CN108840930B - anti-CD 19 monoclonal antibody, preparation method and application thereof - Google Patents

Abstract

The invention provides an anti-CD 19 monoclonal antibody, V thereof_HThe complementarity determining region CDRs of the chain have the amino acid sequences of the CDRs selected from the group consisting of: SEQ ID NO: 3, heavy chain CDR1 shown in SEQ ID NO: 4, heavy chain CDR2 shown in SEQ ID NO: 5 heavy chain CDR 3; it V_LThe complementarity determining region CDRs of the chain have the amino acid sequences of the CDRs selected from the group consisting of: SEQ ID NO: 6, light chain CDR1 shown in SEQ ID NO: 7, light chain CDR2 shown in SEQ ID NO: 8, light chain CDR 3. The invention also provides a preparation method and application of the monoclonal antibody. The monoclonal antibody can specifically recognize human CD19 protein on the surface of cells; binds to human CD19 with a KD of 1X 10-7M or less; has the advantages of low immunogenicity, no HAMA reaction, and the like.

Description

anti-CD 19 monoclonal antibody, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological immunity, in particular to an anti-CD 19 monoclonal antibody and a preparation method and application thereof.

Background

CD19 is a transmembrane protein on the surface of B cells. It is closely related to B cell activation, signal transduction and growth regulation, is a functional receptor molecule on the surface of B lymphocyte, forms a B cell dual antigen binding model when B cell antigen receptor (BCR) recognizes antigen, and participates in Ca in B cell²⁺Regulate the activation and proliferation of B cells. CD19 is widely used as an important marker in the diagnosis and prognosis of leukemia, lymphoma and immune system diseases, and CD19 is also used as an important target point of immunotherapy, and CD19 is an important B cell malignant tumor target point. CD19 is expressed in almost all stages of B cell development, as well as in various B cell tumors, however, it is important that CD19 is not expressed on pluripotent hematopoietic stem cells. These features make CD19 a good target for B cell tumors. Novel antibody drugs have been developed for the treatment of leukemia and lymphoma, including non-binding antibodies, drug-conjugated antibodies, bispecific antibodies and chimeric antigen receptors.

CD19 binding has been shown to both enhance and inhibit B cell activation and proliferation, depending on the amount of cross-linking that occurs (Tedder, supra). CD19 is expressed on more than 90% of B cell lymphomas and is predicted to affect lymphoma growth in vitro and in vivo (Ghetie, supra). anti-CD 19 antibodies have been generated as murine antibodies. One drawback to the use of murine antibodies to treat human subjects is the development of a human anti-mouse (HAMA) response after administration to a patient. The human body has immune response to the murine antibody, which is also a major obstacle to the application of the murine antibody in clinical treatment. The antibody can be modified at the gene level by a gene engineering antibody technology, so that the immunogenicity of the murine antibody is reduced, and the specificity, stability and affinity of the antibody are improved. The humanized modification is carried out on the murine antibody, the fusion of an antibody variable region and a humanized antibody constant region can be reserved, and the affinity of the antibody is improved; or the antibody structure is modified, the scFv only retaining the antibody variable region or the Fab containing the antibody variable region and part of the constant region is constructed, and the absorption percentage and half-life period of the antibody in vivo can be improved. Single-chain antibodies (scFv) are a form of monoclonal antibodies, and form a single-chain variable region by linking the variable regions of the antibody via a hinge region, and are also called single-chain antibodies. The scFv retains the smallest unit of antigen binding ability of the antibody. Because the structure is smaller, the method is more beneficial to genetic engineering operation, and therefore, the method is widely used for construction, screening and application of human antibodies. For example, CAR-T technology uses scFv-structured antibodies as the antigen-recognizing region of the chimeric receptor. Accordingly, there is a need for improved therapeutic anti-CD 19 antibodies that are more effective in the treatment and/or prevention of CD 19-mediated diseases.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an anti-CD 19 monoclonal antibody, a preparation method and application thereof, which can specifically identify human CD19 protein on the cell surface and has obvious killing effect on a CD19 positive Hela cell and a K562 cell; the antibody binds to human CD19 with a KD of 1X 10-7M or less; binding to Raji and daudi b cell tumor cells; has the advantages of low immunogenicity, no HAMA reaction, better stability and safety, and the like.

The invention is realized by the following steps:

in a first aspect of the invention, there is provided an anti-CD 19 monoclonal antibody V_HA chain having the amino acid sequence of the CDRs of the complementarity determining regions selected from the group consisting of:

SEQ ID NO: 3, heavy chain CDR1 shown in SEQ ID NO: 4, heavy chain CDR2 shown in SEQ ID NO: 5, heavy chain CDR 3.

More preferably, the heavy chain variable region comprises a heavy chain variable region that is substantially similar to a light chain variable region selected from seq id nos: 1, an amino acid sequence that is at least 80% homologous to the amino acid sequence of 1;

preferably, the anti-CD 19 monoclonal antibody V_HA chain having the sequence set forth in SEQ ID NO: 1.

In a second aspect of the invention, there is provided an anti-CD 19 monoclonal antibody V_LA chain having complementarity determining region CDRs with amino acid sequences of the CDRs selected from the group consisting of:

SEQ ID NO: 6, light chain CDR1 shown in SEQ ID NO: 7, light chain CDR2 shown in SEQ ID NO: 8, light chain CDR 3.

Preferably, the light chain variable region comprises a heavy chain variable region corresponding to a light chain variable region selected from seq id nos: 2 is at least 80% homologous;

more preferably, the anti-CD 19 monoclonal antibody V_LA chain having the sequence set forth in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In a third aspect of the invention, there is provided an anti-CD 19 monoclonal antibody, V thereof_HChain and V_LThe chains have the respective amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

In other embodiments, V_HAnd/or V_LThe amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequence described above. V having the sequence as described above_HAnd V_LAntibodies with VH and VL regions that are highly (i.e., 80% or more) homologous in region can be obtained as follows: mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) encodes the polypeptide of seq id no: 1. 2, 3, 4, and then detecting the retained function of the encoded altered antibody using the functional assay described herein.

