 and CDR3, numbered consecutively from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. CDR1, CDR2 and CDR3 of the LCVR are LCDR1, LCDR2 and LCDR3 respectively, and CDR1, CDR2 and CDR3 of the HCVR are HCDR1, HCDR2 and HCDR3 respectively. Furthermore, the monoclonal antibody of the present invention also includes functional variants of the monoclonal antibody, which can bind to CD19.

Specifically, the functional variants include, but are not limited to: chemically and/or biochemically modified derivatives that are substantially similar in primary structural sequence but that are not included in the parent monoclonal antibody of the invention, e.g., in vitro or in vivo. These modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, formation of disulfide bonds, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolytic processing, phosphorylation.

Alternatively, the functional variant may be a monoclonal antibody as follows: amino acid sequences comprising substitutions, insertions, deletions or combinations thereof of one or more amino acids compared to the amino acid sequence of a parent monoclonal antibody. Further, the functional variant may comprise a truncation of the amino acid sequence at one or both of the amino terminus or carboxy terminus. Functional variants according to the invention may have the same or different, higher or lower binding properties compared to the parent monoclonal antibody, but are still capable of binding to CD19.

In the present invention, the term "nucleic acid molecule" may comprise natural, non-natural or altered nucleotides; and which may comprise natural, non-natural or altered internucleotide linkages, such as phosphoramidate linkages or phosphorothioate linkages, in place of the phosphodiester present between nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleic acid does not comprise any insertions, deletions, inversions, and/or substitutions. However, in some cases it may be suitable for the nucleic acid to comprise one or more insertions, deletions, inversions and/or substitutions, and thus nucleic acids formed by such insertions, deletions, inversions and/or substitutions are also within the scope of the present invention.

In particular embodiments of the invention, the nucleic acid molecules include nucleic acid molecules that can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to enhance the biological stability of the molecule or to enhance the physical stability of the diad formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Some examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylguanosine (queosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine, dihydrouracil 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosylstevioside, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxysine (wybutoxosine), pseudouracil, stevioside, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil and 2, 6-diaminopurine.

In the present invention, suitable promoters contained in the expression vector include natural promoters, non-natural promoters. The choice of promoter (e.g., strong, weak, inducible, tissue-specific, and development-specific) is within the ordinary skill of the artisan. Similarly, combinations of nucleotide sequences and promoters are also within the skill of the artisan. The promoter may be a non-viral promoter or a viral promoter, such as the Cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or a promoter found in the long terminal repeats of murine stem cell viruses.

Specific illustrative examples of the pharmaceutically acceptable excipient in the pharmaceutical composition provided by the present invention include, but are not limited to: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; powdered tragacanth; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, for example sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. Such pharmaceutically acceptable excipients are described in detail in Remington's Pharmaceutical Sciences (19 th ed., 1995).

The pharmaceutical composition provided by the present invention can be prepared into various dosage forms according to the needs, and can be administered by a physician in a dose beneficial to a patient according to factors such as the type, age, weight and general disease condition of the patient, administration mode, and the like. Administration may be by injection or other therapeutic means, for example. Suitable modes of administration include those suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion, intravenous, inhalable or subcutaneous form. Where the product is for injection or infusion, it may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, preservative, stabilising and/or dispersing agents. Alternatively, monoclonal antibodies provided according to the invention can be in dry form for reconstitution with a suitable sterile liquid prior to use. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The pharmaceutical compositions provided herein for intravenous administration comprise from about 0.01mg/kg to about 30mg/kg of a monoclonal antibody according to the first aspect of the invention per subject per day. The monoclonal antibodies described herein may be provided in lyophilized form and rehydrated with sterile water prior to administration, but they are also provided in sterile solutions of known concentration. The monoclonal antibody solution was then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 to 15mg/kg body weight. In one embodiment, a higher loading dose is administered followed by a maintenance dose at a lower level. For example, an initial loading dose of 4mg/kg antibody, antigen-binding fragment, antibody-drug conjugate (or a corresponding dose comprising antibody, antigen-binding fragment, antibody-drug conjugate) may be infused over a period of approximately 90 minutes, followed by a weekly maintenance dose of 2mg/kg over a period of 30 minutes for 4 to 8 weeks if the previous dose was well tolerated.

The monoclonal antibodies provided by the invention can be used in an unseparated or isolated form. Furthermore, the monoclonal antibodies of the invention may be used alone or in a mixture comprising at least one of the monoclonal antibodies of the invention (or variants or fragments thereof). In other words, the monoclonal antibodies may be used in combination, for example as a pharmaceutical composition comprising two or more monoclonal antibodies, variants or fragments thereof of the invention. For example, monoclonal antibodies having different but complementary activities can be combined in a therapeutic regimen to achieve a desired prophylactic, therapeutic or diagnostic effect, but alternatively monoclonal antibodies having the same activity can be combined in a therapeutic regimen to achieve a desired prophylactic, therapeutic or diagnostic effect.

