CN105348388B - The antibody L3H3 of anti-CD20 antigens and its application - Google Patents

Abstract

The invention discloses a kind of antibody L3H3 of anti-CD20 antigens and its applications.Anti- CD20 antigens provided by the invention, are named as L3H3 antibody, are a kind of IgG, are made of light chain and heavy chain；CDR1, CDR2 and CDR3 in light chain variable region in the light chain is followed successively by the sequence 1 of sequence table from the 61st 79 amino acids residue of N-terminal, the 89th 102 amino acids residue and the 112nd 123 amino acids residue；CDR1, CDR2 and CDR3 in heavy chain variable region in the heavy chain is followed successively by the sequence 3 of sequence table from the 50th 60 amino acids residue of N-terminal, the 82nd 97 amino acids residue and the 107th 120 amino acids residue.The present invention also protects application of the L3H3 antibody in preparing for B cell lymphoma medicine.The present invention is worth the treatment of B cell lymphoma with major application.

Description

Anti-CD20 Antigen Antibody L3H3 and Its Application

technical field

The invention relates to an anti-CD20 antigen antibody L3H3 and its application.

Background technique

Non-Hodgkin's lymphoma (NHL) is a malignant tumor of the lymphatic system with different biological characteristics and histological forms, and it is one of the top ten malignant tumors that threaten human life. % are derived from B cells.

CD20 molecule is a B cell differentiation antigen, which is expressed in more than 95% of B cell lymphomas, but not expressed in hematopoietic stem cells, plasma cells and other normal tissues. The molecular weight of CD20 is 33KD. It is a non-glycosylated protein composed of 279 amino acid residues. It is initially expressed in the pre-B cell stage and ends when the B cell terminal differentiates into plasma cells. It has always been considered as the surface of B lymphocytes. Unique logo. The CD20 molecule has four transmembrane regions, and there is a loop region composed of 43 amino acid residues between the third and fourth transmembrane regions, which constitutes its main antigenic epitope. CD20 molecules are stably expressed on the surface of malignant B lymphocyte membranes, and will not fall off from the surface of tumor cell membranes after binding to antibodies, and no free CD20 molecules exist in the blood circulation. The above-mentioned unique expression methods and biological functions determine that CD20 is a B cell. Ideal target antigen for lymphoma monoclonal antibody therapy.

In 1997, the US Food and Drug Administration (FDA) approved the anti-CD20 human-mouse chimeric antibody rituximab (Rituximab, RTX) for the treatment of NHL. In 1998, the European Union also approved RTX for marketing. The combination of RTX and CD20 molecules can trigger the immune response of lymphoma cell lysis. The possible mechanism of action includes: antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), Induces apoptosis. Although RTX can enhance the biological effects of antibodies, because it is a human-mouse chimeric antibody, it will produce severe human anti-mouse antibody reaction (HAMA) in clinical practice, thereby affecting its function. This study plans to screen out humanized antibodies with similar conformation to chimeric antibodies but low immunogenicity and high affinity from the anti-human antibody library through computer simulation, and test the ADCC and CDC effects of the humanized antibodies.

Contents of the invention

The object of the present invention is to provide an anti-CD20 antigen antibody L3H3 and its application.

The anti-CD20 antigen provided by the present invention, named as L3H3 antibody, is an IgG consisting of a light chain and a heavy chain; CDR1, CDR2 and CDR3 in the light chain variable region of the light chain are the sequences of the sequence listing in sequence 1 From N-terminal amino acid residues 61-79, amino acid residues 89-102 and amino acid residues 112-123; CDR1, CDR2 and CDR3 in the heavy chain variable region in the heavy chain Sequence 3 of the sequence listing starts from N-terminal amino acid residues 50-60, amino acid residues 82-97 and amino acid residues 107-120.

The light chain can specifically be the following (a) or (b): (a) a protein composed of amino acid residues at positions 21-233 of the N-terminal in sequence 1 of the sequence listing; (b) a protein shown in sequence 1 of the sequence listing protein;

Specifically, the heavy chain can be the following (c) or (d): (c) a protein composed of amino acid residues from N-terminal 20-471 in sequence 3 of the sequence listing; (d) a protein shown in sequence 3 of the sequence listing protein.