The anti-CD 19 monoclonal antibody binds to human CD19 with a KD of 1X 10-7M or less; binding to Raji and daudi b cell tumor cells.

In a fourth aspect of the invention, there is provided a nucleic acid molecule encoding the anti-CD 19 monoclonal antibody of any one of the preceding claims.

The nucleic acid may be present in whole cells, cell lysates, or in partially purified or substantially pure form. When combined with other cellular components or other contaminants by standard techniques including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other methods known in the art. The nucleic acids of the invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA nucleic acid molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), cdnas encoding the light and heavy chains of antibodies prepared by the hybridomas can be obtained using standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), one or more nucleic acids encoding the antibody can be recovered from the library. Can be obtained using standard PCR amplification techniques: amplifying a fragment of the CD19 single-chain antibody by using a primer pair; and (2) carrying out double enzyme digestion, connecting the vector plasmid (the vector is connected with Fc fragment of a fusion expression human IgG1 antibody at the downstream of a multiple cloning site) by using T4 DNA ligase in the same double enzyme digestion vector, transforming the vector into host bacteria, selecting clones, identifying positive clones by PCR (polymerase chain reaction) and confirming the positive clones by sequencing to obtain eukaryotic expression plasmid.

In the present invention, the variable region gene is converted into scFv gene, and once the encoding V is obtained_HAnd V_LFragment DNA fragments, i.e., those DNA fragments which can be further manipulated by standard recombinant DNA techniques, e.g., conversion of the variable region gene to a full-length antibody chain gene, Fab fragment gene or scFv gene.

In these operations, V is encoded_LOr V_HIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein means that two DNA segments are linked together such that the amino acid sequences encoded by the two DNA segments remain in reading frame.

By encoding V_HDNA of (3) and a DNA encoding a heavy chain constant region (C)_H1、C_H2And C_H3) Operably linked to an isolated DNA molecule encoding V_HDNA of the region is converted to the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is well known in the art and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, V is encoded_HThe DNA of (a) may be related to a DNA encoding only heavy chain C_H1The other DNA molecule of the constant region is operably linked.

By encoding V_LDNA of (1) and encoding light chain constant region C_LOperably linked to an isolated DNA molecule encoding V_LThe DNA of the region is converted to the full-length light chain gene (as well as the Fab light chain gene). The sequence of the human light chain constant region gene is known in the art that DNA fragments comprising these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To generate the scFv gene, V will be encoded_HAnd V_LIs operably linked to a further fragment encoding a flexible linker, e.g.an amino acid sequence (Gly4-Ser)3, such that V_HAnd V_LThe sequence may be expressed as a continuous single-chain protein, V_LAnd V_HThe zones are connected by the flexible connector.

In a fifth aspect of the invention, there is provided an expression vector comprising an amino acid sequence encoding any one of said anti-CD 19 monoclonal antibodies, capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell. The carrier may be a conventional carrier. Preferably, the present invention employs a third generation self-inactivating lentiviral vector system having a total of three plasmids, namely, a packaging plasmid psPAX2 encoding the protein Gag/Pol and encoding the Rev protein; envelope plasmid pMD2.G encoding VSV-G protein and recombinant plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro encoding the extracellular region and transmembrane region of human CD19 based on the empty vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro (available from Addgene). According to the human CD19 sequence provided by Genbank accession No. NM-001178098, a gene synthesis method based on PCR bridging is used for synthesizing a signal peptide, a human CD19 extracellular region, a transmembrane region and an intracellular region, wherein the extracellular domain comprises an extracellular recognition region sequence of a CAR gene which is the amino acid sequence of the anti-CD 19 monoclonal antibody.

In a sixth aspect of the invention, there is provided a host cell which is a prokaryotic or eukaryotic host cell comprising said vector. When a human CD19 stable expression cell line is constructed, the successfully constructed lentiviral vector pCDH-CMV-huCD19-EF1-GFP-T2A-Puro is transfected into 293T cell packaging lentivirus; the packaged recombinant lentivirus can be used for infecting leukemia cells Hela or K562 and the like. Flow cytometry experiments show that the antibody can specifically recognize human CD19 protein on the surface of cells. The host cell may be leukemia cell Hela or K562 infected with the packaged lentivirus, or T lymphocyte infected with the packaged lentivirus according to the eighth aspect of the invention.

In a seventh aspect of the present invention, there is provided a method for producing an anti-CD 19 monoclonal antibody, comprising expressing the anti-CD 19 monoclonal antibody in said host cell, and isolating the CD19 antibody from said host cell. The anti-CD 19 monoclonal antibody has better killing effect on a Hela cell and a K562 cell which are positive to CD 19.

In an eighth aspect of the invention, there is provided a CAR-T cell comprising at least one extracellular ligand binding domain, a transmembrane domain and at least one intracellular signaling domain, wherein the extracellular domain comprises an extracellular recognition region sequence of a CAR gene that is the amino acid sequence of the anti-CD 19 monoclonal antibody. The CAR-T technology adopts an antibody with an scFv structure as a region of a chimeric receptor for recognizing an antigen to construct a T cell of a chimeric antigen vector containing the CDR fragment. And (3) infecting T lymphocytes by the packaged lentivirus containing the scFv gene to obtain the CAR-T cell of the invention. CAR-T cells all showed better targeting and killing in the experiment compared to traditional immunotherapy.