The monoclonal antibodies or pharmaceutical compositions provided by the invention can be tested in a suitable animal model system prior to use in humans. Such animal model systems include, but are not limited to, mice, monkeys.

The invention is further illustrated below with reference to specific examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the same. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

EXAMPLE 1 hybridoma monoclonal antibody preparation

1. Immunization of laboratory animals

(1) Antigen emulsification: 0.05mg of CD19 antigen is diluted by PBS and then mixed with Freund's adjuvant according to the volume ratio of 1 (the total volume is 0.8 mL) and emulsified for 3-5min at the temperature of 4 ℃ (taking out the stirrer head, lightly tapping on the mouth of a container filled with water, and dropping a drop of emulsified immunogen adhered on a stirring iron wire into the water, wherein the immunogen is emulsified when the immunogen does not diffuse when meeting water, and if the immunogen does diffuse, the emulsification time is continuously prolonged). Ensuring that the emulsion is well emulsified using the shortest time.

(2) The first immunization was performed with Freund's complete adjuvant, and the subsequent booster immunization was performed with Freund's incomplete adjuvant.

(3) Injection of antigen: the emulsified antigen was transferred to a 1mL syringe and air bubbles were removed from the syringe. The foamed ice bags were stored (emulsified on the day, immunised on the day). The mice to be immunized are taken out of the cage and placed in a special fixing frame, and multipoint subcutaneous injection is carried out on the back. During injection, the hair of the mouse at the injection site is removed, the exposed skin is disinfected with alcohol, the skin is lifted to form a triangle, and the needle is inserted into the skin at an angle of 15 degrees, the injection depth is 1-2cm, and the mouse is carefully not punctured into the muscle.

(4) The immune cycle: each immunization was 2 weeks apart.

(5) A total of 3-5 immunizations are given.

(6) Blood is taken after one week of three-immunization to detect the antibody titer, and if the titer reaches the impact immunity, the eyeball is picked, blood is taken, the neck is removed, and the spleen is taken.

(7) A total of 6 mice were immunized.

2. Potency assay

(1) Coating: antigen CD19 was diluted to 2. Mu.g/mL with coating CB, 100. Mu.L/well was added to the microplate and incubated at 4 ℃ overnight or 37 ℃ for 2h.

(2) And (3) sealing: taking out the liquid in the dry-punched holes of the ELISA plate, sealing with 5% skimmed milk (PBS dissolved), adding 200 μ L/hole of the ELISA plate, incubating at 37 ℃ for 2h, and washing the plate with TBS for 3 times.

(3) Adding a primary antibody: the immune sera were diluted in duplicate (blank sera as negative control) at 100 μ L per well and incubated at 37 ℃ for 1h at 1. The TBS plate was washed 3 times.

(4) Adding a secondary antibody: HRP-labeled goat anti-mouse IgG (dilution ratio of 1 to 10000) was added to the plate at 100. Mu.L/well, incubated at 37 ℃ for 40min, and the plate was washed 5 times with TBS.

(5) Color development: adding TMB substrate, 90 μ L/well, and keeping away from light at 37 deg.C for 5-20min.

(6) And (4) terminating: adding stop solution, 50 mu L/hole, reading by an enzyme-labeling instrument (the wavelength is 450 nm), and obtaining the maximum dilution of the positive reaction as the serum titer of the immunized mouse.

3. Preparation of feeder cells

(1) Preparation of mouse abdominal cavity macrophage

1) A Balb/C mouse is taken for 8-10 weeks, an eyeball is picked, blood is discharged, the neck is removed, and the mouse is killed by soaking in 75% alcohol for disinfection for 5min.

2) The mouse was moved into a clean bench and fixed supinely on the foam padded with sterilized newspaper using a sterilized needle, the skin of the abdomen was opened with scissors forceps, and the peritoneum was fully exposed by blunt dissection.

3) The peritoneal rinse was filled with 3-5mL DMEM using a 5mL syringe and the media aspirated into a 50mL centrifuge tube.

(2) Mouse spleen cell preparation

1) Aseptically cutting off peritoneum, exposing abdominal cavity, cutting off connective tissue to separate spleen, dividing into four sections, placing in the center of 200 mesh separation net, folding the separation net twice, clamping the open end of the separation net with hemostatic forceps, adding 3-6mL DMEM, grinding, blowing, mixing well, sucking out, and transferring to 50mL centrifuge tube.

2) Add 3-6mL of DMEM again, blow and mix well to form a single spleen cell suspension, aspirate and move to a 50mL centrifuge tube.