The present invention also protects the gene encoding the L3H3 antibody, characterized in that:

The gene encoding the light chain is as follows (1) or (2) or (3):

(1) the DNA molecule shown in the sequence 2 of the sequence listing from the 80th to the 718th nucleotide at the 5' end;

(2) the DNA molecule shown in the 20th-721st nucleotides from the 5' end of Sequence 2 in the sequence listing;

(3) DNA molecules shown in sequence 2 of the sequence listing;

The gene encoding the heavy chain is as follows (4) or (5) or (6):

(4) the DNA molecule shown in the 58th-1413th nucleotide from the 5' end of the sequence 5 of the sequence listing;

(5) DNA molecules shown in sequence 5 of the sequence listing;

(6) The DNA molecule shown in sequence 4 of the sequence listing.

The present invention also protects the application of the L3H3 antibody in the preparation of drugs for killing B-cell lymphoma cells. The B-cell lymphoma cells are B-cell lymphoma cells expressing CD20 antigen. Specifically, the B-cell lymphoma cells may be Daudi cells.

The invention also protects a medicine for killing B cell lymphoma cells, the active ingredient of which is the L3H3 antibody. The B-cell lymphoma cells are B-cell lymphoma cells expressing CD20 antigen. Specifically, the B-cell lymphoma cells may be Daudi cells.

The present invention also protects the application of the L3H3 antibody in the preparation of drugs for treating B-cell lymphoma. The B-cell lymphoma is a B-cell lymphoma expressing CD20 antigen.

The invention has great application value for the treatment of B cell lymphoma.

Description of drawings

Fig. 1 is the elution curve in embodiment 2.

FIG. 2 is the 10% SDS-PAGE pattern in Example 2.

FIG. 3 is the result of ADCC in Example 4.

FIG. 4 is the result of CDC in Example 4.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

pcDNA-3.3 vector (linear plasmid): Invitrogen Company, catalog number K8300-01. pOptiVEC vector (linear plasmid): Invitrogen, catalog number 12744-017. CHO-DG44 cells: Invitrogen, catalog number A13737-01. OptiPRO ^TM SFM culture medium: Invitrogen Company, catalog number 12309-050. Liposomes (FreeStyle ^™ MAX Reagent): Invitrogen, Cat. No. 16447-100. CD DG44 Medium: Invitrogen, Cat. No. 12610-010. CD OptiCHO ^™ Medium: Invitrogen, Cat. No. 12681-011. Rituximab (solution form; 100mg/10ml, colorless transparent liquid; IgG, its light chain is shown in sequence 6 of the sequence listing, and its heavy chain is shown in sequence 7 of the sequence listing): Germany Roche Diagnostics GmbH, Product batch number H0119, query serial number: 10000661731142.

Rituximab has about 30% mouse protein, long-term use will cause mouse anti-human antibody reaction and can not be used. Therefore, it is particularly urgent to seek a more effective anti-CD20 antigen antibody for the treatment of B lymphoma. The inventor designed an anti-CD20 antigen antibody (named L3H3 antibody) based on a lot of exploration, analysis, verification, computer modeling and humanization. The L3H3 antibody is IgG, its light chain (named light chain L3) is shown in sequence 1 of the sequence listing, and its heavy chain (named heavy chain H3) is shown in sequence 3 of the sequence listing.

In Sequence 1 of the sequence listing, amino acid residues 1-20 from the N-terminal form the leader peptide, and amino acid residues 21-131 form the light chain variable region VL (among them, amino acid residues 61-79 form the CDR1 , 89-102 amino acid residues constitute CDR2, 112-123 amino acid residues constitute CDR3), and 132-233 amino acid residues constitute the light chain constant region CL.

In Sequence 3 of the sequence listing, amino acid residues 1-19 from the N-terminal form the leader peptide, and amino acid residues 20-141 form the heavy chain variable region VH (among them, amino acid residues 50-60 form the CDR1 , amino acid residues 82-97 constitute CDR2, amino acid residues 107-120 constitute CDR3), amino acid residues 142-239 constitute the heavy chain constant region CH1, and amino acid residues 240-254 constitute the hinge region Hinge, amino acid residues 255-365 constitute the heavy chain constant region CH2, and amino acid residues 366-471 constitute the heavy chain constant region CH3.

Embodiment 1, construction recombinant plasmid

1. Insert the DNA molecule shown in sequence 2 of the sequence listing into the pcDNA-3.3 vector to obtain the recombinant plasmid pcDNA-3.3-L3.