In the ninth aspect of the invention, the invention provides the application of the anti-CD 19 monoclonal antibody and the CAR-T cell in preparing anti-tumor drugs.

According to the invention, a human cervical cancer cell line Hela and a Hela cell (CD19-Hela) transfected with CD19 are used as target cells, effector cells are the prepared CAR-T cells, when the CAR-T cells kill tumor cells, the T cells firstly identify and verify with the tumor cells carrying tumor antigens, then an immune synapse is formed, and then a killing factor is released to complete immune monitoring and killing functions of the T cells. The CAR-T cell has more remarkable killing effect on CD19 positive Hela cells and K562 cells.

The invention has the beneficial effects that:

1. the anti-CD 19 monoclonal antibody (scFv-3B5-Fc) provided by the invention has an apparent affinity to CD19 of 475pM, can specifically recognize human CD19 protein on the cell surface, and has a remarkable killing effect on a Hela cell and a K562 cell which are positive to CD 19;

2. compared with the traditional immunotherapy, the CAR-T cell provided by the invention has better targeting property and killing property in the experiment, and has more obvious killing effect on CD19 positive Hela cells and K562 cells;

3. the CAR-T cell has the advantages of low immunogenicity, no HAMA reaction and the like, and has better stability and safety.

Drawings

FIG. 1 shows that the fluorescence peaks of isotype control IgG and the anti-CD 19 monoclonal antibody (scFv-3B5-Fc) of the present invention are significantly different compared with those of Raji cells positive for CD19 expression;

FIG. 2 is a graph showing that the selected phage expressing scfv can bind to the natural antigen of CD19, and the primarily purified phage stain and flow-analyze Raji cells;

FIG. 3 shows the results of antibody-Fc fusion protein scFv-3B5-Fc, polyacrylamide gel electrophoresis;

FIG. 4 is a Scfv-3B5 fusion expressing CAR-T cell specific killing CD19-Hela cells of the invention;

FIG. 5 is the in vitro anti-tumor cytotoxicity of Scfv-3B5 fusion-expressed CAR-T cells of the invention;

FIG. 6 is an exemplary binding curve obtained in a concentration gradient ELISA assay for an anti-CD 19 monoclonal antibody (scFv-3B5-Fc) of the present invention;

FIG. 7 is a diagram of the plasmid structure of CAR-T cells provided by the invention.

Detailed Description

Example 1 preparation of specific Single chain antibody (scFv) that binds human CD19

Screening of anti-human CD19 specific antibody based on phage display

1. To achieve this, 400ml of 2 XYT/ampicillin medium was inoculated with glycerol bacteria (self-constructed library) displaying a natural pool of fully human single-chain antibodies to achieve an OD600 of 0.1, and cultured with shaking at 37 ℃ and 200rpm until the cell density reached an OD600 of 0.5. By 10¹²Pfu's M13KO7 helper phage (purchased from ThermoFisher) was infected and incubated at 30 ℃ and 50rpm for 30 minutes. After adding 50mg/L kanamycin and shaking culture at 37 ℃ and 200rpm for 30 minutes, the precipitate was separated by centrifugation (15 minutes, 1600 Xg, 4 ℃), resuspended in 400ml of 2 XYT/ampicillin/kanamycin medium, and shaking culture at 37 ℃ and 200rpm for 16 hours. Finally the pellet was separated by centrifugation (20 min, 5000 Xg, 4 ℃) and discarded, and the supernatant was filtered through a 0.45 μ M format filter, 1/4 vol of 20% (w/v) PEG8000, 2.5M NaCl solution was added and incubated in an ice bath for 1 hour to pellet the phage particles. Followed by centrifugation (20 min, 8000 Xg, 4 ℃ C.), discarding the supernatant and resuspending the phage pellet in 25ml of precooled PBS solution (137mM NaCl, 2.7mM KCl, 8mM Na)₂HPO₄，2mM KH₂PO₄) In (1), centrifugation (5 min, 20000 Xg, 4 ℃ C.). 1/4 volumes of 20% (w/v) PEG8000, 2.5M NaCl solution were added to the supernatant and the phage particles were again precipitated in ice bath for 30 minutes. The pellet was centrifuged (30 min, 20000 Xg, 4 ℃), and the phage pellet resuspended in 2ml of pre-cooled PBS, kept on ice for 30min and centrifuged (30 min, 17000 Xg, 4 ℃). The supernatant was mixed with 4% (w/v) BSA in PBS at 1:1, placed on a rotary mixer, incubated at room temperature for 30 minutes, and then used directly for screening.

2. Using the phage antibody library described above, 3 rounds of directed screening were performed against biotin-labeled human Fc-CD19 recombinant protein (purchased from Acrobiosystems, Inc.), and the screening protocol was as follows:

3. the phage antibody library was incubated with biotin-labeled recombinant human CD19 antigen at room temperature for 2 hours, and then with streptavidin-labeled with 2% BSA solution

Magnetic beads (purchased from ThermoFisher) were incubated at room temperature for 30 minutes. The beads were then washed with PBST (0.1% Tween-20) buffer to remove non-specifically bound or weakly bound phage. The phage with strong binding ability was eluted from the magnetic beads with glycine-hydrochloric acid buffer (pH 2.2), neutralized with Tris neutralization solution (pH 9.1), and usedE.coli ER2738 in logarithmic growth phase was infected and used for the next round of screening. In 3 screening rounds, the amount of magnetic beads was 50. mu.l, 20. mu.l and 10. mu.l, the concentration of biotin-labeled human Fc-CD19 antigen was 100nM, 10nM and 1nM, and the number of PBST washes was 10, 15 and 20, respectively.