(3) Macrophages and spleen cells were collected, centrifuged at 1500rpm for 5min, the supernatant was discarded, cells were suspended in an amount of 1mL fetal bovine serum from one mouse, and stored at 4 ℃.

4. Cell fusion

(1) Activation and preparation of SP2/0

1) Recovering SP2/0 cells before fusion, subculturing in 10% FBS DMEM medium for one week, and washing with 1 × 10 DMEM medium for 2 times in sterile condition when cell growth state is good ⁶ One cell/mouse was injected subcutaneously on the back. After the tumor grows to 1-3cm in diameter, the mice which are qualified for immunization are subjected to impact immunization and fused after 3 days.

2) Aseptically cutting off the back skin, cutting off tumor mass, cutting into small pieces, transferring to the center of a 200-mesh filter screen, placing in a 10cm dish, adding 5-10mL of DMEM, grinding into single cell suspension, and completely sucking out into a 50mL centrifugal tube.

3) Then 5mL of DMEM was added, the mixture was vortexed and aspirated into a 50mL centrifuge tube.

4) And then 5mL of DMEM is added, the mixture is uniformly blown and sucked out to a 50mL centrifuge tube.

5) Gently adding 20mL of myeloma cell suspension into a 50mL centrifuge tube which is added with 20mL of lymphocyte separation liquid in advance along the tube wall, centrifuging for 15min at 2000r/min, removing the upper layer of cell suspension, transferring the middle-layer white myeloma cell suspension into another 50mL centrifuge tube, suspending by using 10mL of DMEM medium, centrifuging for 5min at 1500r/min, and washing cells twice. The supernatant was discarded, and myeloma cells were resuspended in 10mL DMEM medium and counted for use.

(2) Preparation of immune spleen cells

One immune qualified Balb/C mouse is taken, the eyeball is removed, the neck is removed and the mouse is killed after bloodletting, blood is collected and separated, and serum is used as positive control serum during antibody detection. The spleen is taken out by preparing the trophoblast, and immune spleen cell suspension is separated and prepared.

(3) Cell fusion

1) 1mL PEG and 40mL DMEM medium was incubated at 37 ℃ with 5% CO ₂ The incubator is preheated.

2) The number of myeloma cells and spleen cells were mixed at a ratio of 1.

3) 1mL of PEG preheated at 37 ℃ was added slowly over 60 seconds, and the mixture was allowed to stand for 60 seconds.

4) 40mL of DMEM medium was added to the fusion system to stop the action of PEG, slowly and then rapidly, gently shaking the tube while adding, 1mL (3 sec/drop) dropwise at 1min, 2mL more at 2min, and 7mL of the remaining solution (completed within 5 min). After the first 10mL was added, DMEM medium was then added along the tube wall to 40mL, the cap was tightened, inverted slowly several times, and mixed well. Centrifuge at 800rpm for 5min and discard the supernatant.

5) Suspending the fused mixed cells by 2-4mL of fetal calf serum, adding the fused cells into a semisolid culture medium containing 25% of fetal calf serum of trophoblasts, glutamine, double antibody and HAT according to the amount of the fused cells, and gently mixing the cells. Pouring the mixed semi-solid into 3.5cm dishes (2-3 mL), placing all dishes in a sterilized wet box, at 37 deg.C, 5% CO ₂ Culturing in an incubator.

(4) Selective culture

The number of days of fusion was 0d, and the plate was not moved as much as possible in the preceding 3d, and it was only necessary to pay attention to the presence or absence of contamination of the cells and to maintain the internal environment of the incubator stable. On day 7, 0.5-0.8mL of HT complete medium per dish may be added if the medium becomes more yellow. The colonies can be picked into 96-well plates (200. Mu.L of HT complete medium containing feeder cells per well) and cultured for 2-3 days according to the size of the cell colony by the tenth day of the growth of the cells, and cell culture supernatants can be taken and screened by an indirect ELISA method and an indirect competitive ELISA method. Generally, cells in the plate are picked out in 2-3 batches for culture detection.

5. Subcloning and subcloning assays

(1) Subcloning

1) Feeder cells were prepared the day before subcloning and plated in 96-well plates.

2) And (3) lightly blowing and beating the cells needing subcloning to prepare single cell suspension, adding a counting plate for counting, and calculating the cell density.

3) 100 μ L of cells to be subcloned were diluted to 1X 10 ³ one/mL.

4) 5/well subclone concentration 1X 10 ³ To each mL of cell suspension, 200. Mu.l was added to 3.8mL of medium and mixed (100. Mu.l was added to a 96-well plate), and 100. Mu.l/well was plated 4 pieces to a 96-well plate containing trophoblasts.