The DNA molecule shown in Sequence 2 of the sequence listing encodes a leader peptide from the 20-79th nucleotide at the 5' end, the 80-412th nucleotide codes VL, the 413-718th nucleotide codes CL, and the 719th nucleotide Nucleotide -721 is a stop codon.

2. Insert the DNA molecule shown in sequence 4 in the sequence listing into the pOptiVEC vector to obtain the recombinant plasmid pOptiVEC-H3.

The DNA molecule shown in sequence 4 of the sequence listing encodes a leader peptide from the 14th to 70th nucleotides at the 5' end, the 71st to 436th nucleotides encode VH, the 437th to 730th nucleotides encode CH1, and the 731st -1118 nucleotides are introns, 1119-1163 nucleotides code Hinge, 1164-1281 nucleotides are introns, 1282-1614 nucleotides code CH2, 1615-1711 The first nucleotide is an intron, the 1712th-2029th nucleotide encodes CH3, the 2030th-2032th nucleotide is a stop codon, and the 2033-2231st nucleotide is an intron. That is, after the intron is removed, the coding region in sequence 4 of the sequence listing is shown in sequence 5 of the sequence listing.

Example 2, preparation of L3H3 antibody

1. Take 10 μg of recombinant plasmid pcDNA-3.3-L3 and 10 μg of recombinant plasmid pOptiVEC-H3, dilute to 500 μl with OptiPRO ^TM SFM culture medium, and place at room temperature for 5 minutes.

2. Take 20 μl of liposomes, dilute to 500 μl with OptiPRO ^TM SFM culture medium, and place at room temperature for 5 minutes.

3. Mix the solution of step 1 and step 2, and let it stand at room temperature for 20 minutes.

4. Inoculate CHO-DG44 cells (about ³ ×106 cells/flask) in a 125ml cell culture flask with a vent filter (30ml culture medium in the flask), centrifuge at 800rpm for 4min before transfection, and discard the culture medium. To clear, add 4ml of fresh CDDG44 Medium to each bottle to resuspend.

5. Add the solution obtained in step 3 dropwise into the cell culture bottle that completed step 4, mix well, and culture in a shaker containing 8% _CO at 37°C and 150rpm for 5 days (first cultivate for 6-8 hours, then Centrifuge at 800rpm for 4min, discard the supernatant, re-add 30ml of fresh CD OptiCHO ^™ Medium, and continue to cultivate), centrifuge at 12000rpm for 15min, collect the supernatant, adjust the pH to 6.0-7.0, and then filter with a 0.45μm filter membrane to collect the filtrate.

6. Take the filtrate obtained in step 5, and use rProtein A Sepharose 4B affinity chromatography column for purification.

rProtein A Sepharose 4B affinity chromatography column (Pharmacia, XK16 column): the column volume is 40 ml, and the inner diameter is 16 mm.

Binding buffer (pH7.0): 20mM phosphate buffer containing 0.15M NaCl.

Elution buffer (pH3.0): 0.1M citrate buffer.

Flow rate: 1-3ml/min.

Process: (1) equilibrate the column with 400ml binding buffer; (2) load the sample; (3) wash the column with 400ml binding buffer; (4) elute the target protein with 200ml elution buffer, the elution curve is shown in Figure 1 , Collect the post-column solution with a retention volume of 36-80ml.

7. Take the post-column solution obtained in step 6, adjust the pH to 7.0 with Tris aqueous solution (pH 9.0), and then use an ultrafiltration tube (Millipore Company) with a molecular weight cut-off of 30kd to concentrate, and the obtained solution contains L3H3 antibody solution, named L3H3 solution. The 10% SDS-PAGE profile of the L3H3 solution is shown in Figure 2.

8. Replace the recombinant plasmid pcDNA-3.3-L3 with the pcDNA-3.3 vector, replace the recombinant plasmid pOptiVEC-H3 with the pOptiVEC vector, and carry out steps 1 to 6 in sequence, and no elution peak appears during the elution process.

Embodiment 3, affinity detection

Entrust Beijing Kenuo Xincheng Technology Co., Ltd. to complete the process. The brief process is as follows: using Fortebio instrument, the biotinylated CD20 epitope (the amino acid sequence of the CD20 epitope is: CEP ANPS EKNSPSTQ YCYSI Q) is immobilized onto the Steptavidin chip 5 different concentrations of antibody solutions to be tested (dilute the L3H3 solution or rituximab prepared in Example 1 with pH 7.2, 0.01M PBS buffer solution to obtain different concentrations of antibody solutions to be tested, The protein concentration in each antibody solution to be tested is 0, 25, 50, 75 or 100nmol/L), and the binding (Kon) and dissociation (Koff) values are detected to calculate the affinity (Kd).