Identification of human CD 19-specific binding antibodies

1. From the clones obtained in the third round of screening, single clones were randomly selected and analyzed for their ability to bind to human Fc-CD19 by a single phage ELISA (enzyme-linked immunosorbent assay). For this purpose, each single colony was inoculated with 300. mu.l of 2 XYT/ampicillin medium (containing 2% glucose) in a 96-well deep-well plate and cultured at 37 ℃ and 250rpm for 16 hours with shaking. Mu.l of the culture was inoculated into 500. mu.l of 2 XYT/ampicillin medium (containing 0.1% glucose), and cultured at 37 ℃ and 250rpm for 1.5 hours with shaking. The helper phage solution was prepared and 75. mu.l of M13KO7 (titer 3X 10)¹²pfu/ml) was mixed into 15ml of 2 XYT medium and 50. mu.l/well was added to the plate. After incubation at 37 ℃ and 150rpm for 30 minutes, the prepared kanamycin solution was added to 50. mu.l/well (180. mu.l of 50mg/ml kanamycin was added to 15ml of 2 XYT medium), and shaking culture was performed at 37 ℃ and 250rpm for 16 hours. The cells were finally pelleted by centrifugation (30 min, 5000 Xg, 4 ℃ C.) and the supernatant was transferred to a new 96-well deep-well plate.

2. As shown in fig. 2, to preliminarily determine whether the selected phage expressing scfv can bind to the natural antigen of CD19, we stained Raji cells with the preliminarily purified phage and flow-analyzed. The results of the analysis of figure 2 indicate that the selected scfv-expressing phage can bind to the native antigen of CD 19. In FIG. 2, (A) is the phage expressing scfv obtained by screening, and (B) is the negative control group.

3. For single phage ELISA, a 96-well MediSorp ELISA plate (purchased from ThermoFisher) was coated overnight at 4 ℃ with 100 ng/well of human Fc-CD19 recombinant antigen and negative control protein Fc (100. mu.l/well), respectively. Each well was blocked with a PBST solution containing 2% BSA. The wells were then washed three times with PBST and blotted clean. Then 100. mu.l/well of each phage solution prepared above was addedInto each well in the plate. After incubation at 37 ℃ for 2 hours, the cells were washed three times with PBST. To detect bound phage, anti-M13 antibody superoxide dismutase conjugate (purchased from wuhan sanying) was diluted 1:5000 in PBST and 100 μ l was added to each well. After incubation at 37 ℃ for 1 hour, three rinses were performed with PBST and then three rinses with PBS. Finally 50. mu.l of TMB substrate was pipetted into the wells and developed for 10 minutes at room temperature, followed by 50. mu.l of 2M H per well₂SO₄The color reaction was terminated. The extinction was measured at 450nm with an enzyme linked immunosorbent assay (Bio-Rad). And (5) judging the wells as positive according to the extinction value, extracting phage plasmids and carrying out sequencing verification on the scfv sequence.

4. Binding sequencing analysis showed that the single-chain antibody 3B5(SEQ ID NO:9) had a very strong binding signal to human CD19 in an ELISA assay and NO binding to Fc recombinant protein.

5. Monoclonal antibody 3B5 (i.e., a monoclonal antibody of the invention) comprising: (a) comprises the amino acid sequence shown as SEQ ID NO: 3, the heavy chain CDR1 of the amino acid sequence set forth in seq id no; (b) comprises the amino acid sequence shown as SEQ ID NO: 4, the heavy chain CDR2 of the amino acid sequence set forth in seq id No. 4; (c) comprises the amino acid sequence shown as SEQ ID NO: 5, the heavy chain CDR3 of the amino acid sequence set forth in seq id no; (d) comprises the amino acid sequence shown as SEQ ID NO: 6, a light chain CDR1 of the amino acid sequence set forth in seq id no; (e) comprises the amino acid sequence shown as SEQ ID NO: 7, a light chain CDR2 of the amino acid sequence set forth in seq id No. 7; and (f) comprises the amino acid sequence set forth as SEQ ID NO: 8, wherein the antibody specifically binds to human CD 19.

Example 2 expression and purification of anti-CD 19 Single chain antibody

1. Expression of human CD19 Single chain antibody

And (3) carrying out recombination construction of eukaryotic expression vectors on the selected single-chain antibodies, and transfecting HEK293F to induce recombination expression and purifying. An ScFv-3B5 fragment was amplified from the clone pCantab-3B5 obtained in example 1 using the primer pair 3B5-F (SEQ ID NO:10) and 3B5-R (SEQ ID NO: 11); the plasmid pCMV-V5-Fc (vector expressing Fc fragment of human IgG1 antibody fused downstream of the multiple cloning site, hereinafter referred to as V5-Fc) was ligated with T4 DNA ligase by double digestion with NheI/BamHI (purchased from Takara) and transformed into the host bacterium TOP10, and the clones were selected to identify positive clones by PCR and confirmed by sequencing, to obtain the scFv-3B5-Fc eukaryotic expression plasmid.

2. Purification of human CD19 Single chain antibody

The expression plasmids are respectively transfected into HEK-293F cells with good growth, the HEK-293F cells are continuously cultured for 7 days at 37 ℃, 5% CO2 and a shaking table at 125rpm, the HEK-293F cells are centrifuged at 4000rpm for 10min, precipitates are removed, supernatant is collected and filtered by a 0.45-micron filter membrane, a treated sample is subjected to affinity purification by a Protein A (purchased from GE company) affinity column, and finally, purified antibody-Fc fusion Protein scFv-3B5-Fc is obtained, and the polyacrylamide gel electrophoresis identification result is shown in figure 3. FIG. 3 shows the success of the construction of the purified antibody-Fc fusion protein scFv-3B 5-Fc.