5) 0.5/well subclone was mixed in 4mL (5/well) of cell suspension, 700. Mu.L was added to 6.3mL of medium and mixed (100. Mu.L was added to a 96 well plate). 100 u L/hole spread 8 to containing trophoblast 96 well plate. It is also possible to use only 1/well subclone at a concentration of 1X 10 ³ And adding 50 mu L of the cell suspension into 4.95mL of culture medium, uniformly mixing, and paving 6 strips per 100 mu L of the cell suspension into a 96-well plate containing the trophoblasts.

(2) Subclone detection

1) Coating: the antigen was diluted to 2. Mu.g/mL with coating CB, 100. Mu.L/well was added to the microplate, and incubated at 4 ℃ overnight or 37 ℃ for 2h.

2) And (3) sealing: taking out the liquid in the dry-punched holes of the ELISA plate, sealing with 5% skimmed milk (PBS dissolved), adding 200 μ L/hole of the ELISA plate, incubating at 37 ℃ for 2h, and washing the plate with TBS for 3 times.

3) Sample adding: the cell supernatant was applied at 100. Mu.L/well, incubated at 37 ℃ for 1h, and the plate was washed 3 times with TBS.

4) Adding a secondary antibody: HRP-labeled goat anti-mouse IgG (dilution ratio of 1 to 10000) was added to the wells at 100. Mu.L/well, incubated at 37 ℃ for 40min, and the plates were washed 5 times with TBS.

5) Color development: adding TMB substrate, 90 μ L/well, and keeping away from light at 37 deg.C for 20-30min.

6) And (4) terminating: adding stop solution, 50 mu L/hole, reading by an enzyme-labeling instrument (wavelength 450 nm), and obtaining the corresponding dilution value when the OD value is 2.0, namely the titer of the cell.

6. Freezing and restoring expanded culture of cells

(1) Freezing and storing: the number of cells is more than 1 × 10 ⁶ The cells were frozen and centrifuged at 1500rpm for 5min. The cells are frozen by using a freezing medium with the ratio of DMSO to fetal bovine serum being 1. The cell freezing tube is placed in a gradient freezing box at room temperature and then directly placed at-80 ℃ overnight. And taking out the cells in the gradient cryopreservation box, and placing the cells in a liquid nitrogen tank for long-term storage.

(2) And (3) resuscitation: and (3) quickly taking the cell freezing tube out of the liquid nitrogen tank, and quickly shaking the tube in a constant-temperature water bath kettle at 37 ℃ until the cells are melted. Adding 5mL of DMEM medium, mixing, centrifuging at 1200rpm for 5min, discarding the supernatant, adding 20% of complete medium, reacting at 37 deg.C with 5% CO ₂ Culturing in the incubator, and subculturing.

7. Preparation and purification of anti-CD19 monoclonal antibodies

(1) Preparation of anti-CD19 monoclonal antibody

1) The liquid paraffin or incomplete adjuvant is injected into the abdominal cavity of the mouse, and each mouse is injected with 0.5mL of the incomplete adjuvant.

2) After 7-10 days, hybridoma cells were diluted to a concentration of 1X 10 in sterile PBS or DMEM basal medium ⁶ one/mL, 1mL of each mouse was injected into the mouse abdominal cavity using a sterile syringe.

3) After 6 days, the ascites generation condition of the mice is observed every day, if the abdomen is obviously enlarged and the mice feel tense when touched by hands, the ascites can be collected by using a sterile syringe ascites needle.

4) Centrifuging at 10000rpm for 2min, removing cell components and other precipitate, collecting supernatant, and storing at-20 deg.C.

(2) anti-CD19 monoclonal antibody purification

1) And (3) taking the affinity chromatography column, washing the column by 10 times of the volume of the column bed with water, and washing the column by 10 times of the volume of the column bed with sodium acetate buffer solution.

2) Serum was collected, centrifuged at 12000rpm for 10min at 4 ℃ and the supernatant collected, filtered and mixed with sodium acetate buffer.

3) And (4) applying a Protein G column at the speed of 0.5mL/min, collecting breakthrough, and after the sample application is finished, continuously washing the column by using sodium acetate buffer until the column is colorless for G250 detection.

4) Eluting with glacial acetic acid, buffering and washing the column bed, collecting the elution peak, adjusting the pH of the elution peak to be neutral by saturated sodium carbonate, and washing with water for 10 times of the volume of the column bed.

5) The column was then column-packed with 10mL NaCl-sodium azide buffer and placed at 4 ℃.

6) The eluted peak is ultrafiltered and concentrated to equal volume of serum, and is filled into a dialysis bag for dialysis overnight.

7) The liquid is changed once after 12 hours. Taking out the sample, centrifuging at 12000rpm at 4 deg.C for 10min, collecting supernatant, storing at 4 deg.C, and performing SDS-PAGE detection.