The affinity (Kd) of rituximab was 6.48×10 ^-9 M, and the affinity (Kd) of L3H3 antibody was 3.28×10 ^-9 M.

Embodiment 4, cell experiment

ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) and CDC (complement dependent cytotoxicity, complement-dependent cytotoxicity) are important ways in which antibody drugs can target and destroy target cells. These two mechanisms of action can be applied to in vitro research, evaluation, and screening of high-performance antibody drugs in the process of antibody drug development, which is of great significance. ADCC refers to the effect that effector cells expressing IgG Fc receptors (NK cells, macrophages, neutrophils, etc.) kill these target cells by binding to the Fc fragment of IgG antibodies that have bound to the surface of tumor cells. CDC activates the classic pathway of complement through the combination of specific antibodies and corresponding antigens on the cell membrane surface to form a complex, and the formed membrane attack complex exerts a lytic effect on target cells. Daudi cells (human lymphoma cell line): ATCC catalog number CCL-213, detected by flow cytometry (the labeled antibody used to detect CD20 antigen is FITC Mouse Anti-HumanCD20 purchased from BD Company with catalog number 556632), The expression rate of CD20 antigen was 92%. DIO cell membrane green fluorescent probe: Beijing Zhongsheng Ruitai Technology Co., Ltd., catalog number 60011. PI staining solution: Sigma Company, catalog number P4170-250MG.

1. Determine the optimal dosage of rituximab

Rituximab was reacted with Daudi cells at 37°C for 30 minutes (the reaction system was RPMI 1640 medium; the concentrations of rituximab in the reaction system were 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, respectively. ml or 50 μg/ml, in terms of protein concentration; the concentration of Daudi cells in the reaction system is 1×10 ⁶ /ml), and then the expression of CD20 antigen on Daudi cells was detected by flow cytometry, and the expression was close to zero The concentration of rituximab is the most suitable concentration.

The most suitable in vitro concentration of rituximab is 30 μg/ml.

2. ADCC

1. Use Ficoll-Hapaque to separate peripheral blood lymphocytes (PBL).

2. Label Daudi cells with DIO cell membrane green fluorescent probe (operate according to the instructions of DIO cell membrane green fluorescent probe) to obtain DIO-labeled cells.

3. Take the DIO-labeled cells obtained in step 2, and incubate with the antibody to be tested for 30 minutes in a 37° C. incubator (the reaction system is RPMI 1640 medium; the antibody to be tested is the L3H3 solution prepared in Example 1 or rituximab , the concentration of the antibody to be tested in the reaction system is 30 μg/ml, in terms of protein concentration; the concentration of cells in the reaction system is 1×10 ⁶ /ml), and then the cells are collected and buffered with PBS with pH 7.2 and 0.01M Liquid wash 2 times.

4. Take the cells (target cells) obtained in step 3 and incubate them with the PBL (effector cells) obtained in step 1 for 4 hours in a 37° C. incubator (the reaction system is RPMI 1640 medium; the concentration of target cells in the reaction system is 1×10 ⁴ /ml; the concentration of effector cells in the reaction system is 5×10 ⁵ /ml or 2.5×10 ⁵ /ml respectively, that is, the target-to-effect ratio is 1:50 or 1:25), and then add PI to stain solution and detected by flow cytometry.

Three replicate samples were set for each treatment, and the results were averaged.

DIO-labeled intact cells appear green. Under the action of effector cells, the target cells are broken, and PI enters the nucleus and stains, showing red. Lysis rate of target cells = number of red cells ÷ (number of red cells + number of green cells) × 100%.

The results are shown in Figure 3. The results show that, under the condition of equal target-efficiency ratio, the target cell lysis rate of adding L3H3 antibody is higher than that of adding rituximab, that is, the effect of L3H3 antibody is better than that of rituximab, and can be more effective. Significantly promotes target cell lysis.

3. CDC

1. Label Daudi cells with DIO cell membrane green fluorescent probe (operate according to the instructions of DIO cell membrane green fluorescent probe) to obtain DIO-labeled cells.