Example 3 analysis of antigen binding Activity of Single chain antibody against human CD19

1. ELISA assay for binding Activity of recombinant antibodies to human CD19 antigen

The binding activity of the screened antibodies to the antigen human CD19 was determined by a concentration gradient ELISA assay. For this purpose, antigen human Fc-CD19 was diluted with 0.1M NaHCO3(pH 9.6) coating solution, 100ng per well, 50. mu.l per well, overnight at 4 ℃ and blocked with PBST blocking solution containing 2% BSA and 0.01% (v/v) Tween-20 for 2 hours at room temperature. The plates were then rinsed three times with PBST and removed. Subsequently, 100 μ l of PBST solution containing a range of concentrations (starting concentration 10nM, 3-fold gradient dilutions until dilution to 1:729) of each antibody protein was added to each well plate and each sample assayed using parallel three-well assays. After incubation at 37 ℃ for 2 hours, the cells were rinsed three times with PBST, 100. mu.l/well of horseradish peroxidase-labeled goat anti-human antibody (purchased from Wuhan Sanying) diluted 1:20000 was added, and the reaction was carried out at 37 ℃ for 1 hour. For detection, the wells were rinsed three times with PBST, then three times with PBS, and finally TMB was added to show 15 minutes, the color reaction was stopped with 50. mu.l of 2M H2SO4 per well, and the extinction was measured at 450nm with an enzyme linked immunosorbent assay (Bio-Rad). The resulting absorbance values were evaluated using GraphPad software and the binding strength of the antibody was calculated. For this purpose, the measured extinction values are plotted in each case against the corresponding antibody concentration, and the resulting curves are fitted using the following non-linear regression.

Wherein the association/dissociation equilibrium between the immobilized antigen and the antibody protein is identified as:

x ═ concentration of antibody protein;

y-the concentration of the antigen/antibody complex (measured indirectly by absorbance after chromogenic reaction);

a is the total concentration of immobilized antigen;

b-dissociation constant (KD).

An exemplary binding curve obtained for the antibody ScFv-3B5-Fc in a concentration gradient ELISA assay is shown in FIG. 6, in which ScFv-3B5-Fc has an apparent affinity of 475 pM.

2. FACS analysis of the binding Activity of recombinant antibodies against Raji cell surface CD19

The binding activity of the screened antibody to the cell surface CD19 antigen of the tumor cell line Raji is detected by FACS analysis experiments.

Example 4 flow cytometry analysis of binding of stably expressing cell lines of human CD19 to anti-CD 19 Single chain antibody

Construction of human CD19 stable expression cell line

1. Construction of plasmid vector

(1) The vector system used in this example belongs to the third generation of self-inactivating lentiviral vector system, which comprises three plasmids, namely, a packaging plasmid psPAX2 encoding protein Gag/Pol and encoding Rev protein; envelope plasmid pMD2.G encoding VSV-G protein and recombinant plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro encoding the extracellular region and transmembrane region of human CD19 based on the empty vector pCDH-CMV-MCS-EF1-GFP-T2A-Puro (available from Addgene).

(2) According to the human CD19 sequence provided by Genbank accession NM-001178098, a gene synthesis method based on PCR bypass is used to synthesize a gene synthesis sequence containing a signal peptide, an extracellular region, a transmembrane region and an intracellular region of human CD19, and PCR amplification is carried out on huCD19F (SEQ ID NO: 12)/R (SEQ ID NO: 13) by primers under the conditions of pre-denaturation: 94 ℃ for 4 min; denaturation: 30s at 94 ℃; annealing: at 58 ℃ for 30 s; extension: 68 ℃ for 80 s; 30 cycles. The theoretical size of the obtained fragment is 1716bp, and the amplified product is confirmed to be consistent with the theoretical size through agarose electrophoresis. Wherein Xba I and BamH I cleavage sites are introduced upstream and downstream of the open reading frame. The obtained target gene is subjected to double enzyme digestion by Xba I and BamH I and is connected into a pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector subjected to double enzyme digestion in the same way, a successfully constructed lentiviral vector pCDH-CMV-huCD19-EF1-GFP-T2A-Puro is constructed, and lentiviral packaging is carried out after Xba I and BamH I enzyme digestion identification and sequence determination are correct.

2. Plasmid transfected 293T cell packaging lentivirus

(1) At 6X 10⁶The density of (A) was inoculated into 293T cells (ATCC: CRL-11268) of 6 th to 10 th passages in a 10cm dish at 37 ℃ with 5% CO₂The culture was prepared overnight for transfection. The culture medium was DMEM (thermo fisher corporation) containing 10% phage-free fetal bovine serum (hangzhou holly), and the next day, the culture medium was changed to serum-free DMEM about 2 hours before transfection.

The transfection procedure was as follows:

dissolving 5 μ g of the target gene plasmid pCDH-CMV-huCD19-EF1-GFP-T2A-Puro and 7.5 μ g of the packaging plasmid pSPAX2 and 2.5 μ g of the envelope plasmid pMD2.G in 500 μ L of Mill Q water, and mixing;

62. mu.L of 2.5M CaCl2(Sigma Co.) was added dropwise, mixed at 1200rpm/min vortex,

finally, 500. mu.L of 2 XHBS (280mM NaCl, 10mM KCl, 1.5mM Na2HPO4, 12mM glucose, 50mM Hepes (Sigma Co.), pH7.05, 0.22. mu.M filter sterilized) was added dropwise thereto, mixed by shaking at 1200rpm/min for 10 seconds,

immediately dropwise adding the mixture into a culture dish, gently shaking, culturing at 37 ℃ for 4-6 h with 5% CO2, and then replacing with DMEM containing 10% fetal calf serum.

(2) After transfection for 48h or 72h, cell debris was removed by centrifugation, and the virus was collected by filtration through a 0.45 μm filter (Millipore Corp.).