8. Results of the experiment

The results of antibody subtype determination are shown in table 1, and show that the antibody subtype prepared by the present invention is IgG1. The SDS detection results of antibody preparation by ascites of CD19 cells and antibody preparation by supernatant of collected CD19 cells are shown in FIG. 1, and the results show that the ascites is subjected to Pro G affinity purification to obtain 95% of CD19-6E1B8 antibody, and the cell supernatant is subjected to Pro G affinity purification to obtain 95% of CD19-6E1B8 antibody. The result of the flow verification of the CD19 antibody prepared by the invention is shown in figure 2, and the result shows that the antibody purified from the cell supernatant and the antibody purified from ascites are not much different from the result of the hybridoma cell supernatant and can be used as final antibodies, and the CD19 antibody prepared by the invention can be specifically combined with CD19 protein on the cell surface.

TABLE 1 statistics of CD19 antibody subtype identification results

The sequencing results of the anti-CD19 antibody prepared by the invention are as follows:

(1) Amino acid sequence of the heavy chain variable region of the CD19 antibody CD19-VH (HCVR):

EVQLKESGPGLVAPSQSLSITCTVSGFSLTSHGVSWVRQPPGKGLEWLGVIWGDGRTSYHSALISRLSISKSDSKSQVFLKLNSLQTDDTATYYCAKKGAYYGSDYRYYAMDSWGQGTSVTVSS(SEQ ID NO:7)；

wherein the amino acid sequence of HCDR 1: GFSLTSHG (SEQ ID NO: 1), the amino acid sequence of HCDR 2: IWGDGRT (SEQ ID NO: 2), amino acid sequence of HCDR 3: AKKGAYYGSDYRYYAMDS (SEQ ID NO: 3);

(2) Amino acid sequence of the variable region of the CD19 antibody light chain CD19-VL (LCVR):

DIQLTQTPLSLPVSLGDQASISCRSSQSLVYSNGNTYLHWYLQKPGQSPKLLIYKASNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKR(SEQ ID NO:8)；

wherein, the amino acid sequence of LCDR 1: QSLVYSNGNTY (SEQ ID NO: 4), the amino acid sequence of LCDR 2: KAS (SEQ ID NO: 5), amino acid sequence of LCDR 3: SQSTHVPWT (SEQ ID NO: 6);

(3) Nucleotide sequence of the heavy chain variable region of the CD19 antibody CD19-VH (HCVR):

GAGGTCCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCAGGTTTCTCATTAACCAGCCATGGTGTAAGCTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTGACGGGAGGACAAGTTATCATTCAGCTCTCATATCCAGACTGAGCATCAGCAAGTCTGACTCCAAGAGCCAAGTTTTCTTAAAACTGAACAGTCTACAAACTGATGACACAGCCACGTACTACTGTGCCAAAAAGGGGGCTTACTACGGTAGTGACTACCGTTACTATGCTATGGACTCCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO:9)；

(4) Nucleotide sequence of the variable region of the CD19 antibody light chain CD19-VL (LCVR):

GACATCCAGCTGACACAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTATACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGCTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG(SEQ ID NO:10)。

example 2 construction of CD19-CAR

The DNA sequence of restriction enzyme cleavage site Nde I (CATATG), initiation codon (ATG), hCD8 signal peptide (GCCTTACCAGGGACCGCCTTGCCTCTGCCTGGCCTGTGCTCCACGCCAGGCCG) and C-myc label (GAGCAGAAGCTGATCAGCGAGGAGGACCTG) are added in sequence at the 5 'end of the DNA sequence encoding CD19-CAR, and the DNA sequence of termination codon (TAA) and restriction endonuclease Spe I (ACTAGT) are added in sequence at the 3' end. The DNA sequences described above were encoded by total gene synthesis. The synthesized DNA fragment was inserted into the lentiviral vector pRRL through restriction enzyme sites Nde I and Spe I to construct a pRRL-CD19-CAR expression plasmid.

The structural schematic diagram of the constructed CD19-CAR is shown in figure 3, the CD19-CAR is obtained by sequentially connecting an EF1 alpha promoter, an hCD8 Signal Peptide (SP), a C-myc tag, an anti-CD19 antibody prepared by the invention, a CD8 hinge region, a transmembrane region, an HVEM costimulatory signal domain and a CD3 zeta intracellular signal conduction domain, wherein the amino acid sequence of the anti-CD19 antibody is shown in SEQ ID NO. 11, the amino acid sequences of the CD8 hinge region and the transmembrane region are shown in SEQ ID NO. 12, the amino acid sequence of the HVEM costimulatory signal domain is shown in SEQ ID NO. 13, the amino acid sequence of the CD3 intracellular signal conduction domain is shown in SEQ ID NO. 14, the nucleotide sequence of the anti-CD19 antibody is shown in SEQ ID NO. 15, the nucleotide sequences of the CD8 hinge region and the transmembrane region are shown in SEQ ID NO. 16, the nucleotide sequence of the EM costimulatory signal domain is shown in SEQ ID NO. 17, and the nucleotide sequence of the CD3 zeta signal domain is shown in SEQ ID NO. 18. The amino acid sequence of the CD19-CAR is shown as SEQ ID NO. 19, and the nucleotide sequence of the CD19-CAR is shown as SEQ ID NO. 20. The nucleotide sequence of the EF1 alpha promoter is shown as SEQ ID NO. 21, the amino acid sequence of the hCD8 signal peptide is shown as SEQ ID NO. 22, the nucleotide sequence of the hCD8 signal peptide is shown as SEQ ID NO. 23, the amino acid sequence of the C-myc tag is shown as SEQ ID NO. 24, and the nucleotide sequence of the C-myc tag is shown as SEQ ID NO. 25.