2. Take healthy human serum and inactivate it to obtain inactivated serum.

3. Get the DIO-labeled cells obtained in step 1, and incubate them with the antibody to be tested (the L3H3 solution or rituximab prepared in Example 1) and the inactivated serum obtained in step 2 in a 37° C. incubator for 4 hours (reaction The system is RPMI1640 medium; the concentration of target cells in the reaction system is 1×10 ⁶ /ml; the concentration of the antibody to be tested in the reaction system is 30 μg/ml, calculated as protein concentration; the volume of inactivated serum in the reaction system The percentage concentrations were 20% and 10% respectively; a control group without adding inactivated serum was set, represented by 0% serum concentration), and then the cells were collected and washed twice with PBS buffer solution of pH 7.2 and 0.01M.

4. Take the DIO-labeled cells obtained in step 1, and incubate them with the antibody to be tested (the L3H3 solution or rituximab prepared in Example 1) and healthy human serum in a 37° C. incubator for 4 hours (the reaction system is RPMI 1640 Culture medium; the concentration of target cells in the reaction system is 1×10 ⁶ /ml; the concentration of the antibody to be tested in the reaction system is 30 μg/ml, expressed as protein concentration; the volume percent concentration of healthy human serum in the reaction system 20% and 10% respectively; a control group without addition of healthy human serum was set up, represented by 0% serum concentration), and then the cells were collected and washed twice with PBS buffer solution of pH 7.2 and 0.01M.

5. Take the cells obtained in step 3 or step 4, and resuspend them with pH 7.2, 0.01M PBS buffer to obtain a cell suspension with a cell concentration of 1×10 ⁵ /ml, then add PI staining solution and pass flow cytometry Cytometry detection.

Three replicate samples were set for each treatment, and the results were averaged.

DIO-labeled intact cells appear green. Under the action of effector cells, the target cells are broken, and PI enters the nucleus and stains, showing red. Lysis rate of target cells = number of red cells ÷ (number of red cells + number of green cells) × 100%.

The results are shown in Figure 4. The results showed that in the case of adding healthy human serum, the target cell lysis rate of adding L3H3 antibody was higher than that of adding rituximab, that is, the effect of L3H3 antibody was better than that of rituximab, and it could be more effective. Significantly promotes target cell lysis.

In summary, the L3H3 antibody has a good anti-Daudi cell (B-cell lymphoma cell expressing CD20 antigen) growth effect, and is expected to become an antibody drug for clinical treatment.

Claims (7)

1. a kind of IgG, is made of light chain and heavy chain；

The light chain is for following (a) or (b)：(a) sequence 1 of sequence table is formed from N-terminal 21-233 amino acids residues Protein；(b) protein shown in the sequence 1 of sequence table；

The heavy chain is for following (c) or (d)：(c) sequence 3 of sequence table is formed from N-terminal 20-471 amino acids residues Protein；(d) protein shown in the sequence 3 of sequence table.

2. encoding the gene of IgG described in claim 1, it is characterised in that：

The gene for encoding the light chain is following (1) or (2) or (3)：

(1) sequence 2 of sequence table DNA molecular shown in the nucleotide of 5 ' end 80-718；

(2) sequence 2 of sequence table DNA molecular shown in the nucleotide of 5 ' end 20-721；

(3) DNA molecular shown in the sequence 2 of sequence table；

The gene for encoding the heavy chain is following (4) or (5) or (6)：

(4) sequence 5 of sequence table DNA molecular shown in the nucleotide of 5 ' end 58-1413；

(5) DNA molecular shown in the sequence 5 of sequence table；

(6) DNA molecular shown in the sequence 4 of sequence table.

3. applications of the IgG described in claim 1 in preparing the drug for killing B cell lymphoma cell；The B cell leaching Bar oncocyte is the B cell lymphoma cell for expressing CD20 antigens.

4. application as claimed in claim 3, it is characterised in that：The B cell lymphoma cell is Daudi cells.

5. a kind of drug for killing B cell lymphoma cell, active constituent is IgG described in claim 1；The B is thin Born of the same parents' lymphoma cell is the B cell lymphoma cell for expressing CD20 antigens.

6. drug as claimed in claim 5, it is characterised in that：The B cell lymphoma cell is Daudi cells.

7. applications of the IgG described in claim 1 in preparing for B cell lymphoma medicine；The B cell lymphoma is thin Born of the same parents are the B cell lymphoma cell for expressing CD20 antigens.