3. Recombinant lentivirus infects leukemia cell Hela or K562

The collected virus liquid is concentrated and titrated, and then Hela cells or K562 cells paved in a 6-well plate are respectively infected. Three days after infection, cells were harvested, and a portion of the pooled clones was used to detect cell surface CD19 expression using flow cytometry using the same procedure as in example 5, and using a fluorescently labeled antibody directed to CD 19. After the other cells are expanded and cultured, one part of the cells are frozen and stored, the other part of the cells are passed in a 6-well plate, 2ug/ml of Puromycin (purchased from Sigma) antibiotic is added, the cells are screened for 1-2 weeks and observed under a microscope until all the cells in a visual field express GFP, and at the moment, the frozen and stored cells are used for subsequent flow cytometry detection and killing experiments.

Two, flow cytometry analysis

1. The binding ability of the single-chain antibody scFv-3B5-Fc against human CD19 obtained in example 2 to human CD19 on the cell surface was analyzed by flow cytometry (CytoFLEX, Beckman).

The specific method comprises the following steps:

(1) taking Raji cells, K562-CD19 and K562 cells in logarithmic growth phase, respectively inoculating the cells into a T25 cell culture bottle, wherein the density of the inoculated cells is about 5x10⁵Incubate at 37 ℃ overnight at a concentration of one cell per ml.

(2) The culture medium in the dish was gently shaken and the cells were collected by centrifugation at 200 g.times.5 min. At 2 to 3 x10⁶The concentration per mL was resuspended in 1% phosphate buffered saline containing calf serum (NBS PBS) and added to the flow-through dedicated tube at 100 ul/tube.

(3) Centrifuge at 200g × 5min, discard the supernatant.

(4) The two experimental groups are respectively added with scFv-3B5-Fc and FMC63 positive antibodies to be detected, and the other control group is a PBS blank control without the antibodies. The final concentration of each antibody was 20. mu.g/ml, 100ul per tube. Ice-bath for 45 min.

(5) 2ml of 1% NBS PBS was added to each tube and centrifuged at 200 g.times.5 min twice.

(6) Discard the supernatant and add 1:100 dilution of goat anti-human antibody-FITC (Triweb eagle, Wuhan), 100ul per tube. Ice-bath for 45 min.

(7) 2ml of 1% NBS PBS was added to each tube and centrifuged at 200 g.times.5 min twice.

(8) The supernatant was discarded and resuspended in 300ul of 1% NBS PBS and examined by flow cytometry.

(9) Data were analyzed using flow cytometer data analysis software cytoexpert2.0.

2. As shown in FIG. 1, the flow cytometry analysis result shows that the fluorescence peak of isotype control IgG and scFv-3B5-Fc is significantly different on Raji cells positive to CD19 expression and has no obvious difference on K562 cells negative to human CD19 expression, and the result shows that scFv-3B5-Fc can specifically recognize Raji cells positive to human CD19 expression but does not bind to K562 cells negative to human CD19 expression. Thus, the antibody scFv-3B5-Fc specifically recognizes human CD19 protein on the cell surface. Since the flow chart of K562 is the same as the preceding and following charts, there is no difference, so that it is not shown.

Example 5 preparation of CAR-T cells

Construction of lentiviral plasmid of chimeric antigen receptor protein

1. The lentiviral plasmid vector system used in this example belongs to the third generation lentiviral four-plasmid system, which has a total of four plasmids, namely, envelope plasmid pCMV-VSV-G (purchased from addrene), which encodes VSV-G protein, packaging plasmid pRSV-Rev (purchased from addrene), which encodes Rev protein, pMDLg/pRRE (purchased from addrene), which encodes Gal and Pol, and a recombinant expression vector encoding the CAR gene of interest based on empty vector pLVX-EF1 (constructed by the same company), and is effective in reducing the risk of forming replicating lentiviral particles.

2. All constructed gene promoters of CAR expression lentiviral vectors adopt the on-vector elongation factor-1 alpha (EF-1 alpha) in the published patent 201310164725. X. The construction method comprises the following steps:

(1) designing full-Length amino acid sequences of CAR genes

Typically, a typical CAR structure will comprise a cell membrane protein transport signal peptide, a tumor antigen binding region, a hinge region, a transmembrane region, and a T cell costimulatory signal. Specifically, in this example, as shown in FIG. 7, we used the cell membrane transport signal peptide of human CD8 to link scfv-3B5 as the binding domain of tumor antigen CD19, and then to link the membrane-proximal Hinge region of human CD8 and the transmembrane region of CD8, and the cytoplasmic domain portion links the intracellular domain of 4-1BB and the intracellular domain of CD3 zeta as the co-stimulatory domain of T cells. To facilitate the detection of CAR, we linked GFP co-expression behind the CAR structure by self-splicing peptides. The whole fusion gene and the corresponding amino acid sequence are as follows:

(2) nucleic acid sequence optimization

In order to enhance the expression of the CAR gene in T cells and increase the translation of protein, the codon of the CAR gene is optimized by using a humanized codon optimization algorithm of website http:// www.jcat.de/, wherein the optimized sequence is as follows:

(3) complete sequence synthesis and sequencing verification

And (3) for an optimized codon sequence, submitting the optimized codon sequence to a DNA synthesis supplier (Wuhan Kingkunrei) for fragment synthesis and full sequence splicing, performing sequencing verification after the splicing is completed, performing double enzyme digestion through EcoR I/BamH I restriction enzyme after verification is error-free, and connecting the product into a pLVX-EF1 vector subjected to the same double enzyme digestion, thereby constructing lentiviral vectors pLVX-EF1-3B5-CAR and pLVX-EF1-FMC63-CAR for expressing various chimeric antigen receptors. The successfully constructed vector can be used for packaging lentiviruses after being subjected to EcoR I/BamH I enzyme digestion identification and sequence determination. As previously described, the CAR gene of interest is transcriptionally translated into a peptide chain, and the anti-CD 19 chimeric antigen receptor will be localized on the cell membrane of T cells under the direction of the GM-CSF signal peptide. This example constructed CAR fusion genes consisting of a scfv-3B5 fragment and a scfv-FMC63 fragment assembly using a similar strategy.