Example 3 preparation of CD19-CAR-T cells

1. Packaging Lentiviral pRRL-CD19-CAR

(1) Pretreating a culture dish: adding 5mL of 10% polylysine solution into a 15cm cell culture dish, uniformly covering the bottom of the culture dish, standing in an ultra-clean workbench for 5min, recovering, washing with PBS, and air drying;

(2) Selecting pre-cultured 293T cells, determining the condition to be good, digesting, counting, and collecting 1.8 × 10 ⁷ Plating, and putting the plates in an incubator for overnight incubation;

(3) Before plasmid transfection, the PBS, the plasmid and the PEI are heated to room temperature;

(4) Preparation of transfection reagents: PEI (1. Mu.g/. Mu.L) was removed from the freezer at-20 ℃ and allowed to rewarming at room temperature until PEI was completely dissolved. The plasmid is taken out from a refrigerator at the temperature of-20 ℃, thawed at room temperature and mixed evenly (Vector plasmid, packing plasmid delta8.9, envelope plasmid VSVG);

(5) Preparation of PEI/DNA complexes (the volumes or plasmids used below are scaled up in an amount of 15cm, and the same), (1) PEI mixtures: 75 μ L PEI (1 μ g/. Mu.L) was diluted with 500 μ L PBS; (2) DNA mixture: adding 500 mu L of PBS into an EP tube, respectively adding 27 mu g of vector plasmid, 18 mu g of packaging plasmid and 9 mu g of VSVG plasmid, and carefully and uniformly mixing by blowing up and down a pipette gun; (3) while gently vortexing the plasmid mixture, the PEI mixture was added thereto and allowed to stand at room temperature for 15min; (4) the DNA/PEI complex is slowly and dropwise added into a 15cm culture dish in a dispersing way, the culture dish is immediately shaken in a shape like a Chinese character 'mi', and the mixture is fully and evenly mixed. After culturing the culture dish at 37 ℃ for 6 hours, removing the culture medium containing PEI, and replacing with fresh DMEM complete culture medium containing 1% pyruvic acid;

(6) After transfection for 24h, collecting supernatant, changing liquid, and placing the supernatant in an incubator for continuous culture;

(7) Centrifuging the supernatant collected after transfection for 24h, 48h and 72h at 2000rpm for 5min, and thoroughly removing cell debris;

(8) Sucking the culture medium containing virus solution by using a 20mL syringe, filtering the culture medium through a 0.45 mu m filter membrane, and sterilizing;

(9) After trimming, centrifuging by using an ultracentrifuge at 25000rpm for 3 hours;

(10) After centrifugation, the supernatant was discarded, resuspended in L500 basal medium, and left overnight at 4 ℃. Subpackaging with EP tube, and storing at-80 deg.C.

2. Detection of titre of the prepared Virus

(1) Using well-conditioned 293T cells in 24-well plates, 1X 10 ⁵ Putting the culture medium in an incubator for overnight culture;

(2) Adding 1. Mu.L, 2. Mu.L, 4. Mu.L and 8. Mu.L of virus solution and Polybrene (final concentration 8. Mu.g/mL) as transfection assisting agent on the next day according to the predetermined lentivirus concentration gradient;

(3) On day four 293T cells were digested with 5mM EDTA and washed with PBS;

(4) Incubating 30min with a primary anti-c-myc antibody (using 2% FPBS 400-fold dilution) in the dark, 2% FPBS washing;

(5) Incubation with secondary antibody Alexa Fluor647 (using 2% FPBS 800 fold dilution) in the dark for 30min,2% FPBS wash;

(6) Cells were resuspended in 200. Mu.L of 1% formaldehyde and examined on a flow machine. Calculating the virus titer;

(7) Selecting the concentration with the positive rate of 10% -20% to calculate the virus titer, and adopting the formula: number of cells positive rate/optimal positive rate corresponds to concentration.

3. Activation of T cells

Peripheral blood from healthy donors was collected from the subsidiary hospital of xuzhou medical university, all subjects had signed informed consent, and peripheral blood mononuclear cells PBMCs were isolated and cryopreserved for use. The PBMC cell suspension prepared above was taken and plated on a 10cm dish. Standing in an incubator for 2h to remove adherent cells such as MDSC, collecting suspended cells, and counting. Adjusting the cell density to 1X 10 using T cell complete medium ⁶ and/mL. And cleaning the magnetic beads, calculating the volume of the required CD3/CD28 magnetic beads according to the equivalent amount of the cells, placing the magnetic beads on a magnetic frame, cleaning the CD3/CD28 magnetic beads by using a basic culture medium, and suspending the magnetic beads by using a complete culture medium after the cleaning is finished. Mixing the magnetic beads and the cells, and uniformly blowing and stirring. The mixture of magnetic beads and cells was seeded in 48-well plates.

4. Lentivirus infection

(1) After 24 hours of stimulation of the T cells with anti-human CD3/CD28 antibody-coupled magnetic beads, the medium was discarded in half. The grouping is as follows: untreated, CAR-T group. Taking out the virus from-80 ℃, placing the virus on ice for slow thawing, adding lentivirus solution according to MOI =5, and adding transfection-promoting reagent Polybrene to the virus solution with the final concentration of 5 mug/mL;

(2) Centrifuging with horizontal centrifuge, setting the centrifuge to rise and

fall

4 and 1, 1500g,2h, placing in incubator overnight, and supplementing 500 μ L of complete culture medium to each well on day 2;

(3) After 72h of infection, 400g of cells were harvested and centrifuged for 5min. The supernatant was discarded, resuspended in complete medium, and then placed on a magnetic rack to remove the beads. And continuously culturing until 10 days, and detecting the positive rate.

5. Results of the experiment

The structural schematic diagram of Ctrl-CAR and CD19-CAR is shown in figure 3, the preparation flow diagram of CAR-T cells is shown in figure 4, the result diagram of the flow detection of the positive rate of CAR expression is shown in figure 5, and the result shows that the CD19-CAR-T cells have higher positive rate of CAR expression.

Example 4 ELISA assay to examine the cytokine Release Capacity of CD19-CAR-T cells

1. Experimental methods

Respectively taking 1 × 10 ⁴ The Ctrl-T or CD19-CAR-T cells were incubated with human leukemia cells Raji or Nalm-6 cells, respectively, in a ratio of 2 for 24 hours in a U-bottom 96-well plate, the supernatants were centrifuged and the amount of IFN-. Gamma.and Granzyme B released by CAR-T cells after stimulation of target cells was measured by ELISA.

2. Results of the experiment

The result shows that after the group of CD19-CAR-T cells and human leukemia cells Raji or Nalm-6 cells are incubated together, the release amount of cytokines IFN-gamma and Granzyme B in the cell supernatant is obviously superior to that of the Ctrl-T group, namely the CD19-CAR-T cells constructed by the invention secrete higher cytokines IFN-gamma and Granzyme B in the cell supernatant (see figure 6).

Example 5 flow assay of the killing Effect of CD19-CAR-T on human leukemia cells

1. Experimental methods

2 x 10 to ⁵ A target cell (human leukemia cell Raji or Nalm-6) and 4X 10 ⁵ And mixing the Ctrl-T cells or the CD19-CAR-T cells according to the effective target ratio of 1 to 2, culturing the mixture in a 24-well plate, putting the plate into an incubator for incubation for 48h, centrifuging the cells, taking out the cells for staining, and detecting the killing effect of the CAR-T cells on target cells by using a flow cytometer. CD45 ⁺ CD3 ⁺ Being CAR-T cells, CD45 ^- CD3 ^- Is a target cell and is subjected to statistical analysis.

2. Results of the experiment

The result shows that the CD19-CAR-T cell treatment group constructed by the invention has a better killing effect on human leukemia cells Raji or Nalm-6 and is remarkably superior to a Ctrl-T cell control group, namely CD19-CAR-T cells containing CAR genes have specific killing activity on cells expressing CD19, and the difference is remarkable compared with the control group, so that the CD19-CAR-T cells constructed by the invention have a stronger killing effect on CD19 positive cells (see figure 7), and the CD19-CAR-T cells constructed by the invention can be further used for effectively treating B lymphocyte malignant tumors.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