Second, plasmid transfection 293T packaging lentivirus

1. At 6X 10⁶The density of (2) was inoculated into HEK-293T cells (ATCC: CRL-11268) of 6 th to 10 th generations in a 10cm dish at 37 ℃ with 5% CO₂The culture was prepared overnight for transfection. The medium was 10% fetal bovine serum and DMEM (both from Hyclone).

2. The transfection procedure was as follows:

preparing a solution A: mu.g of the plasmid pLVX-EF1-3B5-CAR of each target gene, 6.2. mu.g of the packaging plasmids pMDLg RRE and pRSV-REV, and 2.4. mu.g of the envelope plasmid pCMV-VSV-G were dissolved in 800. mu.L of serum-free DMEM medium and mixed well.

Preparing a solution B: mu.g PEI (polyethyleneimine 1. mu.g/. mu.l, from Polysciences) was dissolved in 800. mu.L serum-free DMEM medium, gently mixed, and incubated at room temperature for 5 min.

Formation of transfection complexes: and adding the solution A into the solution B, mixing gently, immediately vortex mixing or mixing gently, and incubating at room temperature for 20 min.

1.6ml of the transfection complex was added dropwise to HEK-293T cells, and after 4-5h, the transfected 293T cells were replated with 2% FBS in DMEM medium.

3. After 72h of transfection incubation, the virus was collected by filtration using a 0.45 μm filter (available from Millipore), centrifuged at 28000rpm using a Beckman Optima L-100XP ultracentrifuge at 4 ℃ for 2 hours, the centrifuged supernatant was discarded, and the resulting pellet was centrifuged and resuspended in AIM-V medium (available from Life) at 1/10-1/50 stock volumes and frozen in 100 μ L/tube at-80 ℃ to await virus titration or infection of T lymphocytes.

Preparation of CAR-T cell by infecting T cell with recombinant lentivirus

1. Human peripheral blood mononuclear cells (supplied from the blood center of Wuhan city) were obtained from healthy human peripheral blood by density gradient centrifugation, and the CD3 positive rate was confirmed by FACS analysis, followed by culture with the addition of magnetic beads (ThermoFisher Co.) coated with both anti-CD 3 and CD28 antibodies at a CD3 positive T cell to magnetic bead ratio of 1:2 and a final concentration of 300U/mL of recombinant human IL-2 (purchased from New Biotech, Shanghai, Ltd.) for 24 hours under stimulation. T cells were then infected with the recombinant lentivirus described above at an MOI of 5. Infected cells were used every other day at 5X10⁵Passaging was performed at a density of/mL while the lymphocyte culture broth was supplemented with recombinant human IL-2 at a final concentration of 300U/mL.

2. Infected T cells were tested for chimeric antigen receptor expression by flow cytometry at day 5 in culture. First, infected CAR-T cells were incubated with biotin-labeled human Fc-CD19 protein at 37 ℃ for 30min, washed 2 times with D-PBS, and then incubated with PE-labeled Streptavidin at 37 ℃ for 30 min. The positive cell rate was detected by flow cytometry after 3 washes with D-PBS. Uninfected T lymphocytes and PE-labeled Streptavidin-stained T cells alone were used as negative controls to identify virus-infected T cells expressing chimeric antigen receptors and their level of positive rate. Meanwhile, FACS analysis can be used for confirming the positive rate of the T cells expressing GFP, the positive level of the T cells expressing the CAR gene detected by the Fc-CD19 recombinant antigen is contrasted, and the positive rate result that the CAR structure can be successfully displayed on the cell membrane of the T cells by the lentivirus infected T cells carrying the CAR gene can be confirmed shows, and the T cells expressing the chimeric antigen receptor can be obtained by the lentivirus infection method.

3. T cells were infected with the chimeric antigen receptor-packaged virus at a cell density of 5X10⁵Every other day, the cells are passaged and counted, IL-2 (the final concentration is 300U/ml) is added to the cell culture solution after the passage, and the amplification is about 50-200 times at the 11 th day of the culture, which shows that the CAR-T cells expressing the chimeric antigen receptor can be amplified in vitro in a certain amount, and provides guarantee for subsequent in vitro killing tests and in vivo tests.

Example 6 in vitro toxicity test

First, in an in vitro suitable cell culture system, target cells with specific tumor antigens or CAR-T cells prepared in example 5 were mixed, and after a period of time, recognition (aggregation) and killing (reduction in tumor cell number) of the target cells by the T cells or CAR-T cells was observed. For example, this experiment was performed by mixing HeLa cells overexpressing CD19 (obtained in the construction of the human CD19 stably expressing cell line in example 4) with general expansion cultured T cells or FMC63 CAR-T (positive control), 3B5 CAR-T. Cells used in the experiment are human cervical cancer cell line Hela and Hela cells (CD19-Hela) transfected with CD19 as target cells, effector cells are the CAR-T cells prepared in the previous step, the effective-target ratio is respectively 2.5, 12.5:1 and 25:1, the number of the target cells is 10000 per hole, and appropriate number of the effector cells are added according to different effective-target ratios. All cells were cultured in 5% CO₂In a cell culture chamber at 37 ℃.

Second, microscopic imaging analysis of in vitro toxicity effect

1. The method comprises the following steps: at the beginning of the experiment, 10000/well CD19-Hela cells were inoculated into a 6-well plate, cultured for 12h, and then added with appropriate amount of T cells or CAR-T cells according to the effective-target ratio, and then continuously treated with 5% CO₂After culturing in an incubator at 37 ℃ for 4 hours, the culture plate is taken out and observed under an inverted microscope for imaging.