1. An anti-CD19 monoclonal antibody comprising a heavy chain variable region HCVR, a light chain variable region LCVR;

preferably, the HCVR comprises HCDR1, HCDR2, HCDR3;

preferably, the LCVR comprises LCDR1, LCDR2, LCDR3;

more preferably, the HCDR1 comprises an amino acid sequence as shown in SEQ ID No. 1 or an amino acid sequence having at least 90% identity to SEQ ID No. 1;

more preferably, the HCDR2 comprises an amino acid sequence as shown in SEQ ID NO. 2 or an amino acid sequence having at least 90% identity to SEQ ID NO. 2;

more preferably, the HCDR3 comprises an amino acid sequence as shown in SEQ ID NO. 3 or an amino acid sequence having at least 90% identity with SEQ ID NO. 3;

more preferably, the LCDR1 comprises an amino acid sequence as shown in SEQ ID No. 4 or an amino acid sequence having at least 90% identity to SEQ ID No. 4;

2. A pharmaceutical composition comprising an effective amount of the monoclonal antibody of claim 1;

3. An antibody-drug conjugate, wherein the antibody-drug conjugate comprises:

(1) The monoclonal antibody of claim 1;

(2) A small molecule substance conjugated to the monoclonal antibody;

more preferably, the therapeutic agent comprises an antimetabolite, an antitumor antibiotic, an inhibitor of mitosis, an inhibitor of chromatin function, an antiangiogenic agent, an antiestrogen, an antiandrogen, an immunomodulator.

4. A nucleic acid molecule comprising a nucleotide sequence encoding a HCVR of a monoclonal antibody as recited in claim 1, a nucleotide sequence encoding a LCVR of a monoclonal antibody as recited in claim 1, or a nucleotide sequence encoding a monoclonal antibody as recited in claim 1;

preferably, the nucleotide sequence encoding HCVR is as shown in SEQ ID NO 9;

preferably, the nucleotide sequence for encoding LCVR is shown in SEQ ID NO. 10;

5. An expression vector comprising the nucleic acid molecule of claim 4;

6. An engineered host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5;

preferably, the host cell includes prokaryotic cells, eukaryotic cells;

7. A population of host cells, wherein the population of host cells comprises the engineered host cell of claim 6;

preferably, the population of host cells further comprises host cells that do not comprise the nucleic acid molecule of claim 4 or the expression vector of claim 5;

more preferably, the host cell includes prokaryotic cells, eukaryotic cells;

8. A kit comprising the monoclonal antibody of claim 1, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7.

9. Any of the following methods, wherein the method comprises:

(1) A method of producing the monoclonal antibody of claim 1, comprising the steps of: culturing the engineered host cell of claim 6 or the population of host cells of claim 7, and isolating the monoclonal antibody of claim 1 from the culture;

(2) A method for detecting CD19 in a test sample at a non-diagnostic and non-therapeutic destination, comprising the steps of: contacting a test sample with the monoclonal antibody of claim 1, and detecting the formation of a complex of the monoclonal antibody and CD 19;

most preferably, the avidin is biotin, avidin, streptavidin, vitellin, avidin;

(3) A method of making the engineered host cell of claim 6, comprising the steps of: introducing the expression vector of claim 5 into a host cell;

more preferably, the biological method comprises DNA vectors, RNA vectors, lentiviral vectors, poxviral vectors, herpes simplex viral vectors, adenoviral vectors, adeno-associated viral vectors;

(4) A method of specifically inhibiting CD19 activity in vitro, comprising the steps of: the nucleic acid molecule of claim 4 is introduced into a cell of an organism, and the activity of CD19 is inhibited by expressing the monoclonal antibody of claim 1.

10. The use of any of the following aspects, wherein said use comprises:

(1) Use of the monoclonal antibody of claim 1, the pharmaceutical composition of claim 2, the antibody-drug conjugate of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7 for the manufacture of a medicament for the prevention and/or treatment of a CD 19-associated hematologic malignancy;

(2) Use of the monoclonal antibody of claim 1, the pharmaceutical composition of claim 2, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7 for the preparation of an antibody-drug conjugate for the prevention and/or treatment of a CD 19-associated hematologic malignancy;

(3) Use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7, the kit of claim 8 for detecting a CD19 protein or antigenic fragment thereof;

(4) Use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7, the kit of claim 8 in the preparation of a product for detecting CD19 protein or antigenic fragment thereof;

(5) Use of the monoclonal antibody of claim 1, the nucleic acid molecule of claim 4, the expression vector of claim 5, the engineered host cell of claim 6, the population of host cells of claim 7, the kit of claim 8 in the manufacture of a product for the diagnosis of a CD 19-associated hematologic malignancy;

(6) Use of the monoclonal antibody of claim 1 in the preparation of a nucleic acid molecule, an expression vector, an engineered host cell, a population of host cells;

(7) Use of the nucleic acid molecule of claim 4 in the preparation of an expression vector, an engineered host cell, a population of host cells;

(8) Use of the expression vector of claim 5 in the preparation of an engineered host cell, population of host cells;

(9) Use of the engineered host cell of claim 6 in the preparation of a population of host cells;