2. Results as shown in figure 4, 3B5-CAR-T had a significant killing effect on Hela cells positive for CD 19.

Third, real-time unmarked cell analysis technique for in vitro toxicity effect analysis

1. The principle is as follows: when the CAR-T cell kills tumor cells, the T cells firstly identify and verify with the tumor cells carrying tumor antigens, then form immune synapses, and further release killing factors to complete immune monitoring and killing functions of the T cells, so that the process is a continuous process. The traditional method for detecting the killing of the tumor cells by the T cells usually adopts a time point to carry out end point detection, the obtained result has larger error, and the complete process of killing the tumor cells by the T cells cannot be truly reflected. In order to break through the limitation of the technical bottleneck of the detection means, the Real-Time unlabeled cell Analysis (RTCA) technology can realize Real-Time, unlabeled, and continuous and dynamic monitoring of cytotoxic effects caused by small molecular compounds, antibody drugs, T cells and the like based on the detection of the electrical impedance value generated after the cells adhere to the wall in the culture plate with the microelectrode. Based on the real-time cell killing analysis technology of the electrical impedance, researchers can obtain high-sensitivity quantitative data, and the technology is helpful for researching and disclosing a specific action mechanism of an anti-tumor compound or a cell, and can also obviously reduce the cost of an experiment and improve the accuracy of a result. The RTCA technique continuously and dynamically monitors the processes of attachment, extension and propagation of target cells, and uses the Cell Index to indicate the growth state of target cells, or equivalently, the number of target cells.

2. The method comprises the following steps: except for adding effector cells in the whole experimental process, the culture plate and the detection instrument are ensured to be in a stable culture environment, and the Cell Index data is prevented from generating large fluctuation. In order to obtain a time-dependent cellular effect characteristic curve, 90ul of culture medium is added into a culture well of an E-plate with 16 wells, 100ul of cell suspension is added after a background baseline is measured, and the cell suspension is placed in CO after being mixed uniformly₂And (4) continuously obtaining an electrical impedance value Cell Index reflecting the growth state of the cells on a detection table in the incubator. Adding equal volume of effector cell suspension after the target cells grow completely adherent for a period of time 18h, then placing on a detection table in an incubator, and continuously collecting the growth state of the reaction cellsElectrical impedance value Cell Index data.

3. As a result: the whole experiment was completed after 48h and the data collected was derived as shown in figure 5. In the figure, SKOV-3-CD19 is a stable cell line stably expressing CD19 in the examples. The Cell Index in FIG. 5 visually indicates the number of tumor cells growing on the plate by measuring the electrical impedance of the plate. The added effector cells are suspension cells, so the electrical impedance value of the culture plate is not obviously influenced. The gradual increase of the Cell Index when no effector Cell is added in the initial stage of the experiment indicates that the Cell proliferates and grows under proper culture conditions, and the decrease of the Cell Index after the effector Cell is added indicates that the number of target cells CD19-SKOV3 growing adherently is reduced, namely apoptosis occurs due to killing of the effector Cell. The 3B5-CAR-T provided by the invention is shown to have a remarkable killing effect on a CD19 positive Hela cell.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

The nucleic acid sequence is as follows:

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Claims (9)

1. An anti-CD 19 monoclonal antibody, wherein V is_HThe amino acid sequence of the CDR of (a): SEQ ID NO: 3, heavy chain CDR1 shown in SEQ ID NO: 4, and the heavy chain CDR2 of SEQ ID NO: 5 heavy chain CDR 3;

it V_LThe amino acid sequence of the CDR of (a): SEQ ID NO: 6, light chain CDR1 shown in SEQ ID NO: 7, and the light chain CDR2 shown in SEQ ID NO: 8, light chain CDR 3.

2. An anti-CD 19 monoclonal antibody, wherein V is_HThe amino acid sequence of (a) is as shown in SEQ ID NO:1 is shown in the specification; it V_LThe amino acid sequence of (a) is as shown in SEQ ID NO: 2, respectively.

3. A nucleic acid molecule encoding the anti-CD 19 monoclonal antibody of any one of claims 1-2.

4. An expression vector comprising the nucleic acid of claim 3, capable of expressing said nucleic acid in a prokaryotic or eukaryotic host cell.

5. A host cell, which is a prokaryotic or eukaryotic host cell comprising the expression vector of claim 4.

6. A method for producing an anti-CD 19 monoclonal antibody, comprising expressing the anti-CD 19 monoclonal antibody in the host cell of claim 5, and isolating the anti-CD 19 monoclonal antibody from the host cell.

7. A CAR-T cell comprising at least one extracellular ligand binding domain, a transmembrane domain, and at least one intracellular signaling domain, wherein the extracellular domain comprises an extracellular recognition region sequence of a CAR gene that is the amino acid sequence of the anti-CD 19 monoclonal antibody of any one of claims 1-2.

8. A method of making the CAR-T cell of claim 7, comprising the steps of:

s1, isolation, activation and expansion of T lymphocytes: culturing and expanding the separated CD3+ T lymphocytes after activation;

s2, infecting the T lymphocyte treated by the step S1 with a recombinant lentivirus carrying CAR to prepare the CAR-T cell, wherein the sequence of the extracellular recognition region of the CAR gene carried by the recombinant lentivirus carrying CAR is the amino acid sequence of the anti-CD 19 monoclonal antibody of any one of claims 1-2.

9. Use of the anti-CD 19 monoclonal antibody of any one of claims 1-2 and the CAR-T cell of claim 7 for the preparation of a medicament against B cell tumors.

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